Medical Devices; Hematology and Pathology Devices; Classification of the Flow Cytometric Test System for Hematopoietic Neoplasms. Final order.

PubMed

2017-12-27

The Food and Drug Administration (FDA or we) is classifying the flow cytometric test system for hematopoietic neoplasms into class II (special controls). The special controls that apply to the device type are identified in this order and will be part of the codified language for the flow cytometric test system for hematopoietic neoplasms' classification. We are taking this action because we have determined that classifying the device into class II (special controls) will provide a reasonable assurance of safety and effectiveness of the device. We believe this action will also enhance patients' access to beneficial innovative devices, in part by reducing regulatory burdens.

Development of an automated analysis system for data from flow cytometric intracellular cytokine staining assays from clinical vaccine trials

PubMed Central

Shulman, Nick; Bellew, Matthew; Snelling, George; Carter, Donald; Huang, Yunda; Li, Hongli; Self, Steven G.; McElrath, M. Juliana; De Rosa, Stephen C.

2008-01-01

Background Intracellular cytokine staining (ICS) by multiparameter flow cytometry is one of the primary methods for determining T cell immunogenicity in HIV-1 clinical vaccine trials. Data analysis requires considerable expertise and time. The amount of data is quickly increasing as more and larger trials are performed, and thus there is a critical need for high throughput methods of data analysis. Methods A web based flow cytometric analysis system, LabKey Flow, was developed for analyses of data from standardized ICS assays. A gating template was created manually in commercially-available flow cytometric analysis software. Using this template, the system automatically compensated and analyzed all data sets. Quality control queries were designed to identify potentially incorrect sample collections. Results Comparison of the semi-automated analysis performed by LabKey Flow and the manual analysis performed using FlowJo software demonstrated excellent concordance (concordance correlation coefficient >0.990). Manual inspection of the analyses performed by LabKey Flow for 8-color ICS data files from several clinical vaccine trials indicates that template gates can appropriately be used for most data sets. Conclusions The semi-automated LabKey Flow analysis system can analyze accurately large ICS data files. Routine use of the system does not require specialized expertise. This high-throughput analysis will provide great utility for rapid evaluation of complex multiparameter flow cytometric measurements collected from large clinical trials. PMID:18615598

RNA flow cytometric FISH for investigations into HIV immunology, vaccination and cure strategies.

PubMed

Baxter, Amy E; Niessl, Julia; Morou, Antigoni; Kaufmann, Daniel E

2017-09-12

Despite the tremendous success of anti-retroviral therapy (ART) no current treatment can eradicate latent HIV reservoirs from HIV-infected individuals or generate, effective HIV-specific immunity. Technological limitations have hampered the identification and characterization of both HIV-infected cells and HIV-specific responses in clinical samples directly ex vivo. RNA-flow cytometric fluorescence in situ hybridisation (RNA Flow-FISH) is a powerful technique, which enables detection of mRNAs in conjunction with proteins at a single-cell level. Here, we describe how we are using this technology to address some of the major questions remaining in the HIV field in the era of ART. We discuss how CD4 T cell responses to HIV antigens, both following vaccination and HIV infection, can be characterized by measurement of cytokine mRNAs. We describe how our development of a dual HIV mRNA/protein assay (HIV RNA/Gag assay) enables high sensitivity detection of very rare HIV-infected cells and aids investigations into the translation-competent latent reservoir in the context of HIV cure.

Flow cytometric method for measuring chromatin fragmentation in fixed sperm from yellow perch (Perca flavescens).

PubMed

Jenkins, J A; Draugelis-Dale, R O; Pinkney, A E; Iwanowicz, L R; Blazer, V S

2015-03-15

Declining harvests of yellow perch, Perca flavescens, in urbanized watersheds of Chesapeake Bay have prompted investigations of their reproductive fitness. The purpose of this study was to establish a flow cytometric technique for DNA analysis of fixed samples sent from the field to provide reliable gamete quality measurements. Similar to the sperm chromatin structure assay, measures were made on the susceptibility of nuclear DNA to acid-induced denaturation, but used fixed rather than live or thawed cells. Nuclei were best exposed to the acid treatment for 1 minute at 37 °C followed by the addition of cold (4 °C) propidium iodide staining solution before flow cytometry. The rationale for protocol development is presented graphically through cytograms. Field results collected in 2008 and 2009 revealed DNA fragmentation up to 14.5%. In 2008, DNA fragmentation from the more urbanized watersheds was significantly greater than from reference sites (P = 0.026) and in 2009, higher percentages of haploid testicular cells were noted from the less urbanized watersheds (P = 0.032) indicating better reproductive condition at sites with less urbanization. For both years, total and progressive live sperm motilities by computer-assisted sperm motion analysis ranged from 19.1% to 76.5%, being significantly higher at the less urbanized sites (P < 0.05). This flow cytometric method takes advantage of the propensity of fragmented DNA to be denatured under standard conditions, or 1 minute at 37 °C with 10% buffered formalin-fixed cells. The study of fixed sperm makes possible the restrospective investigation of germplasm fragmentation, spermatogenic ploidy patterns, and chromatin compaction levels from samples translocated over distance and time. The protocol provides an approach that can be modified for other species across taxa. Published by Elsevier Inc.

Flow cytometric method for measuring chromatin fragmentation in fixed sperm from yellow perch (Perca flavescens)

USGS Publications Warehouse

Jenkins, Jill A.; Draugelis-Dale, Rassa O.; Pinkney, Alfred E.; Iwanowicz, Luke R.; Blazer, Vicki

2015-01-01

Declining harvests of yellow perch, Perca flavescens, in urbanized watersheds of Chesapeake Bay have prompted investigations of their reproductive fitness. The purpose of this study was to establish a flow cytometric technique for DNA analysis of fixed samples sent from the field to provide reliable gamete quality measurements. Similar to the sperm chromatin structure assay, measures were made on the susceptibility of nuclear DNA to acid-induced denaturation, but used fixed rather than live or thawed cells. Nuclei were best exposed to the acid treatment for 1 minute at 37 °C followed by the addition of cold (4 °C) propidium iodide staining solution before flow cytometry. The rationale for protocol development is presented graphically through cytograms. Field results collected in 2008 and 2009 revealed DNA fragmentation up to 14.5%. In 2008, DNA fragmentation from the more urbanized watersheds was significantly greater than from reference sites (P = 0.026) and in 2009, higher percentages of haploid testicular cells were noted from the less urbanized watersheds (P = 0.032) indicating better reproductive condition at sites with less urbanization. For both years, total and progressive live sperm motilities by computer-assisted sperm motion analysis ranged from 19.1% to 76.5%, being significantly higher at the less urbanized sites (P < 0.05). This flow cytometric method takes advantage of the propensity of fragmented DNA to be denatured under standard conditions, or 1 minute at 37 °C with 10% buffered formalin–fixed cells. The study of fixed sperm makes possible the restrospective investigation of germplasm fragmentation, spermatogenic ploidy patterns, and chromatin compaction levels from samples translocated over distance and time. The protocol provides an approach that can be modified for other species across taxa.

Flow cytometry, morphometry and histopathology as biomarkers of benzo[a]pyrene exposure in brown bullheads (ameiurus nebulosus)

USGS Publications Warehouse

Grady, Andrew W.; McLaughlin, Ronald M.; Caldwell, Charles W.; Schmitt, Christopher J.; Stalling, David L.

1992-01-01

Brown bullheads were given a single intraperitoneal dose of 0, 5, 25 or 125 mg kg−1 benzo[a]pyrene (BaP), a carcinogenic polycyclic aromatic hydrocarbon, and evaluated over 18 months. Flow cytometric analyses of hepatocyte DNA content indicated an increase in DNA synthesis in BaP-exposed fish prior to day 14 post-exposure. Thereafter, all flow cytometric variables returned to initial levels. Histopathological evaluation of livers from fish sampled at 18 months revealed significant differences among treatments in the amount of hepatic macrophage ceroid pigmentation and basophilic staining intensity. No neoplasms or changes in blood cell DNA content were detected. Significant morphometric variations existed among fish, but differences between sexes overshadowed differences attributable to dose. Flow cytometry yielded no evidence of long-term DNA alterations from a single exposure to BaP; however, the differences detected by DNA analysis shortly after the toxic event suggest that flow cytometric cell cycle analysis may be useful for documenting continuing exposures.

Rapid flow cytometric measurement of protein inclusions and nuclear trafficking

PubMed Central

Whiten, D. R.; San Gil, R.; McAlary, L.; Yerbury, J. J.; Ecroyd, H.; Wilson, M. R.

2016-01-01

Proteinaceous cytoplasmic inclusions are an indicator of dysfunction in normal cellular proteostasis and a hallmark of many neurodegenerative diseases. We describe a simple and rapid new flow cytometry-based method to enumerate, characterise and, if desired, physically recover protein inclusions from cells. This technique can analyse and resolve a broad variety of inclusions differing in both size and protein composition, making it applicable to essentially any model of intracellular protein aggregation. The method also allows rapid quantification of the nuclear trafficking of fluorescently labelled molecules. PMID:27516358

Cytometric analysis of shape and DNA content in mammalian sperm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gledhill, B.L.

1983-10-10

Male germ cells respond dramatically to a variety of insults and are important reproductive dosimeters. Semen analyses are very useful in studies on the effects of drugs, chemicals, and environmental hazards on testicular function, male fertility and heritable germinal mutations. Sperm were analyzed by flow cytometry and slit-scan flow analysis for injury following the exposure of testes to mutagens. The utility of flow cytometry in genotoxin screening and monitoring of occupational exposure was evaluated. The technique proved valuable in separation of X- and Y-chromosome bearing sperm and the potential applicability of this technique in artificial insemination and a solution, ofmore » accurately assessing the DNA content of sperm were evaluated-with reference to determination of X- and Y-chromosome bearing sperm.« less

Flow cytometric osmotic fragility test and eosin-5'-maleimide dye-binding tests are better than conventional osmotic fragility tests for the diagnosis of hereditary spherocytosis.

PubMed

Arora, R D; Dass, J; Maydeo, S; Arya, V; Radhakrishnan, N; Sachdeva, A; Kotwal, J; Bhargava, M

2018-06-01

Hereditary spherocytosis (HS) is the most common inherited hemolytic anemia with heterogeneous clinico-laboratory manifestations. We evaluated the flow-cytometric tests: eosin-5'-maleimide (EMA) and flow-cytometric osmotic fragility test (FOFT) and the conventional osmotic fragility tests (OFT) for the diagnosis of hereditary spherocytosis (HS). One hundred two suspected HS patients underwent EMA, FOFT, incubated OFT (IOFT), and room temperature OFT (RT-OFT). In addition, 10 cases of immune hemolytic anemia (IHA) were included, and performance of the above 4 tests was evaluated. For EMA and FOFT, 5 normal controls were assessed together with the patients and cutoffs were calculated using receiver-operator-characteristics curve (ROC) analysis. The best cutoff for %EMA decrease was 12.5%, and for FOFT, %residual red cells (%RRC) was 25.6%. The sensitivity and specificity of RT-OFT was 62.06% and 86.3%, respectively, while that of IOFT was 79.31% and 87.67%, respectively. Both flow cytometric tests performed better. Sensitivity and specificity of EMA was 86.2% and 93.9% respectively, and that of FOFT was 96.6% and 98.63%, respectively. The combination of the FOFT with IOFT or EMA dye-binding test yields a sensitivity of 100%, but with EMA, it had a higher specificity. Hb/MCHC was a predictor of the severity of the disease while %EMA decrease and %RRC did not correlate with severity of the disease. Flow-cytometric osmotic fragility test is the best possible single test followed by EMA for diagnosis of HS. A combination of FOFT and EMA can correctly diagnose 100% patients. These tests are likely to replace conventional OFTs in future. © 2018 John Wiley & Sons Ltd.

Recommendations for the evaluation of specimen stability for flow cytometric testing during drug development.

PubMed

Brown, Lynette; Green, Cherie L; Jones, Nicholas; Stewart, Jennifer J; Fraser, Stephanie; Howell, Kathy; Xu, Yuanxin; Hill, Carla G; Wiwi, Christopher A; White, Wendy I; O'Brien, Peter J; Litwin, Virginia

2015-03-01

The objective of this manuscript is to present an approach for evaluating specimen stability for flow cytometric methods used during drug development. While this approach specifically addresses stability assessment for assays to be used in clinical trials with centralized testing facilities, the concepts can be applied to any stability assessment for flow cytometric methods. The proposed approach is implemented during assay development and optimization, and includes suggestions for designing a stability assessment plan, data evaluation and acceptance criteria. Given that no single solution will be applicable in all scenarios, this manuscript offers the reader a roadmap for stability assessment and is intended to guide the investigator during both the method development phase and in the experimental design of the validation plan. Copyright © 2015 Elsevier B.V. All rights reserved.

Flow Cytometry with Gold Nanoparticles and their Clusters as scattering Contrast Agents: FDTD Simulation of Light-Cell Interaction

PubMed Central

Tanev, Stoyan; Sun, Wenbo; Pond, James; Tuchin, Valery V.; Zharov, Vladimir P.

2010-01-01

The formulation of the Finite-Difference Time-Domain (FDTD) approach is presented in the framework of its potential applications to in vivo flow cytometry based on light scattering. The consideration is focused on comparison of light scattering by a single biological cell alone in controlled refractive index matching conditions and by cells labeled by gold nanoparticles. The optical schematics including phase contrast (OPCM) microscopy as a prospective modality for in vivo flow cytometry is also analyzed. The validation of the FDTD approach for the simulation of flow cytometry may open a new avenue in the development of advanced cytometric techniques based on scattering effects from nanoscale targets. PMID:19670359

Flow cytometry of duodenal intraepithelial lymphocytes improves diagnosis of celiac disease in difficult cases.

PubMed

Valle, Julio; Morgado, José Mario T; Ruiz-Martín, Juan; Guardiola, Antonio; Lopes-Nogueras, Miriam; García-Vela, Almudena; Martín-Sacristán, Beatriz; Sánchez-Muñoz, Laura

2017-10-01

Diagnosis of celiac disease is difficult when the combined results of serology and histology are inconclusive. Studies using flow cytometry of intraepithelial lymphocytes (IELs) have found that celiac patients have increased numbers of γδ IELs, along with a decrease in CD3-CD103 + IELs. The objective of this article is to assess the role of flow cytometric analysis of IELs in the diagnosis of celiac disease in difficult cases. A total of 312 patients with suspicion of celiac disease were included in the study. Duodenal biopsy samples were used for histological assessment and for flow cytometric analysis of IELs. In 46 out of 312 cases (14.7%) the combination of serology and histology did not allow the confirmation or exclusion of celiac disease. HLA typing had been performed in 42 of these difficult cases. Taking into account HLA typing and the response to a gluten-free diet, celiac disease was excluded in 30 of these cases and confirmed in the remaining 12. Flow cytometric analysis of IELs allowed a correct diagnosis in 39 out of 42 difficult cases (92.8%) and had a sensitivity of 91.7% (95% CI: 61.5% to 99.8%) and a specificity of 93.3% (95% CI: 77.9% to 99.2%) for the diagnosis of celiac disease in this setting. Flow cytometric analysis of IELs is useful for the diagnosis of celiac disease in difficult cases.

Flow cytometry of duodenal intraepithelial lymphocytes improves diagnosis of celiac disease in difficult cases

PubMed Central

Morgado, José Mario T; Ruiz-Martín, Juan; Guardiola, Antonio; Lopes-Nogueras, Miriam; García-Vela, Almudena; Martín-Sacristán, Beatriz; Sánchez-Muñoz, Laura

2016-01-01

Background Diagnosis of celiac disease is difficult when the combined results of serology and histology are inconclusive. Studies using flow cytometry of intraepithelial lymphocytes (IELs) have found that celiac patients have increased numbers of γδ IELs, along with a decrease in CD3-CD103 + IELs. Objective The objective of this article is to assess the role of flow cytometric analysis of IELs in the diagnosis of celiac disease in difficult cases. Methods A total of 312 patients with suspicion of celiac disease were included in the study. Duodenal biopsy samples were used for histological assessment and for flow cytometric analysis of IELs. Results In 46 out of 312 cases (14.7%) the combination of serology and histology did not allow the confirmation or exclusion of celiac disease. HLA typing had been performed in 42 of these difficult cases. Taking into account HLA typing and the response to a gluten-free diet, celiac disease was excluded in 30 of these cases and confirmed in the remaining 12. Flow cytometric analysis of IELs allowed a correct diagnosis in 39 out of 42 difficult cases (92.8%) and had a sensitivity of 91.7% (95% CI: 61.5% to 99.8%) and a specificity of 93.3% (95% CI: 77.9% to 99.2%) for the diagnosis of celiac disease in this setting. Conclusion Flow cytometric analysis of IELs is useful for the diagnosis of celiac disease in difficult cases. PMID:29026596

Detection of early changes in lung cell cytology by flow-systems analysis techniques. [Rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steinkamp, J.A.; Wilson, J.S.; Svitra, Z.V.

1980-03-01

Ongoing experiments designed to develop automated flow-analysis methods for assaying damage to pulmonary lavage cells in experimental animals exposed by inhalation to environmental pollutants are summarized. Pulmonary macrophages were characterized on their ability to phagocytize polystyrene latex fluorescent spheres. Lung cells consisting primarily of macrophages and leukocytes were analyzed for fluorescence (phagocytosis of spheres) and size using flow cytometric methods. Studies also concentrated on combining phagocytosis with other cellular parameters (DNA content, cell viability, and B-glucuronidase activity). As baseline studies are completed in normal animals, experimental animals will be exposed to gaseous and particulate environmental pollutants. (ERB

Wide-field imaging and flow cytometric analysis of cancer cells in blood by fluorescent nanodiamond labeling and time gating

NASA Astrophysics Data System (ADS)

Hui, Yuen Yung; Su, Long-Jyun; Chen, Oliver Yenjyh; Chen, Yit-Tsong; Liu, Tzu-Ming; Chang, Huan-Cheng

2014-07-01

Nanodiamonds containing high density ensembles of negatively charged nitrogen-vacancy (NV-) centers are promising fluorescent biomarkers due to their excellent photostability and biocompatibility. The NV- centers in the particles have a fluorescence lifetime of up to 20 ns, which distinctly differs from those (<10 ns) of cell and tissue autofluorescence, making it possible to achieve background-free detection in vivo by time gating. Here, we demonstrate the feasibility of using fluorescent nanodiamonds (FNDs) as optical labels for wide-field time-gated fluorescence imaging and flow cytometric analysis of cancer cells with a nanosecond intensified charge-coupled device (ICCD) as the detector. The combined technique has allowed us to acquire fluorescence images of FND-labeled HeLa cells in whole blood covered with a chicken breast of ~0.1-mm thickness at the single cell level, and to detect individual FND-labeled HeLa cells in blood flowing through a microfluidic device at a frame rate of 23 Hz, as well as to locate and trace FND-labeled lung cancer cells in the blood vessels of a mouse ear. It opens a new window for real-time imaging and tracking of transplanted cells (such as stem cells) in vivo.

Physiology of spermatozoa at high dilution rates: the influence of seminal plasma.

PubMed

Maxwell, W M; Johnson, L A

1999-12-01

Extensive dilution of spermatozoa, as occurs during flow-cytometric sperm sorting, can reduce their motility and viability. These effects may be minimized by the use of appropriate dilution and collection media, containing balanced salts, energy sources, egg yolk and some protein. Dilution and flow-cytometric sorting of spermatozoa, which involves the removal of seminal plasma, also destabilizes sperm membranes leading to functional capacitation. This membrane destabilization renders the spermatozoa immediately capable of fertilization in vitro, or in vivo after deposition close to the site of fertilization, but shortens their lifespan, resulting in premature death if the cells are deposited in the female tract distant from the site of fertilization or are held in vitro at standard storage temperatures. This functional capacitation can be reversed in boar spermatozoa by inclusion of seminal plasma in the medium used to collect the cells from the cell sorter and, consequently, reduces their in vitro fertility. It has yet to be determined whether seminal plasma would have similar effects on flow cytometrically sorted spermatozoa of other species, and what its effects might be on the in vivo fertility of flow sorted boar.

Flow karyotyping and sorting of human chromosomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gray, J.W.; Lucas, J.; Peters, D.

1986-07-16

Flow cytometry and sorting are becoming increasingly useful as tools for chromosome classfication and for the detection of numerical and structural chromosome aberrations. Chromosomes of a single type can be purified with these tools to facilitate gene mapping or production of chromosome specific recombinant DNA libraries. For analysis of chromosomes with flow cytometry, the chromosomes are extracted from mitotic cells, stained with one or more fluorescent dyes and classified one-by-one according to their dye content(s). Thus, the flow approach is fundamentally different than conventional karyotyping where chromosomes are classified within the context of a metaphase spread. Flow sorting allows purificationmore » of chromosomes that can be distinguished flow cytometrically. The authors describe the basic principles of flow cytometric chromosome classification i.e. flow karyotyping, and chromosome sorting and describe several applications. 30 refs., 8 figs.« less

Novel flow cytometric analysis of the progress and route of internalization of a monoclonal anti-carcinoembryonic antigen (CEA) antibody.

PubMed

Ford, C H; Tsaltas, G C; Osborne, P A; Addetia, K

1996-03-01

A flow cytometric method of studying the internalization of a monoclonal antibody (Mab) directed against carcinoembryonic antigen (CEA) has been compared with Western blotting, using three human colonic cancer cell lines which express varying amounts of the target antigen. Cell samples incubated for increasing time intervals with fluoresceinated or unlabelled Mab were analyzed using flow cytometry or polyacrylamide gel electrophoresis and Western blotting. SDS/PAGE analysis of cytosolic and membrane components of solubilized cells from the cell lines provided evidence of non-degraded internalized anti-CEA Mab throughout seven half hour intervals, starting at 5 min. Internalized anti-CEA was detected in the case of high CEA expressing cell lines (LS174T, SKCO1). Very similar results were obtained with an anti-fluorescein flow cytometric assay. Given that these two methods consistently provided comparable results, use of flow cytometry for the detection of internalized antibody is suggested as a rapid alternative to most currently used methods for assessing antibody internalization. The question of the endocytic route followed by CEA-anti-CEA complexes was addressed by using hypertonic medium to block clathrin mediated endocytosis.

Viability and membrane integrity of spermatozoa after dilution and flow cytometric sorting in the presence or absence of seminal plasma.

PubMed

Maxwell, W M; Welch, G R; Johnson, L A

1996-01-01

Boar, bull and ram spermatozoa were examined after staining with the DNA-permeant Hoechst 33342 fluorochrome and flow cytometric sorting in the presence or absence of seminal plasma. Spermatozoa were assessed for viability with flow cytometry using the live cell nucleic acid stain SYBR-14 and propidium iodide (PI), and for membrane integrity using fluorescein isothiocyanate-conjugated Pisum sativum (FITC-PSA) and PI; motility and acrosome integrity were estimated by microscopy. Flow cytometric sorting was compared with pipette dilution of boar and bull spermatozoa into: (1) medium [boar: Test buffer containing 2% yolk (TY) or Beltsville thawing solution (BTS); bull: TY or HEPES buffer containing 0.1% bovine serum albumin (HEPES-BSA)] with or without 10% (v/v) seminal plasma; or (2) an empty tube containing no medium. Sorted spermatozoa were either not centrifuged or centrifuged before assessment during a 4-h holding period. The viability, motility and membrane integrity of boar, bull and ram spermatozoa centrifuged after sorting were also examined when seminal plasma was present or absent from the staining extender and/or the TY collection medium. The results indicate that the viability and membrane integrity of spermatozoa in vitro would be improved if: (1) seminal plasma (10%) was routinely included in the BTS and HEPES-BSA staining extenders for boar spermatozoa and ram spermatozoa, respectively, when used in preparation for flow cytometric sorting; and (2) 10% and 50% seminal plasma were included in the TY collection medium for boar or bull spermatozoa and ram spermatozoa respectively.

Flow Cytometric Quantification of Peripheral Blood Cell β-Adrenergic Receptor Density and Urinary Endothelial Cell-Derived Microparticles in Pulmonary Arterial Hypertension.

PubMed

Rose, Jonathan A; Wanner, Nicholas; Cheong, Hoi I; Queisser, Kimberly; Barrett, Patrick; Park, Margaret; Hite, Corrine; Naga Prasad, Sathyamangla V; Erzurum, Serpil; Asosingh, Kewal

2016-01-01

Pulmonary arterial hypertension (PAH) is a heterogeneous disease characterized by severe angiogenic remodeling of the pulmonary artery wall and right ventricular hypertrophy. Thus, there is an increasing need for novel biomarkers to dissect disease heterogeneity, and predict treatment response. Although β-adrenergic receptor (βAR) dysfunction is well documented in left heart disease while endothelial cell-derived microparticles (Ec-MPs) are established biomarkers of angiogenic remodeling, methods for easy large clinical cohort analysis of these biomarkers are currently absent. Here we describe flow cytometric methods for quantification of βAR density on circulating white blood cells (WBC) and Ec-MPs in urine samples that can be used as potential biomarkers of right heart failure in PAH. Biotinylated β-blocker alprenolol was synthesized and validated as a βAR specific probe that was combined with immunophenotyping to quantify βAR density in circulating WBC subsets. Ec-MPs obtained from urine samples were stained for annexin-V and CD144, and analyzed by a micro flow cytometer. Flow cytometric detection of alprenolol showed that βAR density was decreased in most WBC subsets in PAH samples compared to healthy controls. Ec-MPs in urine was increased in PAH compared to controls. Furthermore, there was a direct correlation between Ec-MPs and Tricuspid annular plane systolic excursion (TAPSE) in PAH patients. Therefore, flow cytometric quantification of peripheral blood cell βAR density and urinary Ec-MPs may be useful as potential biomarkers of right ventricular function in PAH.

Flow Cytometric Quantification of Peripheral Blood Cell β-Adrenergic Receptor Density and Urinary Endothelial Cell-Derived Microparticles in Pulmonary Arterial Hypertension

PubMed Central

Rose, Jonathan A.; Wanner, Nicholas; Cheong, Hoi I.; Queisser, Kimberly; Barrett, Patrick; Park, Margaret; Hite, Corrine; Naga Prasad, Sathyamangla V.; Erzurum, Serpil; Asosingh, Kewal

2016-01-01

Pulmonary arterial hypertension (PAH) is a heterogeneous disease characterized by severe angiogenic remodeling of the pulmonary artery wall and right ventricular hypertrophy. Thus, there is an increasing need for novel biomarkers to dissect disease heterogeneity, and predict treatment response. Although β-adrenergic receptor (βAR) dysfunction is well documented in left heart disease while endothelial cell-derived microparticles (Ec-MPs) are established biomarkers of angiogenic remodeling, methods for easy large clinical cohort analysis of these biomarkers are currently absent. Here we describe flow cytometric methods for quantification of βAR density on circulating white blood cells (WBC) and Ec-MPs in urine samples that can be used as potential biomarkers of right heart failure in PAH. Biotinylated β-blocker alprenolol was synthesized and validated as a βAR specific probe that was combined with immunophenotyping to quantify βAR density in circulating WBC subsets. Ec-MPs obtained from urine samples were stained for annexin-V and CD144, and analyzed by a micro flow cytometer. Flow cytometric detection of alprenolol showed that βAR density was decreased in most WBC subsets in PAH samples compared to healthy controls. Ec-MPs in urine was increased in PAH compared to controls. Furthermore, there was a direct correlation between Ec-MPs and Tricuspid annular plane systolic excursion (TAPSE) in PAH patients. Therefore, flow cytometric quantification of peripheral blood cell βAR density and urinary Ec-MPs may be useful as potential biomarkers of right ventricular function in PAH. PMID:27270458

The importance of platelets in the expression of monocyte tissue factor antigen measured by a new whole blood flow cytometric assay.

PubMed

Amirkhosravi, A; Alexander, M; May, K; Francis, D A; Warnes, G; Biggerstaff, J; Francis, J L

1996-01-01

Previous methods for the determination of monocyte tissue factor (TF) have been technically complex, difficult to standardize, prone to spuriously elevated results and difficult to implement in a clinical laboratory environment. We report the development of a two-color whole blood cytometric technique that overcomes many of these disadvantages. The assay uses small volumes of citrated blood (1.0 ml), can be performed in under one hour (if endotoxin stimulation is not performed), is reproducible (CV = 5%) and uses methodology commonly available in clinical laboratories. Baseline (mean +/- SD) expression of monocyte TF in normal subjects was very low (1.1 +/- 0.95%, Mean Fluorescence [Mean FL] 0.20 +/- 0.01) making relatively small increases easy to detect. Monocyte TF expression following endotoxin (LPS) stimulation for 1 h was 34.6 +/- 11.2% (Mean FL 0.32 +/- 0.04). LPS-stimulated activity varied between subjects (21-68%) but was remarkably consistent for individual subjects (CV = 5.4%). Stimulated monocyte TF expression was directly proportional to the platelet count and was reduced by platelet protective anticoagulants and by ingestion of aspirin. Non LPS-stimulated monocyte TF was markedly increased, in a dose-dependent manner, by adding collagen to whole blood. This was apparently associated with platelet-monocyte binding and could be abolished by anti-P-Selectin. We conclude that the whole blood flow cytometric assay of monocyte TF may be a valuable tool for clinical use and a useful model system for evaluating the humoral and cellular factors governing monocyte TF expression in a natural environment.

Flow cytometry with gold nanoparticles and their clusters as scattering contrast agents: FDTD simulation of light-cell interaction.

PubMed

Tanev, Stoyan; Sun, Wenbo; Pond, James; Tuchin, Valery V; Zharov, Vladimir P

2009-09-01

The formulation of the finite-difference time-domain (FDTD) approach is presented in the framework of its potential applications to in-vivo flow cytometry based on light scattering. The consideration is focused on comparison of light scattering by a single biological cell alone in controlled refractive-index matching conditions and by cells labeled by gold nanoparticles. The optical schematics including phase contrast (OPCM) microscopy as a prospective modality for in-vivo flow cytometry is also analyzed. The validation of the FDTD approach for the simulation of flow cytometry may open up a new avenue in the development of advanced cytometric techniques based on scattering effects from nanoscale targets. 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Flow cytometric detection of human immunodeficiency virus type 1 proviral DNA by the polymerase chain reaction incorporating digoxigenin- or fluorescein-labeled dUTP

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Gang; Olson, J.C.; Pu, R.

1995-10-01

Serological assays are routinely used in the laboratory diagnosis of human immunodeficiency virus type-1 (HrV-1) infection, but the polymerase chain reaction (PCR) is ultimately the most sensitive and direct method for establishing definitive diagnosis. As an alternative to the conventional radioactive PCR procedure we have developed and evaluated a pair of rapid nonradioisotopic flow cytometric detection methods. Using heminested PCR we directly incorporated fluorescein-12-dUTP (fluo-dUTP) or digoxigenin-11-dUTP (dig-dUTP) into the PCR-amplicons. The labeled amplicons were hybridized with biotinylated antisense and sense probes, followed by capture of the hybrid DNA using streptavidin-coated beads which were finally analyzed in a flow cytometermore » by (1) direct detection of the fluorescence intensity of the amplicons incorporating fluo-dUTP and (2) immunodetection of the amplicons incorporating dig-dUTP by anti-digoxigenin IgG labeled with fluorescein isothiocyanate (FITC). Although both assays were functionally comparable with radiolabeled probe in reliably detecting as low as five copies of HIV-1 proviral DNA sequences, the immunodetection of dig-dUTP consistently yielded higher mean channel fluorescence and gave a stable signal over an extended period of 12-14 weeks. In testing a panel of 20 pedigreed PBMC specimens from blood donors with or without HIV-1 infection, the results of both flow cytometric assays were identical with those of the conventional radioactive procedure. Therefore, we conclude that the dig-dUTP incorporation in amplicons, hybridization with a pair of sense-antisense biotinylated probes and immunodetection of hybrids by flow cytometric analyses is the nonisotopic method of choice for PCR-diagnosis of HIV-1 infection. 21 refs., 2 figs., 4 tabs.« less

Systematic misestimation of cell subpopulations by flow cytometry: a mathematical analysis.

PubMed

Petrunkina, A M; Harrison, R A P

2010-04-15

Various sources of variability in flow cytometric determination of cell concentration have previously been investigated with respect to andrologic applications. Although common aspects related to the variation between samples, variation between operators, and accuracy have been extensively studied, specific sources of false-count estimation have found less attention. In particular, a major and well-recognized source of misestimation of cell counts (i.e., contamination of the sample by non-sperm particles) has not to date been characterized in detail. We show here by means of original mathematical research that not only the cell counts but also the percentages of cells expressing different fluorescence patterns are affected by the presence of alien particles often neglected in studies involving flow cytometric characterization. We demonstrate that there is a systematic overestimation in the proportion of unstained (viable) cells detected by flow cytometry in cases where the non-sperm particles are not excluded from analysis by additional identification other than light-scatter characteristics. Moreover, we provide an exact mathematical estimate for the magnitude of this overestimation, and we discuss the consequences for diagnostic applications and studies on sperm physiology, specifically for studies on sperm capacitation and evaluation of cryopreserved semen. Finally, equations are derived for the correction of the flow cytometric values for use in practical applications. Copyright 2010 Elsevier Inc. All rights reserved.

Kolmogorov-Smirnov statistical test for analysis of ZAP-70 expression in B-CLL, compared with quantitative PCR and IgV(H) mutation status.

PubMed

Van Bockstaele, Femke; Janssens, Ann; Piette, Anne; Callewaert, Filip; Pede, Valerie; Offner, Fritz; Verhasselt, Bruno; Philippé, Jan

2006-07-15

ZAP-70 has been proposed as a surrogate marker for immunoglobulin heavy-chain variable region (IgV(H)) mutation status, which is known as a prognostic marker in B-cell chronic lymphocytic leukemia (CLL). The flow cytometric analysis of ZAP-70 suffers from difficulties in standardization and interpretation. We applied the Kolmogorov-Smirnov (KS) statistical test to make analysis more straightforward. We examined ZAP-70 expression by flow cytometry in 53 patients with CLL. Analysis was performed as initially described by Crespo et al. (New England J Med 2003; 348:1764-1775) and alternatively by application of the KS statistical test comparing T cells with B cells. Receiver-operating-characteristics (ROC)-curve analyses were performed to determine the optimal cut-off values for ZAP-70 measured by the two approaches. ZAP-70 protein expression was compared with ZAP-70 mRNA expression measured by a quantitative PCR (qPCR) and with the IgV(H) mutation status. Both flow cytometric analyses correlated well with the molecular technique and proved to be of equal value in predicting the IgV(H) mutation status. Applying the KS test is reproducible, simple, straightforward, and overcomes a number of difficulties encountered in the Crespo-method. The KS statistical test is an essential part of the software delivered with modern routine analytical flow cytometers and is well suited for analysis of ZAP-70 expression in CLL. (c) 2006 International Society for Analytical Cytology.

Rise of the micromachines: microfluidics and the future of cytometry.

PubMed

Wlodkowic, Donald; Darzynkiewicz, Zbigniew

2011-01-01

The past decade has brought many innovations to the field of flow and image-based cytometry. These advancements can be seen in the current miniaturization trends and simplification of analytical components found in the conventional flow cytometers. On the other hand, the maturation of multispectral imaging cytometry in flow imaging and the slide-based laser scanning cytometers offers great hopes for improved data quality and throughput while proving new vistas for the multiparameter, real-time analysis of cells and tissues. Importantly, however, cytometry remains a viable and very dynamic field of modern engineering. Technological milestones and innovations made over the last couple of years are bringing the next generation of cytometers out of centralized core facilities while making it much more affordable and user friendly. In this context, the development of microfluidic, lab-on-a-chip (LOC) technologies is one of the most innovative and cost-effective approaches toward the advancement of cytometry. LOC devices promise new functionalities that can overcome current limitations while at the same time promise greatly reduced costs, increased sensitivity, and ultra high throughputs. We can expect that the current pace in the development of novel microfabricated cytometric systems will open up groundbreaking vistas for the field of cytometry, lead to the renaissance of cytometric techniques and most importantly greatly support the wider availability of these enabling bioanalytical technologies. Copyright © 2011 Elsevier Inc. All rights reserved.

Rise of the Micromachines: Microfluidics and the Future of Cytometry

PubMed Central

Wlodkowic, Donald; Darzynkiewicz, Zbigniew

2011-01-01

The past decade has brought many innovations to the field of flow and image-based cytometry. These advancements can be seen in the current miniaturization trends and simplification of analytical components found in the conventional flow cytometers. On the other hand, the maturation of multispectral imaging cytometry in flow imaging and the slide-based laser scanning cytometers offers great hopes for improved data quality and throughput while proving new vistas for the multiparameter, real-time analysis of cells and tissues. Importantly, however, cytometry remains a viable and very dynamic field of modern engineering. Technological milestones and innovations made over the last couple of years are bringing the next generation of cytometers out of centralized core facilities while making it much more affordable and user friendly. In this context, the development of microfluidic, lab-on-a-chip (LOC) technologies is one of the most innovative and cost-effective approaches toward the advancement of cytometry. LOC devices promise new functionalities that can overcome current limitations while at the same time promise greatly reduced costs, increased sensitivity, and ultra high throughputs. We can expect that the current pace in the development of novel microfabricated cytometric systems will open up groundbreaking vistas for the field of cytometry, lead to the renaissance of cytometric techniques and most importantly greatly support the wider availability of these enabling bioanalytical technologies. PMID:21704837

Collection, Storage, and Preparation of Human Blood Cells

PubMed Central

Dagur, Pradeep K.; McCoy, J. Philip

2015-01-01

Human peripheral blood is often studied by flow cytometry in both the research and clinical laboratories. The methods for collection, storage, and preparation of peripheral blood will vary depending on the cell lineage to be examined as well as the type of assay to be performed. This unit presents protocols for collection of blood, separation of leukocytes from whole blood by lysis of erythrocytes, isolating mononuclear cells by density gradient separation, and assorted non-flow sorting methods, such as magnetic bead separations, for enriching specific cell populations, including monocytes, T lymphocytes, B lymphocytes, neutrophils,, , and platelets prior to flow cytometric analysis. A protocol is also offered for cryopreservation of cells since clinical research often involves retrospective flow cytometric analysis of samples stored over a period of months or years. PMID:26132177

Air sampling to assess potential generation of aerosolized viable bacteria during flow cytometric analysis of unfixed bacterial suspensions

PubMed Central

Carson, Christine F; Inglis, Timothy JJ

2018-01-01

This study investigated aerosolized viable bacteria in a university research laboratory during operation of an acoustic-assisted flow cytometer for antimicrobial susceptibility testing by sampling room air before, during and after flow cytometer use. The aim was to assess the risk associated with use of an acoustic-assisted flow cytometer analyzing unfixed bacterial suspensions. Air sampling in a nearby clinical laboratory was conducted during the same period to provide context for the existing background of microorganisms that would be detected in the air. The three species of bacteria undergoing analysis by flow cytometer in the research laboratory were Klebsiella pneumoniae, Burkholderia thailandensis and Streptococcus pneumoniae. None of these was detected from multiple 1000 L air samples acquired in the research laboratory environment. The main cultured bacteria in both locations were skin commensal and environmental bacteria, presumed to have been disturbed or dispersed in laboratory air by personnel movements during routine laboratory activities. The concentrations of bacteria detected in research laboratory air samples were reduced after interventional cleaning measures were introduced and were lower than those in the diagnostic clinical microbiology laboratory. We conclude that our flow cytometric analyses of unfixed suspensions of K. pneumoniae, B. thailandensis and S. pneumoniae do not pose a risk to cytometer operators or other personnel in the laboratory but caution against extrapolation of our results to other bacteria and/or different flow cytometric experimental procedures. PMID:29608197

Novel nuclei isolation buffer for flow cytometric genome size estimation of Zingiberaceae: a comparison with common isolation buffers

PubMed Central

Sadhu, Abhishek; Bhadra, Sreetama; Bandyopadhyay, Maumita

2016-01-01

Background and Aims Cytological parameters such as chromosome numbers and genome sizes of plants are used routinely for studying evolutionary aspects of polyploid plants. Members of Zingiberaceae show a wide range of inter- and intrageneric variation in their reproductive habits and ploidy levels. Conventional cytological study in this group of plants is severely hampered by the presence of diverse secondary metabolites, which also affect their genome size estimation using flow cytometry. None of the several nuclei isolation buffers used in flow cytometry could be used very successfully for members of Zingiberaceae to isolate good quality nuclei from both shoot and root tissues. Methods The competency of eight nuclei isolation buffers was compared with a newly formulated buffer, MB01, in six different genera of Zingiberaceae based on the fluorescence intensity of propidium iodide-stained nuclei using flow cytometric parameters, namely coefficient of variation of the G0/G1 peak, debris factor and nuclei yield factor. Isolated nuclei were studied using fluorescence microscopy and bio-scanning electron microscopy to analyse stain–nuclei interaction and nuclei topology, respectively. Genome contents of 21 species belonging to these six genera were determined using MB01. Key Results Flow cytometric parameters showed significant differences among the analysed buffers. MB01 exhibited the best combination of analysed parameters; photomicrographs obtained from fluorescence and electron microscopy supported the superiority of MB01 buffer over other buffers. Among the 21 species studied, nuclear DNA contents of 14 species are reported for the first time. Conclusions Results of the present study substantiate the enhanced efficacy of MB01, compared to other buffers tested, in the generation of acceptable cytograms from all species of Zingiberaceae studied. Our study facilitates new ways of sample preparation for further flow cytometric analysis of genome size of other members belonging to this highly complex polyploid family. PMID:27594649

Novel nuclei isolation buffer for flow cytometric genome size estimation of Zingiberaceae: a comparison with common isolation buffers.

PubMed

Sadhu, Abhishek; Bhadra, Sreetama; Bandyopadhyay, Maumita

2016-11-01

Cytological parameters such as chromosome numbers and genome sizes of plants are used routinely for studying evolutionary aspects of polyploid plants. Members of Zingiberaceae show a wide range of inter- and intrageneric variation in their reproductive habits and ploidy levels. Conventional cytological study in this group of plants is severely hampered by the presence of diverse secondary metabolites, which also affect their genome size estimation using flow cytometry. None of the several nuclei isolation buffers used in flow cytometry could be used very successfully for members of Zingiberaceae to isolate good quality nuclei from both shoot and root tissues. The competency of eight nuclei isolation buffers was compared with a newly formulated buffer, MB01, in six different genera of Zingiberaceae based on the fluorescence intensity of propidium iodide-stained nuclei using flow cytometric parameters, namely coefficient of variation of the G 0 /G 1 peak, debris factor and nuclei yield factor. Isolated nuclei were studied using fluorescence microscopy and bio-scanning electron microscopy to analyse stain-nuclei interaction and nuclei topology, respectively. Genome contents of 21 species belonging to these six genera were determined using MB01. Flow cytometric parameters showed significant differences among the analysed buffers. MB01 exhibited the best combination of analysed parameters; photomicrographs obtained from fluorescence and electron microscopy supported the superiority of MB01 buffer over other buffers. Among the 21 species studied, nuclear DNA contents of 14 species are reported for the first time. Results of the present study substantiate the enhanced efficacy of MB01, compared to other buffers tested, in the generation of acceptable cytograms from all species of Zingiberaceae studied. Our study facilitates new ways of sample preparation for further flow cytometric analysis of genome size of other members belonging to this highly complex polyploid family. © The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Automatic cytometric device using multiple wavelength excitations

NASA Astrophysics Data System (ADS)

Rongeat, Nelly; Ledroit, Sylvain; Chauvet, Laurence; Cremien, Didier; Urankar, Alexandra; Couderc, Vincent; Nérin, Philippe

2011-05-01

Precise identification of eosinophils, basophils, and specific subpopulations of blood cells (B lymphocytes) in an unconventional automatic hematology analyzer is demonstrated. Our specific apparatus mixes two excitation radiations by means of an acousto-optics tunable filter to properly control fluorescence emission of phycoerythrin cyanin 5 (PC5) conjugated to antibodies (anti-CD20 or anti-CRTH2) and Thiazole Orange. This way our analyzer combining techniques of hematology analysis and flow cytometry based on multiple fluorescence detection, drastically improves the signal to noise ratio and decreases the spectral overlaps impact coming from multiple fluorescence emissions.

Flow cytometric analyses of CD34+ cells with inclusion of internal positive controls.

PubMed

Gutensohn, Kai; Jessen, Maria; Ketels, Andrea; Gramatzki, Martin; Humpe, Andreas

2012-02-01

Flow cytometric measurement of CD34+ events is used to ensure the quality of human progenitor cell grafts. This study was conducted to evaluate whether the spiking of routine samples from peripheral blood and apheresis products with CD34+ positive controls is feasible. A total of 42 samples from 32 patients and one healthy donor were stained in duplicate for CD34+ cells. Before flow cytometric analysis, one tube was spiked with stabilized CD34+ cells at a defined concentration. Median numbers of viable CD34+ cells/µL did not differ between unspiked and spiked tubes (median 37, range 0-714; and median 34, range 0-719, respectively). The 95% confidence interval (CI) of the mean showed a broad overlap between these samples (41.9-119.1 and 41.4-119.3, respectively). In addition, the 95% CI of the mean for CD45+ cells/µL overlapped broadly and median numbers did not differ. Median viability of all CD45+ cells was significantly lower in the spiked tubes (96.75, range 64-98.8 vs. 99.25, range 97.5-99.8) with no overlap of the 95% CI of the mean viability. The results of this study show that spiking of routine samples with internal positive controls does not affect CD34+ cell analyses, but does support the reliability of important clinical data. The inclusion of positive controls is expedient for laboratories that perform analyses with low CD34+ numbers and laboratories that use different flow cytometric analyzers and may also become a requirement to meet statutory regulations. © 2012 American Association of Blood Banks.

Flow cytometry of mammalian sperm: progress in DNA and morphology measurement.

PubMed

Pinkel, D; Dean, P; Lake, S; Peters, D; Mendelsohn, M; Gray, J; Van Dilla, M; Gledhill, B

1979-01-01

Variability in DNA content and head shape of mammalian sperm are potentially useful markers for flow cytometric monitoring of genetic damage in spermatogenic cells. The high refractive index and extreme flatness of the sperm heads produce an optical effect which interferes with DNA measurements in flow cytometers which have dye excitation and fluorescence light collection normal to the axis of flow. Orientation of sperm in flow controls this effect and results in coefficients of variation of 2.5% and 4.2%, respectively, for DNA measurements of mouse and human sperm. Alternatively, the optical effect can be used to generate shape-related information. Measurements on randomly oriented sperm from three mammalian species using a pair of fluorescence detectors indicate that large shape differences are detectable. Acriflavine-Feulgen stained sperm nuclei are significantly bleached during flow cytometric measurements at power levels routinely used in many flow cytometers. Dual beam studies of this phenomenon indicate it may be useful in detecting abnormally shaped sperm.

A flow cytometric method for assessing viability of intraerythrocytic hemoparasites.

PubMed

Wyatt, C R; Goff, W; Davis, W C

1991-06-24

We have developed a rapid, reliable method of evaluating growth and viability of intraerythrocytic protozoan hemoparasites. The assay involves the selective uptake and metabolic conversion of hydroethidine to ethidium by live parasites present in intact erythrocytes. The red fluorescence imparted by ethidium intercalated into the DNA of the parasite permits the use of flow cytometry to distinguish infected erythrocytes with viable parasites from uninfected erythrocytes and erythrocytes containing dead parasites. Comparison of the fluorochromasia technique of enumerating the number and viability of hemoparasites in cultured erythrocytes with enumeration in Giemsa-stained films and uptake of [3H]hypoxanthine demonstrated the fluorochromasia technique yields comparable results. Studies with the hemoparasite, Babesia bovis, have shown the fluorochromasia technique can also be used to monitor the effect of parasiticidal drugs on parasites in vitro. The cumulative studies with the fluorochromasia assay suggest the assay will also prove useful in investigations focused on analysis of the immune response to hemoparasites and growth in vitro.

Optimization and Standardization of Fluorescent Cell Barcoding for Multiplexed Flow Cytometric Phenotyping

PubMed Central

Giudice, Valentina; Feng, Xingmin; Kajigaya, Sachiko; Young, Neal S.; Biancotto, Angélique

2017-01-01

Fluorescent cell barcoding (FCB) is a cell-based multiplexing technique for high-throughput flow cytometry. Barcoded samples can be stained and acquired collectively, minimizing staining variability and antibody consumption, and decreasing required sample volumes. Combined with functional measurements, FCB can be used for drug screening, signaling profiling, and cytokine detection, but technical issues are present. We optimized the FCB technique for routine utilization using DyLight 350, DyLight 800, Pacific Orange, and CBD500 for barcoding six, nine, or 36 human peripheral blood specimens. Working concentrations of FCB dyes ranging from 0 to 500 μg/ml were tested, and viability dye staining was optimized to increase robustness of data. A five-color staining with surface markers for Vβ usage analysis in CD4+ and CD8+ T cells was achieved in combination with nine sample barcoding. We provide improvements of the FCB technique that should be useful for multiplex drug screening and for lymphocyte characterization and perturbations in the diagnosis and during the course of disease. PMID:28692789

Bacterial screening by flow cytometry offers potential for extension of platelet storage: results of 14 months of active surveillance.

PubMed

Vollmer, T; Engemann, J; Kleesiek, K; Dreier, J

2011-06-01

Bacterial contamination is currently the major infectious hazard of platelet transfusion in developed countries. It has been demonstrated that a significant transfusion risk remains, in particular with older platelet concentrates (PCs). In 2009, the shelf life of PCs was therefore reduced in Germany to 4 days after the day of production according to Vote 38. The aim of the present study was the application and implementation of a recently developed flow cytometry-based rapid screening method (BactiFlow) for bacterial contamination at the end of PC shelf life as a routine in-process control. A total of 472 apheresis-derived PCs were tested using the BactiFlow flow cytometric assay to detect and count bacteria based on esterase activity in viable bacterial cells, while the BacT/Alert automated culture system served as the reference method. The automation potential of the flow cytometric assay was analysed by applying the semi-automated BactiFlow ALS system. An algorithm was developed for use in routine blood bank operations to extend the storage period of PCs. Two of the 472 apheresis PCs tested were positive in culture and identified as Propionibacterium species. One PC was positive for Staphylococcus aureus by both methods. All remaining specimens were tested negative by both methods. Our study demonstrates that routine bacterial testing of PCs was successfully implemented and the established algorithm proved efficient. The BactiFlow flow cytometric assay is the first rapid screening method which is suitable for a routine application combined with a high sensitivity. © 2011 The Authors. Transfusion Medicine © 2011 British Blood Transfusion Society.

Flow cytometric measurement of total DNA and incorporated halodeoxyuridine

DOEpatents

Dolbeare, Frank A.; Gray, Joe W.

1986-01-01

A method for the simultaneous flow cytometric measurement of the total DNA content and the level of DNA synthesis in normal and malignant cells is disclosed. The sensitivity of the method allows a study of cell cycle traverse rates for large scale cell populations as well as single cell measurements. A DNA stain such as propidium iodide is used as the probe for the measurement of total DNA content and a monoclonal antibody reactive with a DNA precursor such as bromodeoxyuridine (BrdU) is used as a probe for the measurement of BrdU uptake by the cells as a measure of DNA synthesis.

Separation of platelets from whole blood using standing surface acoustic waves in a microchannel.

PubMed

Nam, Jeonghun; Lim, Hyunjung; Kim, Dookon; Shin, Sehyun

2011-10-07

Platelet separation from blood is essential for biochemical analyses and clinical diagnosis. In this article, we propose a method to separate platelets from undiluted whole blood using standing surface acoustic waves (SSAWs) in a microfluidic device. A polydimethylsiloxane (PDMS) microfluidic channel was fabricated and integrated with interdigitated transducer (IDT) electrodes patterned on a piezoelectric substrate. To avoid shear-induced activation of platelets, the blood sample flow was hydrodynamically focused by introducing sheath flow from two side-inlets and pressure nodes were designed to locate at side walls. By means of flow cytometric analysis, the RBC clearance ratio from whole blood was found to be over 99% and the purity of platelets was close to 98%. Conclusively, the present technique using SSAWs can directly separate platelets from undiluted whole blood with higher purity than other methods.

Flow cytometric analysis of microbial contamination in food industry technological lines--initial study.

PubMed

Józwa, Wojciech; Czaczyk, Katarzyna

2012-04-02

Flow cytometry constitutes an alternative for traditional methods of microorganisms identification and analysis, including methods requiring cultivation step. It enables the detection of pathogens and other microorganisms contaminants without the need to culture microbial cells meaning that the sample (water, waste or food e.g. milk, wine, beer) may be analysed directly. This leads to a significant reduction of time required for analysis allowing monitoring of production processes and immediate reaction in case of contamination or any disruption occurs. Apart from the analysis of raw materials or products on different stages of manufacturing process, the flow cytometry seems to constitute an ideal tool for the assessment of microbial contamination on the surface of technological lines. In the present work samples comprising smears from 3 different surfaces of technological lines from fruit and vegetable processing company from Greater Poland were analysed directly with flow cytometer. The measured parameters were forward and side scatter of laser light signals allowing the estimation of microbial cell contents in each sample. Flow cytometric analysis of the surface of food industry production lines enable the preliminary evaluation of microbial contamination within few minutes from the moment of sample arrival without the need of sample pretreatment. The presented method of fl ow cytometric initial evaluation of microbial state of food industry technological lines demonstrated its potential for developing a robust, routine method for the rapid and labor-saving detection of microbial contamination in food industry.

The extended leukocyte differential count using the Cytodiff flow cytometric system reveals that higher CD16+ cytotoxic NK+T lymphocyte levels predict superior survival outcomes in patients with metastatic carcinoma.

PubMed

Park, Borae G; Park, Chan-Jeoung; Yoon, Chan-Hee; Jang, Seongsoo; Chi, Hyun-Sook; Ryu, Min-Hee; Kim, Sang-We

2013-05-01

The recently developed Cytodiff flow cytometric system (Beckman Coulter, Miami, FL) enables leukocyte analysis using a single immunophenotyping panel tube composed of six markers and five colors and that can detect 16 leukocyte subpopulations. We performed a preliminary investigation of whether changes in any of 16 leukocyte differentials were associated with survival and treatment outcomes in patients with metastatic carcinoma or not. We measured 16 leukocyte differential counts using the Cytodiff flow cytometric system in peripheral blood samples from 40 patients with metastatic malignancy (27 stomach cancer and 13 lung cancer) before chemotherapy and at 15 day intervals after chemotherapy for 2 months. A higher percentage of CD16+ cytotoxic NK+T lymphocytes was found to be the only significant prognostic factor among by Cox regression analysis and a higher percentage of CD16+ cytotoxic NK+T lymphocytes (>5.0%) showed significantly longer survival outcomes by Kaplan-Meier analysis (P = 0.003). The Cytodiff system enables 16 leukocyte subpopulations in a one tube assay and also can operate with only small amounts of sample, although it cannot differentiate NK cells from T lymphocytes. Hence, the monitoring of all leukocyte subpopulations using Cytodiff flow cytometry may be a helpful prognostic tool for patients with metastatic carcinoma. Copyright © 2012 International Clinical Cytometry Society.

Flow cytometric analysis of extracellular vesicle subsets in plasma: impact of swarm by particles of non-interest.

PubMed

Libregts, S F W M; Arkesteijn, G J A; Németh, A; Nolte-'t Hoen, E N M; Wauben, M H M

2018-05-20

Essentials Extracellular vesicles (EVs) in biological fluids are promising biomarkers for disease. Fluorescence-based flow cytometric analysis is suitable to detect low abundant EV subsets. Particles of non-interest can induce false-positive light scatter and fluorescent signals. Interference of particles of non-interest can be monitored by analyzing serial dilutions. Background Extracellular vesicles (EVs) in plasma are increasingly being recognized as potential biomarkers. EV analysis for diagnostic purposes should be robust and should allow analysis of EV subsets with a wide range of abundance and in a large number of patient samples. Flow cytometry offers possibilities to meet these criteria, as it allows multiparameter analysis of individual EVs. However, analysis of plasma EVs is challenging, because of their size and heterogeneity, and the presence of other submicrometer-sized particles in plasma that could interfere with EV analysis. Objectives To explore whether fluorescence-based flow cytometric analysis of EV subsets is suitable when the EVs of interest are present in low abundance in a background of non-labeled or differently labeled EVs and particles. Methods Fluorescently labeled EVs of interest were spiked at different ratios in full plasma, purified plasma components, or (non-)fluorescent polystyrene beads, and subsequently analyzed by flow cytometry with fluorescence threshold triggering. Results We found that light scatter detection of low-abundance or rare EV subsets during fluorescence threshold triggering was severely affected by particles of non-interest, owing to coincidence and swarming. Importantly, we show that interfering particles labeled with different fluorophores induced false-positive fluorescent signals on the particles of interest. These unwanted effects could only be discerned and controlled by performing serial dilutions and analyzing light scatter and fluorescence parameters. Conclusions We demonstrate how particles of non-interest in plasma can impact on the light scatter and fluorescence detection of low-abundance EVs of interest during fluorescence-based flow cytometric analysis, and provide a means to prevent erroneous data interpretation. © 2018 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.

Flow cytometric measurement of total DNA and incorporated halodeoxyuridine

DOEpatents

Dolbeare, Frank A.; Gray, Joe W.

1988-01-01

A method for the simultaneous flow cytometric measurement of the total DNA content and the level of DNA synthesis in normal and malignant cells is disclosed. The sensitivity of the method allows a study of cell cycle traverse rates for large scale cell populations as well as single cell measurements. A DNA stain such as propidium iodide or Hoechst 33258 is used as the probe for the measurement of total DNA content and a monoclonal antibody reactive with a DNA precursor such as halodeoxy-uridine (HdU), more specifically bromodeoxyuridine (BrdU) is used as a probe for the measurement of HdU or BrdU uptake by the cells as a measure of DNA synthesis.

Analytical cytology applied to detection of prognostically important cytogenetic aberrations: Current status and future directions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gray, J.W.; Pinkel, D.; Trask, B.

1987-07-24

This paper discusses the application of analytical cytology to the detection of clinically important chromosome abnormalities in human tumors. Flow cytometric measurements of DNA distributions have revealed that many human tumors have abnormal (usually elevated) DNA contents and that the occurrence of DNA abnormality may be diagnostically or prognostically important. However, DNA indices (ratio of tumor DNA content to normal DNA content) provide little information about the specific chromosome(s) involved in the DNA content abnormality. Fluorescence in situ hybridization with chromosome specific probes is suggested as a technique to facilitate detection of specific chromosome aneuploidy in interphase and metaphase humanmore » tumor cells. Fluorescence hybridization to nuclei on slides allows enumeration of brightly fluorescent nuclear domains as an estimate of the number of copies of the chromosome type for which the hybridization probe is specific. Fluorescence hybridization can also be made to nuclei in suspension. The fluorescence intensity can then be measured flow cytometrically as an indication of the number of chromosomes in each nucleus carrying the DNA sequence homologous to the probe. In addition, quantitative image analysis may be used to explore the position of chromosomes in interphase nuclei and to look for changes in the order that may eventually permit detection of clinicaly important conditions. 55 refs., 8 figs., 1 tab.« less

A novel rapid and reproducible flow cytometric method for optimization of transfection efficiency in cells

PubMed Central

Homann, Stefanie; Hofmann, Christian; Gorin, Aleksandr M.; Nguyen, Huy Cong Xuan; Huynh, Diana; Hamid, Phillip; Maithel, Neil; Yacoubian, Vahe; Mu, Wenli; Kossyvakis, Athanasios; Sen Roy, Shubhendu; Yang, Otto Orlean

2017-01-01

Transfection is one of the most frequently used techniques in molecular biology that is also applicable for gene therapy studies in humans. One of the biggest challenges to investigate the protein function and interaction in gene therapy studies is to have reliable monospecific detection reagents, particularly antibodies, for all human gene products. Thus, a reliable method that can optimize transfection efficiency based on not only expression of the target protein of interest but also the uptake of the nucleic acid plasmid, can be an important tool in molecular biology. Here, we present a simple, rapid and robust flow cytometric method that can be used as a tool to optimize transfection efficiency at the single cell level while overcoming limitations of prior established methods that quantify transfection efficiency. By using optimized ratios of transfection reagent and a nucleic acid (DNA or RNA) vector directly labeled with a fluorochrome, this method can be used as a tool to simultaneously quantify cellular toxicity of different transfection reagents, the amount of nucleic acid plasmid that cells have taken up during transfection as well as the amount of the encoded expressed protein. Finally, we demonstrate that this method is reproducible, can be standardized and can reliably and rapidly quantify transfection efficiency, reducing assay costs and increasing throughput while increasing data robustness. PMID:28863132

Automated analysis of flow cytometric data for measuring neutrophil CD64 expression using a multi-instrument compatible probability state model.

PubMed

Wong, Linda; Hill, Beth L; Hunsberger, Benjamin C; Bagwell, C Bruce; Curtis, Adam D; Davis, Bruce H

2015-01-01

Leuko64™ (Trillium Diagnostics) is a flow cytometric assay that measures neutrophil CD64 expression and serves as an in vitro indicator of infection/sepsis or the presence of a systemic acute inflammatory response. Leuko64 assay currently utilizes QuantiCALC, a semiautomated software that employs cluster algorithms to define cell populations. The software reduces subjective gating decisions, resulting in interanalyst variability of <5%. We evaluated a completely automated approach to measuring neutrophil CD64 expression using GemStone™ (Verity Software House) and probability state modeling (PSM). Four hundred and fifty-seven human blood samples were processed using the Leuko64 assay. Samples were analyzed on four different flow cytometer models: BD FACSCanto II, BD FACScan, BC Gallios/Navios, and BC FC500. A probability state model was designed to identify calibration beads and three leukocyte subpopulations based on differences in intensity levels of several parameters. PSM automatically calculates CD64 index values for each cell population using equations programmed into the model. GemStone software uses PSM that requires no operator intervention, thus totally automating data analysis and internal quality control flagging. Expert analysis with the predicate method (QuantiCALC) was performed. Interanalyst precision was evaluated for both methods of data analysis. PSM with GemStone correlates well with the expert manual analysis, r(2) = 0.99675 for the neutrophil CD64 index values with no intermethod bias detected. The average interanalyst imprecision for the QuantiCALC method was 1.06% (range 0.00-7.94%), which was reduced to 0.00% with the GemStone PSM. The operator-to-operator agreement in GemStone was a perfect correlation, r(2) = 1.000. Automated quantification of CD64 index values produced results that strongly correlate with expert analysis using a standard gate-based data analysis method. PSM successfully evaluated flow cytometric data generated by multiple instruments across multiple lots of the Leuko64 kit in all 457 cases. The probability-based method provides greater objectivity, higher data analysis speed, and allows for greater precision for in vitro diagnostic flow cytometric assays. © 2015 International Clinical Cytometry Society.

A Flow Cytometric and Computational Approaches to Carbapenems Affinity to the Different Types of Carbapenemases

PubMed Central

Pina-Vaz, Cidália; Silva, Ana P.; Faria-Ramos, Isabel; Teixeira-Santos, Rita; Moura, Daniel; Vieira, Tatiana F.; Sousa, Sérgio F.; Costa-de-Oliveira, Sofia; Cantón, Rafael; Rodrigues, Acácio G.

2016-01-01

The synergy of carbapenem combinations regarding Enterobacteriaceae producing different types of carbapenemases was study through different approaches: flow cytometry and computational analysis. Ten well characterized Enterobacteriaceae (KPC, verona integron-encoded metallo-β-lactamases –VIM and OXA-48-like enzymes) were selected for the study. The cells were incubated with a combination of ertapenem with imipenem, meropenem, or doripenem and killing kinetic curves performed with and without reinforcements of the drugs. A cephalosporin was also used in combination with ertapenem. A flow cytometric assay with DiBAC4-(3), a membrane potential dye, was developed in order to evaluate the cellular lesion after 2 h incubation. A chemical computational study was performed to understand the affinity of the different drugs to the different types of enzymes. Flow cytometric analysis and time-kill assays showed a synergic effect against KPC and OXA-48 producing-bacteria with all combinations; only ertapenem with imipenem was synergic against VIM producing-bacteria. A bactericidal effect was observed in OXA-48-like enzymes. Ceftazidime plus ertapenem was synergic against ESBL-negative KPC producing-bacteria. Ertapenem had the highest affinity for those enzymes according to chemical computational study. The synergic effect between ertapenem and others carbapenems against different carbapenemase-producing bacteria, representing a therapeutic choice, was described for the first time. Easier and faster laboratorial methods for carbapenemase characterization are urgently needed. The design of an ertapenem derivative with similar affinity to carbapenemases but exhibiting more stable bonds was demonstrated as highly desirable. PMID:27555844

ICCS/ESCCA consensus guidelines to detect GPI-deficient cells in paroxysmal nocturnal hemoglobinuria (PNH) and related disorders part 4 - assay validation and quality assurance.

PubMed

Oldaker, Teri; Whitby, Liam; Saber, Maryam; Holden, Jeannine; Wallace, Paul K; Litwin, Virginia

2018-01-01

Over the past six years, a diverse group of stakeholders have put forth recommendations regarding the analytical validation of flow cytometric methods and described in detail the differences between cell-based and traditional soluble analyte assay validations. This manuscript is based on these general recommendations as well as the published experience of experts in the area of PNH testing. The goal is to provide practical assay-specific guidelines for the validation of high-sensitivity flow cytometric PNH assays. Examples of the reports and validation data described herein are provided in Supporting Information. © 2017 International Clinical Cytometry Society. © 2017 International Clinical Cytometry Society.

Flow Cytometric Analysis of Presynaptic Nerve Terminals Isolated from Rats Subjected to Hypergravity

NASA Astrophysics Data System (ADS)

Borisova, Tatiana

2008-06-01

Flow cytometric studies revealed an insignificant decrease in cell size heterogeneity and cytoplasmic granularity of rat brain nerve terminals (synaptosomes) isolated from animals subjected to centrifuge-induced hypergravity as compared to control ones. The analysis of plasma membrane potential using the potentiometric optical dye rhodamine 6G showed a decrease in fluorescence intensity by 10 % at steady state level in hypergravity synaptosomes. To monitor synaptic vesicle acidification we used pH-sensitive fluorescent dye acridine orange and demonstrated a lower fluorescence intensity level at steady state (10%) after hypergravity as compared to controls. Thus, exposure to hypergravity resulted in depolarization of the synaptosomal plasma membrane and diminution in synaptic vesicle acidification that may be a cause leading to altered synaptic neurotransmission.

Applications of Flow Cytometry to Clinical Microbiology†

PubMed Central

Álvarez-Barrientos, Alberto; Arroyo, Javier; Cantón, Rafael; Nombela, César; Sánchez-Pérez, Miguel

2000-01-01

Classical microbiology techniques are relatively slow in comparison to other analytical techniques, in many cases due to the need to culture the microorganisms. Furthermore, classical approaches are difficult with unculturable microorganisms. More recently, the emergence of molecular biology techniques, particularly those on antibodies and nucleic acid probes combined with amplification techniques, has provided speediness and specificity to microbiological diagnosis. Flow cytometry (FCM) allows single- or multiple-microbe detection in clinical samples in an easy, reliable, and fast way. Microbes can be identified on the basis of their peculiar cytometric parameters or by means of certain fluorochromes that can be used either independently or bound to specific antibodies or oligonucleotides. FCM has permitted the development of quantitative procedures to assess antimicrobial susceptibility and drug cytotoxicity in a rapid, accurate, and highly reproducible way. Furthermore, this technique allows the monitoring of in vitro antimicrobial activity and of antimicrobial treatments ex vivo. The most outstanding contribution of FCM is the possibility of detecting the presence of heterogeneous populations with different responses to antimicrobial treatments. Despite these advantages, the application of FCM in clinical microbiology is not yet widespread, probably due to the lack of access to flow cytometers or the lack of knowledge about the potential of this technique. One of the goals of this review is to attempt to mitigate this latter circumstance. We are convinced that in the near future, the availability of commercial kits should increase the use of this technique in the clinical microbiology laboratory. PMID:10755996

Detection of immunoglobulins on bacterial surface by laser flow cytometry: analysis between Haemophilus influenzae type b and Vibrio cholerae O1 of healthy mother-full term newborn sera.

PubMed

Cano-Morales, S; Luna-Guerrero, R; Mendez-Cuevas, G; Alvarado-Aleman, F J

1996-01-01

The identification of human IgG immunoglobulins on the surface of Vibrio cholerae O1, and Haemophilus influenzae type b microorganisms was assessed via a flow cytometric technique. A group of 31 healthy mother-full term newborn duo sera from a non-endemic cholera area was assayed. The sera of mothers and full-term newborns against both microorganisms were compared. The mean fluorescent intensity of the samples was not different at the 0.05 significance level by paired t-test. On the other hand, the immunoglobulins of newborn and mothers for V. cholerae O1 was notably lower when compared with H. influenzae type b microorganisms (p < 0.05 by paired t-test, t = -5.570 for mothers' sera, and t = -7.496 for the sera of the newborns). These data provide circumstantial evidence that LFC technique would be useful on bacteria-related serology.

A simplified plastic embedding and immunohistologic technique for immunophenotypic analysis of human hematopoietic and lymphoid tissues.

PubMed Central

Casey, T. T.; Cousar, J. B.; Collins, R. D.

1988-01-01

Routine fixation and paraffin embedding destroys many hematopoietic and lymphoid differentiation antigens detected by flow cytometry or frozen section immunohistochemistry. On the other hand, morphologic evaluation is difficult in flow cytometric or frozen section studies. A simplified three-step plastic embedding system using acetone-fixed tissues embedded in glycol-methacrylate (GMA) resin has been found to provide both excellent morphologic and antigenic preservation. With our system, a wide variety of antigens are detected in plastic sections without trypsinization or prolonged embedding procedures; pan-B (CD19, CD22), pan-T (CD7, CD5, CD3, CD2), T-subset (CD4, CD8, CD1, CD25) markers as well as surface immunoglobulin and markers for myeloid and mononuclear-phagocyte cells are preserved. In summary, modifications of plastic embedding techniques used in this study simplify the procedure, apparently achieve excellent antigenic preservation, and facilitate evaluation of morphologic details in relation to immunocytochemical markers. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:3282442

A cluster analysis method for identification of subpopulations of cells in flow cytometric list-mode arrays

NASA Technical Reports Server (NTRS)

Li, Z. K.

1985-01-01

A specialized program was developed for flow cytometric list-mode data using an heirarchical tree method for identifying and enumerating individual subpopulations, the method of principal components for a two-dimensional display of 6-parameter data array, and a standard sorting algorithm for characterizing subpopulations. The program was tested against a published data set subjected to cluster analysis and experimental data sets from controlled flow cytometry experiments using a Coulter Electronics EPICS V Cell Sorter. A version of the program in compiled BASIC is usable on a 16-bit microcomputer with the MS-DOS operating system. It is specialized for 6 parameters and up to 20,000 cells. Its two-dimensional display of Euclidean distances reveals clusters clearly, as does its 1-dimensional display. The identified subpopulations can, in suitable experiments, be related to functional subpopulations of cells.

High speed flow cytometric separation of viable cells

DOEpatents

Sasaki, D.T.; Van den Engh, G.J.; Buckie, A.M.

1995-11-14

Hematopoietic cell populations are separated to provide cell sets and subsets as viable cells with high purity and high yields, based on the number of original cells present in the mixture. High-speed flow cytometry is employed using light characteristics of the cells to separate the cells, where high flow speeds are used to reduce the sorting time.

High speed flow cytometric separation of viable cells

DOEpatents

Sasaki, Dennis T.; Van den Engh, Gerrit J.; Buckie, Anne-Marie

1995-01-01

Hematopoietic cell populations are separated to provide cell sets and subsets as viable cells with high purity and high yields, based on the number of original cells present in the mixture. High-speed flow cytometry is employed using light characteristics of the cells to separate the cells, where high flow speeds are used to reduce the sorting time.

Flow cytometric characterization of freshwater crayfish hemocytes for the examination of physiological status in wild and captive animals.

PubMed

Taylor, Sean; Landman, Michael J; Ling, Nicholas

2009-09-01

Enumeration of invertebrate hemocytes is a potentially powerful tool for the determination of physiological effects of extrinsic stressors, such as hypoxia, disease, and toxicant exposure. A detailed flow cytometric method of broad application was developed for the objective characterization and enumeration of the hemocytes of New Zealand freshwater crayfish Paranephrops planifrons for the purpose of physiological health assessment. Hemocyte populations were isolated by flow cytometric sorting based on differential light scatter properties followed by morphological characterization via light microscopy and software image analysis. Cells were identified as hyaline, semigranular, and granular hemocytes based on established invertebrate hemocyte classification. A characteristic decrease in nuclear size, an increase in granularity between the hyaline and granular cells, and the eccentric location of nuclei in granular cells were also observed. The granulocyte subpopulations were observed to possess varying degrees of granularity. The developed methodology was used to perform total and differential hemocyte counts from three lake populations and between wild and captive crayfish specimens. Differences in total and differential hemocyte counts were not observed among the wild populations. However, specimens held in captivity for 14 d exhibited a significant 63% reduction in total hemocyte count, whereas the relative hemocyte proportions remained the same. These results demonstrate the utility of this method for the investigation of subacute stressor effects in selected decapod crustaceans.

The G6PD flow-cytometric assay is a reliable tool for diagnosis of G6PD deficiency in women and anaemic subjects.

PubMed

Bancone, Germana; Kalnoky, Michael; Chu, Cindy S; Chowwiwat, Nongnud; Kahn, Maria; Malleret, Benoit; Wilaisrisak, Pornpimon; Rénia, Laurent; Domingo, Gonzalo J; Nosten, Francois

2017-08-29

Glucose-6-phosphate dehydrogenase (G6PD) activity is essential for redox equilibrium of red blood cells (RBCs) and, when compromised, the RBCs are more susceptible to haemolysis. 8-aminoquinolines (primaquine and tafenoquine) are used for the radical curative treatment of Plasmodium vivax malaria and can cause haemolysis in G6PD deficient subjects. Haemolytic risk is dependent on treatment dose and patient G6PD status but ultimately it correlates with the number of G6PD deficient RBCs. The G6PD spectrophotometric assay reliably identifies deficient subjects but is less reliable in heterozygous females, especially when other blood conditions are present. In this work we analysed samples with a range of G6PD phenotypes and haematologic conditions from 243 healthy volunteers of Asian or African-American heritage using both the spectrophotomeric assay and the G6PD flow-cytometric assay. Overall 18.5% of subjects (29.3% of Asian females) presented with anaemia, associated with decreased RBCs volume (MCV) and reticulocytosis; the flow-cytometric assay showed good correlation with the spectrophotometric assay (Pearson's r 0.918-0.957) and was less influenced by haemoglobin concentration, number of RBCs and number of reticulocytes. This resulted in more precise quantification of the number of G6PD deficient RBCs and presumably higher predictive power of drug induced haemolytic risk.

Continuous separation of microparticles in a microfluidic channel via the elasto-inertial effect of non-Newtonian fluid.

PubMed

Nam, Jeonghun; Lim, Hyunjung; Kim, Dookon; Jung, Hyunwook; Shin, Sehyun

2012-04-07

Pure separation and sorting of microparticles from complex fluids are essential for biochemical analyses and clinical diagnostics. However, conventional techniques require highly complex and expensive labeling processes for high purity separation. In this study, we present a simple and label-free method for separating microparticles with high purity using the elasto-inertial characteristic of a non-Newtonian fluid in microchannel flow. At the inlet, particle-containing sample flow was pushed toward the side walls by introducing sheath fluid from the center inlet. Particles of 1 μm and 5 μm in diameter, which were suspended in viscoelastic fluid, were successfully separated in the outlet channels: larger particles were notably focused on the centerline of the channel at the outlet, while smaller particles continued flowing along the side walls with minimal lateral migration towards the centerline. The same technique was further applied to separate platelets from diluted whole blood. Through cytometric analysis, we obtained a purity of collected platelets of close to 99.9%. Conclusively, our microparticle separation technique using elasto-inertial forces in non-Newtonian fluid is an effective method for separating and collecting microparticles on the basis of size differences with high purity. This journal is © The Royal Society of Chemistry 2012

Digital Analysis and Sorting of Fluorescence Lifetime by Flow Cytometry

PubMed Central

Houston, Jessica P.; Naivar, Mark A.; Freyer, James P.

2010-01-01

Frequency-domain flow cytometry techniques are combined with modifications to the digital signal processing capabilities of the Open Reconfigurable Cytometric Acquisition System (ORCAS) to analyze fluorescence decay lifetimes and control sorting. Real-time fluorescence lifetime analysis is accomplished by rapidly digitizing correlated, radiofrequency modulated detector signals, implementing Fourier analysis programming with ORCAS’ digital signal processor (DSP) and converting the processed data into standard cytometric list mode data. To systematically test the capabilities of the ORCAS 50 MS/sec analog-to-digital converter (ADC) and our DSP programming, an error analysis was performed using simulated light scatter and fluorescence waveforms (0.5–25 ns simulated lifetime), pulse widths ranging from 2 to 15 µs, and modulation frequencies from 2.5 to 16.667 MHz. The standard deviations of digitally acquired lifetime values ranged from 0.112 to >2 ns, corresponding to errors in actual phase shifts from 0.0142° to 1.6°. The lowest coefficients of variation (<1%) were found for 10-MHz modulated waveforms having pulse widths of 6 µs and simulated lifetimes of 4 ns. Direct comparison of the digital analysis system to a previous analog phase-sensitive flow cytometer demonstrated similar precision and accuracy on measurements of a range of fluorescent microspheres, unstained cells and cells stained with three common fluorophores. Sorting based on fluorescence lifetime was accomplished by adding analog outputs to ORCAS and interfacing with a commercial cell sorter with a radiofrequency modulated solid-state laser. Two populations of fluorescent microspheres with overlapping fluorescence intensities but different lifetimes (2 and 7 ns) were separated to ~98% purity. Overall, the digital signal acquisition and processing methods we introduce present a simple yet robust approach to phase-sensitive measurements in flow cytometry. The ability to simply and inexpensively implement this system on a commercial flow sorter will both allow better dissemination of this technology and better exploit the traditionally underutilized parameter of fluorescence lifetime. PMID:20662090

Analysis of synthetic and biological microparticles on several flow cytometric platforms

EPA Science Inventory

Microparticles (MPs) are membrane vesicles (0.1 to 1 urn) released from cells upon activation. The limit of detection ofmost standard flow cytometers is just below 1 urn. Recent advances enable detection of particles lower than 0.5 urn, Synthetic. beads are used to define size ra...

Genome-size variation in switchgrass (Panicum virgatum): flow cytometry and cytology reveal rampant aneuploidy

USDA-ARS?s Scientific Manuscript database

Switchgrass (Panicum virgatum L.), a native perennial dominant of the prairies of North America, has been targeted as a model herbaceous species for biofeedstock development. A flow-cytometric survey of a core set of 11 primarily upland polyploid switchgrass accessions indicated that there was con...

Multivariate analysis of flow cytometric data using decision trees.

PubMed

Simon, Svenja; Guthke, Reinhard; Kamradt, Thomas; Frey, Oliver

2012-01-01

Characterization of the response of the host immune system is important in understanding the bidirectional interactions between the host and microbial pathogens. For research on the host site, flow cytometry has become one of the major tools in immunology. Advances in technology and reagents allow now the simultaneous assessment of multiple markers on a single cell level generating multidimensional data sets that require multivariate statistical analysis. We explored the explanatory power of the supervised machine learning method called "induction of decision trees" in flow cytometric data. In order to examine whether the production of a certain cytokine is depended on other cytokines, datasets from intracellular staining for six cytokines with complex patterns of co-expression were analyzed by induction of decision trees. After weighting the data according to their class probabilities, we created a total of 13,392 different decision trees for each given cytokine with different parameter settings. For a more realistic estimation of the decision trees' quality, we used stratified fivefold cross validation and chose the "best" tree according to a combination of different quality criteria. While some of the decision trees reflected previously known co-expression patterns, we found that the expression of some cytokines was not only dependent on the co-expression of others per se, but was also dependent on the intensity of expression. Thus, for the first time we successfully used induction of decision trees for the analysis of high dimensional flow cytometric data and demonstrated the feasibility of this method to reveal structural patterns in such data sets.

Use of LysoTracker dyes: a flow cytometric study of autophagy.

PubMed

Chikte, Shaheen; Panchal, Neelam; Warnes, Gary

2014-02-01

The flow cytometric use of LysoTracker dyes was employed to investigate the autophagic process and to compare this with the upregulation of autophagy marker, the microtubule-associated protein LC3B. Although the mechanism of action of LysoTracker dyes is not fully understood, they have been used in microscopy to image acidic spherical organelles, and their use in flow cytometry has not been thoroughly investigated in the study of autophagy. This investigation uses numerous autophagy-inducing agents including chloroquine (CQ), rapamycin, low serum (<1%) RPMI, and nutrient starvation to induce autophagy in Jurkat T-cell leukemia and K562 erythromyeloid cell lines. LC3B showed an increase with CQ treatment although this was different to LysoTracker signals in terms of dose and time. Rapamycin, low serum (<1%) RPMI, and nutrient starvation induction of autophagy also induced an increase in LysoTracker and LC3B signals. CQ also induced apoptosis in cell lines, which was blocked by pan-caspase inhibitor z-VAD resulting in a reduction in cells undergoing apoptosis and a subsequent upregulation of autophagic markers LC3B and lysosomal dye signals. Given that LC3B and LysoTracker are measuring different biological events in the autophagic process, they surprisingly both upregulated during autophagic process. This study, however, shows that although LysoTracker dyes do not specifically label lysosomes or autophagosomes within the cell, they allow the simultaneous measurement of an autophagy-related process and other live-cell functions, which are not possible with the standard LC3B antibody-labeling technique. This method has the advantage of other live-cell LCB-GFP-tagged experiments in that be used to analyze patient cells as well as easier to use and significantly less costly. Copyright © 2013 International Society for Advancement of Cytometry.

Induction and disappearance of thymine dimers in human skin exposed to UVB radiation: flow cytometric measurements in replicating and nonreplicating epidermal cells.

PubMed

Berg, R J; de Bueger, S C; Guikers, K; van Weelden, H; van Vloten, W A; van der Leun, J C; de Gruijl, F R

1995-12-01

We have earlier reported on determining UV-induced DNA damage in murine epidermal cell suspensions by flow cytometric analysis of the fluorescence from a fluorescein isothiocyanate-labeled antibody (H3) directed against thymine dimers (T < > T). Here we present an optimization of the technique for analysis of epidermal cell suspensions from 4 mm biopsies from human skin. Cells with different DNA contents can easily be distinguished in flow cytometry by the intensity of DNA-specific 7-amino-actinomycin D fluorescence. Genuine G2-M-phase cells can further be distinguished from cell doublets by pulse-shape discrimination. Thus, T < > T levels in individual cells with different DNA contents (i.e. G0-G1, S or G2-M phases) can be determined after in vivo exposure of human skin to environmentally relevant UVB (280-315 nm) doses. The method was applied to measure the decrease of T < > T in nonreplicating cells (G0-G1 phase) and replicating cells (S phase or G2-M phase) from seven volunteers exposed to twice their minimal erythema dose. The reduction in the average T < > T-specific fluorescence at 24 h after exposure was 46% (ranging between 16% and 66%) for the G0-G1 cells and 70% (ranging between 37% and 100%) for the S + G2-M cells. The difference was statistically highly significant. Determination of individual DNA repair capacities with this method can become a convenient diagnostic tool for patients with DNA repair disorders, or it may even be used to identify individuals with low repair proficiencies and increased risk of developing skin cancers.

A flow cytometric approach to the study of crustacean cellular immunity

USGS Publications Warehouse

Cardenas, W.; Jenkins, J.A.; Dankert, J.R.

2000-01-01

Responses of hemocytes from the crayfish Procambarus zonangulus to stimulation by fungal cell walls (Zymosan A) were measured by flow cytometry. Changes in hemocyte physical characteristics were assessed flow cytometrically using forward- and sidescatter light parameters, and viability was measured by two-color fluorescent staining with calcein-AM and ethidium homodimer 1. The main effects of zymosan A on crayfish hemocytes were reduction in cell size and viability compared to control mixtures (hemocytes in buffer only). Adding diethyldithiocarbamic acid, an inhibitor of phenoloxidase, to hemocyte to zymosan mixtures delayed the time course of cell size reduction and cell death compared to zymosan-positive controls. The inclusion of trypsin inhibitor in reaction mixtures further delayed the reduction in hemocyte size and cell death, thereby indicating that a proteolytic cascade, along with prophenoloxidase activation, played a key role in generating signal molecules which mediate these cellular responses. In addition to traditional methods such as microscopy and protein chemistry, flow cytometry can provide a simple, reproducible, and sensitve method for evaluating invertebrate hemocyte responses to immunological stimuli.

Bronchoalveolar Lavage Fluid Characteristics of Patients With Sarcoidosis and Nonsarcoidosis Interstitial Lung Diseases: Ten-Year Experience of a Single Center in Turkey

PubMed Central

Tanriverdi, Hakan; Erboy, Fatma; Altinsoy, Bulent; Uygur, Firat; Arasli, Mehmet; Ozel Tekin, Ishak; Tor, Muge Meltem; Atalay, Figen

2015-01-01

Background: Bronchoalveolar lavage (BAL) is a noninvasive and useful technique for evaluating interstitial lung diseases (ILDs). Flow cytometric analysis of BAL fluid reveals specific diagnostic information in some unusual ILDs, and helps to narrow down the possible causes of interstitial diseases in most patients with more common disorders. A high BAL CD4/CD8 ratio is highly specific for sarcoidosis but can also be seen in other ILDs. Objectives: In this retrospective, descriptive, cross-sectional study, we compared BAL fluid characteristics and clinical variables in patients with sarcoidosis and non-sarcoidosis ILDs in a large cohort. Patients and Methods: The study was conducted in a tertiary university hospital in Zonguldak, the biggest city of the western Black Sea region of Turkey. Between 2004 and 2014, all patients who underwent both fiberoptic bronchoscopy and BAL with a suspicion of ILD were included in the study, retrospectively. Patients were divided into two main groups: sarcoidosis and non-sarcoidosis ILDs. Non-sarcoidosis ILDs were further divided into subgroups: pneumoconiosis, tuberculosis (TB), collagen vascular diseases, idiopathic interstitial pneumonias, malignancies, and unclassified ILDs. The clinical data of patients, including age, gender, smoking status, pulmonary function tests, and BAL flow cytometric analysis results, were compared among groups. Results: In total, 261 patients (119 sarcoidosis and 142 non-sarcoidosis ILDs) were enrolled. The median (interquartile range) BAL CD4/CD8 ratio and lymphocyte fraction were significantly higher in sarcoidosis than in non-sarcoidosis ILDs: 3.88 (3.76) versus 0.88 (1.01), respectively, and 20.6 (28.3) versus 6.0 (13.7), respectively. T cell receptor γ delta, CD16+56+, CD103+, CD8+103+, and CD3+16+56+ cells were significantly lower in sarcoidosis than in non-sarcoidosis ILDs. The median BAL CD4/CD8 ratios were significantly higher in patients with TB (1.87, P = 0.01) and malignancies (1.69, P = 0.03) than in other non-sarcoidosis ILDs. Conclusions: Among BAL fluid flow cytometric parameters, CD4/CD8 and lymphocyte fraction may be helpful for distinguishing sarcoidosis from other ILDs, but they are neither specific nor diagnostic for any lung disease. Thus, a multidisciplinary diagnostic discussion is required to differentiate various ILDs. PMID:26566455

An efficient and rapid transgenic pollen screening and detection method using flow cytometry.

PubMed

Moon, Hong S; Eda, Shigetoshi; Saxton, Arnold M; Ow, David W; Stewart, C Neal

2011-01-01

Assaying for transgenic pollen, a major vector of transgene flow, provides valuable information and essential data for the study of gene flow and assessing the effectiveness of transgene containment. Most studies have employed microscopic screening methods or progeny analyses to estimate the frequency of transgenic pollen. However, these methods are time-consuming and laborious when large numbers of pollen grains must be analyzed to look for rare transgenic pollen grains. Thus, there is an urgent need for the development of a simple, rapid, and high throughput analysis method for transgenic pollen analysis. In this study, our objective was to determine the accuracy of using flow cytometry technology for transgenic pollen quantification in practical application where transgenic pollen is not frequent. A suspension of non-transgenic tobacco pollen was spiked with a known amount of verified transgenic tobacco pollen synthesizing low or high amounts of green fluorescent protein (GFP). The flow cytometric method detected approximately 75% and 100% of pollen grains synthesizing low and high amounts of GFP, respectively. The method is rapid, as it is able to count 5000 pollen grains per minute-long run. Our data indicate that this flow cytometric method is useful to study gene flow and assessment of transgene containment.

Functional characterization of neotropical snakes peripheral blood leukocytes subsets: Linking flow cytometry cell features, microscopy images and serum corticosterone levels.

PubMed

de Carvalho, Marcelo Pires Nogueira; Queiroz-Hazarbassanov, Nicolle Gilda Teixeira; de Oliveira Massoco, Cristina; Sant'Anna, Sávio Stefanini; Lourenço, Mariana Mathias; Levin, Gabriel; Sogayar, Mari Cleide; Grego, Kathleen Fernandes; Catão-Dias, José Luiz

2017-09-01

Reptiles are the unique ectothermic amniotes, providing the key link between ectothermic anamniotes fish and amphibians, and endothermic birds and mammals; becoming an important group to study with the aim of providing significant knowledge into the evolutionary history of vertebrate immunity. Classification systems for reptiles' leukocytes have been described by their appearance rather than function, being still inconsistent. With the advent of modern techniques and the establishment of analytical protocols for snakes' blood by flow cytometry, we bring a qualitative and quantitative assessment of innate activities presented by snakes' peripheral blood leukocytes, thereby linking flow cytometric features with fluorescent and light microscopy images. Moreover, since corticosterone is an important immunomodulator in reptiles, hormone levels of all blood samples were measured. We provide novel and additional information which should contribute to better understanding of the development of the immune system of reptiles and vertebrates. Copyright © 2017 Elsevier Ltd. All rights reserved.

Transient myeloproliferative disease of the newborn: case report with placental, cytogenetic, and flow cytometric findings.

PubMed

de Tar, M W; Dittman, W; Gilbert, J

2000-03-01

Transient myeloproliferative disease (TMD) of the newborn is a rare hematologic abnormality associated with trisomy 21. It is frequently difficult to distinguish the disorder from true congenital leukemia (TCL). Unlike leukemia, which has a clinically aggressive course, TMD generally resolves within weeks to months. We present a case of TMD of the newborn diagnosed on the basis of peripheral blood studies and describe the pertinent pathological findings within the placenta. Flow cytometric analysis of the blasts in the peripheral blood showed phenotypic heterogeneity with features consistent with megakaryocytic differentiation. Cytogenetic studies showed trisomy 21 within the blastic cells. The placenta showed villous dysmaturity with associated chorangiosis and prominent intravascular aggregates of primitive-appearing cells with focal, early vascular wall invasion. The neonate recovered fully and shows no evidence of disease at 2 years of age.

Standardizing flow cytometric assays in long-term population-based studies

NASA Astrophysics Data System (ADS)

Melzer, Susanne; Bocsi, Jozsef; Tárnok, Attila

2015-03-01

Quantification of leukocyte subpopulations and characterization of antigen-expression pattern on the cellular surface can play an important role in diagnostics. The state of cellular immunology on the single-cell level was analyzed by polychromatic flow cytometry in a recent comparative study within the average Leipzig population (LIFE - Leipzig Research Centre for Civilization Diseases). Data of 1699 subjects were recorded over a long-time period of three years (in a total of 1126 days). To ensure compatibility of such huge data sets, quality-controls on many levels (stability of instrumentation, low intra-laboratory variance and reader independent data analysis) are essential. The LIFE study aims to analyze various cytometric pattern to reveal the relationship between the life-style, the environmental effects and the individual health. We therefore present here a multi-step quality control procedure for long-term comparative studies.

Rapid Detection and Enumeration of Giardia lamblia Cysts in Water Samples by Immunomagnetic Separation and Flow Cytometric Analysis ▿ †

PubMed Central

Keserue, Hans-Anton; Füchslin, Hans Peter; Egli, Thomas

2011-01-01

Giardia lamblia is an important waterborne pathogen and is among the most common intestinal parasites of humans worldwide. Its fecal-oral transmission leads to the presence of cysts of this pathogen in the environment, and so far, quantitative rapid screening methods are not available for various matrices, such as surface waters, wastewater, or food. Thus, it is necessary to establish methods that enable reliable rapid detection of a single cyst in 10 to 100 liters of drinking water. Conventional detection relies on cyst concentration, isolation, and confirmation by immunofluorescence microscopy (IFM), resulting in low recoveries and high detection limits. Many different immunomagnetic separation (IMS) procedures have been developed for separation and cyst purification, so far with variable but high losses of cysts. A method was developed that requires less than 100 min and consists of filtration, resuspension, IMS, and flow cytometric (FCM) detection. MACS MicroBeads were used for IMS, and a reliable flow cytometric detection approach was established employing 3 different parameters for discrimination from background signals, i.e., green and red fluorescence (resulting from the distinct pattern emitted by the fluorescein dye) and sideward scatter for size discrimination. With spiked samples, recoveries exceeding 90% were obtained, and false-positive results were never encountered for negative samples. Additionally, the method was applicable to naturally occurring cysts in wastewater and has the potential to be automated. PMID:21685159

A Review of Current Methods for Analysis of Mycotoxins in Herbal Medicines

PubMed Central

Zhang, Lei; Dou, Xiao-Wen; Zhang, Cheng; Logrieco, Antonio F.; Yang, Mei-Hua

2018-01-01

The presence of mycotoxins in herbal medicines is an established problem throughout the entire world. The sensitive and accurate analysis of mycotoxin in complicated matrices (e.g., herbs) typically involves challenging sample pretreatment procedures and an efficient detection instrument. However, although numerous reviews have been published regarding the occurrence of mycotoxins in herbal medicines, few of them provided a detailed summary of related analytical methods for mycotoxin determination. This review focuses on analytical techniques including sampling, extraction, cleanup, and detection for mycotoxin determination in herbal medicines established within the past ten years. Dedicated sections of this article address the significant developments in sample preparation, and highlight the importance of this procedure in the analytical technology. This review also summarizes conventional chromatographic techniques for mycotoxin qualification or quantitation, as well as recent studies regarding the development and application of screening assays such as enzyme-linked immunosorbent assays, lateral flow immunoassays, aptamer-based lateral flow assays, and cytometric bead arrays. The present work provides a good insight regarding the advanced research that has been done and closes with an indication of future demand for the emerging technologies. PMID:29393905

In vitro interactions between splenocytes and dansylamide dye-embedded nanoparticles detected by flow cytometry

PubMed Central

Nyland, Jennifer F.; Bai, Jennifer J. K.; Katz, Howard E.; Silbergeld, Ellen K.

2009-01-01

Engineered nanoparticles (NPs) possess a range of biological activity. In vitro methods for assessing toxicity and efficacy would be enhanced by simultaneous quantitative information on the behavior of NPs in culture systems and signals of cell response. We have developed a method for visualizing NPs within cells using standard flow cytometric techniques and uniquely designed spherical siloxane NPs with an embedded (covalently bound) dansylamide dye. This method allowed NP visualization without obscuring detection of relevant biomarkers of cell subtype, activation state, and other events relevant to assessing bioactivity. We determined that NPs penetrated cells and induced a range of biological signals consistent with activation and costimulation. These results indicate that NPs may affect cell function at concentrations below those inducing cytotoxicity or apoptosis and demonstrate a novel method to image both localization of NPs and cell-level effects. PMID:19523425

Navigation to the graveyard-induction of various pathways of necrosis and their classification by flow cytometry.

PubMed

Janko, Christina; Munoz, Luis; Chaurio, Ricardo; Maueröder, Christian; Berens, Christian; Lauber, Kirsten; Herrmann, Martin

2013-01-01

Apoptosis and necrosis reflect the program of cell death employed by a dying cell and the final stage of death, respectively. Whereas apoptosis is defined as a physiological, highly organized cell death process, necrosis is commonly considered to be accidental and uncontrolled. Physiological and weak pathological death stimuli preferentially induce apoptosis, while harsh non-physiological insults often immediately instigate (primary) necrosis. If an apoptosing cell transits into a phase of plasma membrane disintegration, this stage of death is referred to as secondary or post-apoptotic necrosis.Here, we present several conditions that stimulate primary and/or secondary necrosis and show that necrosis displays considerably different time courses. For subclassification of necrotic phenotypes we employed a flow cytometric single-tube 4-color staining technique including annexin A5-FITC, propidium iodide, DiIC1(5), and Hoechst 33342.

Enzymatic signal amplification for sensitive detection of intracellular antigens by flow cytometry.

PubMed

Karkmann, U; Radbruch, A; Hölzel, V; Scheffold, A

1999-11-19

Flow cytometry is the method of choice for the analysis of single cells with respect to the expression of specific antigens. Antigens can be detected with specific antibodies either on the cell surface or within the cells, after fixation and permeabilization of the cell membrane. Using conventional fluorochrome-labeled antibodies several thousand antigens are required for clear-cut separation of positive and negative cells. More sensitive reagents, e.g., magnetofluorescent liposomes conjugated to specific antibodies permit the detection of less than 200 molecules per cell but cannot be used for the detection of intracellular antigens. Here, we describe an enzymatic amplification technique (intracellular tyramine-based signal amplification, ITSA) for the sensitive cytometric analysis of intracellular cytokines by immunofluorescence. This approach results in a 10 to 15-fold improvement of the signal-to-noise ratio compared to conventional fluorochrome labeled antibodies and permits the detection of as few as 300-400 intracellular antigens per cell.

Flow cytometric method for scoring rat liver micronuclei with simultaneous assessments of hepatocyte proliferation.

PubMed

Avlasevich, Svetlana L; Khanal, Sumee; Singh, Priyanka; Torous, Dorothea K; Bemis, Jeffrey C; Dertinger, Stephen D

2018-04-01

The current report describes a newly devised method for automatically scoring the incidence of rat hepatocyte micronuclei (MNHEP) via flow cytometry, with concurrent assessments of hepatocyte proliferation-frequency of Ki-67-positive nuclei, and the proportion of polyploid nuclei. Proof-of-concept data are provided from experiments performed with 6-week old male Crl:CD(SD) rats exposed to diethylnitrosamine (DEN) or quinoline (QUIN) for 3 or 14 consecutive days. Non-perfused liver tissue was collected 4 days after cessation of treatment in the case of 3-day studies, or 1 day after last administration in the case of 14-day studies for processing and flow cytometric analysis. In addition to livers, blood samples were collected one day after final treatment for micronucleated reticulocyte (MN-RET) measurements. Dose-dependent increases in MNHEP, Ki-67-positive nuclei, and polyploidy were observed in 3- and 14-day DEN studies. Both treatment schedules resulted in elevated %MNHEP for QUIN-exposed rats, and while cell proliferation effects were subtle, appreciable increases to normalized liver weights were observed. Whereas DEN caused markedly higher %MNHEP when exposure was extended to two weeks, QUIN-induced MNHEP were slightly increased with protracted dosing. Parallel microscopy-based MNHEP frequencies were highly correlated with flow cytometry-based measurements (four study/aggregate R 2  = 0.80). No increases in MN-RET were seen in any of the four studies. Collectively, these results suggest liver micronuclei are amenable to an automated scoring technique that provides objective analyses and higher information content relative to conventional microscopy. Additional work is needed to expand the number and types of chemicals tested, identify the most advantageous treatment schedules, and test the transferability of the method. Environ. Mol. Mutagen. 59:176-187, 2018. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

A Study of the Effects of High Power Pulsed 2450 MHz Microwaves, ELF modulated Microwaves, and ELF Fields on Human Lymphocytes and Selected Cell Lines

DTIC Science & Technology

1993-01-27

Considerable effect was expended in investigating shifts in intercellular calcium of one particular cell line, Jurket, using flow cytometry methods. No...culture. The following analysis were used to characterize the immortalized cell lines: flow cytometry , electron microscopy, two-dimensional protein gel...further characterized by flow cytometry , electron microscopy, two dimensional protein electrophoresis and nuclear run-off assay. Flow cytometric analysis of

Annual Progress Report FY-92. Volume 1

DTIC Science & Technology

1993-01-21

Billups, L Flow Cytom Resh Psychologist 12 0180 CS Hamm, C DCI 7 DESCRIPTION GRADE MOS BRANCH NAME ACTIVITY Kyle Metabolic Unit Nursing Service Supv...3349 Salata, Kalman PhD. Mitogen-Inducible T Suppressor Cell 13 Assay by Flow Cytometry (12/89) 3350 Salata, Kalman PhD. Flow Cytometric Analysis of...17 Immunotherapy (3/90) 3354 Salata, Kalman PhD. Two Way Mixed Lymphocyte Culture: 18 Analysis by Two Color Flow Cytometry (4/90) 3355 Salata, Kalman

Simultaneous use of multiplex ligation-dependent probe amplification assay and flow cytometric DNA ploidy analysis in patients with acute leukemia.

PubMed

Reyes-Núñez, Virginia; Galo-Hooker, Evelyn; Pérez-Romano, Beatriz; Duque, Ricardo E; Ruiz-Arguelles, Alejandro; Garcés-Eisele, Javier

2018-01-01

The aim of this work was to simultaneously use multiplex ligation-dependent probe amplification (MLPA) assay and flow cytometric DNA ploidy analysis (FPA) to detect aneuploidy in patients with newly diagnosed acute leukemia. MLPA assay and propidium iodide FPA were used to test samples from 53 consecutive patients with newly diagnosed acute leukemia referred to our laboratory for immunophenotyping. Results were compared by nonparametric statistics. The combined use of both methods significantly increased the rate of detection of aneuploidy as compared to that obtained by each method alone. The limitations of one method are somehow countervailed by the other and vice versa. MPLA and FPA yield different yet complementary information concerning aneuploidy in acute leukemia. The simultaneous use of both methods might be recommended in the clinical setting. © 2017 International Clinical Cytometry Society. © 2017 International Clinical Cytometry Society.

Method for screening inhibitors of the toxicity of Bacillus anthracis

DOEpatents

Cirino, Nick M.; Jackson, Paul J.; Lehnert, Bruce E.

2001-01-01

The protective antigen (PA) of Bacillus anthracis is integral to the mechanism of anthrax poisoning. The cloning, expression and purification of a 32 kDa B. anthracis PA fragment (PA32) is described. This fragment has also been expressed as a fusion construct to stabilized green fluorescent protein (EGFP-PA32). Both proteins were capable of binding to specific cell surface receptors as determined by fluorescent microscopy and a flow cytometric assay. To confirm binding specificity in the flow cytometric assay, non-fluorescent PA83 or PA32 was used to competitively inhibit fluorescent EGFP-PA32 binding to cell receptors. This assay can be employed as a rapid screen for compounds which disrupts binding of PA to cells. Additionally, the high intracellular expression levels and ease of purification make this recombinant protein an attractive vaccine candidate or therapeutic treatment for anthrax poisoning.

Integrating molecular diagnostic and flow cytometric reporting for improved longitudinal monitoring of HIV patients.

PubMed Central

Asare, A. L.; Huda, H.; Klimczak, J. C.; Caldwell, C. W.

1998-01-01

Studies have shown that monitoring HIV-infected patients undergoing antiretroviral therapy is best represented by combined measurement of plasma HIV-1 RNA and CD4+ T-lymphocytes [1]. This pilot study at the University of Missouri-Columbia integrates molecular diagnostic and flow cytometric data reporting to provide current and historical HIV-1 RNA levels and CD4+ T-cell counts. The development of a single database for storage and retrieval of these values facilitates composite report generation that includes longitudinal HIV-1 RNA levels and CD4+ T-cell counts for all patients. Results are displayed in tables and plotted graphically within a web browser. This method of data presentation converts individual data points to more useful medical information and could provide clinicians with decision support for improved monitoring of HIV patients undergoing antiretroviral therapy. Images Figure 2 Figure 3 Figure 4 PMID:9929359

Flow cytometric detection of micronuclei by combined staining of DNA and membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wessels, J.M.; Nuesse, M.

1995-03-01

A new staining method is presented for flow cytometric measurement of micronuclei (MN) in cell cultures and human lymphocytes using membrane-specific fluorescent dyes in addition to DNA staining. Several combinations of fluorescent membrane and DNA dyes were studied for a better discrimination of MN from debris in a suspension of nuclei and micronuclei. For staining of membranes, the lipophilic dyes 2-hydroxyethyl-7,12,17-tris(methoxyethyl)porphycene (HEPn) and 1,6-diphenyl-1,3,5-hexatriene (DPH) were used in combination with ethidium bromide (EB), proflavine (PF), and Hoechst 33258 (HO). Due to their spectral properties, HO or EB combined with HEPn were not as suitable for the discrimination of MN frommore » debris as was HEPn in combination with PF. With HEPn in combination with PF, however, additional noise was found at low fluorescence intensities, probably due to free fluorescent dye molecules in the solution. The optimal simultaneous staining of membranes and DNA was obtained using a combination of DPH and EB. The induction of MN in Chinese hamster and mouse NIH-3T3 cells by UV-B illumination was studied with this new staining technique. UV-B illumination (280-360 nm) induced MN in both cell lines. Chinese hamster cells were found to be more sensitive to these wavelengths. Illumination with wavelengths above 360 nm did not induce MN in either cell line. The results obtained from human lymphocytes using the combination of EB or DPH were comparable to the results obtained with the combination of EB and HO. 23 refs., 7 figs.« less

DNA flow cytometry of human spermatozoa: consistent stoichiometric staining of sperm DNA using a novel decondensation protocol.

PubMed

Kovács, Tamás; Békési, Gyöngyi; Fábián, Akos; Rákosy, Zsuzsa; Horváth, Gábor; Mátyus, László; Balázs, Margit; Jenei, Attila

2008-10-01

Rapid flow cytometric measurement of the frequency of aneuploid human sperms is in increasing demand but development of an exploitable method is hindered by difficulties of stoichiometric staining of sperm DNA. An aggressive decondensation protocol is needed after which cell integrity still remains intact. We used flow cytometry to examine the effect of lithium diiodosalicylate (LIS, chaotropic agent) on fluorescence intensity of propidium iodide-treated human spermatozoa from 10 normozoospermic men. When flow cytometric identification of diploid spermatozoa was achieved, validation was performed after sorting by three-color FISH. In contrast with the extremely variable histograms of nondecondensed sperms, consistent identification of haploid and diploid spermatozoa was possible if samples were decondensed with LIS prior to flow cytometry. A 76-fold enrichment of diploid sperms was observed in the sorted fractions by FISH. A significant correlation was found between the proportion of sorted cells and of diploid sperms by FISH. Application of LIS during the preparation of sperm for flow cytometry appears to ensure the stoichiometric staining of sperm DNA, making quantification of aneuploid sperm percentage possible. To our knowledge this is the first report in terms of separating spermatozoa with confirmedly abnormal chromosomal content. High correlation between the proportion of cells identified as having double DNA content by flow cytometry and diploid sperm by FISH allows rapid calculation of diploidy rate. Copyright 2008 International Society for Advancement of Cytometry.

Cell proliferation assessment in oncology.

PubMed

Hofstädter, F; Knüchel, R; Rüschoff, J

1995-01-01

A review of the current knowledge on cell cycle control and the techniques used to assess proliferation of normal and neoplastic cells was the focus of a workshop in Regensburg, Germany, held under the joint auspices of the Graduiertenkolleg: Therapieforschung Onkologie and the Committee on AgNOR Quantification. An overview of the recently discovered group of cyclins and their specific kinases, and of other proliferation-associated antigens, such as Ki67, PCNA and topoiseromase II alpha, was given. The topics continued with a reappraisal of modern imaging and flow-cytometric techniques. An update of the relation of AgNORs to cellular proliferation and differentiation was the link to presentations on clinical data, problems and strategies for standardization, as well as guidelines to establish the prognostic value of marker molecules. These lectures were supported by posters. Bringing together researchers from life sciences, technically oriented workers, pathologists, and clinicians resulted in a lively and constructive discussion, which is briefly summarized in the Concluding remarks.

Novel flow cytometric method for the detection of podocalyxin-positive elements in urine of patients with glomerulonephritides - first promising results.

PubMed

Habara, P; Marečková, H; Malíčková, K; Potyšová, Z; Hrušková, Z; Zima, T; Tesař, V

2012-01-01

Glomerulonephritides together create a heterogenic group of supposedly immunologically mediated diseases of glomeruli. They still belong among the most frequent causes of chronic renal failure. Detection of podocytes in urine might serve as an important marker of glomerulonephritides activity. The aim of this study was to develop a novel flow cytometric method for the detection of podocyte fragments and podocytes in urine and assess its possible use in clinical practice. We placed emphasis on the improvement of pre-analytic phase. To suppress the autofluorescence of the background, blocking solutions and magnetic separation were used. An additional surface marker CD10 (nephrilysin) was used together with routinely used podocalyxin (PCX) in order to achieve better identification of podocytes. Based on the surface marker expression, three different element types were identified in the urine samples: PCX+/CD10+ elements (EL) (supposedly podocytes), PCX-/CD10+ EL (supposedly parietal epithelial cells) and PCX+ EL. We examined a total of 36 patients who underwent renal biopsy (non-glomerular nephropathy, MGN, FSGS, IgAN, AAV and MPGN) and 27 healthy controls. Negative results were found in non-glomerular nephropathy and in MGN. In patients with FSGS and IgAN, the levels of urine elements were slightly increased. The highest levels of all elements were found in AAV and MPGN. Our first results suggest that flow cytometric detection may distinguish between glomerular and nonglomerular diseases and that the levels of urine elements might correlate with the degree of glomerular destruction.

2006 Bethesda International Consensus recommendations on the immunophenotypic analysis of hematolymphoid neoplasia by flow cytometry: optimal reagents and reporting for the flow cytometric diagnosis of hematopoietic neoplasia.

PubMed

Wood, Brent L; Arroz, Maria; Barnett, David; DiGiuseppe, Joseph; Greig, Bruce; Kussick, Steven J; Oldaker, Teri; Shenkin, Mark; Stone, Elizabeth; Wallace, Paul

2007-01-01

Immunophenotyping by flow cytometry has become standard practice in the evaluation and monitoring of patients with hematopoietic neoplasia. However, despite its widespread use, considerable variability continues to exist in the reagents used for evaluation and the format in which results are reported. As part of the 2006 Bethesda Consensus conference, a committee was formed to attempt to define a consensus set of reagents suitable for general use in the diagnosis and monitoring of hematopoietic neoplasms. The committee included laboratory professionals from private, public, and university hospitals as well as large reference laboratories that routinely operate clinical flow cytometry laboratories with an emphasis on lymphoma and leukemia immunophenotyping. A survey of participants successfully identified the cell lineage(s) to be evaluated for each of a variety of specific medical indications and defined a set of consensus reagents suitable for the initial evaluation of each cell lineage. Elements to be included in the reporting of clinical flow cytometric results for leukemia and lymphoma evaluation were also refined and are comprehensively listed. The 2006 Bethesda Consensus conference represents the first successful attempt to define a set of consensus reagents suitable for the initial evaluation of hematopoietic neoplasia. Copyright 2007 Clinical Cytometry Society.

Is flow cytometric evaluation of DNA degradation a reliable method to investigate the early postmortem period?

PubMed

Di Nunno, N R; Costantinides, F; Bernasconi, P; Bottin, C; Melato, M

1998-03-01

The time of death can be established by determining the length of the postmortem interval. Many methods have been proposed to achieve this goal. Flow cytometric evaluation of DNA degradation seems to be reliable for the first 72 hours after death. Our study evaluated the correspondence of the corruption process between in vitro and corpse tissues. We chose spleen tissue to perform our investigation because it is rich in nucleated cells. Results showed a precise correspondence between the two kinds of samples in the time period between 24 and 36 hours. The period from 36 to 72 hours is characterized by a much looser correspondence than that found in the first period. After the first 72 hours, DNA denaturation is massive and does not allow useful cytofluorimetric readings. The spleen does not seem to be the most suitable organ for this type of investigation because it tends to colliquate very rapidly. We therefore are evaluating other organs to identify a more suitable tissue source for the investigation of longer postmortem period using flow cytometry.

Usefulness of bronchoalveolar lavage and flow cytometry in patients with hematological malignancies and respiratory failure.

PubMed

Ferrà, Christelle; Xicoy, Blanca; Castillo, Nerea; Morgades, Mireia; Juncà, Jordi; Andreo, Felipe; Millá, Fuensanta; Feliu, Evarist; Ribera, Josep-María

2017-04-07

Strategies to improve the efficiency of bronchoalveolar lavage (BAL) are needed. We conducted a study to establish the diagnostic value of BAL in patients with hematological malignancies and pulmonary infiltrates. The correlation of cytologic and flow cytometric study of BAL with the microbiological findings and the clinical evolution was determined. Seventy BAL were performed and flow cytometric study was analyzed in 23 of them. Fifty-three patients did not present any adverse event attributable to BAL. Anti-infectious therapy was modified in 64 (91%) patients. T lymphocyte count >0.3×10 9 /l in peripheral blood was associated with longer OS at 3 years (53 vs. 22%, p=.009). Higher CD4 (>20/μL) and CD8 (>35/μL) lymphocyte counts in the BAL were associated with a longer OS at 3 years: 82 vs. 21% (p=.030) and 80 vs. 23% (p=.059). Our study confirms the clinical value of BAL for treatment decision making in patients with hematological malignancies and acute respiratory failure. Copyright © 2016 Elsevier España, S.L.U. All rights reserved.

Analysis of synthetic and biological microparticles on several flow cytometric platforms***

EPA Science Inventory

Biological microparticles (MPs) are potentially important biomarkers for thrombosis, cancer, glomerulonephritis and other disease states. These MPs are generally accepted to be membrane vesicles extruded following cellular activation. While human blood cells range from 10-15 micr...

Genome size variation in the pine fusiform rust pathogen Cronartium quercuum f.sp. fusiforme as determined by flow cytometry

Treesearch

Claire L Anderson; Thomas L Kubisiak; C Dana Nelson; Jason A Smith; John M Davis

2010-01-01

The genome size of the pine fusiform rust pathogen Cronartium quercuum f.sp. fusiforme (Cqf) was determined by flow cytometric analysis of propidium iodide-stained, intact haploid pycniospores with haploid spores of two genetically well characterized fungal species, Sclerotinia sclerotiorum and Puccinia graminis f.sp. tritici, as size standards. The Cqf haploid genome...

Comparative analysis of minimal residual disease detection using four-color flow cytometry, consensus IgH-PCR, and quantitative IgH PCR in CLL after allogeneic and autologous stem cell transplantation.

PubMed

Böttcher, S; Ritgen, M; Pott, C; Brüggemann, M; Raff, T; Stilgenbauer, S; Döhner, H; Dreger, P; Kneba, M

2004-10-01

The clinically most suitable method for minimal residual disease (MRD) detection in chronic lymphocytic leukemia is still controversial. We prospectively compared MRD assessment in 158 blood samples of 74 patients with CLL after stem cell transplantation (SCT) using four-color flow cytometry (MRD flow) in parallel with consensus IgH-PCR and ASO IgH real-time PCR (ASO IgH RQ-PCR). In 25 out of 106 samples (23.6%) with a polyclonal consensus IgH-PCR pattern, MRD flow still detected CLL cells, proving higher sensitivity of flow cytometry over PCR-genescanning with consensus IgH-primers. Of 92 samples, 14 (15.2%) analyzed in parallel by MRD flow and by ASO IgH RQ-PCR were negative by our flow cytometric assay but positive by PCR, thus demonstrating superior sensitivity of RQ-PCR with ASO primers. Quantitative MRD levels measured by both methods correlated well (r=0.93). MRD detection by flow and ASO IgH RQ-PCR were equally suitable to monitor MRD kinetics after allogeneic SCT, but the PCR method detected impending relapses after autologous SCT earlier. An analysis of factors that influence sensitivity and specificity of flow cytometry for MRD detection allowed to devise further improvements of this technique.

Telomere content measurement in human hematopoietic cells: Comparative analysis of qPCR and Flow-FISH techniques.

PubMed

Wand, Taylor; Fang, Mike; Chen, Christina; Hardy, Nathan; McCoy, J Philip; Dumitriu, Bogdan; Young, Neal S; Biancotto, Angélique

2016-10-01

Abnormal telomere lengths have been linked to cancer and other hematologic disorders. Determination of mean telomere content (MTC) is traditionally performed by Southern blotting and densitometry, giving a mean telomere restriction fragment (TRF) value for the total cell population studied. Here, we compared a quantitative Polymerase Chain Reaction approach (qPCR) and a flow cytometric approach, fluorescence in situ hybridization (Flow-FISH), to evaluate telomere content distribution in total patient peripheral blood mononuclear cells or specific cell populations. Flow-FISH is based on in situ hybridization using a fluorescein-labeled peptide nucleic acid (PNA) (CCCTAA) 3 probe and DNA staining with propidium iodide. We showed that both qPCR and Flow-FISH provide a robust measurement, with Flow-FISH measuring a relative content longer than qPCR at a single cell approach and that TRF2 fluorescence intensity did not correlate with MTC. Both methods showed comparable telomere content reduction with age, and the rate of relative telomere loss was similar. Published 2016 Wiley Periodicals Inc. This article is a US government work and, as such, is in the public domain in the United States of America. Published 2016 Wiley Periodicals Inc. This article is a US government work and, as such, is in the public domain in the United States of America.

FLOW CYTOMETRIC DETECTION OF CRYPTOSPORIDIUM OOCYSTS IN HUMAN STOOL SAMPLES. (R824759)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Flow Cytometric Immunobead Assay for Detection of BCR-ABL1 Fusion Proteins in Chronic Myleoid Leukemia: Comparison with FISH and PCR Techniques

PubMed Central

Recchia, Anna Grazia; Caruso, Nadia; Bossio, Sabrina; Pellicanò, Mariavaleria; De Stefano, Laura; Franzese, Stefania; Palummo, Angela; Abbadessa, Vincenzo; Lucia, Eugenio; Gentile, Massimo; Vigna, Ernesto; Caracciolo, Clementina; Agostino, Antolino; Galimberti, Sara; Levato, Luciano; Stagno, Fabio; Molica, Stefano; Martino, Bruno; Vigneri, Paolo; Di Raimondo, Francesco; Morabito, Fortunato

2015-01-01

Chronic Myeloid Leukemia (CML) is characterized by a balanced translocation juxtaposing the Abelson (ABL) and breakpoint cluster region (BCR) genes. The resulting BCR-ABL1 oncogene leads to increased proliferation and survival of leukemic cells. Successful treatment of CML has been accompanied by steady improvements in our capacity to accurately and sensitively monitor therapy response. Currently, measurement of BCR-ABL1 mRNA transcript levels by real-time quantitative PCR (RQ-PCR) defines critical response endpoints. An antibody-based technique for BCR-ABL1 protein recognition could be an attractive alternative to RQ-PCR. To date, there have been no studies evaluating whether flow-cytometry based assays could be of clinical utility in evaluating residual disease in CML patients. Here we describe a flow-cytometry assay that detects the presence of BCR-ABL1 fusion proteins in CML lysates to determine the applicability, reliability, and specificity of this method for both diagnosis and monitoring of CML patients for initial response to therapy. We show that: i) CML can be properly diagnosed at onset, (ii) follow-up assessments show detectable fusion protein (i.e. relative mean fluorescent intensity, rMFI%>1) when BCR-ABL1IS transcripts are between 1–10%, and (iii) rMFI% levels predict CCyR as defined by FISH analysis. Overall, the FCBA assay is a rapid technique, fully translatable to the routine management of CML patients. PMID:26111048

Highly multiparametric analysis by mass cytometry.

PubMed

Ornatsky, Olga; Bandura, Dmitry; Baranov, Vladimir; Nitz, Mark; Winnik, Mitchell A; Tanner, Scott

2010-09-30

This review paper describes a new technology, mass cytometry, that addresses applications typically run by flow cytometer analyzers, but extends the capability to highly multiparametric analysis. The detection technology is based on atomic mass spectrometry. It offers quantitation, specificity and dynamic range of mass spectrometry in a format that is familiar to flow cytometry practitioners. The mass cytometer does not require compensation, allowing the application of statistical techniques; this has been impossible given the constraints of fluorescence noise with traditional cytometry instruments. Instead of "colors" the mass cytometer "reads" the stable isotope tags attached to antibodies using metal-chelating labeling reagents. Because there are many available stable isotopes, and the mass spectrometer provides exquisite resolution between detection channels, many parameters can be measured as easily as one. For example, in a single tube the technique allows for the ready detection and characterization of the major cell subsets in blood or bone marrow. Here we describe mass cytometric immunophenotyping of human leukemia cell lines and leukemia patient samples, differential cell analysis of normal peripheral and umbilical cord blood; intracellular protein identification and metal-encoded bead arrays. Copyright © 2010 Elsevier B.V. All rights reserved.

Measurement of lipocortin 1 levels in murine peripheral blood leukocytes by flow cytometry: modulation by glucocorticoids and inflammation.

PubMed

Perretti, M; Flower, R J

1996-06-01

1. Lipocortin 1 (LC1) immunoreactivity in murine peripheral blood leukocytes was quantified by use of a flow cytometric technique associated with a permeabilisation protocol with saponin. Using specific antisera raised against the whole protein or against its N-terminus peptide, cell-associated LC1-like immunoreactivity was easily detected in circulating neutrophils and monocytes, whereas very low levels were found in lymphocytes. Of the total protein measured 17.6% and 36% were associated with the external plasma membrane in neutrophils and monocytes, as assessed in the absence of cell permeabilisation, whereas no signal was detected on lymphocyte plasma membrane. 2. Treatment of mice with dexamethasone (Dex; 0.5-5 micrograms per mouse corresponding to approximately 0.015-1.5 mg kg-1) increased LC1 levels in neutrophils and monocytes. The 2-3 fold increase in LC1 levels was time-dependent with a peak at 2 h. Treatment of mice with the steroid antagonist, RU486 (two doses of 20 mg kg-1 orally) decreased LC1-like immunoreactivity in all three types of circulating leukocytes by > or = 50%. 3. Extravasation of blood neutrophils into inflamed tissue sites resulted in a consistent reduction (> or = 50%) in LC1 levels compared with circulating neutrophils. A high LC1-like immunoreactivity was also measured in resident macrophages, of which approximately one third was membrane-associated. Induction of an acute inflammatory response in the murine peritoneal cavity did not modify total LC1 levels measured in macrophages, but reduced membrane-associated LC1 to a significant extent, i.e. up to 70%. 4. In conclusion, flow cytometric analysis is a rapid and convenient method for detecting and measuring LC1 in murine leukocytes. We confirmed that LC1 protein expression is controlled by exogenous and endogenous glucocorticoids. Amongst other factor(s) influencing protein concentrations, extravasation was found to be associated with a reduced LC1 expression in the emigrated cells.

The role of RhD agglutination for the detection of weak D red cells by anti-D flow cytometry.

PubMed

Grey, D E; Davies, J I; Connolly, M; Fong, E A; Erber, W N

2005-04-01

Anti-D flow cytometry is an accurate method for quantifying feto-maternal haemorrhage (FMH). However, weak D red cells with <1000 RhD sites are not detectable using this methodology but are immunogenic. As quantitation of RhD sites is not practical, an alternative approach is required to identify those weak D fetal red cells where anti-D flow cytometry is inappropriate. We describe a simple algorithm based on RhD agglutination and flow cytometry peak separation. All weak D (n = 34) gave weak agglutination with RUM-1 on immediate spin (grading

Quantification of circulating mature endothelial cells using a whole blood four-color flow cytometric assay.

PubMed

Jacques, Nathalie; Vimond, Nadege; Conforti, Rosa; Griscelli, Franck; Lecluse, Yann; Laplanche, Agnes; Malka, David; Vielh, Philippe; Farace, Françoise

2008-09-15

Circulating endothelial cells (CEC) are currently proposed as a potential biomarker for measuring the impact of anti-angiogenic treatments in cancer. However, the lack of consensus on the appropriate method of CEC measurement has led to conflicting data in cancer patients. A validated assay adapted for evaluating the clinical utility of CEC in large cohorts of patients undergoing anti-angiogenic treatments is needed. We developed a four-color flow cytometric assay to measure CEC as CD31(+), CD146(+), CD45(-), 7-amino-actinomycin-D (7AAD)(-) events in whole blood. The distinctive features of the assay are: (1) staining of 1 ml whole blood, (2) use of a whole blood IgPE control to measure accurately background noise, (3) accumulation of a large number of events (almost 5 10(6)) to ensure statistical analysis, and (4) use of 10 microm fluorescent microbeads to evaluate the event size. Assay reproducibility was determined in duplicate aliquots of samples drawn from 20 metastatic cancer patients. Assay linearity was tested by spiking whole blood with low numbers of HUVEC. Five-color flow cytometric experiments with CD144 were performed to confirm the endothelial origin of the cells. CEC were measured in 20 healthy individuals and 125 patients with metastatic cancer. Reproducibility was good between duplicate aliquots (r(2)=0.948, mean difference between duplicates of 0.86 CEC/ml). Detected HUVEC correlated with spiked HUVEC (r(2)=0.916, mean recovery of 100.3%). Co-staining of CD31, CD146 and CD144 confirmed the endothelial nature of cells identified as CEC. Median CEC levels were 6.5/ml (range, 0-15) in healthy individuals and 15.0/ml (range, 0-179) in patients with metastatic carcinoma (p<0.001). The assay proposed here allows reproducible and sensitive measurement of CEC by flow cytometry and could help evaluate CEC as biomarkers of anti-angiogenic therapies in large cohorts of patients.

In vitro effects of nonesterified fatty acids on bovine neutrophils oxidative burst and viability.

PubMed

Scalia, D; Lacetera, N; Bernabucci, U; Demeyere, K; Duchateau, L; Burvenich, C

2006-01-01

An in vitro study was conducted to examine the influence of nonesterified fatty acids (NEFA) on bovine polymorphonuclear leukocytes (PMN). Eight healthy, midlactating Holstein cows were used as blood donors. Blood PMN were isolated and incubated with a mixture of NEFA, reflecting composition of bovine plasma NEFA at concentrations that were intended to mimic those found in blood of cows undergoing high, moderate, or low lipomobilization intensity (2, 1, 0.5, 0.25, 0.125, and 0.0625 mM). Control samples were incubated in absence of NEFA. Phagocytosis and oxidative burst activities were assessed by a 2-color flow cytometric method, which was based on oxidation of intracellular dihydrorhodamine 123 to green fluorescent rhodamine 123. Oxidative burst products were generated by incubating PMN with Staphylococcus aureus labeled with propidium iodide. A flow cytometric technique was used to detect PMN viability, necrosis, and apoptosis using fluorescein isothiocyanate-labeled annexin-V and propidium iodide. Phagocytic activity was not affected by NEFA. The highest concentration of NEFA (2 mM) was associated with a dramatic increase of phagocytosis-associated oxidative burst activities with a reduction in cell viability (48.0 vs. 97.5% in control samples) and with a marked increase of necrosis (49.4 vs. 0.5% in control samples). Conversely, the mixture of NEFA did not affect the occurrence of apoptosis. Enhancement of the oxidative burst associated with the highest concentration of NEFA might explain the reduced viability and higher percentage of necrosis observed under the same conditions. This study demonstrated a substantial resistance of bovine PMN to an overload of fatty acids. However, observation that the highest concentration of NEFA regulated some PMN functions encourages the possibility of in vivo studies to assess the relationships between intensity of lipomobilization, plasma NEFA, and bovine PMN functions.

Cr(VI) induces DNA damage, cell cycle arrest and polyploidization: a flow cytometric and comet assay study in Pisum sativum.

PubMed

Rodriguez, Eleazar; Azevedo, Raquel; Fernandes, Pedro; Santos, Conceição

2011-07-18

Chromium(VI) is recognized as the most toxic valency of Cr, but its genotoxicity and cytostaticity in plants is still poorly studied. In order to analyze Cr(VI) cyto- and gentotoxicity, Pisum sativum L. plants were grown in soil and watered with solutions with different concentrations of Cr up to 2000 mg/L. After 28 days of exposure, leaves showed no significant variations in either cell cycle dynamics or ploidy level. As for DNA damage, flow cytometric (FCM) histograms showed significant differences in full peak coefficient of variation (FPCV) values, suggesting clastogenicity. This is paralleled by the Comet assay results, showing an increase in DNA damage for 1000 and 2000 mg/L. In roots, exposure to 2000 mg/L resulted in cell cycle arrest at the G(2)/M checkpoint. It was also verified that under the same conditions 40% of the individuals analyzed suffered polyploidization having both 2C and 4C levels. DNA damage analysis by the Comet assay and FCM revealed dose-dependent increases in DNA damage and FPCV. Through this, we have unequivocally demonstrated for the first time in plants that Cr exposure can result in DNA damage, cell cycle arrest, and polyploidization. Moreover, we critically compare the validity of the Comet assay and FCM in evaluating cytogenetic toxicity tests in plants and demonstrate that the data provided by both techniques complement each other and present high correlation levels. In conclusion, the data presented provides new insight on Cr effects in plants in general and supports the use of the parameters tested in this study as reliable endpoints for this metal toxicity in plants. © 2011 American Chemical Society

Does fluorescence diagnosis have a role in follow up of response to therapy in mycosis fungoides?

PubMed

Bosseila, Manal; Mahgoub, Doaa; El-Sayed, Abeer; Salama, Dina; Abd El-Moneim, Marwa; Al-Helf, Fatma

2014-12-01

Monitoring of tumor burden during mycosis fungoides (MF) treatment, is crucial to adjust therapy accordingly. This is usually achieved through combined by clinical assessment with histopathological and immunohistochemical evaluation. To assess the validity of fluorescence diagnosis (FD) in the measurement of response to therapy in early MF, using in comparison flow cytometric technique of skin biopsies for CD4+/CD7- malignant T-cell count before and after therapy. Twenty-two patients of histologically proven early MF (stages Ia, Ib, IIa) were subjected to fluorescence diagnosis of their most affected skin lesion before and after 12 weeks of phototherapy with or without combination therapy. In comparison flow cytometric assessment of skin biopsies for CD4+/CD7- malignant T-cell count was evaluated before and after therapy from skin biopsy of the same lesion. All tested MF lesions showed varying degrees of fluorescence by FD at week zero, with a mean accumulation factor (AF), which is the fluorescence ratio between the tumor tissue and normal skin, of 2.2. After 12 weeks of therapy, the mean AF showed significant reduction to 1.94 (p=0.009). The percent of CD4+/CD7- cells dropped significantly after treatment (p=0.029). No correlation between CD4+/CD7- cell counts and the mean AF could be deduced. In cases of mycosis fungoides, fluorescence diagnosis can represent an effective tool for evaluating the response to therapy. Changes in accumulation factor values can be used for follow-up of therapy in the same patient, but it should not be used as an absolute value. Copyright © 2014 Elsevier B.V. All rights reserved.

[Prokaryotic expression, purification and biological activity analysis of recombinant β-Lactamase protein].

PubMed

Zhou, Xiao-liang; Shi, Pei-ji; Wang, Hao

2011-01-01

To prepare RGD4CβL fusion protein using prokaryotic expression system and evaluate the biological activity of the RGD4CβL. RGD4CβL gene was cloned into pColdII to contruct β-Lactamase prokaryotic expression vector. After transformation, the recombinant vector was induced to express recombinant protein RGD4CβL by IPTG in E.coli BL(DE3). The recombinant protein was purified by Ni-NTA resin under denaturing condition and then dialyzed to renature. The tumor cell targeting ability of the recombinant protein was analyzed by flow cytometric analysis. After cleavage and purification, β-Lactamase moiety showed the expected size of 42 000 on Tricine-SDS-PAGE, and was further confirmed by Western blotting. Based on flow cytometric analysis, the purified protein specially targeted breast cancer cell line MCF-7. This research successfully estiblished a method for prokaryotic expression and purification of β-lactamase. These results suggest the potential use of the protein as an agent for ADEPT.

Chemo Resistance of Breast Cancer Stem Cells

DTIC Science & Technology

2006-05-01

for stem cell markers , including CD44+ CD24- lin- by flow cytometry following chemotherapy residual tumor is assayed. As shown in Figure 3 below...the tumor and thus may contribute to relapse following therapy. This was to be accomplished by utilizing mouse xenograft models as well as markers ...limitation of utilizing these markers is that they are not suitable for immunochemical detection of tumor stem cells since they require flow cytometric

The cytometric future: it ain't necessarily flow!

PubMed

Shapiro, Howard M

2011-01-01

Initial approaches to cytometry for classifying and characterizing cells were based on microscopy; it was necessary to collect relatively high-resolution images of cells because only a few specific reagents usable for cell identification were available. Although flow cytometry, now the dominant cytometric technology, typically utilizes lenses similar to microscope lenses for light collection, improved, more quantitative reagents allow the necessary information to be acquired in the form of whole-cell measurements of the intensities of light transmission, scattering, and/or fluorescence.Much of the cost and complexity of both automated microscopes and flow cytometers arises from the necessity for them to measure one cell at a time. Recent developments in digital camera technology now offer an alternative in which one or more low-magnification, low-resolution images are made of a wide field containing many cells, using inexpensive light-emitting diodes (LEDs) for illumination. Minimalist widefield imaging cytometers can provide a smaller, less complex, and substantially less expensive alternative to flow cytometry, critical in systems intended for in resource-poor areas. Minimalism is, likewise, a good philosophy in developing instrumentation and methodology for both clinical and large-scale research use; it simplifies quality assurance and compliance with regulatory requirements, as well as reduces capital outlays, material costs, and personnel training requirements. Also, importantly, it yields "greener" technology.

Identification of microbes from the surfaces of food-processing lines based on the flow cytometric evaluation of cellular metabolic activity combined with cell sorting.

PubMed

Juzwa, W; Duber, A; Myszka, K; Białas, W; Czaczyk, K

2016-09-01

In this study the design of a flow cytometry-based procedure to facilitate the detection of adherent bacteria from food-processing surfaces was evaluated. The measurement of the cellular redox potential (CRP) of microbial cells was combined with cell sorting for the identification of microorganisms. The procedure enhanced live/dead cell discrimination owing to the measurement of the cell physiology. The microbial contamination of the surface of a stainless steel conveyor used to process button mushrooms was evaluated in three independent experiments. The flow cytometry procedure provided a step towards monitoring of contamination and enabled the assessment of microbial food safety hazards by the discrimination of active, mid-active and non-active bacterial sub-populations based on determination of their cellular vitality and subsequently single cell sorting to isolate microbial strains from discriminated sub-populations. There was a significant correlation (r = 0.97; p < 0.05) between the bacterial cell count estimated by the pour plate method and flow cytometry, despite there being differences in the absolute number of cells detected. The combined approach of flow cytometric CRP measurement and cell sorting allowed an in situ analysis of microbial cell vitality and the identification of species from defined sub-populations, although the identified microbes were limited to culturable cells.

Genetic analyses of endoreduplication in Zea mays endosperm: evidence of sporophytic and zygotic maternal control.

PubMed

Dilkes, Brian P; Dante, Ricardo A; Coelho, Cintia; Larkins, Brian A

2002-03-01

Flow cytometry was used to assess the variability of endoreduplication in endosperms of maize inbred lines. Little variation was found between midwestern dent types, and high levels of endoreduplication were observed in popcorns. Endoreduplication is different between inbred lines by 13-18 days after pollination, and flow cytometric analysis of ploidy level was feasible until 20 DAP. To study the genetic regulation of endoreduplication, four inbreds were crossed to B73 and developing endosperms from both parental, reciprocal F(1), and backcross generations were subjected to flow cytometric analysis. Three measurements of endoreduplication were calculated from these data and analyzed as quantitative genetic traits. Multiple models of trait inheritance were considered including triploid, diploid, sporophytic maternal, and maternal and paternal zygotic nuclear inheritance. Maternal zygotic effects, often considered a form of parental imprinting, and maternal sporophytic effects were detected. To test the feasibility of introgressing a high endoreduplication phenotype into a midwestern dent inbred line, a backcross population was generated from B73 x Sg18. Parental and progeny endoreduplication levels were compared and heritabilities assessed. The heritabilities calculated from these data generally agree with the values calculated in the larger crossing experiments.

Flow cytometric and morphological analyses of Pinus pinaster somatic embryogenesis.

PubMed

Marum, Liliana; Loureiro, João; Rodriguez, Eleazar; Santos, Conceição; Oliveira, M Margarida; Miguel, Célia

2009-09-25

An approach combining morphological profiling and flow cytometric analysis was used to assess genetic stability during the several steps of somatic embryogenesis in Pinus pinaster. Embryogenic cell lines of P. pinaster were established from immature zygotic embryos excised from seeds obtained from open-pollinated trees. During the maturation stage, phenotype of somatic embryos was characterized as being either normal or abnormal. Based upon the prevalent morphological traits, different types of abnormal embryos underwent further classification and quantification. Nuclear DNA content of maritime pine using the zygotic embryos was estimated to be 57.04 pg/2C, using propidium iodide flow cytometry. According to the same methodology, no significant differences (P< or =0.01) in DNA ploidy were detected among the most frequently observed abnormal phenotypes, embryogenic cell lines, zygotic and normal somatic embryos, and somatic embryogenesis-derived plantlets. Although the differences in DNA ploidy level do not exclude the occurrence of a low level of aneuploidy, the results obtained point to the absence of major changes in ploidy level during the somatic embryogenesis process of this economically important species. Therefore, our primary goal of true-to-typeness was assured at this level.

Supercontinuum white light lasers for flow cytometry

PubMed Central

Telford, William G.; Subach, Fedor V.; Verkhusha, Vladislav V.

2009-01-01

Excitation of fluorescent probes for flow cytometry has traditionally been limited to a few discrete laser lines, an inherent limitation in our ability to excite the vast array of fluorescent probes available for cellular analysis. In this report, we have used a supercontinuum (SC) white light laser as an excitation source for flow cytometry. By selectively filtering the wavelength of interest, almost any laser wavelength in the visible spectrum can be separated and used for flow cytometric analysis. The white light lasers used in this study were integrated into a commercial flow cytometry platform, and a series of high-transmission bandpass filters used to select wavelength ranges from the blue (~480 nm) to the long red (>700 nm). Cells labeled with a variety of fluorescent probes or expressing fluorescent proteins were then analyzed, in comparison with traditional lasers emitting at wavelengths similar to the filtered SC source. Based on a standard sensitivity metric, the white light laser bandwidths produced similar excitation levels to traditional lasers for a wide variety of fluorescent probes and expressible proteins. Sensitivity assessment using fluorescent bead arrays confirmed that the SC laser and traditional sources resulted in similar levels of detection sensitivity. Supercontinuum white light laser sources therefore have the potential to remove a significant barrier in flow cytometric analysis, namely the limitation of excitation wavelengths. Almost any visible wavelength range can be made available for excitation, allowing access to virtually any fluorescent probe, and permitting “fine-tuning” of excitation wavelength to particular probes. PMID:19072836

Effect of salt on cell viability and membrane integrity of Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium longum as observed by flow cytometry.

PubMed

Gandhi, Akanksha; Shah, Nagendra P

2015-08-01

The aim of the current study was to investigate the effect of varying sodium chloride concentrations (0-5%) on viability and membrane integrity of three probiotic bacteria, Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium longum, using conventional technique and flow cytometry. Double staining of cells by carboxyfluorescein diacetate (cFDA) and propidium iodide (PI) enabled to evaluate the effect of NaCl on cell esterase activity and membrane integrity. Observations from conventional culture technique were compared with findings from flow cytometric analysis on the metabolic activities of the cells and a correlation was observed between culturability and dye extrusion ability of L. casei and B. longum. However, a certain population of L. acidophilus was viable as per the plate count method but its efflux activity was compromised. Esterase activity of most bacteria reduced significantly (P < 0.05) during one week storage at NaCl concentrations greater than 3.5%. The study revealed that L. casei was least affected by higher NaCl concentrations among the three probiotic bacteria, as opposed to B. longum where the cF extrusion performance was greatly reduced during 1 wk storage. The metabolic activity and salt resistance of L. casei was found to be highest among the bacteria studied. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

Ciliate ingestion and digestion: flow cytometric measurements and regrowth of a digestion-resistant campylobacter jejuni

USDA-ARS?s Scientific Manuscript database

We developed a method to measure ingestion and digestion rates of bacterivorous protists feeding on pathogenic bacteria. We tested this method using the enteric bacteria Campylobacter jejuni and a freshwater colpodid ciliate. Campylobacter and a non-pathogenic bacteria isolated from the environment ...

Food sensing: selection and characterization of DNA aptamers to Alicyclobacillus spores for trapping and detection from orange juice.

PubMed

Hünniger, Tim; Fischer, Christin; Wessels, Hauke; Hoffmann, Antonia; Paschke-Kratzin, Angelika; Haase, Ilka; Fischer, Markus

2015-03-04

The quality of the beverage industry's products has to be constantly monitored to fulfill consumers' high expectations. The thermo-acidophilic Gram-positive Alicyclobacillus spp. are not pathogenic, but their heat-resistant endospores can survive juice-processing conditions and have become a major economic concern for the fruit juice industry. Current detection methods rely on cultivation, isolation, and organism identification, which can take up to a week, resulting in economic loss. This work presents the selection and identification of DNA aptamers targeting Alicyclobacillus spores by spore-SELEX (systematic evolution of ligands by exponential enrichment) in orange-juice-simulating buffer. The selection process was verified by various techniques, including flow cytometric binding assays, radioactive binding assays, and agarose gel electrophoresis. The subsequent aptamer characterization included the determination of dissociations constants and selectivity by different techniques, such as surface plasmon resonance spectroscopy and fluorescence microscopy. In summary, 10 different aptamers with an affinity to Alicyclobacillus spp. have been developed, analyzed, and characterized in terms of affinity and specificity.

Tools for Visualizing HIV in Cure Research.

PubMed

Niessl, Julia; Baxter, Amy E; Kaufmann, Daniel E

2018-02-01

The long-lived HIV reservoir remains a major obstacle for an HIV cure. Current techniques to analyze this reservoir are generally population-based. We highlight recent developments in methods visualizing HIV, which offer a different, complementary view, and provide indispensable information for cure strategy development. Recent advances in fluorescence in situ hybridization techniques enabled key developments in reservoir visualization. Flow cytometric detection of HIV mRNAs, concurrently with proteins, provides a high-throughput approach to study the reservoir on a single-cell level. On a tissue level, key spatial information can be obtained detecting viral RNA and DNA in situ by fluorescence microscopy. At total-body level, advancements in non-invasive immuno-positron emission tomography (PET) detection of HIV proteins may allow an encompassing view of HIV reservoir sites. HIV imaging approaches provide important, complementary information regarding the size, phenotype, and localization of the HIV reservoir. Visualizing the reservoir may contribute to the design, assessment, and monitoring of HIV cure strategies in vitro and in vivo.

Clonogenic colony-forming ability of flow cytometrically isolated hepatic progenitor cells in the murine fetal liver.

PubMed

Taniguchi, H; Kondo, R; Suzuki, A; Zheng, Y W; Takada, Y; Fukunaga, K; Seino, K; Yuzawa, K; Otsuka, M; Fukao, K; Nakauchi, H

2000-01-01

Stem cells are defined as cells having multilineage differentiation potential and self-renewal capability. Hepatic stem cells have aroused considerable interest not only because of their developmental importance but also for their therapeutic potential. However, their presence in the liver has not yet been demonstrated. With the use of a fluorescence-activated cell sorter (FACS) and monoclonal antibodies, we attempted to ascertain whether hepatic stem cells are present in the murine fetal liver. For this purpose, we optimized a cell isolation technique for FACS sorting of fetal liver cells. When isolated CD45 TER119 cells (the non-blood cell fraction in the fetal liver) were tested for their clonogenic colony-forming ability, mechanical dissociation (pipetting) was the most suitable cell isolation technique for FACS sorting. We confirmed that these colonies contained not only cells expressing hepatocyte markers but also cells expressing cholangiocyte markers. To identify hepatic stem cells, studies must focus on CD45TER119- cells in the murine fetal liver.

Clinical cytometry and progress in HLA antibody detection.

PubMed

Bray, Robert A; Tarsitani, Christine; Gebel, Howard M; Lee, Jar-How

2011-01-01

For most solid organ and selected stem cell transplants, antibodies against mismatched HLA antigens can lead to early and late graft failure. In recognition of the clinical significance of these antibodies, HLA antibody identification is one of the most critical functions of histocompatibility laboratories. Early methods employed cumbersome and insensitive complement-dependent cytotoxicity assays with a visual read-out. A little over 20 years ago flow cytometry entered the realm of antibody detection with the introduction of the flow cytometric crossmatch. Cytometry's increased sensitivity and objectivity quickly earned it popularity as a preferred crossmatch method especially for sensitized recipients. Although a sensitive method, the flow crossmatch was criticized as being "too sensitive" as false positive reactions were a know drawback. In part, the shortcomings of the flow crossmatch were due to the lack of corresponding sensitive and specific HLA antibody screening assays. However, in the mid 1990s, solid phase assays, capable of utilizing standard flow cytometers, were developed. These assays used microparticles coated with purified HLA molecules. Hence, the era of solid-phase, microparticle technology for HLA antibody detection was born permitting the sensitive and specific detection of HLA antibody. It was now possible to provide better correlation between HLA antibody detection and the flow cytometric crossmatch. This flow-based technology was soon followed by adaptation to the Luminex platform permitting a mutltiplexed approach for the identification and characterization of HLA antibodies. It is hoped that these technologies will ultimately lead to the identification of parameters that best correlate with and/or predict transplant outcomes. Copyright © 2011 Elsevier Inc. All rights reserved.

Flow cytometric HyPer-based assay for hydrogen peroxide.

PubMed

Lyublinskaya, O G; Antonov, S A; Gorokhovtsev, S G; Pugovkina, N A; Kornienko, Ju S; Ivanova, Ju S; Shatrova, A N; Aksenov, N D; Zenin, V V; Nikolsky, N N

2018-05-30

HyPer is a genetically encoded fluorogenic sensor for hydrogen peroxide which is generally used for the ratiometric imaging of H 2 O 2 fluxes in living cells. Here, we demonstrate the advantages of HyPer-based ratiometric flow cytometry assay for H 2 O 2 , by using K562 and human mesenchymal stem cell lines expressing HyPer. We show that flow cytometry analysis is suitable to detect HyPer response to submicromolar concentrations of extracellularly added H 2 O 2 that is much lower than concentrations addressed previously in the other HyPer-based assays (such as cell imaging or fluorimetry). Suggested technique is also much more sensitive to hydrogen peroxide than the widespread flow cytometry assay exploiting H 2 O 2 -reactive dye H 2 DCFDA and, contrary to the H 2 DCFDA-based assay, can be employed for the kinetic studies of H 2 O 2 utilization by cells, including measurements of the rate constants of H 2 O 2 removal. In addition, flow cytometry multi-parameter ratiometric measurements enable rapid and high-throughput detection of endogenously generated H 2 O 2 in different subpopulations of HyPer-expressing cells. To sum up, HyPer can be used in multi-parameter flow cytometry studies as a highly sensitive indicator of intracellular H 2 O 2 . Copyright © 2018. Published by Elsevier Inc.

Naphthol AS-BI (7-bromo-3-hydroxy-2-naphtho-o-anisidine) phosphatase and naphthol AS-BI. beta. -D-glucuronidase in Chinese hamster ovary cells: biochemical and flow cytometric studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dolbeare, F.A.; Phares, W.

1979-01-01

Conditions for the biochemical and flow cytometric assay of 7-bromo-3-hydroxy-2-naphtho-o-anisidine phosphatase and ..beta..-D-glucuronidase activities in Chinese hamster ovary cells were studied. In the biochemical assays, the pH optimum for the phosphatase activity was pH 4.6 with a Km of 10/sup -5/ M; the pH optimum for ..beta..-D-glucuronidase activity was pH 5.0 with a Km of 2 x 10/sup -5/ M. For intact cells the derived constants were 3 to 10 times higher. The rate of hydrolysis of both substrates was also examined by flow cytometry. Cellular fluorescence increased linearly for only about 15 min. Diffusion of the fluorescent product probablymore » caused nonlinearity of the fluorescence increase and was demonstrated by mixing cells incubated with substrate with those that had not been incubated. After 15 min, cells that had not been exposed previously to product or substrate contained the fluorescent product. Cells fractionated into size classes by centrifugal elutriation also were analyzed by flow cytometry for ..beta..-D-glucuronidase activity. The activity increased linearly with the increase in cell size corresponding to the progression from G/sub 1/ through S and into G/sub 2/-M phases of the cell cycle.« less

Interference of blood leucocytes in the measurements of immature red cells (reticulocytes) by two different (semi-) automated flow-cytometry technologies.

PubMed

Villamor, N; Kirsch, A; Huhn, D; Vives-Corrons, J L; Serke, S

1996-06-01

Flow cytometrical methods have been introduced recently as an alternative to the enumeration of reticulocytes by microscopy. Two of these methods have gained widespread use in haematological practice; the multiparametric flow cytometer using thiazole orange staining (Retic-Count, FACScan) and the single-application reticulocyte counter using auramine-O staining (R-series, Sysmex). Several studies have emphasized the excellent correlations between microscopy and these techniques. The purpose of our study has been to examine the specificity of these automated devices with regard to cells classified as 'reticulocytes' and the effect that this may have on measures of reticulocyte maturity. Our results indicate that the specificity of reticulocyte measurements by both the Sysmex R-1000/-3000 and the Retic-Count system is relatively low. This is due to the presence of leucocytes amongst cells classified as reticulocytes. These leucocytes display intense staining with either dye, leading to an erroneous estimation of RMI (thiazole orange) and high fluorescence count (R-1000/-3000). This error is directly correlated with the leucocyte count. The basis for reticulocyte identification should be improved before automated estimation of reticulocyte maturation can be used in clinical practice.

Multiple Strategies Reveal a Bidentate Interaction between the Nipah Virus Attachment and Fusion Glycoproteins.

PubMed

Stone, Jacquelyn A; Vemulapati, Bhadra M; Bradel-Tretheway, Birgit; Aguilar, Hector C

2016-12-01

The paramyxoviral family contains many medically important viruses, including measles virus, mumps virus, parainfluenza viruses, respiratory syncytial virus, human metapneumovirus, and the deadly zoonotic henipaviruses Hendra and Nipah virus (NiV). To both enter host cells and spread from cell to cell within infected hosts, the vast majority of paramyxoviruses utilize two viral envelope glycoproteins: the attachment glycoprotein (G, H, or hemagglutinin-neuraminidase [HN]) and the fusion glycoprotein (F). Binding of G/H/HN to a host cell receptor triggers structural changes in G/H/HN that in turn trigger F to undergo a series of conformational changes that result in virus-cell (viral entry) or cell-cell (syncytium formation) membrane fusion. The actual regions of G/H/HN and F that interact during the membrane fusion process remain relatively unknown though it is generally thought that the paramyxoviral G/H/HN stalk region interacts with the F head region. Studies to determine such interactive regions have relied heavily on coimmunoprecipitation approaches, whose limitations include the use of detergents and the micelle-mediated association of proteins. Here, we developed a flow-cytometric strategy capable of detecting membrane protein-protein interactions by interchangeably using the full-length form of G and a soluble form of F, or vice versa. Using both coimmunoprecipitation and flow-cytometric strategies, we found a bidentate interaction between NiV G and F, where both the stalk and head regions of NiV G interact with F. This is a new structural-biological finding for the paramyxoviruses. Additionally, our studies disclosed regions of the NiV G and F glycoproteins dispensable for the G and F interactions. Nipah virus (NiV) is a zoonotic paramyxovirus that causes high mortality rates in humans, with no approved treatment or vaccine available for human use. Viral entry into host cells relies on two viral envelope glycoproteins: the attachment (G) and fusion (F) glycoproteins. Binding of G to the ephrinB2 or ephrinB3 cell receptors triggers conformational changes in G that in turn cause F to undergo conformational changes that result in virus-host cell membrane fusion and viral entry. It is currently unknown, however, which specific regions of G and F interact during membrane fusion. Past efforts to determine the interacting regions have relied mainly on coimmunoprecipitation, a technique with some pitfalls. We developed a flow-cytometric assay to study membrane protein-protein interactions, and using this assay we report a bidentate interaction whereby both the head and stalk regions of NiV G interact with NiV F, a new finding for the paramyxovirus family. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Two-Color Flow Cytometric Analysis of Intraerythrocytic Malaria Parasite DNA and Surface Membrane-Associated Antigen in Erythrocytes Infected with Plasmodium falciparum

DTIC Science & Technology

1993-01-01

Antigen in Erythrocytes Infected With Plasmodium falciparum 1 Kovit Pattanapanyasat,2 Rachanee Udomsangpetch, and H. Kyle Webster The Thalassemia Center... Thalassemia Center, Division of Hematology., Siriraj Hospital, 15). Identification of conserved epitopes has important Bangkok 10700. Thailan 93-10832 93 5 4

A Method for Identification and Analysis of Non-Overlapping Myeloid Immunophenotypes in Humans

PubMed Central

Gustafson, Michael P.; Lin, Yi; Maas, Mary L.; Van Keulen, Virginia P.; Johnston, Patrick B.; Peikert, Tobias; Gastineau, Dennis A.; Dietz, Allan B.

2015-01-01

The development of flow cytometric biomarkers in human studies and clinical trials has been slowed by inconsistent sample processing, use of cell surface markers, and reporting of immunophenotypes. Additionally, the function(s) of distinct cell types as biomarkers cannot be accurately defined without the proper identification of homogeneous populations. As such, we developed a method for the identification and analysis of human leukocyte populations by the use of eight 10-color flow cytometric protocols in combination with novel software analyses. This method utilizes un-manipulated biological sample preparation that allows for the direct quantitation of leukocytes and non-overlapping immunophenotypes. We specifically designed myeloid protocols that enable us to define distinct phenotypes that include mature monocytes, granulocytes, circulating dendritic cells, immature myeloid cells, and myeloid derived suppressor cells (MDSCs). We also identified CD123 as an additional distinguishing marker for the phenotypic characterization of immature LIN-CD33+HLA-DR- MDSCs. Our approach permits the comprehensive analysis of all peripheral blood leukocytes and yields data that is highly amenable for standardization across inter-laboratory comparisons for human studies. PMID:25799053

Biological behaviour of human umbilical artery smooth muscle cell grown on nickel-free and nickel-containing stainless steel for stent implantation

PubMed Central

Li, Liming; An, Liwen; Zhou, Xiaohang; Pan, Shuang; Meng, Xin; Ren, Yibin; Yang, Ke; Guan, Yifu

2016-01-01

To evaluate the clinical potential of high nitrogen nickel-free austenitic stainless steel (HNNF SS), we have compared the cellular and molecular responses of human umbilical artery smooth muscle cells (HUASMCs) to HNNF SS and 316L SS (nickel-containing austenitic 316L stainless steel). CCK-8 analysis and flow cytometric analysis were used to assess the cellular responses (proliferation, apoptosis, and cell cycle), and quantitative real-time PCR (qRT-PCR) was used to analyze the gene expression profiles of HUASMCs exposed to HNNF SS and 316L SS, respectively. CCK-8 analysis demonstrated that HUASMCs cultured on HNNF SS proliferated more slowly than those on 316L SS. Flow cytometric analysis revealed that HNNF SS could activate more cellular apoptosis. The qRT-PCR results showed that the genes regulating cell apoptosis and autophagy were up-regulated on HNNF SS. Thus, HNNF SS could reduce the HUASMC proliferation in comparison to 316L SS. The findings furnish valuable information for developing new biomedical materials for stent implantation. PMID:26727026

Neural cell adhesion molecule-180 expression as a prognostic criterion in colorectal carcinoma: Feasible or not?

PubMed Central

Tascilar, Oge; Cakmak, Güldeniz Karadeniz; Tekin, Ishak Ozel; Emre, Ali Ugur; Ucan, Bulent Hamdi; Irkorucu, Oktay; Karakaya, Kemal; Gül, Mesut; Engin, Hüseyin Bülent; Comert, Mustafa

2007-01-01

AIM: To evaluate the frequency of neural cell adhesion molecule (NCAM)-180 expression in fresh tumor tissue samples and to discuss the prognostic value of NCAM-180 in routine clinical practice. METHODS: Twenty-six patients (16 men, 10 women) with colorectal cancer were included in the study. Fresh tumor tissue samples and macroscopically healthy proximal margins of each specimen were subjected to flow-cytometric analysis for NCAM-180 expression. RESULTS: Flow-cytometric analysis determined NCAM-180 expression in whole tissue samples of macroscopically healthy colorectal tissues. However, NCAM-180 expression was positive in only one case (3.84%) with well-differentiated Stage II disease who experienced no active disease at 30 mon follow-up. CONCLUSION: As a consequence of the limited number of cases in our series, it might not be possible to make a generalisation, nevertheless the routine use of NCAM-180 expression as a prognostic marker for colorectal carcinoma seems to be unfeasible and not cost-effective in clinical practice due to its very low incidence. PMID:17907291

Flow cytometric analysis of immunoglobulin heavy chain expression in B-cell lymphoma and reactive lymphoid hyperplasia

PubMed Central

Grier, David D; Al-Quran, Samer Z; Cardona, Diana M; Li, Ying; Braylan, Raul C

2012-01-01

The diagnosis of B-cell lymphoma (BCL) is often dependent on the detection of clonal immunoglobulin (Ig) light chain expression. In some BCLs, the determination of clonality based on Ig light chain restriction may be difficult. The aim of our study was to assess the utility of flow cytometric analysis of surface Ig heavy chain (HC) expression in lymphoid tissues in distinguishing lymphoid hyperplasias from BCLs, and also differentiating various BCL subtypes. HC expression on B-cells varied among different types of hyperplasias. In follicular hyperplasia, IgM and IgD expression was high in mantle cells while germinal center cells showed poor HC expression. In other hyperplasias, B cell compartments were blurred but generally showed high IgD and IgM expression. Compared to hyperplasias, BCLs varied in IgM expression. Small lymphocytic lymphomas had lower IgM expression than mantle cell lymphomas. Of importance, IgD expression was significantly lower in BCLs than in hyperplasias, a finding that can be useful in differentiating lymphoma from reactive processes. PMID:22400070

Mitomycin C-induced apoptosis in cultured human Tenon's capsule fibroblasts.

PubMed

Kim, J W; Kim, S K; Song, I H; Kim, I T

1999-06-01

To investigate the mitomycin C-induced apoptotic cell death of fibroblasts, the primarily cultured human Tenon's capsule fibroblasts were exposed to a clinically used dosage of 0.4 mg/ml of mitomycin C for 5 minutes. TUNEL (TdT-mediated dUTP-biotin nick end labeling) assay and electron microscopic studies were performed to determine the extent of mitomycin C-induced apoptosis. A flow cytometric study was performed to quantify the apoptotic cell population over time. The TUNEL stains were positive and electron microscopy showed features of apoptotic cell death in some fibroblasts 3 and 5 days after treatment. Flow cytometric analysis using Annexin V-propidium iodide double staining detected apoptotic cells 3 days after treatment. These apoptotic cell populations increased at 4 days and were sustained for one week. This study revealed that the clinical effects of mitomycin C on fibroblasts may be mediated not only by antiproliferative but also apoptotic cell death to some degree. Therefore, the apoptotic cell death of fibroblasts induced by mitomycin C should be considered to properly understand the mechanism of wound healing after trabeculectomy with adjunctive mitomycin C.

Biological behaviour of human umbilical artery smooth muscle cell grown on nickel-free and nickel-containing stainless steel for stent implantation

NASA Astrophysics Data System (ADS)

Li, Liming; An, Liwen; Zhou, Xiaohang; Pan, Shuang; Meng, Xin; Ren, Yibin; Yang, Ke; Guan, Yifu

2016-01-01

To evaluate the clinical potential of high nitrogen nickel-free austenitic stainless steel (HNNF SS), we have compared the cellular and molecular responses of human umbilical artery smooth muscle cells (HUASMCs) to HNNF SS and 316L SS (nickel-containing austenitic 316L stainless steel). CCK-8 analysis and flow cytometric analysis were used to assess the cellular responses (proliferation, apoptosis, and cell cycle), and quantitative real-time PCR (qRT-PCR) was used to analyze the gene expression profiles of HUASMCs exposed to HNNF SS and 316L SS, respectively. CCK-8 analysis demonstrated that HUASMCs cultured on HNNF SS proliferated more slowly than those on 316L SS. Flow cytometric analysis revealed that HNNF SS could activate more cellular apoptosis. The qRT-PCR results showed that the genes regulating cell apoptosis and autophagy were up-regulated on HNNF SS. Thus, HNNF SS could reduce the HUASMC proliferation in comparison to 316L SS. The findings furnish valuable information for developing new biomedical materials for stent implantation.

Prospective identification of erythroid elements in cultured peripheral blood.

PubMed

Miller, J L; Njoroge, J M; Gubin, A N; Rodgers, G P

1999-04-01

We have developed a prospective approach to identify the generation of erythroid cells derived from cultured peripheral blood mononuclear cells (PBMC) by monitoring the expression of the cell surface protein CD48. Unpurified populations of PBMC obtained from the buffy coats of normal volunteers were grown in suspension culture in the absence or presence of erythropoietin. A profile of surface CD48 expression permitted a flow cytometric identification of erythropoietin responsive populations at various stages of their maturation. In the absence of erythropoietin (EPO) supplemented media, the CD48- cells represented <5% of the total population of PBMC remaining in culture. In cultures supplemented with 1 U/mL EPO, the mean percentage of CD48- cells increased to 34.7 + 14.9% (p < 0.01) after 14 days in culture. Coordinated CD34 and CD71 (transferrin receptor) expression, morphology, gamma-globin transcription, and colony formation in methylcellulose were observed during the 14-day culture period. Flow cytometric monitoring of bulk cultured PBMC provides a simple and reliable means for the prospective or real-time study of human erythropoiesis.

Apoptosis triggered by pyropheophorbide-α methyl ester-mediated photodynamic therapy in a giant cell tumor in bone

NASA Astrophysics Data System (ADS)

Li, K.-T.; Zhang, J.; Duan, Q.-Q.; Bi, Y.; Bai, D.-Q.; Ou, Y.-S.

2014-06-01

A giant cell tumor in bone is the common primary bone tumor with aggressive features, occurring mainly in young adults. Photodynamic therapy is a new therapeutic technique for tumors. In this study, we investigated the effects of Pyropheophorbide-α methyl ester (MPPa)-mediated photodynamic therapy on the proliferation of giant cell tumor cells and its mechanism of action. Cell proliferation was evaluated using an MTT assay. Cellular apoptosis was detected by Hoechst nuclear staining, and flow cytometric assay. Mitochondrial membrane potential changes and cytochrome c, caspase-9, caspase-3, and Bcl-2 expression was assessed. Finally, we found that MPPa-mediated photodynamic therapy could effectively suppress the proliferation of human giant cell tumor cells and induce apoptosis. The mitochondrial pathway was involved in the MPPa-photodynamic therapy-induced apoptosis.

Plant regeneration from haploid cell suspension-derived protoplasts of Mediterranean rice (Oryza sativa L. cv. Miara).

PubMed

Guiderdoni, E; Chaïr, H

1992-11-01

More than 750 plants were regenerated from protoplasts isolated from microspore callus-derived cell suspensions of the Mediterranean japonica rice Miara, using a nurse-feeder technique and N6-based culture medium. The mean plating efficiency and the mean regeneration ability of the protocalluses were 0.5% and 49% respectively. Flow cytometric evaluation of the DNA contents of 7 month old-cell and protoplast suspensions showed that they were still haploid. Contrastingly, the DNA contents of leaf cell nuclei of the regenerated protoclones ranged from 1C to 5C including 60% 2C plants. This was consistent with the morphological type and the fertility of the mature plants. These results and the absence of chimeric plants suggest that polyploidization occurred during the early phase of protoplast culture.

HLA-targeted flow cytometric sorting of blood cells allows separation of pure and viable microchimeric cell populations.

PubMed

Drabbels, Jos J M; van de Keur, Carin; Kemps, Berit M; Mulder, Arend; Scherjon, Sicco A; Claas, Frans H J; Eikmans, Michael

2011-11-10

Microchimerism is defined by the presence of low levels of nonhost cells in a person. We developed a reliable method for separating viable microchimeric cells from the host environment. For flow cytometric cell sorting, HLA antigens were targeted with human monoclonal HLA antibodies (mAbs). Optimal separation of microchimeric cells (present at a proportion as low as 0.01% in artificial mixtures) was obtained with 2 different HLA mAbs, one targeting the chimeric cells and the other the background cells. To verify purity of separated cell populations, flow-sorted fractions of 1000 cells were processed for DNA analysis by HLA-allele-specific and Y-chromosome-directed real-time quantitative PCR assays. After sorting, PCR signals of chimeric DNA markers in the positive fractions were significantly enhanced compared with those in the presort samples, and they were similar to those in 100% chimeric control samples. Next, we demonstrate applicability of HLA-targeted FACS sorting after pregnancy by separating chimeric maternal cells from child umbilical cord mononuclear cells. Targeting allelic differences with anti-HLA mAbs with FACS sorting allows maximal enrichment of viable microchimeric cells from a background cell population. The current methodology enables reliable microchimeric cell detection and separation in clinical specimens.

Fluorescence lifetime measurements in flow cytometry

NASA Astrophysics Data System (ADS)

Beisker, Wolfgang; Klocke, Axel

1997-05-01

Fluorescence lifetime measurements provide insights int eh dynamic and structural properties of dyes and their micro- environment. The implementation of fluorescence lifetime measurements in flow cytometric systems allows to monitor large cell and particle populations with high statistical significance. In our system, a modulated laser beam is used for excitation and the phase shift of the fluorescence signal recorded with a fast computer controlled digital oscilloscope is processed digitally to determine the phase shift with respect to a reference beam by fast fourier transform. Total fluorescence intensity as well as other parameters can be determined simultaneously from the same fluorescence signal. We use the epi-illumination design to allow the use of high numerical apertures to collect as much light as possible to ensure detection of even weak fluorescence. Data storage and processing is done comparable to slit-scan flow cytometric data using data analysis system. The results are stored, displayed, combined with other parameters and analyzed as normal listmode data. In our report we discuss carefully the signal to noise ratio for analog and digital processed lifetime signals to evaluate the theoretical minimum fluorescence intensity for lifetime measurements. Applications to be presented include DNA staining, parameters of cell functions as well as different applications in non-mammalian cells such as algae.

Flow cytometric evaluation of physico-chemical impact on Gram-positive and Gram-negative bacteria

PubMed Central

Fröhling, Antje; Schlüter, Oliver

2015-01-01

Since heat sensitivity of fruits and vegetables limits the application of thermal inactivation processes, new emerging inactivation technologies have to be established to fulfill the requirements of food safety without affecting the produce quality. The efficiency of inactivation treatments has to be ensured and monitored. Monitoring of inactivation effects is commonly performed using traditional cultivation methods which have the disadvantage of the time span needed to obtain results. The aim of this study was to compare the inactivation effects of peracetic acid (PAA), ozonated water (O3), and cold atmospheric pressure plasma (CAPP) on Gram-positive and Gram-negative bacteria using flow cytometric methods. E. coli cells were completely depolarized after treatment (15 s) with 0.25% PAA at 10°C, and after treatment (10 s) with 3.8 mg l−1 O3 at 12°C. The membrane potential of CAPP treated cells remained almost constant at an operating power of 20 W over a time period of 3 min, and subsequently decreased within 30 s of further treatment. Complete membrane permeabilization was observed after 10 s O3 treatment, but treatment with PAA and CAPP did not completely permeabilize the cells within 2 and 4 min, respectively. Similar results were obtained for esterase activity. O3 inactivates cellular esterase but esterase activity was detected after 4 min CAPP treatment and 2 min PAA treatment. L. innocua cells and P. carotovorum cells were also permeabilized instantaneously by O3 treatment at concentrations of 3.8 ± 1 mg l−1. However, higher membrane permeabilization of L. innocua and P. carotovorum than of E. coli was observed at CAPP treatment of 20 W. The degree of bacterial damage due to the inactivation processes is highly dependent on treatment parameters as well as on treated bacteria. Important information regarding the inactivation mechanisms can be obtained by flow cytometric measurements and this enables the definition of critical process parameters. PMID:26441874

Deep sequencing and flow cytometric characterization of expanded effector memory CD8+CD57+ T cells frequently reveals T-cell receptor Vβ oligoclonality and CDR3 homology in acquired aplastic anemia.

PubMed

Giudice, Valentina; Feng, Xingmin; Lin, Zenghua; Hu, Wei; Zhang, Fanmao; Qiao, Wangmin; Ibanez, Maria Del Pilar Fernandez; Rios, Olga; Young, Neal S

2018-05-01

Oligoclonal expansion of CD8 + CD28 - lymphocytes has been considered indirect evidence for a pathogenic immune response in acquired aplastic anemia. A subset of CD8 + CD28 - cells with CD57 expression, termed effector memory cells, is expanded in several immune-mediated diseases and may have a role in immune surveillance. We hypothesized that effector memory CD8 + CD28 - CD57 + cells may drive aberrant oligoclonal expansion in aplastic anemia. We found CD8 + CD57 + cells frequently expanded in the blood of aplastic anemia patients, with oligoclonal characteristics by flow cytometric Vβ usage analysis: skewing in 1-5 Vβ families and frequencies of immunodominant clones ranging from 1.98% to 66.5%. Oligoclonal characteristics were also observed in total CD8 + cells from aplastic anemia patients with CD8 + CD57 + cell expansion by T-cell receptor deep sequencing, as well as the presence of 1-3 immunodominant clones. Oligoclonality was confirmed by T-cell receptor repertoire deep sequencing of enriched CD8 + CD57 + cells, which also showed decreased diversity compared to total CD4 + and CD8 + cell pools. From analysis of complementarity-determining region 3 sequences in the CD8 + cell pool, a total of 29 sequences were shared between patients and controls, but these sequences were highly expressed in aplastic anemia subjects and also present in their immunodominant clones. In summary, expansion of effector memory CD8 + T cells is frequent in aplastic anemia and mirrors Vβ oligoclonal expansion. Flow cytometric Vβ usage analysis combined with deep sequencing technologies allows high resolution characterization of the T-cell receptor repertoire, and might represent a useful tool in the diagnosis and periodic evaluation of aplastic anemia patients. (Registered at clinicaltrials.gov identifiers: 00001620, 01623167, 00001397, 00071045, 00081523, 00961064 ). Copyright © 2018 Ferrata Storti Foundation.

Flow cytometric detection of endothelial microparticles (EMP): effects of centrifugation and storage alter with the phenotype studied.

PubMed

van Ierssel, Sabrina H; Van Craenenbroeck, Emeline M; Conraads, Viviane M; Van Tendeloo, Viggo F; Vrints, Christiaan J; Jorens, Philippe G; Hoymans, Vicky Y

2010-04-01

Endothelial microparticles (EMP) are released into the circulation in case of endothelial disturbance, and are therefore increasingly investigated as a biomarker reflecting disease activity. Numerous pre-analytic methods have been proposed for their flow cytometric enumeration, but standardization is still lacking. In this study we evaluated the influence of centrifugation and storage conditions on EMP quantification. Platelet-poor plasma (PPP) from 10 healthy volunteers was prepared by centrifugation at 1,550 g for 20 minutes twice. A first aliquot of PPP was analyzed immediately, a second after storage at 4 degrees C for 7 hours. A third and fourth aliquot were snap-frozen and stored at -80 degrees C for 7 and 28 days. A final aliquot was further centrifuged at 10,000g for 10 minutes and analyzed immediately. EMP were defined as CD31+CD42b-, CD62E+, CD144+ or CD144+CD105+ particles, smaller than 1.0 microm. High speed centrifugation led to a significant loss of CD31+CD42b- EMP (p=0.004). A good correlation between PPP and high speed centrifuged PPP was only found for CD144+ EMP (Kendall tau b=0.611, p=0.025). Storage at 4 degrees C did not affect EMP quantification. However, freezing at -80 degrees C increased CD31+CD42b- and CD62E+ EMP counts, and lowered CD144+ EMP (p<0.05). Nevertheless, the agreement among the different storage conditions was relatively good (Kendall coefficient of concordance >0.487; p<0.05). The flow cytometric detection of EMP varies with the centrifugation protocol and the storage method used, and these changes also depend on the phenotype studied. The results of this study caution against comparing study results gathered with different EMP laboratory protocols. (c) 2010 Elsevier Ltd. All rights reserved.

Japanese Society for Laboratory Hematology flow cytometric reference method of determining the differential leukocyte count: external quality assurance using fresh blood samples.

PubMed

Kawai, Y; Nagai, Y; Ogawa, E; Kondo, H

2017-04-01

To provide target values for the manufacturers' survey of the Japanese Society for Laboratory Hematology (JSLH), accurate standard data from healthy volunteers were needed for the five-part differential leukocyte count. To obtain such data, JSLH required an antibody panel that achieved high specificity (particularly for mononuclear cells) using simple gating procedures. We developed a flow cytometric method for determining the differential leukocyte count (JSLH-Diff) and validated it by comparison with the flow cytometric differential leukocyte count of the International Council for Standardization in Haematology (ICSH-Diff) and the manual differential count obtained by microscopy (Manual-Diff). First, the reference laboratory performed an imprecision study of JSLH-Diff and ICSH-Diff, as well as performing comparison among JSLH-Diff, Manual-Diff, and ICSH-Diff. Then two reference laboratories and seven participating laboratories performed imprecision and accuracy studies of JSLH-Diff, Manual-Diff, and ICSH-Diff. Simultaneously, six manufacturers' laboratories provided their own representative values by using automated hematology analyzers. The precision of both JSLH-Diff and ICSH-Diff methods was adequate. Comparison by the reference laboratory showed that all correlation coefficients, slopes and intercepts obtained by the JSLH-Diff, ICSH-Diff, and Manual-Diff methods conformed to the criteria. When the imprecision and accuracy of JSLH-Diff were assessed at seven laboratories, the CV% for lymphocytes, neutrophils, monocytes, eosinophils, and basophils was 0.5~0.9%, 0.3~0.7%, 1.7~2.6%, 3.0~7.9%, and 3.8~10.4%, respectively. More than 99% of CD45 positive leukocytes were identified as normal leukocytes by JSLH-Diff. When JSLH-Diff method were validated by comparison with Manual-Diff and ICSH-Diff, JSLH-Diff showed good performance as a reference method. © 2016 John Wiley & Sons Ltd.

Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses.

PubMed Central

Davey, H M; Kell, D B

1996-01-01

The most fundamental questions such as whether a cell is alive, in the sense of being able to divide or to form a colony, may sometimes be very hard to answer, since even axenic microbial cultures are extremely heterogeneous. Analyses that seek to correlate such things as viability, which is a property of an individual cell, with macroscopic measurements of culture variables such as ATP content, respiratory activity, and so on, must inevitably fail. It is therefore necessary to make physiological measurements on individual cells. Flow cytometry is such a technique, which allows one to analyze cells rapidly and individually and permits the quantitative analysis of microbial heterogeneity. It therefore offers many advantages over conventional measurements for both routine and more exploratory analyses of microbial properties. While the technique has been widely applied to the study of mammalian cells, is use in microbiology has until recently been much more limited, largely because of the smaller size of microbes and the consequently smaller optical signals obtainable from them. Since these technical barriers no longer hold, flow cytometry with appropriate stains has been used for the rapid discrimination and identification of microbial cells, for the rapid assessment of viability and of the heterogeneous distributions of a wealth of other more detailed physiological properties, for the analysis of antimicrobial drug-cell interactions, and for the isolation of high-yielding strains of biotechnological interest. Flow cytometric analyses provide an abundance of multivariate data, and special methods have been devised to exploit these. Ongoing advances mean that modern flow cytometers may now be used by nonspecialists to effect a renaissance in our understanding of microbial heterogeneity. PMID:8987359

Accurate measurement of volume and shape of resting and activated blood platelets from light scattering.

PubMed

Moskalensky, Alexander E; Yurkin, Maxim A; Konokhova, Anastasiya I; Strokotov, Dmitry I; Nekrasov, Vyacheslav M; Chernyshev, Andrei V; Tsvetovskaya, Galina A; Chikova, Elena D; Maltsev, Valeri P

2013-01-01

We introduce a novel approach for determination of volume and shape of individual blood platelets modeled as an oblate spheroid from angle-resolved light scattering with flow-cytometric technique. The light-scattering profiles (LSPs) of individual platelets were measured with the scanning flow cytometer and the platelet characteristics were determined from the solution of the inverse light-scattering problem using the precomputed database of theoretical LSPs. We revealed a phenomenon of parameter compensation, which is partly explained in the framework of anomalous diffraction approximation. To overcome this problem, additional a priori information on the platelet refractive index was used. It allowed us to determine the size of each platelet with subdiffraction precision and independent of the particular value of the platelet aspect ratio. The shape (spheroidal aspect ratio) distributions of platelets showed substantial differences between native and activated by 10 μM adenosine diphosphate samples. We expect that the new approach may find use in hematological analyzers for accurate measurement of platelet volume distribution and for determination of the platelet activation efficiency.

Flow Cytometric Measurement of Cellular Ionized Calcium Concentration

DTIC Science & Technology

1988-01-01

AD-A207 011 ORT DOCUMENTATION PAGE lb. RESTRICTIVE MARKINGS i. .a. SECRIrY CL.AS.FICA𔃻iON AUTHORITY -- 3 DISTRIBUTION/AVAILA3BLIrY OF REPORT...Cytometry 8: 396-404 K.E.; Ledbetter, iA.; Rabinovitch, P.S.; Morishi- (1987). -ta, Y.; Hellstrom, I.; Hansen, J.A.: Induction of 14 Cobbold , P.H.; Rink

A novel tool for high-throughput screening of granulocyte-specific antibodies using the automated flow cytometric granulocyte immunofluorescence test (Flow-GIFT).

PubMed

Nguyen, Xuan Duc; Dengler, Thomas; Schulz-Linkholt, Monika; Klüter, Harald

2011-02-03

Transfusion-related acute lung injury (TRALI) is a severe complication related with blood transfusion. TRALI has usually been associated with antibodies against leukocytes. The flow cytometric granulocyte immunofluorescence test (Flow-GIFT) has been introduced for routine use when investigating patients and healthy blood donors. Here we describe a novel tool in the automation of the Flow-GIFT that enables a rapid screening of blood donations. We analyzed 440 sera from healthy female blood donors for the presence of granulocyte antibodies. As positive controls, 12 sera with known antibodies against anti-HNA-1a, -b, -2a; and -3a were additionally investigated. Whole-blood samples from HNA-typed donors were collected and the test cells isolated using cell sedimentation in a Ficoll density gradient. Subsequently, leukocytes were incubated with the respective serum and binding of antibodies was detected using FITC-conjugated antihuman antibody. 7-AAD was used to exclude dead cells. Pipetting steps were automated using the Biomek NXp Multichannel Automation Workstation. All samples were prepared in the 96-deep well plates and analyzed by flow cytometry. The standard granulocyte immunofluorescence test (GIFT) and granulocyte agglutination test (GAT) were also performed as reference methods. Sixteen sera were positive in the automated Flow-GIFT, while five of these sera were negative in the standard GIFT (anti-HNA 3a, n = 3; anti-HNA-1b, n = 1) and GAT (anti-HNA-2a, n = 1). The automated Flow-GIFT was able to detect all granulocyte antibodies, which could be only detected in GIFT in combination with GAT. In serial dilution tests, the automated Flow-GIFT detected the antibodies at higher dilutions than the reference methods GIFT and GAT. The Flow-GIFT proved to be feasible for automation. This novel high-throughput system allows an effective antigranulocyte antibody detection in a large donor population in order to prevent TRALI due to transfusion of blood products.

A single-platform approach using flow cytometry and microbeads to evaluate immune reconstitution in mice after bone marrow transplantation.

PubMed

Perruche, Sylvain; Kleinclauss, François; Lienard, Agnès; Robinet, Eric; Tiberghien, Pierre; Saas, Philippe

2004-11-01

The monitoring of immune reconstitution in murine models of HC transplantation, using accurate and automated methods, is necessary in view of the recent developments of hematopoietic cell (HC) transplantation (including reduced intensity conditioning regimens) as well as emerging immunological concepts (such as the involvement of dendritic cells or regulatory T cells). Here, we describe the use of a single-platform approach based on flow cytometry and tubes that contain a defined number of microbeads to evaluate absolute blood cell counts in mice. This method, previously used in humans to quantify CD34+ stem cells or CD4+ T cells in HIV infected patients, was adapted for mouse blood samples. A CD45 gating strategy in this "lyse no wash" protocol makes it possible to discriminate erythroblasts or red blood cell debris from CD45+ leukocytes, thus avoiding cell loss. Tubes contain a lyophilized brightly fluorescent microbead pellet permitting the acquisition of absolute counts of leukocytes after flow cytometric analysis. We compared this method to determine absolute counts of circulating cells with another method combining Unopette reservoir diluted blood samples, hemocytometer, microscopic examination and flow cytometry. The sensitivity of this single-platform approach was evaluated in different situations encountered in allogeneic HC transplantation, including immune cell depletion after different conditioning regimens, activation status of circulating cells after transplantation, evaluation of in vivo cell depletion and hematopoietic progenitor mobilization in the periphery. This single-platform flow cytometric assay can also be proposed to standardize murine (or other mammalian species) leukocyte count determination for physiological, pharmacological/toxicological and diagnostic applications in veterinary practice.

Flow cytometry: a promising technique for the study of silicone oil-induced particulate formation in protein formulations.

PubMed

Ludwig, D Brett; Trotter, Joseph T; Gabrielson, John P; Carpenter, John F; Randolph, Theodore W

2011-03-15

Subvisible particles in formulations intended for parenteral administration are of concern in the biopharmaceutical industry. However, monitoring and control of subvisible particulates can be complicated by formulation components, such as the silicone oil used for the lubrication of prefilled syringes, and it is difficult to differentiate microdroplets of silicone oil from particles formed by aggregated protein. In this study, we demonstrate the ability of flow cytometry to resolve mixtures comprising subvisible bovine serum albumin (BSA) aggregate particles and silicone oil emulsion droplets with adsorbed BSA. Flow cytometry was also used to investigate the effects of silicone oil emulsions on the stability of BSA, lysozyme, abatacept, and trastuzumab formulations containing surfactant, sodium chloride, or sucrose. To aid in particle characterization, the fluorescence detection capabilities of flow cytometry were exploited by staining silicone oil with BODIPY 493/503 and model proteins with Alexa Fluor 647. Flow cytometric analyses revealed that silicone oil emulsions induced the loss of soluble protein via protein adsorption onto the silicone oil droplet surface. The addition of surfactant prevented protein from adsorbing onto the surface of silicone oil droplets. There was minimal formation of homogeneous protein aggregates due to exposure to silicone oil droplets, although oil droplets with surface-adsorbed trastuzumab exhibited flocculation. The results of this study demonstrate the utility of flow cytometry as an analytical tool for monitoring the effects of subvisible silicone oil droplets on the stability of protein formulations. Copyright © 2010 Elsevier Inc. All rights reserved.

CD33 monoclonal antibody conjugated Au cluster nano-bioprobe for targeted flow-cytometric detection of acute myeloid leukaemia

NASA Astrophysics Data System (ADS)

Retnakumari, Archana; Jayasimhan, Jasusri; Chandran, Parwathy; Menon, Deepthy; Nair, Shantikumar; Mony, Ullas; Koyakutty, Manzoor

2011-07-01

Protein stabilized gold nanoclusters (Au-NCs) are biocompatible, near-infrared (NIR) emitting nanosystems having a wide range of biomedical applications. Here, we report the development of a Au-NC based targeted fluorescent nano-bioprobe for the flow-cytometric detection of acute myeloid leukaemia (AML) cells. Au-NCs with ~ 25-28 atoms showing bright red-NIR fluorescence (600-750 nm) and average size of ~ 0.8 nm were prepared by bovine serum albumin assisted reduction-cum-stabilization in aqueous phase. The protein protected clusters were conjugated with monoclonal antibody against CD33 myeloid antigen, which is overexpressed in ~ 99.2% of the primitive population of AML cells, as confirmed by immunophenotyping using flow cytometry. Au-NC-CD33 conjugates having average size of ~ 12 nm retained bright fluorescence over an extended duration of ~ a year, as the albumin protein protects Au-NCs against degradation. Nanotoxicity studies revealed excellent biocompatibility of Au-NC conjugates, as they showed no adverse effect on the cell viability and inflammatory response. Target specificity of the conjugates for detecting CD33 expressing AML cells (KG1a) in flow cytometry showed specific staining of ~ 95.4% of leukaemia cells within 1-2 h compared to a non-specific uptake of ~ 8.2% in human peripheral blood cells (PBMCs) which are CD33low. The confocal imaging also demonstrated the targeted uptake of CD33 conjugated Au-NCs by leukaemia cells, thus confirming the flow cytometry results. This study demonstrates that novel nano-bioprobes can be developed using protein protected fluorescent nanoclusters of Au for the molecular receptor targeted flow cytometry based detection and imaging of cancer cells.

4β-Hydroxywithanolide E from Physalis peruviana (golden berry) inhibits growth of human lung cancer cells through DNA damage, apoptosis and G2/M arrest

PubMed Central

2010-01-01

Background The crude extract of the fruit bearing plant, Physalis peruviana (golden berry), demonstrated anti-hepatoma and anti-inflammatory activities. However, the cellular mechanism involved in this process is still unknown. Methods Herein, we isolated the main pure compound, 4β-Hydroxywithanolide (4βHWE) derived from golden berries, and investigated its antiproliferative effect on a human lung cancer cell line (H1299) using survival, cell cycle, and apoptosis analyses. An alkaline comet-nuclear extract (NE) assay was used to evaluate the DNA damage due to the drug. Results It was shown that DNA damage was significantly induced by 1, 5, and 10 μg/mL 4βHWE for 2 h in a dose-dependent manner (p < 0.005). A trypan blue exclusion assay showed that the proliferation of cells was inhibited by 4βHWE in both dose- and time-dependent manners (p < 0.05 and 0.001 for 24 and 48 h, respectively). The half maximal inhibitory concentrations (IC50) of 4βHWE in H1299 cells for 24 and 48 h were 0.6 and 0.71 μg/mL, respectively, suggesting it could be a potential therapeutic agent against lung cancer. In a flow cytometric analysis, 4βHWE produced cell cycle perturbation in the form of sub-G1 accumulation and slight arrest at the G2/M phase with 1 μg/mL for 12 and 24 h, respectively. Using flow cytometric and annexin V/propidium iodide immunofluorescence double-staining techniques, these phenomena were proven to be apoptosis and complete G2/M arrest for H1299 cells treated with 5 μg/mL for 24 h. Conclusions In this study, we demonstrated that golden berry-derived 4βHWE is a potential DNA-damaging and chemotherapeutic agent against lung cancer. PMID:20167063

4beta-Hydroxywithanolide E from Physalis peruviana (golden berry) inhibits growth of human lung cancer cells through DNA damage, apoptosis and G2/M arrest.

PubMed

Yen, Ching-Yu; Chiu, Chien-Chih; Chang, Fang-Rong; Chen, Jeff Yi-Fu; Hwang, Chi-Ching; Hseu, You-Cheng; Yang, Hsin-Ling; Lee, Alan Yueh-Luen; Tsai, Ming-Tz; Guo, Zong-Lun; Cheng, Yu-Shan; Liu, Yin-Chang; Lan, Yu-Hsuan; Chang, Yu-Ching; Ko, Ying-Chin; Chang, Hsueh-Wei; Wu, Yang-Chang

2010-02-18

The crude extract of the fruit bearing plant, Physalis peruviana (golden berry), demonstrated anti-hepatoma and anti-inflammatory activities. However, the cellular mechanism involved in this process is still unknown. Herein, we isolated the main pure compound, 4beta-Hydroxywithanolide (4betaHWE) derived from golden berries, and investigated its antiproliferative effect on a human lung cancer cell line (H1299) using survival, cell cycle, and apoptosis analyses. An alkaline comet-nuclear extract (NE) assay was used to evaluate the DNA damage due to the drug. It was shown that DNA damage was significantly induced by 1, 5, and 10 microg/mL 4betaHWE for 2 h in a dose-dependent manner (p < 0.005). A trypan blue exclusion assay showed that the proliferation of cells was inhibited by 4betaHWE in both dose- and time-dependent manners (p < 0.05 and 0.001 for 24 and 48 h, respectively). The half maximal inhibitory concentrations (IC50) of 4betaHWE in H1299 cells for 24 and 48 h were 0.6 and 0.71 microg/mL, respectively, suggesting it could be a potential therapeutic agent against lung cancer. In a flow cytometric analysis, 4betaHWE produced cell cycle perturbation in the form of sub-G1 accumulation and slight arrest at the G2/M phase with 1 microg/mL for 12 and 24 h, respectively. Using flow cytometric and annexin V/propidium iodide immunofluorescence double-staining techniques, these phenomena were proven to be apoptosis and complete G2/M arrest for H1299 cells treated with 5 microg/mL for 24 h. In this study, we demonstrated that golden berry-derived 4betaHWE is a potential DNA-damaging and chemotherapeutic agent against lung cancer.

Selection and identification of a DNA aptamer targeted to Vibrio parahemolyticus.

PubMed

Duan, Nuo; Wu, Shijia; Chen, Xiujuan; Huang, Yukun; Wang, Zhouping

2012-04-25

A whole-bacterium systemic evolution of ligands by exponential enrichment (SELEX) method was applied to a combinatorial library of FAM-labeled single-stranded DNA molecules to identify DNA aptamers demonstrating specific binding to Vibrio parahemolyticus . FAM-labeled aptamer sequences with high binding affinity to V. parahemolyticus were identified by flow cytometric analysis. Aptamer A3P, which showed a particularly high binding affinity in preliminary studies, was chosen for further characterization. This aptamer displayed a dissociation constant (K(d)) of 16.88 ± 1.92 nM. Binding assays to assess the specificity of aptamer A3P showed a high binding affinity (76%) for V. parahemolyticus and a low apparent binding affinity (4%) for other bacteria. Whole-bacterium SELEX is a promising technique for the design of aptamer-based molecular probes for microbial pathogens that does not require the labor-intensive steps of isolating and purifying complex markers or targets.

Precision toxicology based on single cell sequencing: an evolving trend in toxicological evaluations and mechanism exploration.

PubMed

Zhang, Boyang; Huang, Kunlun; Zhu, Liye; Luo, Yunbo; Xu, Wentao

2017-07-01

In this review, we introduce a new concept, precision toxicology: the mode of action of chemical- or drug-induced toxicity can be sensitively and specifically investigated by isolating a small group of cells or even a single cell with typical phenotype of interest followed by a single cell sequencing-based analysis. Precision toxicology can contribute to the better detection of subtle intracellular changes in response to exogenous substrates, and thus help researchers find solutions to control or relieve the toxicological effects that are serious threats to human health. We give examples for single cell isolation and recommend laser capture microdissection for in vivo studies and flow cytometric sorting for in vitro studies. In addition, we introduce the procedures for single cell sequencing and describe the expected application of these techniques to toxicological evaluations and mechanism exploration, which we believe will become a trend in toxicology.

Cytometric methods for measuring bacteria in water: advantages, pitfalls and applications.

PubMed

Hammes, Frederik; Egli, Thomas

2010-06-01

Rapid detection of microbial cells is a challenge in microbiology, particularly when complex indigenous communities or subpopulations varying in viability, activity and physiological state are investigated. Flow cytometry (FCM) has developed during the last 30 years into a multidisciplinary technique for analysing bacteria. When used correctly, FCM can provide a broad range of information at the single-cell level, including (but not limited to) total counts, size measurements, nucleic acid content, cell viability and activity, and detection of specific bacterial groups or species. The main advantage of FCM is that it is fast and easy to perform. It is a robust technique, which is adaptable to different types of samples and methods, and has much potential for automation. Hence, numerous FCM applications have emerged in industrial biotechnology, food and pharmaceutical quality control, routine monitoring of drinking water and wastewater systems, and microbial ecological research in soils and natural aquatic habitats. This review focuses on the information that can be gained from the analysis of bacteria in water, highlighting some of the main advantages, pitfalls and applications.

Molecular identification of Armillaria gallica from the Niobrara Valley Preserve in Nebraska

Treesearch

Mee-Sook Kim; Ned B. Klopfenstein

2011-01-01

Armillaria isolates were collected from a unique forest ecosystem in the Niobrara Valley Preserve in Nebraska, USA, which comprises a glacial and early postglacial refugium in the central plains of North America. The isolates were collected from diverse forest trees representing a unique mixture of forest types. Combined methods of rDNA sequencing and flow cytometric...

Study of NGEP expression in androgen sensitive prostate cancer cells: A potential target for immunotherapy

PubMed Central

Mohsenzadegan, Monireh; Tajik, Nader; Madjd, Zahra; Shekarabi, Mehdi; Farajollahi, Mohammad M

2015-01-01

Background: Prostate cancer is one of the leading causes of cancer deaths among men. New gene expressed in prostate (NGEP), is a prostate-specific gene expressed only in normal prostate and prostate cancer tissue. Because of its selective expression in prostate cancer cell surface, NGEP is a potential immunotherapeutic target. To target the NGEP in prostate cancer, it is essential to investigate its expression in prostate cancer cells. Methods: In the present study, we investigated NGEP expression in LNCaP and DU145 cells by real time and RT-PCR, flow cytometric and immunocytochemical analyses. Results: Real time and RT-PCR analyses of NGEP expression showed that NGEP was expressed in the LNCaP cells but not in DU145 cells. The detection of NGEP protein by flow cytometric and immunocytochemistry analyses indicated that NGEP protein was weakly expressed only in LNCaP cell membrane. Conclusion: Our results demonstrate that LNCaP cell line is more suitable than DU145 for NGEP expression studies; however, its low-level expression is a limiting issue. NGEP expression may be increased by androgen supplementation of LNCaP cell culture medium. PMID:26000254

Fine-needle aspiration cytology of malignant hemangiopericytomas with ultrastructural and flow cytometric analyses.

PubMed

Geisinger, K R; Silverman, J F; Cappellari, J O; Dabbs, D J

1990-07-01

A hemangiopericytoma (HPC) is an uncommon soft-tissue neoplasm that may arise in many body sites. The cytologic features of fine-needle aspirates (FNAs) of HPCs have only rarely been described in the literature. We examined FNAs of malignant HPCs from the head and neck region (three) and the retroperitoneum (one) in four adults (aged 38 to 83 years). All four FNAs yielded cellular specimens that consisted of uninuclear tumor cells with high nuclear-cytoplasmic ratios. The cytomorphological spectrum included nuclei that were oval to elongate and had very finely granular, evenly distributed chromatin with one or two small but distinct nucleoli. Hemangiopericytomas yield aspirates that may be considered malignant and may suggest sarcoma. Histologically, all four neoplasms manifested high mitotic activity. The ultrastructural features of all four tumors were supportive of the diagnosis of HPC. Although a specific primary diagnosis of HPC on FNA of a soft-tissue mass is unlikely, cytologic analysis may allow diagnosis of recurrent or metastatic HPC. We were able to perform flow cytometric determinations of tumor DNA content on three of the resected neoplasms. In two, an aneuploid pattern was found, including the neoplasm with the most marked pleomorphism in the FNA. The third was diploid.

Identification of early B cell precursors (stage 1 and 2 hematogones) in the peripheral blood.

PubMed

Kurzer, Jason H; Weinberg, Olga K

2018-05-25

Differentiating malignant B-lymphoblasts from early benign B cell precursors (hematogones) is a vital component of the diagnosis of B-lymphoblastic leukaemia. It has been previously reported that only late-stage B cell precursors circulate in the peripheral blood. Consequently, flow cytometric detection of cells with immunophenotypic findings similar to earlier stage precursors in the peripheral blood justifiably raises concern for involvement by B-lymphoblastic leukaemia. We report here, however, that benign early B cell precursors can indeed be detected in the peripheral blood, thus complicating the interpretation of flow cytometric findings derived from these sample types. A retrospective search of our collective databases identified 13 cases containing circulating early stage B cell precursors. The patients ranged in age from 15 days to 85 years old. All positive cases demonstrated that the earlier B cell precursors were associated with later stage precursors, a finding that could help differentiate these cells from B-lymphoblastic leukaemia. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Flow cytometric evaluation of antibiotic effects on viability and mitochondrial function of refrigerated spermatozoa of Nile tilapia

USGS Publications Warehouse

Segovia, M.; Jenkins, J.A.; Paniagua-Chavez, C.; Tiersch, T.R.

2000-01-01

Improved techniques for storage and evaluation of fish sperm would enhance breeding programs around the world. The goal of this study was to test the effect of antibiotics on refrigerated sperm from Nile tilapia (Oreochromis niloticus) by use of flow cytometry with 2 dual-staining protocols for objective assessment of sperm quality. Concentrations of 1 x 109 sperm/mL were suspended in Ringer's buffer at 318 mOsmol/kg (pH 8.0). The fluorescent stains Sybr 14 (10 ??M), propidium iodide (2.4 mM), and rhodamine 123 (0.13 ??M) were used to assess cell viability and mitochondrial function. Three concentrations of ampicillin, gentamicin, and an antibiotic/antimycotic solution were added to fresh spermatozoa. Motility estimates and flow cytometry measurements were made daily during 7 d of refrigerated storage (4 ??C). The highest concentrations of gentamicin and antibiotic/antimycotic and all 3 concentrations of ampicillin significantly reduced sperm viability. The highest of each of the 3 antibiotic concentrations significantly reduced mitochondrial function. This study demonstrates that objective sperm quality assessments can be made using flow cytometry and that addition of antibiotics at appropriate concentrations can lengthen refrigerated storage time for tilapia spermatozoa. With minor modifications, these protocols can be adapted for use with sperm from other species and with other tissue types.

Effect of freezing of sputum samples on flow cytometric analysis of lymphocyte subsets.

PubMed

Jaksztat, E; Holz, O; Paasch, K; Kelly, M M; Hargreave, F E; Cox, G; Magnussen, H; Jörres, R A

2004-08-01

Sputum samples should be processed shortly after induction to prevent cell degradation. For intermediate storage, freezing of homogenised samples or immediate fixation have been shown to be suitable for cytospins. The aim of this study was to investigate whether freezing or immediate fixation of sputum affect the analysis of lymphocyte subsets by flow cytometry. Selected plugs from 24 sputum samples were homogenised. One aliquot was processed immediately and analysed by flow cytometry. A second aliquot was homogenised, frozen at -20 C after addition of dimethylsulfoxide and stored for a median time of 6 days. In six samples a third aliquot was fixed in formalin after induction and stored for up to 72 h before further processing. Compared to immediate processing, percentages of total lymphocytes and T-suppressor cells were elevated after being frozen, with a minor decrease in the T4/T8 ratio. Proportions of total lymphocytes, T-helper and T-suppressor cells correlated between native and frozen samples, intra-class correlation coefficients being 0.74, 0.85 and 0.70, respectively. The formalin-fixed aliquots could not be analysed with the antibodies used. In conclusion, freezing seems to be a suitable technique to store sputum samples for flow cytometry of CD3, CD4 and CD8 lymphocyte subsets. Its effects were minor compared to the variation between subjects.

Flow cytometry-assisted rapid isolation of recombinant Plasmodium berghei parasites exemplified by functional analysis of aquaglyceroporin

PubMed Central

Kenthirapalan, Sanketha; Waters, Andrew P.; Matuschewski, Kai; Kooij, Taco W.A.

2012-01-01

The most critical bottleneck in the generation of recombinant Plasmodium berghei parasites is the mandatory in vivo cloning step following successful genetic manipulation. This study describes a new technique for rapid selection of recombinant P. berghei parasites. The method is based on flow cytometry to isolate isogenic parasite lines and represents a major advance for the field, in that it will speed the generation of recombinant parasites as well as cut down on animal use significantly. High expression of GFP during blood infection, a prerequisite for robust separation of transgenic lines by flow cytometry, was achieved. Isogenic recombinant parasite populations were isolated even in the presence of a 100-fold excess of wild-type (WT) parasites. Aquaglyceroporin (AQP) loss-of-function mutants and parasites expressing a tagged AQP were generated to validate this approach. aqp− parasites grow normally within the WT phenotypic range during blood infection of NMRI mice. Similarly, colonization of the insect vector and establishment of an infection after mosquito transmission were unaffected, indicating that AQP is dispensable for life cycle progression in vivo under physiological conditions, refuting its use as a suitable drug target. Tagged AQP localized to perinuclear structures and not the parasite plasma membrane. We suggest that flow-cytometric isolation of isogenic parasites overcomes the major roadblock towards a genome-scale repository of mutant and transgenic malaria parasite lines. PMID:23137753

Shifts in the fluorescence lifetime of EGFP during bacterial phagocytosis measured by phase-sensitive flow cytometry

NASA Astrophysics Data System (ADS)

Li, Wenyan; Houston, Kevin D.; Houston, Jessica P.

2017-01-01

Phase-sensitive flow cytometry (PSFC) is a technique in which fluorescence excited state decay times are measured as fluorescently labeled cells rapidly transit a finely focused, frequency-modulated laser beam. With PSFC the fluorescence lifetime is taken as a cytometric parameter to differentiate intracellular events that are challenging to distinguish with standard flow cytometry. For example PSFC can report changes in protein conformation, expression, interactions, and movement, as well as differences in intracellular microenvironments. This contribution focuses on the latter case by taking PSFC measurements of macrophage cells when inoculated with enhanced green fluorescent protein (EGFP)-expressing E. coli. During progressive internalization of EGFP-E. coli, fluorescence lifetimes were acquired and compared to control groups. It was hypothesized that fluorescence lifetimes would correlate well with phagocytosis because phagosomes become acidified and the average fluorescence lifetime of EGFP is known to be affected by pH. We confirmed that average EGFP lifetimes consistently decreased (3 to 2 ns) with inoculation time. The broad significance of this work is the demonstration of how high-throughput fluorescence lifetime measurements correlate well to changes that are not easily tracked by intensity-only cytometry, which is affected by heterogeneous protein expression, cell-to-cell differences in phagosome formation, and number of bacterium engulfed.

A GEIL flow cytometry consensus proposal for quantification of plasma cells: application to differential diagnosis between MGUS and myeloma.

PubMed

Frébet, Elise; Abraham, Julie; Geneviève, Franck; Lepelley, Pascale; Daliphard, Sylvie; Bardet, Valérie; Amsellem, Sophie; Guy, Julien; Mullier, Francois; Durrieu, Francoise; Venon, Marie-Dominique; Leleu, Xavier; Jaccard, Arnaud; Faucher, Jean-Luc; Béné, Marie C; Feuillard, Jean

2011-05-01

Flow cytometry is the sole available technique for quantification of tumor plasma-cells in plasma-cell disorders, but so far, no consensus technique has been proposed. Here, we report on a standardized, simple, robust five color flow cytometry protocol developed to characterize and quantify bone marrow tumor plasma-cells, validated in a multicenter manner. CD36 was used to exclude red blood cell debris and erythroblasts, CD38 and CD138 to detect plasma-cells, immunoglobulin light chains, CD45, CD56, CD19, and CD117 + CD34 to simultaneously characterize abnormal plasma-cells and quantify bone marrow precursors. This approach was applied in nine centers to 229 cases, including 25 controls. Tumor plasma-cells were detected in 96.8% of cases, all exhibiting an immunoglobulin peak over 1g/L. Calculation of a plasma-cells/precursors (PC/P) ratio allowed quantification of the plasma-cell burden independently from bone marrow hemodilution. The PC/P ratio yielded the best results in terms of sensitivity (81%) and specificity (84%) for differential diagnosis between MGUS and myeloma, when compared with other criteria. Combination of both the PC/P ratio and percentage of abnormal plasma-cells allowed the best differential diagnosis, but these criteria were discordant in 25% cases. Indirect calculation of CD19 negative PC/R ratio gave the best results in terms of sensitivity (87%). This standardized multiparameter flow cytometric approach allows for the detection and quantification of bone marrow tumor plasma-cell infiltration in nearly all cases of MGUS and myeloma, independently of debris and hemodilution. This approach may also prove useful for the detection of minimal residual disease. Copyright © 2010 International Clinical Cytometry Society.

A novel fluorescent sensor for measurement of CFTR function by flow cytometry.

PubMed

Vijftigschild, Lodewijk A W; van der Ent, Cornelis K; Beekman, Jeffrey M

2013-06-01

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis. CFTR-dependent iodide transport measured by fluorescent quenching of ectopically expressed halide-sensitive yellow fluorescent protein (YFP) is widely being used to study CFTR function by microscopy or plate readers. Since YFP fluorescence in these systems is dependent on YFP expression levels and iodide concentration, differences in sensor expression level between experimental units are normalized at the start of each experiment. To allow accurate measurement of CFTR function by flow cytometry, we reasoned that co-expression of an iodide insensitive fluorescent protein would allow for normalization of sensor expression levels and more accurate quantification of CFTR function. Our data indicated that dsRed and mKate fluorescence are iodide insensitive, and we determined an optimal format for co-expression of these fluorescent proteins with halide-sensitive YFP. We showed using microscopy that ratiometric measurement (YFP/mKate) corrects for differences in sensor expression levels. Ratiometric measurements were essential to accurately measure CFTR function by flow cytometry that we here describe for the first time. Mixing of wild type or mutant CFTR expressing cells indicated that addition of approximately 10% of wild type CFTR expressing cells could be distinguished by ratiometric YFP quenching. Flow cytometric ratiometric YFP quenching also allowed us to study CFTR mutants associated with differential residual function upon ectopic expression. Compared with conventional plate-bound CFTR function assays, the flow cytometric approach described here can be used to study CFTR function in suspension cells. It may be further adapted to study CFTR function in heterologous cell populations using cell surface markers and selection of cells that display high CFTR function by cell sorting. Copyright © 2013 International Society for Advancement of Cytometry.

Protein expression pattern of PAWP in bull spermatozoa is associated with sperm quality and fertility following artificial insemination.

PubMed

Kennedy, Chelsey E; Krieger, Kari Beth; Sutovsky, Miriam; Xu, Wei; Vargovič, Peter; Didion, Bradley A; Ellersieck, Mark R; Hennessy, Madison E; Verstegen, John; Oko, Richard; Sutovsky, Peter

2014-05-01

Post-acrosomal WW-domain binding protein (PAWP) is a signaling molecule located in the post-acrosomal sheath (PAS) of mammalian spermatozoa. We hypothesized that the proper integration of PAWP in the sperm PAS is reflective of bull-sperm quality and fertility. Cryopreserved semen samples from 298 sires of acceptable, but varied, fertility used in artificial insemination services were analyzed using immunofluorescence microscopy and flow cytometry for PAWP protein. In normal spermatozoa, PAWP fluorescence formed a regular band around the proximal PAS. Anomalies of PAWP labeling in defective spermatozoa were reflected in flow cytometry by varied intensities of PAWP-induced fluorescence. Distinct sperm phenotypes were also identified, including morphologically normal and some defective spermatozoa with moderate levels of PAWP; grossly defective spermatozoa with low/no PAWP; and defective spermatozoa with high PAWP. Analysis by ImageStream flow cytometry confirmed the prevalence of abnormal sperm phenotypes in the spermatozoa with abnormal PAWP content. Live/dead staining and video recording showed that some abnormal spermatozoa are viable and capable of progressive motility. Conventional flow-cytometric measurements of PAWP correlated significantly with semen quality and fertility parameters that reflect the sires' artificial insemination fertility, including secondary sperm morphology, conception rate, non-return rate, and residual value. A multiplex, flow-cytometric test detecting PAWP, aggresomes (ubiquitinated protein aggregates), and acrosomal integrity (peanut-agglutinin-lectin labeling) had a predictive value for conception rate, as demonstrated by step-wise regression analysis. We conclude that PAWP correlates with semen/fertility parameters used in the cattle artificial insemination industry, making PAWP a potential biomarker of bull fertility. © 2014 Wiley Periodicals, Inc.

Detailed immunophenotyping of B-cell precursors in regenerating bone marrow of acute lymphoblastic leukaemia patients: implications for minimal residual disease detection.

PubMed

Theunissen, Prisca M J; Sedek, Lukasz; De Haas, Valerie; Szczepanski, Tomasz; Van Der Sluijs, Alita; Mejstrikova, Ester; Nováková, Michaela; Kalina, Tomas; Lecrevisse, Quentin; Orfao, Alberto; Lankester, Arjan C; van Dongen, Jacques J M; Van Der Velden, Vincent H J

2017-07-01

Flow cytometric detection of minimal residual disease (MRD) in children with B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) requires immunophenotypic discrimination between residual leukaemic cells and B-cell precursors (BCPs) which regenerate during therapy intervals. In this study, EuroFlow-based 8-colour flow cytometry and innovative analysis tools were used to first characterize the immunophenotypic maturation of normal BCPs in bone marrow (BM) from healthy children, resulting in a continuous multiparametric pathway including transition stages. This pathway was subsequently used as a reference to characterize the immunophenotypic maturation of regenerating BCPs in BM from children treated for BCP-ALL. We identified pre-B-I cells that expressed low or dim CD34 levels, in contrast to the classical CD34 high pre-B-I cell immunophenotype. These CD34 -dim pre-B-I cells were relatively abundant in regenerating BM (11-85% within pre-B-I subset), while hardly present in healthy control BM (9-13% within pre-B-I subset; P = 0·0037). Furthermore, we showed that some of the BCP-ALL diagnosis immunophenotypes (23%) overlapped with CD34 -dim pre-B-I cells. Our results indicate that newly identified CD34 -dim pre-B-I cells can be mistaken for residual BCP-ALL cells, potentially resulting in false-positive MRD outcomes. Therefore, regenerating BM, in which CD34 -dim pre-B-I cells are relatively abundant, should be used as reference frame in flow cytometric MRD measurements. © 2017 John Wiley & Sons Ltd.

Expanding the potential of standard flow cytometry by extracting fluorescence lifetimes from cytometric pulse shifts

PubMed Central

Cao, Ruofan; Naivar, Mark A; Wilder, Mark; Houston, Jessica P

2014-01-01

Fluorescence lifetime measurements provide information about the fluorescence relaxation, or intensity decay, of organic fluorophores, fluorescent proteins, and other inorganic molecules that fluoresce. The fluorescence lifetime is emerging in flow cytometry and is helpful in a variety of multiparametric, single cell measurements because it is not impacted by nonlinearity that can occur with fluorescence intensity measurements. Yet time-resolved cytometry systems rely on major hardware modifications making the methodology difficult to reproduce. The motivation of this work is, by taking advantage of the dynamic nature of flow cytometry sample detection and applying digital signal processing methods, to measure fluorescence lifetimes using an unmodified flow cytometer. We collect a new lifetime-dependent parameter, referred to herein as the fluorescence-pulse-delay (FPD), and prove it is a valid representation of the average fluorescence lifetime. To verify we generated cytometric pulses in simulation, with light emitting diode (LED) pulsation, and with true fluorescence measurements of cells and microspheres. Each pulse is digitized and used in algorithms to extract an average fluorescence lifetime inherent in the signal. A range of fluorescence lifetimes is measurable with this approach including standard organic fluorophore lifetimes (∼1 to 22 ns) as well as small, simulated shifts (0.1 ns) under standard conditions (reported herein). This contribution demonstrates how digital data acquisition and signal processing can reveal time-dependent information foreshadowing the exploitation of full waveform analysis for quantification of similar photo-physical events within single cells. © 2014 The Authors. Published by Wiley Periodicals, Inc. PMID:25274073

Evaluation of flow cytometric HIT assays in relation to an IgG-Specific immunoassay and clinical outcome.

PubMed

Kerényi, Adrienne; Beke Debreceni, Ildikó; Oláh, Zsolt; Ilonczai, Péter; Bereczky, Zsuzsanna; Nagy, Béla; Muszbek, László; Kappelmayer, János

2017-09-01

Heparin-induced thrombocytopenia (HIT) is a severe side effect of heparin treatment caused by platelet activating IgG antibodies generated against the platelet factor 4 (PF4)-heparin complex. Thrombocytopenia and thrombosis are the leading clinical symptoms of HIT. The clinical pretest probability of HIT was evaluated by the 4T score system. Laboratory testing of HIT was performed by immunological detection of antibodies against PF4-heparin complex (EIA) and two functional assays. Heparin-dependent activation of donor platelets by patient plasma was detected by flow cytometry. Increased binding of Annexin-V to platelets and elevated number of platelet-derived microparticles (PMP) were the indicators of platelet activation. EIA for IgG isotype HIT antibodies was performed in 405 suspected HIT patients. Based on negative EIA results, HIT was excluded in 365 (90%) of cases. In 40 patients with positive EIA test result functional tests were performed. Platelet activating antibodies were detected in 17 cases by Annexin V binding. PMP count analysis provided nearly identical results. The probability of a positive flow cytometric assay result was higher in patients with elevated antibody titer. 71% of patients with positive EIA and functional assay had thrombosis. EIA is an important first line laboratory test in the diagnosis of HIT; however, HIT must be confirmed by a functional test. Annexin V binding and PMP assays using flow cytometry are functional HIT tests convenient in a clinical diagnostic laboratory. The positive results of functional assays may predict the onset of thrombosis. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

New Advances in Molecular Therapy for Muscle Repair after Diseases and Injuries

DTIC Science & Technology

2011-01-01

members of the broader scientific community . Statement of Work...negative for CD34 (1A). Nuclei were stained blue with Dapi. Scale bars, 100 µm. Flow cytometric analysis indicated percentage of cryopreserveded...muscle cells were also cytocentrifuged on glass slides and stained with antibodies to CD56, CD146, UEA-1(2Q, scale bars, 100 µm), and CD56/UEA-1

Technical advance: liposomal alendronate depletes monocytes and macrophages in the nonhuman primate model of human disease.

PubMed

Burwitz, Benjamin J; Reed, Jason S; Hammond, Katherine B; Ohme, Merete A; Planer, Shannon L; Legasse, Alfred W; Ericsen, Adam J; Richter, Yoram; Golomb, Gershon; Sacha, Jonah B

2014-09-01

Nonhuman primates are critical animal models for the study of human disorders and disease and offer a platform to assess the role of immune cells in pathogenesis via depletion of specific cellular subsets. However, this model is currently hindered by the lack of reagents that safely and specifically ablate myeloid cells of the monocyte/macrophage Lin. Given the central importance of macrophages in homeostasis and host immunity, development of a macrophage-depletion technique in nonhuman primates would open new avenues of research. Here, using LA at i.v. doses as low as 0.1 mg/kg, we show a >50% transient depletion of circulating monocytes and tissue-resident macrophages in RMs by an 11-color flow cytometric analysis. Diminution of monocytes was followed rapidly by emigration of monocytes from the bone marrow, leading to a rebound of monocytes to baseline levels. Importantly, LA was well-tolerated, as no adverse effects or changes in gross organ function were observed during depletion. These results advance the ex vivo study of myeloid cells by flow cytometry and pave the way for in vivo studies of monocyte/macrophage biology in nonhuman primate models of human disease. © 2014 Society for Leukocyte Biology.

Technical Advance: Liposomal alendronate depletes monocytes and macrophages in the nonhuman primate model of human disease

PubMed Central

Burwitz, Benjamin J.; Reed, Jason S.; Hammond, Katherine B.; Ohme, Merete A.; Planer, Shannon L.; Legasse, Alfred W.; Ericsen, Adam J.; Richter, Yoram; Golomb, Gershon; Sacha, Jonah B.

2014-01-01

Nonhuman primates are critical animal models for the study of human disorders and disease and offer a platform to assess the role of immune cells in pathogenesis via depletion of specific cellular subsets. However, this model is currently hindered by the lack of reagents that safely and specifically ablate myeloid cells of the monocyte/macrophage Lin. Given the central importance of macrophages in homeostasis and host immunity, development of a macrophage-depletion technique in nonhuman primates would open new avenues of research. Here, using LA at i.v. doses as low as 0.1 mg/kg, we show a >50% transient depletion of circulating monocytes and tissue-resident macrophages in RMs by an 11-color flow cytometric analysis. Diminution of monocytes was followed rapidly by emigration of monocytes from the bone marrow, leading to a rebound of monocytes to baseline levels. Importantly, LA was well-tolerated, as no adverse effects or changes in gross organ function were observed during depletion. These results advance the ex vivo study of myeloid cells by flow cytometry and pave the way for in vivo studies of monocyte/macrophage biology in nonhuman primate models of human disease. PMID:24823811

Development of a novel flow cytometric approach to evaluate fish sperm chromatin using fixed samples

USGS Publications Warehouse

Jenkins, Jill A.

2013-01-01

The integrity of the paternal DNA is essential for the accurate transmission of genetic information, yet fertilization is not inhibited by chromatin breakage. Some methods are available for the sensitive detection of DNA damage and can be applied in studies of environmental toxicology, carcinogenesis, aging, and assisted reproduction techniques in both clinical and experimental settings. Because semen samples obtained from remote locations undergo chromatin damage prior to laboratory assessment, the present study was undertaken to evaluate treatments for effective chromatin staining in the development of a DNA fragmentation assay using fixed milt from yellow perch (Perca flavescens). Similar to the sperm chromatin structure assay (SCSA), susceptibility of nuclear DNA to acid-induced denaturation was measured by flow cytometry (FCM). Use of 10% buffered formalin for milt fixation allowed easier peak discrimination than 4% paraformaldehyde. The effects of time and temperature of incubation in 0.08 N HCl were evaluated in order to determine the ideal conditions for promoting DNA decondensation and making strand breaks more available for staining and detection by FCM. The best results were obtained with incubation at 37°C for 1 minute, followed by cold propidium iodide staining for 30 minutes.

Myasthenia gravis sera have no effect on cardiomyocytes in vitro.

PubMed

Helgeland, Geir; Luckman, Steven P; Romi, Fredrik R; Jonassen, Anne K; Gilhus, Nils Erik

2008-09-15

Myasthenia gravis (MG) is an autoimmune disorder primarily caused by circulating autoantibodies targeting the nicotinic acetylcholine receptor. Several studies have suggested a link between MG and heart disease. Girardi heart cells were treated with MG sera, measuring cytotoxic effects using flow cytometry, adenylate kinase (AK) release and evaluating morphology. MG sera did not induce morphological changes in the cells. AK release from cells treated with MG sera did not exceed controls and flow cytometric examination did not reveal any increase in dead or apoptotic cells. We conclude that MG sera have no cytotoxic effect in our heart cell culture system.

Simultaneous flow cytometric measurement of antigen attachment to phagocytes and phagocytosis.

PubMed

Laopajon, Witida; Takheaw, Nuchjira; Kasinrerk, Watchara; Pata, Supansa

2016-01-01

The current available assays cannot differentiate the stages of phagocytosis. We, therefore, established methods for concurrent detection of antigen attachment and engulfment by phagocyte using latex beads coated with lipopolysaccharide, rabbit IgG, and carboxyfluorescein diacetate succinimidyl ester. The generated beads were incubated with whole blood at 37°C for 1 hr and stained with PE-Cy5.5 anti-rabbit IgG antibody. By flow cytometry, attachment and phagocytic processes could be detected, simultaneously. The established method is a valuable tool for diagnosis of phagocytic disorder and study of molecules involved in phagocytosis.

Breast epithelium procurement from stereotactic core biopsy washings: flow cytometry-sorted cell count analysis.

PubMed

Stoler, Daniel L; Stewart, Carleton C; Stomper, Paul C

2002-02-01

Molecular studies of breast lesions have been constrained by difficulties in procuring adequate tissues for analyses. Standard procedures are restricted to larger, palpable masses or the use of paraffin-embedded materials, precluding facile procurement of fresh specimens of early lesions. We describe a study to determine the yield and characteristics of sorted cell populations retrieved in core needle biopsy specimen rinses from a spectrum of breast lesions. Cells from 114 consecutive stereotactic core biopsies of mammographic lesions released into saline washes were submitted for flow cytometric analysis. For each specimen, epithelial cells were separated from stromal and blood tissue based on the presence of cytokeratin 8 and 18 markers. Epithelial cell yields based on pathological diagnoses of the biopsy specimen, patient age, and mammographic appearance of the lesion were determined. Biopsies containing malignant lesions yielded significantly higher numbers of cells than were obtained from benign lesion biopsies. Significantly greater cell counts were observed from lesions from women age 50 or above compared with those of younger women. Mammographic density surrounding the biopsy site, the mammographic appearance of the lesion, and the number of cores taken at the time of biopsy appeared to have little effect on the yield of epithelial cells. We demonstrate the use of flow cytometric sorting of stereotactic core needle biopsy washes from lesions spanning the spectrum of breast pathology to obtain epithelial cells in sufficient numbers to meet the requirements of a variety of molecular and genetic analyses.

Flow Cytometric Ploidy Determination of Oral Premalignant and Malignant Lesions

DTIC Science & Technology

1990-01-01

could advantageously influence patient management. REVIEW OF LITERATURE 3 ORAL PRECANCER As early as 1957 the World Health Organization 1 (WHO...euploid lichenoid mucositis was combined with eosinophilia 88 as the diagnosis of the intermediate lesion in case 6 and occurred a year after the...International Histological Classification of Tumours, No. 4. Geneva, World Health Organization, 1971, p. 23. 3. Pindborg, J.J.: Premalignant and malignant

Induction of apoptosis in human promyelocytic leukemia HL-60 cells by Ampelopsis cantoniensis crude extract.

PubMed

Tan, Tzu-Wei; Tsai, Huei-Yann; Chen, Yuh-Fung; Chung, Jing-Gung

2004-01-01

The crude extract of Ampelopsis cantoniensis induced apoptosis in human promyelocytic leukemia HL-60 cells and this induction was investigated by flow cytometric analysis, DNA gel electrophoresis and poly (ADP-ribose) fluorescence staining. The results demonstrated that this extract induced dose-dependent cytotoxicity and apoptosis. The level of active caspase-3 was increased after treatment with the crude extract for 24 hours.

Novel whole blood assay for phenotyping platelet reactivity in mice identifies ICAM-1 as a mediator of platelet-monocyte interaction

PubMed Central

Kirkby, Nicholas S.; Chan, Melissa V.; Finsterbusch, Michaela; Hogg, Nancy; Nourshargh, Sussan; Warner, Timothy D.

2015-01-01

Testing of platelet function is central to the cardiovascular phenotyping of genetically modified mice. Traditional platelet function tests have been developed primarily for testing human samples and the volumes required make them highly unsuitable for the testing of mouse platelets. This limits research in this area. To address this problem, we have developed a miniaturized whole blood aggregometry assay, based on a readily accessible 96-well plate format coupled with quantification of single platelet depletion by flow cytometric analysis. Using this approach, we observed a concentration-dependent loss of single platelets in blood exposed to arachidonic acid, collagen, U46619 or protease activated receptor 4 activating peptide. This loss was sensitive to well-established antiplatelet agents and genetic manipulation of platelet activation pathways. Observations were more deeply analyzed by flow cytometric imaging, confocal imaging, and measurement of platelet releasates. Phenotypic analysis of the reactivity of platelets taken from mice lacking intercellular adhesion molecule (ICAM)-1 identified a marked decrease in fibrinogen-dependent platelet-monocyte interactions, especially under inflammatory conditions. Such findings exemplify the value of screening platelet phenotypes of genetically modified mice and shed further light upon the roles and interactions of platelets in inflammation. PMID:26215112

Flow cytometric determination of quantitative immunophenotypes

NASA Astrophysics Data System (ADS)

Redelman, Douglas; Ensign, Wayne; Roberts, Don

2001-05-01

Immunofluorescent flow cytometric analysis of peripheral blood leucocytes is most commonly used to identify and enumerate cells defined by one or more clusters of differentiation (CD) antigens. Although less widely employed, quantitative tests that measure the amounts of CD antigens expressed per cell are used in some situations such as the characterization of lymphomas and leukocytes or the measurement of CD38 on CD3plu8pluT cells in HIV infected individuals. The CD antigens used to identify leukocyte populations are functionally important molecules and it is known that under- or over-expression of some CD antigens can affect cellular responses. For example, high or low expression of CD19 on B cells is associated with autoimmune conditions or depressed antibody responses, respectively. In the current studies, the quantitative expression of CD antigens on T cells, B cells and monocytes was determined in a group of age and sex-matched Marines at several times before and after training exercises. There was substantial variation among these individuals in the quantitative expression of CD antigens and in the number of cells in various populations. However, there was relatively little variation within individuals during the two months they were examined. Thus, the number of cells in leukocyte sub-populations and the amount of CD antigens expressed per cell appear to comprise a characteristic quantitative immunophenotype.

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

PubMed Central

Wang, Li; Carnegie, Graeme K.

2013-01-01

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells. BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction. PMID:23979513

Flow cytometric analysis of bimolecular fluorescence complementation: a high throughput quantitative method to study protein-protein interaction.

PubMed

Wang, Li; Carnegie, Graeme K

2013-08-15

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells. BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

DNA polymeric films as a support for cell growth as a new material for regenerative medicine: Compatibility and applicability.

PubMed

Jayme, Cristiano Ceron; de Paula, Leonardo Barcelos; Rezende, Nayara; Calori, Italo Rodrigo; Franchi, Leonardo Pereira; Tedesco, Antonio Claudio

2017-11-15

DNA polymeric films (DNA-PFs) are a promising drug delivery system (DDS) in modern medicine. In this study, we evaluated the growth behavior of oral squamous cell carcinoma (OSCC) cells on DNA-PFs. The morphological, biochemical, and cytometric features of OSCC cell adhesion on DNA-PFs were also assessed. An initial, temporary alteration in cell morphology was observed at early time points owing to the inhibition of cell attachment to the film, which then returned to a normal morphological state at later time points. MTT and resazurin assays showed a moderate reduction in cell viability related to increased DNA concentration in the DNA-PFs. Flow cytometry studies showed low cytotoxicity of DNA-PFs, with cell viabilities higher than 90% in all the DNA-PFs tested. Flow cytometric cell cycle analysis also showed average cell cycle phase distributions at later time points, indicating that OSCC cell growth is maintained in the presence of DNA-PFs. These results show high biocompatibility of DNA-PFs and suggest their use in designing "dressing material," where the DNA film acts as a support for cell growth, or with incorporation of active or photoactive compounds, which can induce tissue regeneration and are useful to treat many diseases, especially oral cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

Role of flow-cytometric immunophenotyping in prediction of BCR/ABL1 gene rearrangement in adult B-cell acute lymphoblastic leukemia.

PubMed

Corrente, Francesco; Bellesi, Silvia; Metafuni, Elisabetta; Puggioni, Pier Luigi; Marietti, Sara; Ciminello, Angela Maria; Za, Tommaso; Sorà, Federica; Fianchi, Luana; Sica, Simona; De Stefano, Valerio; Chiusolo, Patrizia

2018-05-01

We performed a retrospective analysis of 88 adult patients with B-ALL diagnosed in our center by a flow-cytometric assessment. Immunophenotypic expression of leukemic cells was explored by simultaneous evaluation of positivity, percentage of expressing cells and median fluorescence intensity (MFI). BCR/ABL1 fusion transcripts were assessed by RT-PCR analysis and were identified in 36 patients (40.9%). CD10 and CD34 were positive in the totality of BCR/ABL1-positive cases. Patients with gene rearrangement had a greater frequency of CD66c, CD13 and CD33 positivity compared with BCR/ABL1-negative cases. Moreover, BCR/ABL1-positive cases exhibited a greater median percentage and MFI values of CD13, CD33, CD66c, CD10, CD34 and CD25 expressions, but a lower median percentage and MFI values of CD38 and CD22 expressions than patients without gene rearrangement. Multivariate logistic regression analysis showed that CD10, CD38 and CD13 expressions were independent predictors for the presence of BCR/ABL1 rearrangement. Predictive probabilities of molecular occurrence based on these markers are proposed. © 2017 International Clinical Cytometry Society. © 2017 International Clinical Cytometry Society.

Empirically Optimized Flow Cytometric Immunoassay Validates Ambient Analyte Theory

PubMed Central

Parpia, Zaheer A.; Kelso, David M.

2010-01-01

Ekins’ ambient analyte theory predicts, counter intuitively, that an immunoassay’s limit of detection can be improved by reducing the amount of capture antibody. In addition, it also anticipates that results should be insensitive to the volume of sample as well as the amount of capture antibody added. The objective of this study is to empirically validate all of the performance characteristics predicted by Ekins’ theory. Flow cytometric analysis was used to detect binding between a fluorescent ligand and capture microparticles since it can directly measure fractional occupancy, the primary response variable in ambient analyte theory. After experimentally determining ambient analyte conditions, comparisons were carried out between ambient and non-ambient assays in terms of their signal strengths, limits of detection, and their sensitivity to variations in reaction volume and number of particles. The critical number of binding sites required for an assay to be in the ambient analyte region was estimated to be 0.1VKd. As predicted, such assays exhibited superior signal/noise levels and limits of detection; and were not affected by variations in sample volume and number of binding sites. When the signal detected measures fractional occupancy, ambient analyte theory is an excellent guide to developing assays with superior performance characteristics. PMID:20152793

Cytotoxicity of four denture adhesives on human gingival fibroblast cells.

PubMed

Lee, Yoon; Ahn, Jin-Soo; Yi, Young-Ah; Chung, Shin-Hye; Yoo, Yeon-Jee; Ju, Sung-Won; Hwang, Ji-Yun; Seo, Deog-Gyu

2015-02-01

The purpose of this study was to compare the cytotoxicity of four denture adhesives on human gingival fibroblast cells. Immortalized human gingival fibroblasts were cultured with one of four different denture adhesives, Polident, Protefix, Staydent or Denfix-A, which was placed in insert dishes (10% w/v concentration) for 48 h. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and flow cytometric apoptosis assay were used to evaluate cell viability and apoptosis rates. The fibroblasts were also examined under a scanning electron microscope. The MTT assay showed that all denture adhesives resulted in a significantly lower cell viability compared to the control cells propagated in normal culture medium (p < 0.05), with Staydent demonstrating the lowest cell viability. According to the flow cytometric apoptosis assay, Staydent and Protefix showed significantly higher apoptosis rates than the control group (p < 0.05), whereas Polident and Denfix-A did not demonstrate any significant differences (p > 0.05). Staydent showed the highest apoptosis rate. Scanning electron microscopy showed that the cells of the Staydent group underwent cytoplasmic membrane shrinkage, with cell free areas containing residual fragments of the membrane of dead cells. The four denture adhesives evaluated in this study imparted cytotoxic effects on human gingival fibroblast cells. Staydent showed the highest toxicity.

Piperine impairs cell cycle progression and causes reactive oxygen species-dependent apoptosis in rectal cancer cells.

PubMed

Yaffe, Paul B; Doucette, Carolyn D; Walsh, Mark; Hoskin, David W

2013-02-01

Piperine, an alkaloid phytochemical found in the fruit of black and long pepper plants, is reported to inhibit the growth of cancer cells; however, the mechanism of action in human cancer cells is not clear. In this study we investigated the effect of piperine on the growth of HRT-18 human rectal adenocarcinoma cells. MTT assays showed that piperine inhibited the metabolic activity of HRT-18 cells in a dose- and time-dependent fashion, suggesting a cytostatic and/or cytotoxic effect. Flow cytometric analysis of Oregon Green 488-stained and propidium iodide-stained HRT-18 cells showed that piperine inhibited cell cycle progression. Piperine also caused HRT-18 cells to die by apoptosis, as determined by Annexin-V-FLUOS staining and characteristic changes in cell morphology. Flow cytometric analysis of dihydroethidium- and 2',7'-dichlorofluorescein diacetate-stained HRT-18 cells showed increased production of reactive oxygen species in piperine-treated cells. Furthermore, the antioxidant N-acetylcysteine reduced apoptosis in cultures of piperine-treated HRT-18 cells, indicating that piperine-induced cytotoxicity was mediated at least in part by reactive oxygen species. The cytostatic and cytotoxic effects of piperine on rectal cancer cells suggest that this dietary phytochemical may be useful in cancer treatment. Copyright © 2012 Elsevier Inc. All rights reserved.

Characterization of surface interleukin-2 receptor expression on gated populations of peripheral blood mononuclear cells from manatees, Trichechus manatus latirostris.

PubMed

Sweat, J M; Johnson, C M; Marikar, Y; Gibbs, E P

2005-12-15

An in vitro system to determine surface interleukin-2 receptor (IL-2R) expression on mitogen-stimulated peripheral blood mononuclear cells (PBMC) from free-ranging manatees, Trichechus manatus latirostris was developed. Human recombinant IL-2, conjugated with a fluorescein dye was used in conjunction with flow cytometric analysis to determine changes in surface expression of IL-2R at sequential times over a 48-h period of in vitro stimulation. Surface expression of IL-2R was detected on manatee PBMC, which also cross-reacted with an anti-feline pan T-cell marker. An expression index (EI) was calculated by comparing mitogen-activated and non-activated PBMC. Based on side- and forward-scatter properties, flow cytometric analysis showed an increase in the number of larger, more granular "lymphoblasts" following concanavalin A (Con A) stimulation. The appearance of lymphoblasts was correlated with an increase in their surface expression of IL-2 receptors. Surface IL-2R expression, in Con A-stimulated PBMC, was detected at 16 h, peaked at 24-36 h, and began to decrease by 48 h. Characterization of the IL-2R expression should provide additional information on the health status of manatees, and the effect of their sub lethal exposure to brevetoxin.

FLOCK cluster analysis of plasma cell flow cytometry data predicts bone marrow involvement by plasma cell neoplasia.

PubMed

Dorfman, David M; LaPlante, Charlotte D; Li, Betty

2016-09-01

We analyzed plasma cell populations in bone marrow samples from 353 patients with possible bone marrow involvement by a plasma cell neoplasm, using FLOCK (FLOw Clustering without K), an unbiased, automated, computational approach to identify cell subsets in multidimensional flow cytometry data. FLOCK identified discrete plasma cell populations in the majority of bone marrow specimens found by standard histologic and immunophenotypic criteria to be involved by a plasma cell neoplasm (202/208 cases; 97%), including 34 cases that were negative by standard flow cytometric analysis that included clonality assessment. FLOCK identified discrete plasma cell populations in only a minority of cases negative for involvement by a plasma cell neoplasm by standard histologic and immunophenotypic criteria (38/145 cases; 26%). Interestingly, 55% of the cases negative by standard analysis, but containing a FLOCK-identified discrete plasma cell population, were positive for monoclonal gammopathy by serum protein electrophoresis and immunofixation. FLOCK-identified and quantitated plasma cell populations accounted for 3.05% of total cells on average in cases positive for involvement by a plasma cell neoplasm by standard histologic and immunophenotypic criteria, and 0.27% of total cells on average in cases negative for involvement by a plasma cell neoplasm by standard histologic and immunophenotypic criteria (p<0.0001; area under the curve by ROC analysis=0.96). The presence of a FLOCK-identified discrete plasma cell population was predictive of the presence of plasma cell neoplasia with a sensitivity of 97%, compared with only 81% for standard flow cytometric analysis, and had specificity of 74%, PPV of 84% and NPV of 95%. FLOCK analysis, which has been shown to provide useful diagnostic information for evaluating patients with suspected systemic mastocytosis, is able to identify neoplastic plasma cell populations analyzed by flow cytometry, and may be helpful in the diagnostic evaluation of bone marrow samples for involvement by plasma cell neoplasia. Copyright © 2016 Elsevier Ltd. All rights reserved.

Detection of acute lymphoblastic leukemia involvement in pleural fluid in an adult patient with ataxia telangiectasia by flow cytometry method.

PubMed

Keklik, Muzaffer; Koker, M Yavuz; Sivgin, Serdar; Camlica, Demet; Pala, Cigdem; Cetin, Mustafa; Kaynar, Leylagul; Unal, Ali; Eser, Bulent

2014-09-01

Ataxia-telangiectasia (AT) is a rare multisystem, neurodegenerative genetic disorder. Patients should be closely monitored due to risk of malignancy development. Due to its wide clinical heterogeneity, it often leads physicians to an inaccurate or missed diagnosis, and insight into this rare disease is important. Pediatric patients may develop lymphomas and acute lymphoblastic leukemia (ALL). However, in adults, there are limited numbers of reports regarding association of AT and ALL. Rarely, ALL cases may present with pleural fluid involvement. In our study, we presented an adult case with AT, in which ALL involvement was detected in pleural fluid by flow cytometry (FC). A 20-years old male presented to emergency department with fever, shortness of breath and cough, as he had been followed with a diagnosis of AT. The following findings were detected in laboratory tests: Hb, 11.5 g/L; WBC, 36 × 10(9)/L; Plt: 140 × 10(9)/L. Blastic cells were observed in peripheral blood smear. On chest radiography, pleural fluid appearance was observed. On thorax CT, pleural fluid was detected in both hemithorax. Cytoplasmic CD3(+) and superficial CD3 (+), CD45 (+), CD5 (+), CD7 (+) and CD38 (+) was found in the flow cytometric evaluation of peripheral blood. Superficial CD3 (+), CD2 (+), CD5 (+) and CD7 (+) were found in flow cytometric evaluation of pleural fluid. These findings were considered as consistent with pleural involvement of T-ALL. FC is a potentially useful diagnostic tool for clinical practice and it is a convenience method which has an important role in detection of ALL in patients with pleural fluid.

An enzyme-free flow cytometric bead assay for the sensitive detection of microRNAs based on click nucleic acid ligation-mediated signal amplification.

PubMed

Qi, Yan; Qiu, Liying; Fan, Wenjiao; Liu, Chenghui; Li, Zhengping

2017-08-07

A versatile flow cytometric bead assay (FCBA) coupled with a completely enzyme-free signal amplification mechanism is developed for the sensitive detection of microRNAs (miRNAs). This new strategy integrates click chemistry-mediated ligation chain reaction (CLCR) with hybridization chain reaction (HCR) for enzyme-free signal amplification on magnetic beads (MBs), and a flow cytometer for the robust fluorescence readout of the MBs. Firstly, target miRNA can initiate CLCR on the surface of MBs based on the click chemical ligation between dibenzocyclooctyne (DBCO)- and azide-modified single-stranded DNA (ssDNA) probes, and the amount of ligated ssDNA sequences on the MBs will be proportional to the dosage of target miRNA. Afterward, each of the ligated ssDNA products can trigger a cascade chain reaction of hybridization events between two alternating fluorophore-tagged hairpin probes, resulting in another signal amplification pathway with an amplified accumulation of fluorophores on the MBs. Finally, the fluorophore-anchored MBs are directly and rapidly analyzed by using a flow cytometer without any separation or elution processes. Herein, the click nucleic acid ligation only occurs on the surface of MBs, so the nonspecific ligations are greatly inhibited compared with that of ligation reaction performed in homogeneous solution. Furthermore, the signal amplification by CLCR-HCR is highly efficient but totally enzyme-free, which may overcome the potential drawbacks of conventional enzyme-catalyzed signal amplification protocols and lead to a high sensitivity. The CLCR-HCR-based FCBA has pushed the detection limit of let-7a miRNA down to the femtomolar (fM) level, showing great potential in miRNA-related biological studies and disease diagnosis.

Sperm sexing in sheep and cattle: the exception and the rule.

PubMed

de Graaf, S P; Beilby, K H; Underwood, S L; Evans, G; Maxwell, W M C

2009-01-01

Flow cytometric sorting for the preselection of sex has progressed considerably in the 20 years since its inception. This technique has allowed the production of pre-sexed offspring in a multitude of species and become a commercial success in cattle around the world. However, due to the stress inherent to the sex-sorting process, sex-sorted spermatozoa are widely recognized as functionally compromised in terms of their fertilizing lifespan within the female reproductive tract as a result of reduced motility and viability and changed functional state. These characteristics, when compared to non-sorted controls, are manifest in vivo as lower fertility. However, improvements to the technology and a greater understanding of its biological impact have facilitated recent developments in sheep, showing sex-sorting is capable of selecting a functionally superior population in terms of both in vitro and in vivo function. These results are reviewed in the context of recent developments in other species and the reasons for success after artificial insemination with sex-sorted ram spermatozoa are discussed.

SPICE: exploration and analysis of post-cytometric complex multivariate datasets.

PubMed

Roederer, Mario; Nozzi, Joshua L; Nason, Martha C

2011-02-01

Polychromatic flow cytometry results in complex, multivariate datasets. To date, tools for the aggregate analysis of these datasets across multiple specimens grouped by different categorical variables, such as demographic information, have not been optimized. Often, the exploration of such datasets is accomplished by visualization of patterns with pie charts or bar charts, without easy access to statistical comparisons of measurements that comprise multiple components. Here we report on algorithms and a graphical interface we developed for these purposes. In particular, we discuss thresholding necessary for accurate representation of data in pie charts, the implications for display and comparison of normalized versus unnormalized data, and the effects of averaging when samples with significant background noise are present. Finally, we define a statistic for the nonparametric comparison of complex distributions to test for difference between groups of samples based on multi-component measurements. While originally developed to support the analysis of T cell functional profiles, these techniques are amenable to a broad range of datatypes. Published 2011 Wiley-Liss, Inc.

Hydroxyhydroquinone, a by-product of coffee bean roasting, increases intracellular Ca2+ concentration in rat thymic lymphocytes.

PubMed

Kamae, Risa; Nojima, Shoko; Akiyoshi, Kenji; Setsu, Shoki; Honda, Sari; Masuda, Toshiya; Oyama, Yasuo

2017-04-01

Hydroxyhydroquinone (HHQ) is generated during coffee bean roasting. A cup of coffee contains 0.1-1.7 mg of HHQ. The actions of HHQ on mammalian DNA were examined because HHQ is a metabolite of benzene, which causes leukemia. Currently, information on the cellular actions of HHQ is limited. We examined the effects of sublethal levels of HHQ on the concentration of intracellular Ca 2+ in rat thymic lymphocytes by using a flow cytometric technique with fluorescent probes. HHQ at 10 μM or more significantly elevated intracellular Ca 2+ levels by increasing the membrane permeability of divalent cations, resulting in hyperpolarization via the activation of Ca 2+ -dependent K + channels. HHQ-induced changes in the intracellular Ca 2+ concentration and membrane potential may affect the cell functions of lymphocytes. HHQ-reduced coffee may be preferable in order to avoid the possible adverse effects of HHQ. Copyright © 2017 Elsevier Ltd. All rights reserved.

Quality assessment program for EuroFlow protocols: summary results of four-year (2010-2013) quality assurance rounds.

PubMed

Kalina, Tomas; Flores-Montero, Juan; Lecrevisse, Quentin; Pedreira, Carlos E; van der Velden, Vincent H J; Novakova, Michaela; Mejstrikova, Ester; Hrusak, Ondrej; Böttcher, Sebastian; Karsch, Dennis; Sędek, Łukasz; Trinquand, Amelie; Boeckx, Nancy; Caetano, Joana; Asnafi, Vahid; Lucio, Paulo; Lima, Margarida; Helena Santos, Ana; Bonaccorso, Paola; van der Sluijs-Gelling, Alita J; Langerak, Anton W; Martin-Ayuso, Marta; Szczepański, Tomasz; van Dongen, Jacques J M; Orfao, Alberto

2015-02-01

Flow cytometric immunophenotyping has become essential for accurate diagnosis, classification, and disease monitoring in hemato-oncology. The EuroFlow Consortium has established a fully standardized "all-in-one" pipeline consisting of standardized instrument settings, reagent panels, and sample preparation protocols and software for data analysis and disease classification. For its reproducible implementation, parallel development of a quality assurance (QA) program was required. Here, we report on the results of four consecutive annual rounds of the novel external QA EuroFlow program. The novel QA scheme aimed at monitoring the whole flow cytometric analysis process (cytometer setting, sample preparation, acquisition and analysis) by reading the median fluorescence intensities (MedFI) of defined lymphocytes' subsets. Each QA participant applied the predefined reagents' panel on blood cells of local healthy donors. A uniform gating strategy was applied to define lymphocyte subsets and to read MedFI values per marker. The MedFI values were compared with reference data and deviations from reference values were quantified using performance score metrics. In four annual QA rounds, we analyzed 123 blood samples from local healthy donors on 14 different instruments in 11 laboratories from nine European countries. The immunophenotype of defined cellular subsets appeared sufficiently standardized to permit unified (software) data analysis. The coefficient of variation of MedFI for 7 of 11 markers performed repeatedly below 30%, average MedFI in each QA round ranged from 86 to 125% from overall median. Calculation of performance scores was instrumental to pinpoint standardization failures and their causes. Overall, the new EuroFlow QA system for the first time allowed to quantify the technical variation that is introduced in the measurement of fluorescence intensities in a multicentric setting over an extended period of time. EuroFlow QA is a proficiency test specific for laboratories that use standardized EuroFlow protocols. It may be used to complement, but not replace, established proficiency tests. © 2014 International Society for Advancement of Cytometry. © 2014 International Society for Advancement of Cytometry.

Evaluation of Premarin in a Rat Model of Mild and Severe Hemorrhage

DTIC Science & Technology

2013-10-04

extracellular-only ligand ) on peripheral blood lymphocytes, as revealed in flow cytometric analysis. In Figure 20 (previous page) it is clear that in...magenta) which dropped to 27% with STSi pretreatment in vivo. This precluded accurate evaluation of EE-3-SO4 as ligand for ER in the context of...unable to enter the cell and engage the intracellular estrogen receptor GPER , where synthetic cell permeable (i.e., hydrophobic constructs) freely

Isolation and characterization of mouse innate lymphoid cells.

PubMed

Halim, Timotheus Y F; Takei, Fumio

2014-08-01

Innate lymphoid cells (ILCs) are rare populations of cytokine-producing lymphocytes and are divided into three groups, namely ILC1, ILC2, and ILC3, based on the cytokines that they produce. They comprise less than 1% of lymphocytes in mucosal tissues and express no unique cell surface markers. Therefore, they can only be identified by combinations of multiple cell surface markers and further characterized by cytokine production in vitro. Thus, multicolor flow cytometry is the only reliable method to purify and characterize ILCs. Here we describe the methods for cell preparation, flow cytometric analysis, and purification of murine ILC2 and ILC3. Copyright © 2014 John Wiley & Sons, Inc.

Flow cytometric analysis of normal and neoplastic mast cells: role in diagnosis and follow-up of mast cell disease.

PubMed

Escribano, Luis; Garcia Montero, Andres C; Núñez, Rosa; Orfao, Alberto

2006-08-01

Human mast cells (MCs) are directly derived from human pluripotent CD34+ stem and progenitor hematopoietic cells with stem cell factor being a critical growth factor supporting human MC proliferation, differentiation, and survival. Because of the advantages that flow cytometry offers (it allows rapid, objective, and sensitive multiparameter analysis of high numbers of cells from a sample, with information being provided on the basis of a single cell), it has become the method of choice in the past decade for immunophenotypic identification, enumeration, and characterization of human MCs in bone marrow and other tissue specimens.

Flow cytometric analysis of crayfish haemocytes activated by lipopolysaccharides

USGS Publications Warehouse

Cardenas, W.; Dankert, J.R.; Jenkins, J.A.

2004-01-01

Lipopolysaccharides (LPS) from Gram-negative bacteria are strong stimulators of white river crayfish, Procambarus zonangulus, haemocytes in vitro. Following haemocyte treatment with LPS and with LPS from rough mutant R5 (LPS Rc) from Salmonella minnesota, flow cytometric analysis revealed a conspicuous and reproducible decrease in cell size as compared to control haemocytes. These LPS molecules also caused a reduction in haemocyte viability as assessed by flow cytometry with the fluorescent dyes calcein-AM and ethidium homodimer. The onset of cell size reduction was gradual and occurred prior to cell death. Haemocytes treated with LPS from S. minnesota without the Lipid A moiety (detoxified LPS) decreased in size without a reduction of viability. The action of LPS on crayfish haemocytes appeared to be related to the activation of the prophenoloxidase system because phenoloxidase (PO)-specific activity in the supernatants from control and detoxified LPS-treated cells was significantly lower than that from LPS and LPS-Rc treated cells (P < 0.05). Furthermore, addition of trypsin inhibitor to the LPS treatments caused noticeable delays in cell size and viability changes. These patterns of cellular activation by LPS formulations indicated that crayfish haemocytes react differently to the polysaccharide and lipid A moieties of LPS, where lipid A is cytotoxic and the polysaccharide portion is stimulatory. These effects concur with the general pattern of mammalian cell activation by LPS, thereby indicting commone innate immune recognition mechanisms to bacterial antigens between cells from mammals and invertebrates. These definitive molecular approaches used to verify and identify mechanisms of invertbrate haemocyte responses to LPS could be applied with other glycoconjugates, soluble mediators, or xenobiotic compounds.

Flow cytometric analysis of lectin binding to in vitro-cultured Perkinsus marinus surface carbohydrates

USGS Publications Warehouse

Gauthier, J.D.; Jenkins, J.A.; La Peyre, Jerome F.

2004-01-01

Parasite surface glycoconjugates are frequently involved in cellular recognition and colonization of the host. This study reports on the identification of Perkinsus marinus surface carbohydrates by flow cytometric analyses of fluorescein isothiocyanate-conjugated lectin binding. Lectin-binding specificity was confirmed by sugar inhibition and Kolmogorov-Smirnov statistics. Clear, measurable fluorescence peaks were discriminated, and no parasite autofluorescence was observed. Parasites (GTLA-5 and Perkinsus-1 strains) harvested during log and stationary phases of growth in a protein-free medium reacted strongly with concanavalin A and wheat germ agglutinin, which bind to glucose-mannose and N-acetyl-D-glucosamine (GlcNAc) moieties, respectively. Both P. marinus strains bound with lower intensity to Maclura pomifera agglutinin, Bauhinia purpurea agglutinin, soybean agglutinin (N-acetyl-D-galactosamine-specific lectins), peanut agglutinin (PNA) (terminal galactose specific), and Griffonia simplicifolia II (GlcNAc specific). Only background fluorescence levels were detected with Ulex europaeus agglutinin I (L-fucose specific) and Limulus polyphemus agglutinin (sialic acid specific). The lectin-binding profiles were similar for the 2 strains except for a greater relative binding intensity of PNA for Perkinsus-1 and an overall greater lectin-binding capacity of Perkinsus-1 compared with GTLA-5. Growth stage comparisons revealed increased lectin-binding intensities during stationary phase compared with log phase of growth. This is the first report of the identification of surface glycoconjugates on a Perkinsus spp. by flow cytometry and the first to demonstrate that differential surface sugar expression is growth phase and strain dependent. ?? American Society of Parasitologists 2004.

Determination of chitin content in fungal cell wall: an alternative flow cytometric method.

PubMed

Costa-de-Oliveira, Sofia; Silva, Ana P; Miranda, Isabel M; Salvador, Alexandre; Azevedo, Maria M; Munro, Carol A; Rodrigues, Acácio G; Pina-Vaz, Cidália

2013-03-01

The conventional methods used to evaluate chitin content in fungi, such as biochemical assessment of glucosamine release after acid hydrolysis or epifluorescence microscopy, are low throughput, laborious, time-consuming, and cannot evaluate a large number of cells. We developed a flow cytometric assay, efficient, and fast, based on Calcofluor White staining to measure chitin content in yeast cells. A staining index was defined, its value was directly related to chitin amount and taking into consideration the different levels of autofluorecence. Twenty-two Candida spp. and four Cryptococcus neoformans clinical isolates with distinct susceptibility profiles to caspofungin were evaluated. Candida albicans clinical isolate SC5314, and isogenic strains with deletions in chitin synthase 3 (chs3Δ/chs3Δ) and genes encoding predicted GlycosylPhosphatidylInositol (GPI)-anchored proteins (pga31Δ/Δ and pga62Δ/Δ), were used as controls. As expected, the wild-type strain displayed a significant higher chitin content (P < 0.001) than chs3Δ/chs3Δ and pga31Δ/Δ especially in the presence of caspofungin. Ca. parapsilosis, Ca. tropicalis, and Ca. albicans showed higher cell wall chitin content. Although no relationship between chitin content and antifungal drug susceptibility phenotype was found, an association was established between the paradoxical growth effect in the presence of high caspofungin concentrations and the chitin content. This novel flow cytometry protocol revealed to be a simple and reliable assay to estimate cell wall chitin content of fungi. Copyright © 2013 International Society for Advancement of Cytometry.

Establishing tools for early diagnosis of congenital toxoplasmosis: Flow cytometric IgG avidity assay as a confirmatory test for neonatal screening.

PubMed

de Castro Zacche-Tonini, Aline; Fonseca, Giuliana Schmidt França; de Jesus, Laura Néspoli Nassar Pansini; Barros, Geisa Baptista; Coelho-Dos-Reis, Jordana Grazziela Alves; Béla, Samantha Ribeiro; Machado, Anderson Silva; Carneiro, Ana Carolina Aguiar Vasconcelos; Andrade, Gláucia Manzan Queiroz; Vasconcelos-Santos, Daniel Vitor; Januário, José Nélio; Teixeira-Carvalho, Andréa; Vitor, Ricardo Wagner Almeida; Ferro, Eloísa Amália Vieira; Mineo, José Roberto; Martins-Filho, Olindo Assis; Lemos, Elenice Moreira

2017-12-01

The aim of this study was to evaluate the performance of conventional serology (Q-Preven™ and ELFAVIDAS™) and flow cytometry-based serologic tools for early serologic diagnosis of congenital toxoplasmosis. The study groups included prospectively confirmed cases of congenital toxoplasmosis (TOXO=88) and age-matching non-infected controls (NI=15).The results demonstrated that all samples tested positive/indeterminate for anti-T. gondii IgM screening at birth using air-dried whole blood samples. Serum samples collected at 30-45days after birth tested positive for ELFAVIDAS™ IgG in both groups. While all NI tested negative for ELFAVIDAS™ IgM and IgA, only 78% and 36% of TOXO tested positive for IgM and IgA, respectively. Flow cytometry-based anti-T. gondii IgM, IgA and IgG reactivity displayed moderate performance with low sensitivity (47.6%, 72.6% and 75.0%, respectively). Regardless the remarkable specificity of IgG1, IgG2 and IgG3 subclasses for early diagnosis, weak or moderate specificity was observed (Se=73.9%, 60.2% and 83.0%, respectively). The analysis of IgG avidity indices (AI) demonstrated the highest performance among the flow cytometry-based methods (Se=96.6%; Sp=93.3%), underscoring the low avidity index (AI<60%) within TOXO (97.0%) in contrast with the high avidity index (AI>60%) in NI (93%). Analysis of anti-T. gondii IgG and IgG3 reactivity for mother:infant paired samples may represent a relevant complementary tests for early diagnosis. In conclusion, a feasible high-standard algorithm (Accuracy=97.1%) was proposed consisting of Q-Preven™ IgM screening at birth, followed by ELFAVIDAS™ IgM and flow cytometric IgG avidity analysis at 30-45days after birth as a high performance tool for early serological diagnosis of congenital toxoplasmosis. Copyright © 2017. Published by Elsevier B.V.

Intra-Prostate Cancer Vaccine Inducer

DTIC Science & Technology

2006-07-01

4 that RNAi technology is possibly a more effective and reliable method to silence expression of a given gene. Therefore, we constructed an Ii...experiments, bone marrow cells were extracted from the femurs of BALB/c mice. Cells were plated at 4x106 cells in 100 mm dishes, in RPMI 1640-medium...permeabilized with saponin in preparation for staining with Ii monoclonal antibodies (data not shown). C1 E1 C2 E2 Figure 3 (C1) represents flow cytometric

Flow cytometric analysis of cell-surface and intracellular antigens in leukemia diagnosis.

PubMed

Knapp, W; Strobl, H; Majdic, O

1994-12-15

New technology allows highly sensitive flow cytometric detection and quantitative analysis of intracellular antigens in normal and malignant hemopoietic cells. With this technology, the earliest stages of myeloid and lymphoid differentiation can easily and reliably be identified using antibodies directed against (pro-)myeloperoxidase/MPO, CD22 and CD3 antigens, respectively. Particularly for the analysis of undifferentiated acute myeloblastic leukemia (AML) cells, the immunological demonstration of intracellular MPO or its enzymatically inactive proforms is highly relevant, since other myeloid marker molecules such as CD33, CD13, or CDw65 are either not restricted to the granulomonocytic lineage or appear later in differentiation. By combining MPO staining with staining for lactoferrin (LF), undifferentiated cells can be distinguished from the granulomonocytic maturation compartment in bone marrow, since LF is selectively expressed from the myelocyte stage of differentiation onward. The list of informative intracellular antigens to be used in leukemia cell analysis will certainly expand in the near future. One candidate, intracellular CD68, has already been tested by us, and results are presented. Also dealt within this article are surface marker molecules not (as yet) widely used in leukemia cell analysis but with the potential to provide important additional information. Among them are the surface structures CD15, CD15s, CDw65, CD79a (MB-1), CD79b (B29), CD87 (uPA-R), and CD117 (c-kit).

Analyses of cell surface molecules on hepatic stem/progenitor cells in mouse fetal liver.

PubMed

Kakinuma, Sei; Ohta, Haruhiko; Kamiya, Akihide; Yamazaki, Yuji; Oikawa, Tsunekazu; Okada, Ken; Nakauchi, Hiromitsu

2009-07-01

Hepatic stem/progenitor cells possess active proliferative ability and the capacity for differentiation into hepatic and cholangiocytic lineages. Our group and others have shown that a prospectively defined population in mid-gestational fetal liver contains hepatic stem/progenitor cells. However, the phenotypes of such cells are incompletely elucidated. We analyzed the profile of cell-surface molecules on primary hepatic stem/progenitor cells. Expression of cell surface molecules on primary hepatic stem/progenitor cells in mouse mid-gestational fetal liver was analyzed using flow cytometric multicolor analyses and colony-formation assays. The potential of the cells for liver repopulation was examined by transplantation assay. We found that CD13 (aminopeptidase N) was detected on the cells of the previously reported (Dlk/Pref-1(+)) hepatic stem/progenitor fraction. Colony-formation assays revealed that the CD13(+) fraction, compared with the Dlk(+) fraction, of non-hematopoietic cells in fetal liver was enriched in hepatic stem/progenitor cells. Transplantation assay showed the former fraction exhibited repopulating potential in regenerating liver. Moreover, flow cytometric analysis for over 90 antigens demonstrated enrichment of hepatic stem/progenitor cells using several positive selection markers, including (hitherto unknown) CD13, CD73, CD106, and CD133. Our data indicated that CD13 is a positive selection marker for hepatic stem/progenitor cells in mid-gestational fetal liver.

Performance evaluation of the new hematology analyzer Sysmex XN-series.

PubMed

Seo, J Y; Lee, S-T; Kim, S-H

2015-04-01

The Sysmex XN-series is a new automated hematology analyzer designed to improve the accuracy of cell counts and the specificity of the flagging events. The basic characteristics and the performance of new measurement channels of the XN were evaluated and compared with the Sysmex XE-2100 and the manual method. Fluorescent platelet count (PLT-F) was compared with the flow cytometric method. The low WBC mode and body fluid mode were also evaluated. For workflow analysis, 1005 samples were analyzed on both the XN and the XE-2100, and manual review rates were compared. All parameters measured by the XN correlated well with the XE-2100. PLT-F showed better correlation with the flow cytometric method (r(2)  = 0.80) compared with optical platelet count (r(2)  = 0.73) for platelet counts <70 × 10(9) /L. The low WBC mode reported accurate leukocyte differentials for samples with a WBC count <0.5 × 10(9) /L. Relatively good correlation was found for WBC counts between the manual method and the body fluid mode (r = 0.88). The XN made less flags than the XE-2100, while the sensitivities of both instruments were comparable. The XN provided reliable results on low cell counts, as well as reduced manual blood film reviews, while maintaining a proper level of diagnostic sensitivity. © 2014 John Wiley & Sons Ltd.

Arctigenin induces apoptosis in colon cancer cells through ROS/p38MAPK pathway.

PubMed

Li, Qing-chun; Liang, Yun; Tian, Yuan; Hu, Guang-rui

2016-01-01

In the current study the antiproliferative effect of arctigenin, plant lignin, was evaluated on human colon cancer cell line HT-29. Furthermore, attempts were made to explore the signaling mechanism which may be responsible for its effect. Cell growth inhibition was assessed by MTT and LDH assays. Flow cytometric analysis was performed to determine cell arrest in the cell cycle phase and apoptosis. Furthermore, to confirm the apoptotic activity of arctigenin, caspase-9 and -3 activities analysis was performed. The levels of reactive oxygen species (ROS) and p38 mitogen activated protein kinase (MAPK) were investigated to determine their role in inducing apoptosis in arctigenin-treated HT-29 colon cancer cell line. MTT and LDH results demonstrated significant cell growth inhibitory effect of arctigenin on HT-29 cells in a dose-dependent manner. Furthermore, increase in cell number arrested at G2/M phase was observed in flow cytometric analysis upon arctigenin treatment. In addition, arctigenin increased the apoptotic ratio in a dose-dependent manner. The involvement of intrinsic apoptotic pathway was indicated by the activation of caspase-9 and -3. Moreover, increased ROS production, activation of p38 MAPK and changes in mitochondrial membrane potential (ΔΨm) also revealed the role of intrinsic apoptotic signaling pathway in cell growth inhibition after arctigenin exposure. Arctigenin induces apoptosis in HT-29 colon cancer cells by regulating ROS and p38 MAPK pathways.

A comparative study of U937 cell size changes during apoptosis initiation by flow cytometry, light scattering, water assay and electronic sizing.

PubMed

Yurinskaya, Valentina; Aksenov, Nikolay; Moshkov, Alexey; Model, Michael; Goryachaya, Tatyana; Vereninov, Alexey

2017-10-01

A decrease in flow cytometric forward light scatter (FSC) is commonly interpreted as a sign of apoptotic cell volume decrease (AVD). However, the intensity of light scattering depends not only on the cell size but also on its other characteristics, such as hydration, which may affect the scattering in the opposite way. That makes estimation of AVD by FSC problematic. Here, we aimed to clarify the relationship between light scattering, cell hydration (assayed by buoyant density) and cell size by the Coulter technique. We used human lymphoid cells U937 exposed to staurosporine, etoposide or hypertonic stress as an apoptotic model. An initial increase in FSC was found to occur in apoptotic cells treated with staurosporine and hypertonic solutions; it is accompanied by cell dehydration and is absent in apoptosis caused by etoposide that is consistent with the lack of dehydration in this case. Thus, the effect of dehydration on the scattering signal outweighs the effect of reduction in cell size. The subsequent FSC decrease, which occurred in parallel to accumulation of annexin-positive cells, was similar in apoptosis caused by all three types of inducers. We conclude that an increase, but not a decrease in light scattering, indicates the initial cell volume decrease associated with apoptotic cell dehydration.

Adaptation of the TdT assay for semi-quantitative flow cytometric detection of DNA strand breaks

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bromidge, T.J.; Howe, D.J.; Johnson, S.A.

The enzyme Terminal Deoxynucleotidyl Transferase (TdT) is a DNA polymerase which can be used to label DNA strand breaks by the incorporation of a labelled nucleotide followed by a fluorescent detection step. The amount of label incorporated can then be assessed by flow cytometry. The mechanism of action of TdT, however, will allow the addition of varying numbers of nucleotides to the free 3{prime} termini produced by DNA strand breaks. The substitution of Digoxigenin (DIG){trademark} labelled dideoxynucleotides for labelled deoxy-nucleotides in the TdT assay will limit the addition of label to a DNA break to a single nucleotide, thus ensuringmore » a direct relationship between an increase in DNA strand breaks and an increase in fluorescence. We have used this adaptation of the TdT assay to evaluate DNA damage incurred in lymphocytes, from patients with Chronic Lymphocytic Leukemia (CLL), on exposure to UV irradiation and apoptosis-inducing drugs, fludarabine and 2-Chloro-2{prime}-deoxyadenosine (2-CdA). This technique may give a good indication of the susceptibility of CLL patients to apoptosis inducing drugs, and hence an indication of the likely response to these therapies. 7 refs., 2 figs., 2 tabs.« less

High-resolution FISH on super-stretched flow-sorted plant chromosomes.

PubMed

Valárik, M; Bartos, J; Kovárová, P; Kubaláková, M; de Jong, J H; Dolezel, J

2004-03-01

A novel high-resolution fluorescence in situ hybridisation (FISH) strategy, using super-stretched flow-sorted plant chromosomes as targets, is described. The technique that allows longitudinal extension of chromosomes of more than 100 times their original metaphase size is especially attractive for plant species with large chromosomes, whose pachytene chromosomes are generally too long and heterochromatin patterns too complex for FISH analysis. The protocol involves flow cytometric sorting of metaphase chromosomes, mild proteinase-K digestion of air-dried chromosomes on microscopic slides, followed by stretching with ethanol:acetic acid (3 : 1). Stretching ratios were assessed in a number of FISH experiments with super-stretched chromosomes from barley, wheat, rye and chickpea, hybridised with 45S and 5S ribosomal DNAs and the [GAA]n microsatellite, the [TTTAGGG]n telomeric repeat and a bacterial artificial chromosome (BAC) clone as probes. FISH signals on stretched chromosomes were brighter than those on the untreated control, resulting from better accessibility of the stretched chromatin and maximum observed sensitivity of 1 kbp. Spatial resolution of neighbouring loci was improved down to 70 kbp as compared to 5-10 Mbp after FISH on mitotic chromosomes, revealing details of adjacent DNA sequences hitherto not obtained with any other method. Stretched chromosomes are advantageous over extended DNA fibres from interphase nuclei as targets for FISH studies because they still retain chromosomal integrity. Although the method is confined to species for which chromosome flow sorting has been developed, it provides a unique system for controlling stretching degree of mitotic chromosomes and high-resolution bar-code FISH.

Deciphering the mechanisms of cellular uptake of engineered nanoparticles by accurate evaluation of internalization using imaging flow cytometry

PubMed Central

2013-01-01

Background The uptake of nanoparticles (NPs) by cells remains to be better characterized in order to understand the mechanisms of potential NP toxicity as well as for a reliable risk assessment. Real NP uptake is still difficult to evaluate because of the adsorption of NPs on the cellular surface. Results Here we used two approaches to distinguish adsorbed fluorescently labeled NPs from the internalized ones. The extracellular fluorescence was either quenched by Trypan Blue or the uptake was analyzed using imaging flow cytometry. We used this novel technique to define the inside of the cell to accurately study the uptake of fluorescently labeled (SiO2) and even non fluorescent but light diffracting NPs (TiO2). Time course, dose-dependence as well as the influence of surface charges on the uptake were shown in the pulmonary epithelial cell line NCI-H292. By setting up an integrative approach combining these flow cytometric analyses with confocal microscopy we deciphered the endocytic pathway involved in SiO2 NP uptake. Functional studies using energy depletion, pharmacological inhibitors, siRNA-clathrin heavy chain induced gene silencing and colocalization of NPs with proteins specific for different endocytic vesicles allowed us to determine macropinocytosis as the internalization pathway for SiO2 NPs in NCI-H292 cells. Conclusion The integrative approach we propose here using the innovative imaging flow cytometry combined with confocal microscopy could be used to identify the physico-chemical characteristics of NPs involved in their uptake in view to redesign safe NPs. PMID:23388071

Flow cytometry for intracellular SPION quantification: specificity and sensitivity in comparison with spectroscopic methods

PubMed Central

Friedrich, Ralf P; Janko, Christina; Poettler, Marina; Tripal, Philipp; Zaloga, Jan; Cicha, Iwona; Dürr, Stephan; Nowak, Johannes; Odenbach, Stefan; Slabu, Ioana; Liebl, Maik; Trahms, Lutz; Stapf, Marcus; Hilger, Ingrid; Lyer, Stefan; Alexiou, Christoph

2015-01-01

Due to their special physicochemical properties, iron nanoparticles offer new promising possibilities for biomedical applications. For bench to bedside translation of super-paramagnetic iron oxide nanoparticles (SPIONs), safety issues have to be comprehensively clarified. To understand concentration-dependent nanoparticle-mediated toxicity, the exact quantification of intracellular SPIONs by reliable methods is of great importance. In the present study, we compared three different SPION quantification methods (ultraviolet spectrophotometry, magnetic particle spectroscopy, atomic adsorption spectroscopy) and discussed the shortcomings and advantages of each method. Moreover, we used those results to evaluate the possibility to use flow cytometric technique to determine the cellular SPION content. For this purpose, we correlated the side scatter data received from flow cytometry with the actual cellular SPION amount. We showed that flow cytometry provides a rapid and reliable method to assess the cellular SPION content. Our data also demonstrate that internalization of iron oxide nanoparticles in human umbilical vein endothelial cells is strongly dependent to the SPION type and results in a dose-dependent increase of toxicity. Thus, treatment with lauric acid-coated SPIONs (SEONLA) resulted in a significant increase in the intensity of side scatter and toxicity, whereas SEONLA with an additional protein corona formed by bovine serum albumin (SEONLA-BSA) and commercially available Rienso® particles showed only a minimal increase in both side scatter intensity and cellular toxicity. The increase in side scatter was in accordance with the measurements for SPION content by the atomic adsorption spectroscopy reference method. In summary, our data show that flow cytometry analysis can be used for estimation of uptake of SPIONs by mammalian cells and provides a fast tool for scientists to evaluate the safety of nanoparticle products. PMID:26170658

Automated flow cytometric analysis across large numbers of samples and cell types.

PubMed

Chen, Xiaoyi; Hasan, Milena; Libri, Valentina; Urrutia, Alejandra; Beitz, Benoît; Rouilly, Vincent; Duffy, Darragh; Patin, Étienne; Chalmond, Bernard; Rogge, Lars; Quintana-Murci, Lluis; Albert, Matthew L; Schwikowski, Benno

2015-04-01

Multi-parametric flow cytometry is a key technology for characterization of immune cell phenotypes. However, robust high-dimensional post-analytic strategies for automated data analysis in large numbers of donors are still lacking. Here, we report a computational pipeline, called FlowGM, which minimizes operator input, is insensitive to compensation settings, and can be adapted to different analytic panels. A Gaussian Mixture Model (GMM)-based approach was utilized for initial clustering, with the number of clusters determined using Bayesian Information Criterion. Meta-clustering in a reference donor permitted automated identification of 24 cell types across four panels. Cluster labels were integrated into FCS files, thus permitting comparisons to manual gating. Cell numbers and coefficient of variation (CV) were similar between FlowGM and conventional gating for lymphocyte populations, but notably FlowGM provided improved discrimination of "hard-to-gate" monocyte and dendritic cell (DC) subsets. FlowGM thus provides rapid high-dimensional analysis of cell phenotypes and is amenable to cohort studies. Copyright © 2015. Published by Elsevier Inc.

High throughput, parallel imaging and biomarker quantification of human spermatozoa by ImageStream flow cytometry.

PubMed

Buckman, Clayton; George, Thaddeus C; Friend, Sherree; Sutovsky, Miriam; Miranda-Vizuete, Antonio; Ozanon, Christophe; Morrissey, Phil; Sutovsky, Peter

2009-12-01

Spermatid specific thioredoxin-3 protein (SPTRX-3) accumulates in the superfluous cytoplasm of defective human spermatozoa. Novel ImageStream technology combining flow cytometry with cell imaging was used for parallel quantification and visualization of SPTRX-3 protein in defective spermatozoa of five men from infertile couples. The majority of the SPTRX-3 containing cells were overwhelmingly spermatozoa with a variety of morphological defects, detectable in the ImageStream recorded images. Quantitative parameters of relative SPTRX-3 induced fluorescence measured by ImageStream correlated closely with conventional flow cytometric measurements of the same sample set and reflected the results of clinical semen evaluation. Image Stream quantification of SPTRX-3 combines and surpasses the informative value of both conventional flow cytometry and light microscopic semen evaluation. The observed patterns of the retention of SPTRX-3 in the sperm samples from infertility patients support the view that SPTRX3 is a biomarker of male infertility.

Flow cytometric measurement of total DNA and incorporated halodeoxyuridine

DOEpatents

Dolbeare, F.A.; Gray, J.W.

1983-10-18

A method for the simultaneous flow cylometric measurement of total cellular DNA content and of the uptake of DNA precursors as a measure of DNA synthesis during various phases of the cell cycle in normal and malignant cells in vitro and in vivo is described. The method comprises reacting cells with labelled halodeoxyuridine (HdU), partially denaturing cellular DNA, adding to the reaction medium monoclonal antibodies (mabs) reactive with HdU, reacting the bound mabs with a second labelled antibody, incubating the mixture with a DNA stain, and measuring simultaneously the intensity of the DNA stain as a measure of the total cellular DNA and the HdU incorporated as a measure of DNA synthesis. (ACR)

Flow cytometric detection method for DNA samples

DOEpatents

Nasarabadi, Shanavaz [Livermore, CA; Langlois, Richard G [Livermore, CA; Venkateswaran, Kodumudi S [Round Rock, TX

2011-07-05

Disclosed herein are two methods for rapid multiplex analysis to determine the presence and identity of target DNA sequences within a DNA sample. Both methods use reporting DNA sequences, e.g., modified conventional Taqman.RTM. probes, to combine multiplex PCR amplification with microsphere-based hybridization using flow cytometry means of detection. Real-time PCR detection can also be incorporated. The first method uses a cyanine dye, such as, Cy3.TM., as the reporter linked to the 5' end of a reporting DNA sequence. The second method positions a reporter dye, e.g., FAM.TM. on the 3' end of the reporting DNA sequence and a quencher dye, e.g., TAMRA.TM., on the 5' end.

Flow cytomeric measurement of DNA and incorporated nucleoside analogs

DOEpatents

Dolbeare, Frank A.; Gray, Joe W.

1989-01-01

A method is provided for simultaneously measuring total cellular DNA and incorporated nucleoside analog. The method entails altering the cellular DNA of cells grown in the presence of a nucleoside analog so that single stranded and double stranded portions are present. Separate stains are used against the two portions. An immunochemical stain is used against the single stranded portion to provide a measure of incorporated nucleoside analog, and a double strand DNA-specific stain is used against the double stranded portion to simultaneously provide a measure of total cellular DNA. The method permits rapid flow cytometric analysis of cell populations, rapid identification of cycling and noncycling subpopulations, and determination of the efficacy of S phase cytotoxic anticancer agents.

Flow cytometric detection method for DNA samples

DOEpatents

Nasarabadi, Shanavaz [Livermore, CA; Langlois, Richard G [Livermore, CA; Venkateswaran, Kodumudi S [Livermore, CA

2006-08-01

Disclosed herein are two methods for rapid multiplex analysis to determine the presence and identity of target DNA sequences within a DNA sample. Both methods use reporting DNA sequences, e.g., modified conventional Taqman.RTM. probes, to combine multiplex PCR amplification with microsphere-based hybridization using flow cytometry means of detection. Real-time PCR detection can also be incorporated. The first method uses a cyanine dye, such as, Cy3.TM., as the reporter linked to the 5' end of a reporting DNA sequence. The second method positions a reporter dye, e.g., FAM, on the 3' end of the reporting DNA sequence and a quencher dye, e.g., TAMRA, on the 5' end.

Whole blood flow cytometric analysis of Ureaplasma-stimulated monocytes from pregnant women.

PubMed

Friedland, Yael D; Lee-Pullen, Tracey F; Nathan, Elizabeth; Watts, Rory; Keelan, Jeffrey A; Payne, Matthew S; Ireland, Demelza J

2015-06-01

We hypothesised that circulating monocytes of women with vaginal colonisation with Ureaplasma spp., genital microorganisms known to cause inflammation-driven preterm birth, would elicit a tolerised cytokine response to subsequent in vitro Ureaplasma parvum serovar 3 (UpSV3) stimulation. Using multi-parameter flow cytometry, we found no differences with regard to maternal colonisation status in the frequency of TNF-α-, IL-6-, IL-8- and IL-1β-expressing monocytes in response to subsequent UpSV3 stimulation (P > 0.10 for all cytokines). We conclude that vaginal Ureaplasma spp. colonisation does not specifically tolerise monocytes of pregnant women towards decreased responses to subsequent stimulation. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Intracellular staining for analysis of the expression and phosphorylation of signal transducers and activators of transcription (STATs) in NK cells.

PubMed

Miyagi, Takuya; Lee, Seung-Hwan; Biron, Christine A

2010-01-01

Cytokines stimulate biological responses by activating intracellular signaling pathways. We have been adapting flow cytometric techniques to measure the levels of expression and activation of signaling molecules within mixed populations containing NK cells and to characterize their differences within NK cell subpopulations. Approaches for evaluating the total levels of the signal transducers and activators of transcription STAT1 and STAT4, of STAT1 in cells expressing IFNgamma, and of the type 1 interferon (type 1 IFN) activation by phosphorylation, i.e., induction of pSTAT1 and pSTAT4, have been developed. The results of experiments using these techniques have demonstrated that an unusual feature of NK cells is high basal expression of STAT4 but reduced STAT1 levels. The condition predisposes for pSTAT4 activation by type 1 IFNs. The work has also shown, however, that total STAT1 levels are induced during viral infections as a result of IFN exposure, and that this change acts to promote the activation of STAT1 but limit both the activation of STAT4 and IFNgamma expression. The intracellular staining approaches used for the studies described here have utility in characterizing other mechanisms regulating cytokine-mediated signaling, and defining additional pathways shaping cellular responses to cytokines.

Efficient monitoring of the blood-stage infection in a malaria rodent model by the rotating-crystal magneto-optical method

NASA Astrophysics Data System (ADS)

Orbán, Ágnes; Rebelo, Maria; Molnár, Petra; Albuquerque, Inês S.; Butykai, Adam; Kézsmárki, István

2016-03-01

Intense research efforts have been focused on the improvement of the efficiency and sensitivity of malaria diagnostics, especially in resource-limited settings for the detection of asymptomatic infections. Our recently developed magneto-optical (MO) method allows the accurate quantification of malaria pigment crystals (hemozoin) in blood by their magnetically induced rotation. First evaluations of the method using β-hematin crystals and in vitro P. falciparum cultures implied its potential for high-sensitivity malaria diagnosis. To further investigate this potential, here we study the performance of the method in monitoring the in vivo onset and progression of the blood-stage infection in a rodent malaria model. Our results show that the MO method can detect the first generation of intraerythrocytic P. berghei parasites 66-76 hours after sporozoite injection, demonstrating similar sensitivity to Giesma-stained light microscopy and exceeding that of flow cytometric techniques. Magneto-optical measurements performed during and after the treatment of P. berghei infections revealed that both the follow up under treatment and the detection of later reinfections are feasible with this new technique. The present study demonstrates that the MO method - besides being label and reagent-free, automated and rapid - has a high in vivo sensitivity and is ready for in-field evaluation.

Endoscopic ultrasound-guided fine-needle aspiration with liquid-based cytologic preparation in the diagnosis of primary pancreatic lymphoma.

PubMed

Rossi, Esther Diana; Larghi, Alberto; Verna, Elizabeth C; Martini, Maurizio; Galasso, Domenico; Carnuccio, Antonella; Larocca, Luigi Maria; Costamagna, Guido; Fadda, Guido

2010-11-01

The diagnosis subtyping of lymphoma on specimens collected by endoscopic ultrasound fine-needle aspiration (EUS-FNA) can be extremely difficult. When a cytopathologist is available for the on-site evaluation, the diagnosis may be achieved by applying flow cytometric techniques. We describe our experience with immunocytochemistry (ICC) and molecular biology studies applied on EUS-FNA specimens processed with a liquid-based cytologic (LBC) preparation for the diagnosis of primary pancreatic lymphoma (PPL). Three patients with a pancreatic mass underwent EUS-FNA. The collected specimens were processed with the ThinPrep method for the cytologic diagnosis and eventual additional investigations. A morphologic picture consistent with PPL was found on the LBC specimens of the 3 patients. Subsequent ICC and molecular biology studies for immunoglobulin heavy chain gene rearrangement established the diagnosis of pancreatic large B-cell non-Hodgkin lymphoma in 2 patients and a non-Hodgkin lymphoma with plasmoblastic/immunoblastic differentiation in the remaining one. An LBC preparation can be used to diagnose and subtype PPL by applying ICC and molecular biology techniques to specimens collected with EUS-FNA. This method can be an additional processing method for EUS-FNA specimens in centers where on-site cytopathologist expertise is not available.

Immunomagnetic cell separation, imaging, and analysis using Captivate ferrofluids

NASA Astrophysics Data System (ADS)

Jones, Laurie; Beechem, Joseph M.

2002-05-01

We have developed applications of CaptivateTM ferrofluids, paramagnetic particles (approximately 200 nm diameter), for isolating and analyzing cell populations in combination with fluorescence-based techniques. Using a microscope-mounted magnetic yoke and sample insertion chamber, fluorescent images of magnetically captured cells were obtained in culture media, buffer, or whole blood, while non-magnetically labeled cells sedimented to the bottom of the chamber. We combined this immunomagnetic cell separation and imaging technique with fluorescent staining, spectroscopy, and analysis to evaluate cell surface receptor-containing subpopulations, live/dead cell ratios, apoptotic/dead cell ratios, etc. The acquired images were analyzed using multi-color parameters, as produced by nucleic acid staining, esterase activity, or antibody labeling. In addition, the immunomagnetically separated cell fractions were assessed through microplate analysis using the CyQUANT Cell Proliferation Assay. These methods should provide an inexpensive alternative to some flow cytometric measurements. The binding capacities of the streptavidin- labled Captivate ferrofluid (SA-FF) particles were determined to be 8.8 nmol biotin/mg SA-FF, using biotin-4- fluorescein, and > 106 cells/mg SA-FF, using several cell types labeled with biotinylated probes. For goat anti- mouse IgG-labeled ferrofluids (GAM-FF), binding capacities were established to be approximately 0.2 - 7.5 nmol protein/mg GAM-FF using fluorescent conjugates of antibodies, protein G, and protein A.

Whole Blood Activation Results in Enhanced Detection of T Cell and Monocyte Cytokine Production by Flow Cytometry

NASA Technical Reports Server (NTRS)

Sams, Clarence F.; Crucian, Brian E.

2001-01-01

An excellent monitor of the immune balance of peripheral circulating cells is to determine their cytokine production patterns in response to stimuli. Using flow cytometry a positive identification of cytokine producing cells in a mixed culture may be achieved. Recently, the ability to assess cytokine production following a wholeblood activation culture has been described. We compared whole blood culture to standard PBMC culture and determined the individual cytokine secretion patterns for both T cells and monocytes via flow cytometry. For T cells cytokine assessment following PMA +ionomycin activation: (1) a significantly greater percentages of T cells producing IFNgamma and IL-2 were observed following whole-blood culture; (2) altered T cell cytokine production kinetics were observed by varying whole blood culture times. In addition, a four-color cytometric analysis was used to allow accurate phenotyping and quantitation of cytokine producing lymphocyte populations. Using this technique we found IFNgamma production to be significantly elevated in the CD3+/CD8+ T cell population as compared to the CD3+/CD8- population following five hours of whole blood activation. Conversely, IL-2 and IL-10 production were significantly elevated in the CD3+/CD8- T cell population as compared to the CD3+/CD8+ population. Monocyte cytokine production was assessed in both culture systems following LPS activation for 24 hours. A three-color flow cytometric was used to assess two cytokines in conjunction with CD 14. The cytokine pairs used for analysis were IL-1a/IL-12, and IL-10ITNFa. Nearly all monocytes were stimulated to produce IL-1a, IL-12 and TNFalpha equally well in both culture systems. Monocyte production of IL-10 was significantly elevated following whole blood culture as compared to PBMC culture. IL-12 producing monocytes appeared to be a distinct subpopulation of the IL-1a producing set, whereas IL-10 and TNFa producing monocytes were largely mutually exclusive. IL-10 and TNFa producing monocytes may represent functionally different monocyte subsets with distinct functions. Whole blood culture eliminates the need to purify cell populations prior to culture and may have significant utility for the routine monitoring of the cytokine balances of the peripheral blood T cell and monocyte populations. In addition, there are distinct advantages to performing whole-blood (WB) activation as compared to PBMC activation. These advantages would include retaining all various cell-cell interactions as well as any soluble factors present in serum that influence cell activation. It is likely that the altered cytokine production observed following whole blood culture more accurately represents the in-vivo immune balance.

Analytic errors in Sysmex-generated hematology results in blood from a dog with chronic lymphocytic leukemia.

PubMed

Novacco, Marilisa; Martini, Valeria; Grande, Carmen; Comazzi, Stefano

2015-09-01

A blood sample from a 14-year-old dog was submitted to the veterinary diagnostic laboratory of the University of Milan for marked leukocytosis with atypical cells. A diagnosis of chronic T-cell lymphocytic leukemia (CLL) was made based on blood smear evaluation and flow cytometric phenotyping. A CBC by Sysmex XT-2000iV revealed a moderate normocytic normochromic anemia. Red blood cells counted by optic flow cytometry (RBC-O) resulted in a higher value than using electrical impedance (RBC-I). The relative reticulocyte count based on RNA content and size was 35.3%, while the manual reticulocyte count was < 1%. The WBC count of 1,562,680 cells/μL was accompanied by a flag. Manual counts for RBC and WBC using the Bürker chamber confirmed the Sysmex impedance results. Finally the manual PCV was lower than HCT by Sysmex. While Sysmex XT can differentiate between RBC and WBC by impedance, even in the face of extreme lymphocytosis due to CLL, RBC-O can be affected by bias, resulting in falsely increased RBC and reticulocyte numbers. Overestimation of RBC-O may be due to incorrect Sysmex classification of leukemic cells or their fragments as reticulocytes. This phenomenon is known as pseudoreticulocytosis and can lead to misinterpretation of regenerative anemia. On the other side PCV can be affected by bias in CLL due to the trapping of RBC in the buffy coat, resulting in a pink hue in the separation area. As HGB concentration is not affected by flow cytometric or other cell-related artifacts it may represent the most reliable variable to assess the degree of anemia in cases of CLL. © 2015 American Society for Veterinary Clinical Pathology.

Laboratory-Scale Simulation and Real-Time Tracking of a Microbial Contamination Event and Subsequent Shock-Chlorination in Drinking Water

PubMed Central

Besmer, Michael D.; Sigrist, Jürg A.; Props, Ruben; Buysschaert, Benjamin; Mao, Guannan; Boon, Nico; Hammes, Frederik

2017-01-01

Rapid contamination of drinking water in distribution and storage systems can occur due to pressure drop, backflow, cross-connections, accidents, and bio-terrorism. Small volumes of a concentrated contaminant (e.g., wastewater) can contaminate large volumes of water in a very short time with potentially severe negative health impacts. The technical limitations of conventional, cultivation-based microbial detection methods neither allow for timely detection of such contaminations, nor for the real-time monitoring of subsequent emergency remediation measures (e.g., shock-chlorination). Here we applied a newly developed continuous, ultra high-frequency flow cytometry approach to track a rapid pollution event and subsequent disinfection of drinking water in an 80-min laboratory scale simulation. We quantified total (TCC) and intact (ICC) cell concentrations as well as flow cytometric fingerprints in parallel in real-time with two different staining methods. The ingress of wastewater was detectable almost immediately (i.e., after 0.6% volume change), significantly changing TCC, ICC, and the flow cytometric fingerprint. Shock chlorination was rapid and detected in real time, causing membrane damage in the vast majority of bacteria (i.e., drop of ICC from more than 380 cells μl-1 to less than 30 cells μl-1 within 4 min). Both of these effects as well as the final wash-in of fresh tap water followed calculated predictions well. Detailed and highly quantitative tracking of microbial dynamics at very short time scales and for different characteristics (e.g., concentration, membrane integrity) is feasible. This opens up multiple possibilities for targeted investigation of a myriad of bacterial short-term dynamics (e.g., disinfection, growth, detachment, operational changes) both in laboratory-scale research and full-scale system investigations in practice. PMID:29085343

Flow cytometric immunobead assay for quantitative detection of platelet autoantibodies in immune thrombocytopenia patients.

PubMed

Zhai, Juping; Ding, Mengyuan; Yang, Tianjie; Zuo, Bin; Weng, Zhen; Zhao, Yunxiao; He, Jun; Wu, Qingyu; Ruan, Changgeng; He, Yang

2017-10-23

Platelet autoantibody detection is critical for immune thrombocytopenia (ITP) diagnosis and prognosis. Therefore, we aimed to establish a quantitative flow cytometric immunobead assay (FCIA) for ITP platelet autoantibodies evaluation. Capture microbeads coupled with anti-GPIX, -GPIb, -GPIIb, -GPIIIa and P-selectin antibodies were used to bind the platelet-bound autoantibodies complex generated from plasma samples of 250 ITP patients, 163 non-ITP patients and 243 healthy controls, a fluorescein isothiocyanate (FITC)-conjugated secondary antibody was the detector reagent and mean fluorescence intensity (MFI) signals were recorded by flow cytometry. Intra- and inter-assay variations of the quantitative FCIA assay were assessed. Comparisons of the specificity, sensitivity and accuracy between quantitative and qualitative FCIA or monoclonal antibody immobilization of platelet antigen (MAIPA) assay were performed. Finally, treatment process was monitored by our quantitative FCIA in 8 newly diagnosed ITPs. The coefficient of variations (CV) of the quantitative FCIA assay were respectively 9.4, 3.8, 5.4, 5.1 and 5.8% for anti-GPIX, -GPIb, -GPIIIa, -GPIIb and -P-selectin autoantibodies. Elevated levels of autoantibodies against platelet glycoproteins GPIX, GPIb, GPIIIa, GPIIb and P-selectin were detected by our quantitative FCIA in ITP patients compared to non-ITP patients or healthy controls. The sensitivity, specificity and accuracy of our quantitative assay were respectively 73.13, 81.98 and 78.65% when combining all 5 autoantibodies, while the sensitivity, specificity and accuracy of MAIPA assay were respectively 41.46, 90.41 and 72.81%. A quantitative FCIA assay was established. Reduced levels of platelet autoantibodies could be confirmed by our quantitative FCIA in ITP patients after corticosteroid treatment. Our quantitative assay is not only good for ITP diagnosis but also for ITP treatment monitoring.

[Establishment of the retrovirus-mediated murine model with MLL-AF9 leukemia].

PubMed

Xu, Si-Miao; Yang, Yang; Zhou, Mi; Zhao, Xue-Jiao; Qin, Yu; Zhang, Pei-Ling; Yuan, Rui-Feng; Zhou, Jian-Feng; Fang, Yong

2013-10-01

This study was purposed to establish a retrovirus-mediated murine model with MLL-AF9 leukemia, so as to provide a basis for further investigation of the pathogenesis and therapeutic strategy of MLL associated leukemia. Murine (CD45.2) primary hematopoietic precursor positively selected for expression of the progenitor marker c-Kit by means of MACS were transduced with a retrovirus carrying MLL-AF9 fusion gene. After cultured in vitro, the transduced cells were injected intravenously through the tail vein into the lethally irradiated mice (CD45.1). PCR, flow cytometry and morphological observation were employed to evaluate the murine leukemia model system. The results showed that MLL-AF9 fusion gene was expressed in the infected cells, and the cells had a dramatically enhanced potential to generate myeloid colonies with primitive and immature morphology. Flow cytometric analysis revealed that the immortalized cells highly expressed myeloid lineage surface markers Gr-1 and Mac-1. Moreover, the expression levels of Hoxa9 and Meis1 mRNA were significantly higher in the MLL-AF9 cells than that in control. The mice transplanted with MLL-AF9 cells displayed typical signs of leukemia within 6-12 weeks. Extensive infiltration leukemic cells was observed in the Wright-Giemsa stained peripheral blood smear and bone marrow, and also in the histology of liver and spleen. Flow cytometric analysis of the bone marrow and spleen cells demonstrated that the CD45.2 populations expressed highly myeloid markers Gr-1 and Mac-1. The leukemic mice died within 12 weeks. It is concluded that the retrovirus-mediated murine model with MLL-AF9 leukemia is successfully established, which can be applied in the subsequent researches.

Interlaboratory Evaluation of a Multiplexed High Information Content In Vitro Genotoxicity Assay

PubMed Central

Bryce, Steven M.; Bernacki, Derek T.; Bemis, Jeffrey C.; Spellman, Richard A.; Engel, Maria E.; Schuler, Maik; Lorge, Elisabeth; Heikkinen, Pekka T.; Hemmann, Ulrike; Thybaud, Véronique; Wilde, Sabrina; Queisser, Nina; Sutter, Andreas; Zeller, Andreas; Guérard, Melanie; Kirkland, David; Dertinger, Stephen D.

2017-01-01

We previously described a multiplexed in vitro genotoxicity assay based on flow cytometric analysis of detergent-liberated nuclei that are simultaneously stained with propidium iodide and labeled with fluorescent antibodies against p53, γH2AX, and phospho-histone H3. Inclusion of a known number of microspheres provides absolute nuclei counts. The work described herein was undertaken to evaluate the interlaboratory transferability of this assay, commercially known as MultiFlow™ DNA Damage Kit— p53, γH2AX, Phospho-histone H3. For these experiments seven laboratories studied reference chemicals from a group of 84 representing clastogens, aneugens, and non-genotoxicants. TK6 cells were exposed to chemicals in 96-well plates over a range of concentrations for 24 hrs. At 4 and 24 hrs cell aliquots were added to the MultiFlow reagent mix and following a brief incubation period flow cytometric analysis occurred, in most cases directly from a 96-well plate via a robotic walk-away data acquisition system. Multiplexed response data were evaluated using two analysis approaches, one based on global evaluation factors (i.e., cutoff values derived from all inter-laboratory data), and a second based on multinomial logistic regression that considers multiple biomarkers simultaneously. Both data analysis strategies were devised to categorize chemicals as predominately exhibiting a clastogenic, aneugenic, or non-genotoxic mode of action (MoA). Based on the aggregate 231 experiments that were performed, assay sensitivity, specificity, and concordance in relation to a priori MoA grouping were ≥ 92%. These results are encouraging as they suggest that two distinct data analysis strategies can rapidly and reliably predict new chemicals’ predominant genotoxic MoA based on data from an efficient and transferable multiplexed in vitro assay. PMID:28370322

Flow cytometric kinetic assay of the activity of Na+/H+ antiporter in mammalian cells.

PubMed

Dolz, María; O'Connor, José-Enrique; Lequerica, Juan L

2004-10-01

The Na(+)/H(+) exchanger (NHE) of mammalian cells is an integral membrane protein that extrudes H(+) ion in exchange for extracellular Na(+) and plays a crucial role in the regulation of intracellular pH (pHi). Thus, when pHi is lowered, NHE extrudes protons at a rate depending of pHi that can be expressed as pH units/s. To abolish the activity of other cellular pH-restoring systems, cells were incubated in bicarbonate-free Dulbecco's modified Eagle's medium buffered with HEPES. Flow cytometry was used to determine pHi with 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester or 5-(and-6)-carboxy SNARF-1 acetoxymethyl ester acetate, and the appropriate fluorescence ratios were measured. The calibration of fluorescence ratios versus pHi was established by using ionophore nigericin. The activity of NHE was calculated by a kinetic flow cytometric assay as the slope at time 0 of the best-fit curve of pHi recovery versus time after intracellular acidification with a pulse of exogenous sodium propionate. The kinetic method allowed determination of the pHi-dependent activity of NHE in cell lines and primary cell cultures. NHE activity values were demonstrated to be up to 0.016 pH units/s within the pHi range of 7.3 to 6.3. The inhibition of NHE activity by the specific inhibitor ethyl isopropyl amiloride was easily detected by this method. The assay conditions can be used to relate variations in pHi with the activity of NHE and provide a standardized method to compare between different cells, inhibitors, models of ischemia by acidification, and other relevant experimental or clinical situations.

Type I IFN-related NETosis in ataxia telangiectasia and Artemis deficiency.

PubMed

Gul, Ersin; Sayar, Esra Hazar; Gungor, Bilgi; Eroglu, Fehime Kara; Surucu, Naz; Keles, Sevgi; Guner, Sukru Nail; Findik, Siddika; Alpdundar, Esin; Ayanoglu, Ihsan Cihan; Kayaoglu, Basak; Geckin, Busra Nur; Sanli, Hatice Asena; Kahraman, Tamer; Yakicier, Cengiz; Muftuoglu, Meltem; Oguz, Berna; Cagdas Ayvaz, Deniz Nazire; Gursel, Ihsan; Ozen, Seza; Reisli, Ismail; Gursel, Mayda

2017-11-16

Pathological inflammatory syndromes of unknown etiology are commonly observed in ataxia telangiectasia (AT) and Artemis deficiency. Similar inflammatory manifestations also exist in patients with STING-associated vasculopathy in infancy (SAVI). We sought to test the hypothesis that the inflammation-associated manifestations observed in patients with AT and Artemis deficiency stem from increased type I IFN signature leading to neutrophil-mediated pathological damage. Cytokine/protein signatures were determined by ELISA, cytometric bead array, or quantitative PCR. Stat1 phosphorylation levels were determined by flow cytometry. DNA species accumulating in the cytosol of patients' cells were quantified microscopically and flow cytometrically. Propensity of isolated polymorhonuclear granulocytes to form neutrophil extracellular traps (NETs) was determined using fluorescence microscopy and picogreen assay. Neutrophil reactive oxygen species levels and mitochondrial stress were assayed using fluorogenic probes, microscopy, and flow cytometry. Type I and III IFN signatures were elevated in plasma and peripheral blood cells of patients with AT, Artemis deficiency, and SAVI. Chronic IFN production stemmed from the accumulation of DNA in the cytoplasm of AT and Artemis-deficient cells. Neutrophils isolated from patients spontaneously produced NETs and displayed indicators of oxidative and mitochondrial stress, supportive of their NETotic tendencies. A similar phenomenon was also observed in neutrophils from healthy controls exposed to patient plasma samples or exogeneous IFN-α. Type I IFN-mediated neutrophil activation and NET formation may contribute to inflammatory manifestations observed in patients with AT, Artemis deficiency, and SAVI. Thus, neutrophils represent a promising target to manage inflammatory syndromes in diseases with active type I IFN signature. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Flow Cytometric Detection of PrPSc in Neurons and Glial Cells from Prion-Infected Mouse Brains.

PubMed

Yamasaki, Takeshi; Suzuki, Akio; Hasebe, Rie; Horiuchi, Motohiro

2018-01-01

In prion diseases, an abnormal isoform of prion protein (PrP Sc ) accumulates in neurons, astrocytes, and microglia in the brains of animals affected by prions. Detailed analyses of PrP Sc -positive neurons and glial cells are required to clarify their pathophysiological roles in the disease. Here, we report a novel method for the detection of PrP Sc in neurons and glial cells from the brains of prion-infected mice by flow cytometry using PrP Sc -specific staining with monoclonal antibody (MAb) 132. The combination of PrP Sc staining and immunolabeling of neural cell markers clearly distinguished neurons, astrocytes, and microglia that were positive for PrP Sc from those that were PrP Sc negative. The flow cytometric analysis of PrP Sc revealed the appearance of PrP Sc -positive neurons, astrocytes, and microglia at 60 days after intracerebral prion inoculation, suggesting the presence of PrP Sc in the glial cells, as well as in neurons, from an early stage of infection. Moreover, the kinetic analysis of PrP Sc revealed a continuous increase in the proportion of PrP Sc -positive cells for all cell types with disease progression. Finally, we applied this method to isolate neurons, astrocytes, and microglia positive for PrP Sc from a prion-infected mouse brain by florescence-activated cell sorting. The method described here enables comprehensive analyses specific to PrP Sc -positive neurons, astrocytes, and microglia that will contribute to the understanding of the pathophysiological roles of neurons and glial cells in PrP Sc -associated pathogenesis. IMPORTANCE Although formation of PrP Sc in neurons is associated closely with neurodegeneration in prion diseases, the mechanism of neurodegeneration is not understood completely. On the other hand, recent studies proposed the important roles of glial cells in PrP Sc -associated pathogenesis, such as the intracerebral spread of PrP Sc and clearance of PrP Sc from the brain. Despite the great need for detailed analyses of PrP Sc -positive neurons and glial cells, methods available for cell type-specific analysis of PrP Sc have been limited thus far to microscopic observations. Here, we have established a novel high-throughput method for flow cytometric detection of PrP Sc in cells with more accurate quantitative performance. By applying this method, we succeeded in isolating PrP Sc -positive cells from the prion-infected mouse brains via fluorescence-activated cell sorting. This allows us to perform further detailed analysis specific to PrP Sc -positive neurons and glial cells for the clarification of pathological changes in neurons and pathophysiological roles of glial cells. Copyright © 2017 American Society for Microbiology.

Prognostic comparative study of S-phase fraction and Ki-67 index in breast carcinoma

PubMed Central

Pinto, A; Andre, S; Pereira, T; Nobrega, S; Soares, J

2001-01-01

Aims—To investigate the prognostic value of recently proposed flow cytometric S-phase fraction (SPF) variables (average SPF and SPF tertiles) compared with conventional SPF, and to compare the one with the best predictive value with the immunohistochemical Ki-67 index in breast carcinoma. Methods—A short term follow up study (median, 39.6 months) of a large series of patients (n = 306) was conducted. DNA ploidy was analysed on fresh/frozen tumour samples by flow cytometry, and the SPF was calculated from the DNA histogram using an algorithm. The Ki-67 index was assessed on paraffin wax embedded material by immunohistochemistry (cut off point, 10%). The two methods were compared by means of κ statistics, and the prognostic significance of both in relation to disease free survival (DFS) and overall survival (OS) was determined. Results—SPF and Ki-67 analysis was performed on 234 (76.5%) and 295 (96.4%) tumours, respectively. The two assessments were simultaneously available in 230 cases. All SPF variables analysed in the whole series significantly correlated with disease evolution, with the conventional median SPF (cut off point, 6.1%) showing the highest predictive value in relation to both DFS (p = 0.0001) and OS (p = 0.0003). SPF tertiles and median SPF evaluated according to DNA ploidy status had no prognostic significance. The Ki-67 index showed a trend in relation to DFS (p = 0.086) that did not reach significance, and no correlation with OS was found (p = 0.264). The comparative analysis of SPF and Ki-67 revealed some agreement between the two methods (agreement, 69.13%; κ statistic, 0.3844; p < 0.001), especially in the subgroup of diploid tumours. Conclusions—Flow cytometric SPF is a better prognosticator than the Ki-67 index, but only SPF variables applied in the whole series show potential clinical usefulness. Key Words: breast carcinoma • DNA flow cytometry • immunohistochemistry • S-phase fraction • Ki-67 • prognosis PMID:11429427

An image cytometric technique is a concise method to detect adenoviruses and host cell proteins and to monitor the infection and cellular responses induced.

PubMed

Morinaga, Takao; Nguyễn, Thảo Thi Thanh; Zhong, Boya; Hanazono, Michiko; Shingyoji, Masato; Sekine, Ikuo; Tada, Yuji; Tatsumi, Koichiro; Shimada, Hideaki; Hiroshima, Kenzo; Tagawa, Masatoshi

2017-11-10

Genetically modified adenoviruses (Ad) with preferential replications in tumor cells have been examined for a possible clinical applicability as an anti-cancer agent. A simple method to detect viral and cellular proteins is valuable to monitor the viral infections and to predict the Ad-mediated cytotoxicity. We used type 5 Ad in which the expression of E1A gene was activated by 5'-regulatory sequences of genes that were augmented in the expression in human tumors. The Ad were further modified to have the fiber-knob region replaced with that derived from type 35 Ad. We infected human mesothelioma cells with the fiber-replaced Ad, and sequentially examined cytotoxic processes together with an expression level of the viral E1A, hexon, and cellular cleaved caspase-3 with image cytometric and Western blot analyses. The replication-competent Ad produced cytotoxicity on mesothelioma cells. The infected cells expressed E1A and hexon 24 h after the infection and then showed cleavage of caspase-3, all of which were detected with image cytometry and Western blot analysis. Image cytometry furthermore demonstrated that increased Ad doses did not enhance an expression level of E1A and hexon in an individual cell and that caspase-3-cleaved cells were found more frequently in hexon-positive cells than in E1A-positive cells. Image cytometry thus detected these molecular changes in a sensitive manner and at a single cell level. We also showed that an image cytometric technique detected expression changes of other host cell proteins, cyclin-E and phosphorylated histone H3 at a single cell level. Image cytometry is a concise procedure to detect expression changes of Ad and host cell proteins at a single cell level, and is useful to analyze molecular events after the infection.

Long-term preservation of Tetraselmis indica (Chlorodendrophyceae, Chlorophyta) for flow cytometric analysis: Influence of fixative and storage temperature.

PubMed

Naik, Sangeeta Mahableshwar; Anil, Arga Chandrashekar

2017-08-01

Immediate enumeration of phytoplankton is seldom possible. Therefore, fixation and subsequent storage are required for delayed analysis. This study investigated the influence of glutaraldehyde (GA) concentrations (0.25%, 0.5%, and 1%) and storage temperatures (-80°C LN2 , -80°C, -20°C, and 5°C) on Tetraselmis indica for flow cytometric analysis. Cell recovery, granularity, and membrane permeability were independent of GA concentration whereas cell size and chlorophyll autofluorescence were concentration dependent. After an initial cell loss (16-19%), no cell loss was observed when samples were stored at 5°C. Cell recovery was not influenced by storage temperature until 4months but later samples preserved at -80°C LN2 , -80°C, and -20°C resulted in ~41% cell loss. Although maximum cell recovery with minimal effect on cell integrity was obtained at 5°C, autofluorescence was retained better at -80°C LN2 and -80°C. This suggests that in addition to fixative, the choice of storage temperature is equally important. Thus for long-term preservation, especially to retain autofluorescence, the use of lower concentration (0.25%) of GA when stored at a lower temperature (-80°C LN2 and -80°C) while a higher concentration (1%) of GA when stored at a higher temperature (5°C) is recommended. Copyright © 2017 Elsevier B.V. All rights reserved.

Sulforaphane Induces Cell Death Through G2/M Phase Arrest and Triggers Apoptosis in HCT 116 Human Colon Cancer Cells.

PubMed

Liu, Kuo-Ching; Shih, Ting-Ying; Kuo, Chao-Lin; Ma, Yi-Shih; Yang, Jiun-Long; Wu, Ping-Ping; Huang, Yi-Ping; Lai, Kuang-Chi; Chung, Jing-Gung

2016-01-01

Sulforaphane (SFN), an isothiocyanate, exists exclusively in cruciferous vegetables, and has been shown to possess potent antitumor and chemopreventive activity. However, there is no available information that shows SFN affecting human colon cancer HCT 116 cells. In the present study, we found that SFN induced cell morphological changes, which were photographed by contrast-phase microscopy, and decreased viability. SFN also induced G2/M phase arrest and cell apoptosis in HCT 116 cells, which were measured with flow cytometric assays. Western blotting indicated that SFN increased Cyclin A, cdk 2, Cyclin B and WEE1, but decreased Cdc 25C, cdk1 protein expressions that led to G2/M phase arrest. Apoptotic cell death was also confirmed by Annexin V/PI and DAPI staining and DNA gel electrophoresis in HCT 116 cells after exposure to SFN. The flow cytometric assay also showed that SFN induced the generation of reactive oxygen species (ROS) and Ca[Formula: see text] and decreased mitochondria membrane potential and increased caspase-8, -9 and -3 activities in HCT 116 cell. Western blotting also showed that SFN induced the release of cytochrome c, and AIF, which was confirmed by confocal microscopy examination. SFN induced ER stress-associated protein expression. Based on those observations, we suggest that SFN may be used as a novel anticancer agent for the treatment of human colon cancer in the future.

Modelling cell population growth with applications to cancer therapy in human tumour cell lines.

PubMed

Basse, Britta; Baguley, Bruce C; Marshall, Elaine S; Wake, Graeme C; Wall, David J N

2004-01-01

In this paper we present an overview of the work undertaken to model a population of cells and the effects of cancer therapy. We began with a theoretical one compartment size structured cell population model and investigated its asymptotic steady size distributions (SSDs) (On a cell growth model for plankton, MMB JIMA 21 (2004) 49). However these size distributions are not similar to the DNA (size) distributions obtained experimentally via the flow cytometric analysis of human tumour cell lines (data obtained from the Auckland Cancer Society Research Centre, New Zealand). In our one compartment model, size was a generic term, but in order to obtain realistic steady size distributions we chose size to be DNA content and devised a multi-compartment mathematical model for the cell division cycle where each compartment corresponds to a distinct phase of the cell cycle (J. Math. Biol. 47 (2003) 295). We then incorporated another compartment describing the possible induction of apoptosis (cell death) from mitosis phase (Modelling cell death in human tumour cell lines exposed to anticancer drug paclitaxel, J. Math. Biol. 2004, in press). This enabled us to compare our model to flow cytometric data of a melanoma cell line where the anticancer drug, paclitaxel, had been added. The model gives a dynamic picture of the effects of paclitaxel on the cell cycle. We hope to use the model to describe the effects of other cancer therapies on a number of different cell lines. Copyright 2004 Elsevier Ltd.

Flow cytometric analysis of lymphocyte proliferative responses to food allergens in dogs with food allergy.

PubMed

Fujimura, Masato; Masuda, Kenichi; Hayashiya, Makio; Okayama, Taro

2011-10-01

Two different allergy tests, antigen-specific immunoglobulin E quantification (IgE test) and flow cytometric analysis of antigen-specific proliferation of peripheral lymphocytes (lymphocyte proliferation test), were performed to examine differences in allergic reactions to food allergens in dogs with food allergy (FA). Thirteen dogs were diagnosed as FA based on clinical findings and elimination diet trials. Seven dogs clinically diagnosed with canine atopic dermatitis (CAD) were used as a disease control group, and 5 healthy dogs were used as a negative control group. In the FA group, 19 and 33 allergen reactions were identified using the serum IgE test and the lymphocyte proliferation test, respectively. Likewise, in the CAD group, 12 and 6 allergen reactions and in the healthy dogs 3 and 0 allergen reactions were identified by each test, respectively. A significant difference was found between FA and healthy dogs in terms of positive allergen detection by the lymphocyte proliferation test, suggesting that the test can be useful to differentiate FA from healthy dogs but not from CAD. Both tests were repeated in 6 of the dogs with FA after a 1.5- to 5-month elimination diet trial. The IgE concentrations in 9 of 11 of the positive reactions decreased by 20-80%, whereas all the positive reactions in the lymphocyte proliferation test decreased to nearly zero (P<0.05), suggesting that lymphocytes against food allergens may be involved in the pathogenesis of canine FA.

Evaluation of synergistic anticandidal and apoptotic effects of ferulic acid and caspofungin against Candida albicans.

PubMed

Canturk, Zerrin

2018-01-01

This study aimed to investigate the synergy between anticandidal and apoptotic effects of ferulic acid and caspofungin against Candida albicans and Candida glabrata, with the help of a quantitative checkerboard microdilution assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as a viability dye. Apoptotic effects of caspofungin and ferulic acid concentrations (alone and combined) were analyzed for C. albicans and C. glabrata based on annexin V-propidium iodide binding capacities using flow cytometric analysis. C. albicans showed a synergistic effect, represented by a fractional inhibitory concentration index of < 0.5, but C. glabrata showed no synergistic effect (fractional inhibitory concentration index > 0.5). Early and late apoptotic effects of caspofungin and ferulic acid concentrations (1 μg/mL and 1000 μg/mL) were calculated as 55.7% and 18.3%, respectively, while their necrotic effects were determined as 5.8% and 51.6%, respectively, using flow cytometric analyses. The apoptotic effects of the combination of caspofungin and ferulic acid at concentrations of 1 μg/mL and 1000 μg/mL on C. albicans and C. glabrata were 73.0% and 48.7%, respectively. Ferulic acid also demonstrated a synergistic effect in combination with caspofungin against C. albicans. Another possibility is to combine the existing anticandidal drug with phytochemicals to enhance the efficacy of anticandidal drug. Copyright © 2017. Published by Elsevier B.V.

Histone deacetylase inhibitors suppress ABO transcription in vitro, leading to reduced expression of the antigens.

PubMed

Takahashi, Yoichiro; Kubo, Rieko; Sano, Rie; Nakajima, Tamiko; Takahashi, Keiko; Kobayashi, Momoko; Handa, Hiroshi; Tsukada, Junichi; Kominato, Yoshihiko

2017-03-01

The ABO system is of fundamental importance in the fields of transfusion and transplantation and has apparent associations with certain diseases, including cardiovascular disorders. ABO expression is reduced in the late phase of erythroid differentiation in vitro, whereas histone deacetylase inhibitors (HDACIs) are known to promote cell differentiation. Therefore, whether or not HDACIs could reduce the amount of ABO transcripts and A or B antigens is an intriguing issue. Quantitative polymerase chain reactions were carried out for the ABO transcripts in erythroid-lineage K562 and epithelial-lineage KATOIII cells after incubation with HDACIs, such as sodium butyrate, panobinostat, vorinostat, and sodium valproate. Flow cytometric analysis was conducted to evaluate the amounts of antigen in KATOIII cells treated with panobinostat. Quantitative chromatin immunoprecipitation (ChIP) assays and luciferase assays were performed on both cell types to examine the mechanisms of ABO suppression. HDACIs reduced the ABO transcripts in both K562 and KATOIII cells, with panobinostat exerting the most significant effect. Flow cytometric analysis demonstrated a decrease in B-antigen expression on panobinostat-treated KATOIII cells. ChIP assays indicated that panobinostat altered the modification of histones in the transcriptional regulatory regions of ABO, and luciferase assays demonstrated reduced activity of these elements. ABO transcription seems to be regulated by an epigenetic mechanism. Panobinostat appears to suppress ABO transcription, reducing the amount of antigens on the surface of cultured cells. © 2016 AABB.

The use of flow cytometry to examine calcium signalling by TRPV1 in mixed cell populations.

PubMed

Assas, Bakri M; Abdulaal, Wesam H; Wakid, Majed H; Zakai, Haytham A; Miyan, J; Pennock, J L

2017-06-15

Flow cytometric analysis of calcium mobilisation has been in use for many years in the study of specific receptor engagement or isolated cell:cell communication. However, calcium mobilisation/signaling is key to many cell functions including apoptosis, mobility and immune responses. Here we combine multiplex surface staining of whole spleen with Indo-1 AM to visualise calcium mobilisation and examine calcium signaling in a mixed immune cell culture over time. We demonstrate responses to a TRPV1 agonist in distinct cell subtypes without the need for cell separation. Multi parameter staining alongside Indo-1 AM to demonstrate calcium mobilization allows the study of real time calcium signaling in a complex environment. Copyright © 2017. Published by Elsevier Inc.

Construction of BAC Libraries from Flow-Sorted Chromosomes.

PubMed

Šafář, Jan; Šimková, Hana; Doležel, Jaroslav

2016-01-01

Cloned DNA libraries in bacterial artificial chromosome (BAC) are the most widely used form of large-insert DNA libraries. BAC libraries are typically represented by ordered clones derived from genomic DNA of a particular organism. In the case of large eukaryotic genomes, whole-genome libraries consist of a hundred thousand to a million clones, which make their handling and screening a daunting task. The labor and cost of working with whole-genome libraries can be greatly reduced by constructing a library derived from a smaller part of the genome. Here we describe construction of BAC libraries from mitotic chromosomes purified by flow cytometric sorting. Chromosome-specific BAC libraries facilitate positional gene cloning, physical mapping, and sequencing in complex plant genomes.

Monitoring of Legionella pneumophila viability after chlorine dioxide treatment using flow cytometry.

PubMed

Mustapha, Pascale; Epalle, Thibaut; Allegra, Séverine; Girardot, Françoise; Garraud, Olivier; Riffard, Serge

2015-04-01

The viability of three Legionella pneumophila strains was monitored after chlorine dioxide (ClO2) treatment using a flow cytometric assay. Suspensions of L. pneumophila cells were submitted to increasing concentrations of ClO2. Culturable cells were still detected when using 4 mg/L, but could no longer be detected after exposure to 6 mg/L of ClO2, although viable but not culturable (VBNC) cells were found after exposure to 4-5 mg/L of ClO2. When testing whether these VBNC were infective, two of the strains were resuscitated after co-culture with Acanthamoeba polyphaga, but neither of them could infect macrophage-like cells. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Systematic Methodological Evaluation of a Multiplex Bead-Based Flow Cytometry Assay for Detection of Extracellular Vesicle Surface Signatures

PubMed Central

Wiklander, Oscar P. B.; Bostancioglu, R. Beklem; Welsh, Joshua A.; Zickler, Antje M.; Murke, Florian; Corso, Giulia; Felldin, Ulrika; Hagey, Daniel W.; Evertsson, Björn; Liang, Xiu-Ming; Gustafsson, Manuela O.; Mohammad, Dara K.; Wiek, Constanze; Hanenberg, Helmut; Bremer, Michel; Gupta, Dhanu; Björnstedt, Mikael; Giebel, Bernd; Nordin, Joel Z.; Jones, Jennifer C.; EL Andaloussi, Samir; Görgens, André

2018-01-01

Extracellular vesicles (EVs) can be harvested from cell culture supernatants and from all body fluids. EVs can be conceptually classified based on their size and biogenesis as exosomes and microvesicles. Nowadays, it is however commonly accepted in the field that there is a much higher degree of heterogeneity within these two subgroups than previously thought. For instance, the surface marker profile of EVs is likely dependent on the cell source, the cell’s activation status, and multiple other parameters. Within recent years, several new methods and assays to study EV heterogeneity in terms of surface markers have been described; most of them are being based on flow cytometry. Unfortunately, such methods generally require dedicated instrumentation, are time-consuming and demand extensive operator expertise for sample preparation, acquisition, and data analysis. In this study, we have systematically evaluated and explored the use of a multiplex bead-based flow cytometric assay which is compatible with most standard flow cytometers and facilitates a robust semi-quantitative detection of 37 different potential EV surface markers in one sample simultaneously. First, assay variability, sample stability over time, and dynamic range were assessed together with the limitations of this assay in terms of EV input quantity required for detection of differently abundant surface markers. Next, the potential effects of EV origin, sample preparation, and quality of the EV sample on the assay were evaluated. The findings indicate that this multiplex bead-based assay is generally suitable to detect, quantify, and compare EV surface signatures in various sample types, including unprocessed cell culture supernatants, cell culture-derived EVs isolated by different methods, and biological fluids. Furthermore, the use and limitations of this assay to assess heterogeneities in EV surface signatures was explored by combining different sets of detection antibodies in EV samples derived from different cell lines and subsets of rare cells. Taken together, this validated multiplex bead-based flow cytometric assay allows robust, sensitive, and reproducible detection of EV surface marker expression in various sample types in a semi-quantitative way and will be highly valuable for many researchers in the EV field in different experimental contexts.

Systematic Methodological Evaluation of a Multiplex Bead-Based Flow Cytometry Assay for Detection of Extracellular Vesicle Surface Signatures.

PubMed

Wiklander, Oscar P B; Bostancioglu, R Beklem; Welsh, Joshua A; Zickler, Antje M; Murke, Florian; Corso, Giulia; Felldin, Ulrika; Hagey, Daniel W; Evertsson, Björn; Liang, Xiu-Ming; Gustafsson, Manuela O; Mohammad, Dara K; Wiek, Constanze; Hanenberg, Helmut; Bremer, Michel; Gupta, Dhanu; Björnstedt, Mikael; Giebel, Bernd; Nordin, Joel Z; Jones, Jennifer C; El Andaloussi, Samir; Görgens, André

2018-01-01

Extracellular vesicles (EVs) can be harvested from cell culture supernatants and from all body fluids. EVs can be conceptually classified based on their size and biogenesis as exosomes and microvesicles. Nowadays, it is however commonly accepted in the field that there is a much higher degree of heterogeneity within these two subgroups than previously thought. For instance, the surface marker profile of EVs is likely dependent on the cell source, the cell's activation status, and multiple other parameters. Within recent years, several new methods and assays to study EV heterogeneity in terms of surface markers have been described; most of them are being based on flow cytometry. Unfortunately, such methods generally require dedicated instrumentation, are time-consuming and demand extensive operator expertise for sample preparation, acquisition, and data analysis. In this study, we have systematically evaluated and explored the use of a multiplex bead-based flow cytometric assay which is compatible with most standard flow cytometers and facilitates a robust semi-quantitative detection of 37 different potential EV surface markers in one sample simultaneously. First, assay variability, sample stability over time, and dynamic range were assessed together with the limitations of this assay in terms of EV input quantity required for detection of differently abundant surface markers. Next, the potential effects of EV origin, sample preparation, and quality of the EV sample on the assay were evaluated. The findings indicate that this multiplex bead-based assay is generally suitable to detect, quantify, and compare EV surface signatures in various sample types, including unprocessed cell culture supernatants, cell culture-derived EVs isolated by different methods, and biological fluids. Furthermore, the use and limitations of this assay to assess heterogeneities in EV surface signatures was explored by combining different sets of detection antibodies in EV samples derived from different cell lines and subsets of rare cells. Taken together, this validated multiplex bead-based flow cytometric assay allows robust, sensitive, and reproducible detection of EV surface marker expression in various sample types in a semi-quantitative way and will be highly valuable for many researchers in the EV field in different experimental contexts.

Detection of early changes in lung cell cytology by flow-systems analysis techniques. Progress report, January 1--December 31, 1978. [Resulting from exposure to toxic agents associated with production and use of synthetic fuels from oil shale and coal

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steinkamp, J.A.; Wilson, J.S.; Svitra, Z.V.

1979-02-01

This report summarizes ongoing experiments to develop cytological and biochemical indicators for measuring damage to respiratory tract cells of experimental animals exposed by inhalation to environmental toxic agents. The specific goal is to apply flow cytometric methods to analyze and detect changes in lung epithelium as a function of exposure to toxic agents associated with the production and utilization of synthetic fuels from oil shale and coal. During the past 6 months, hamsters were exposed to raw and spent oil shale particulates, silica dust, and ozone, and DNA content measurements were performed on lung cell samples. Although initial shale exposuresmore » did not yield the expected results, recent data show atypical changes in DNA content per cell distributions. Ozone exposures also were expanded to include DNA measurements and cytology, ranging up to 72 h postexposure. Progress was achieved in developing a new method for quantitating pulmonary macrophage phagocytosis in rats using micron-sized fluorescent spheres. New methods for determining alkaline phosphatase, DNA content, and protein also were under development. Plans are to continue developing cytological and biochemical markers for measuring atypical cellular changes, including macrophage function, and to emphasize exposing experimental animals to particulates and gaseous agents for studying dose-damage relationships.« less

Monoclonal antibodies for the separate detection of halodeoxyuridines and method for their use

DOEpatents

Vanderlaan, M.; Watkins, B.E.; Stanker, L.H.

1991-10-01

Monoclonal antibodies are described which have specific affinities for halogenated nucleoside analogs and are preferentially selective for one particular halogen. Such antibodies, when incorporated into immunochemical reagents, may be used to identify and independently quantify the cell division character of more than one population or subpopulation in flow cytometric measurements. Independent assessment of division activity in cell sub-populations facilitates selection of appropriate time and dose for administration of anti-proliferative agents. The hybridomas which secrete halogen selective antibodies and the method of making them are described. 14 figures.

Monoclonal antibodies for the separate detection of halodeoxyuridines and method for their use

DOEpatents

Vanderlaan, Martin; Watkins, Bruce E.; Stanker, Larry H.

1991-01-01

Monoclonal antibodies are described which have specific affinities for halogenated nucleoside analogs and are preferentially selective for one particular halogen. Such antibodies, when incorporated into immunochemical reagents, may be used to identify and independently quantify the cell division character of more than one population or subpopulation in flow cytometric measurements. Independent assessment of division activity in cell sub-populations facilitates selection of appropriate time and dose for administration of anti-proliferative agents. The hybridomas which secrete halogen selective antibodies and the method of making them are described.

Improvement of a rapid screening test for chronic granulomatous disease.

PubMed

Iacobini, M; Duse, M; Di Coste, A; Balducci, L

2013-01-01

Diagnosis of CGD is made by demonstrating absent or markedly reduced oxidase activity in stimulated neutrophils. The screening test proposed is based upon the naked eye evaluation of the reduction of NBT on a solid surface. It seems to be a useful tool for rapid and inexpensive detection of CGD patients, especially for large-scale screening purposes. The test was carried out on forty-five subjects: two males affected by CGD, three female carriers and forty healthy donors. The test confirmed the results obtained with flow cytometric and NBT assays.

Multiplexed immunophenotyping of human antigen-presenting cells in whole blood by polychromatic flow cytometry

PubMed Central

Fung, Erik; Esposito, Laura; Todd, John A.; Wicker, Linda S.

2010-01-01

We describe two modular protocols for immunostaining and multiparameter flow cytometric analysis of major human antigen-presenting cells (dendritic cells, monocytes, B lymphocytes) in minimally manipulated whole blood. Simultaneous detection of up to eight colors is enabled by careful selection and testing of cell-subset-defining monoclonal antibodies (anchor markers) in the appropriate fluorochrome combinations, to demonstrate the quantification of surface expression levels of molecules involved in chemotaxis (e.g. CX3CR1, CCR2), adhesion (e.g. CD11b, CD62L), antigen presentation (e.g. CD83, CD86, CD209) and immune regulation (e.g. CD101) on circulating antigen-presenting cells. Each immunostaining reaction requires as little as 50–100 μl of peripheral whole blood, no density-gradient separation, and the entire procedure from preparation of reagents to flow cytometry can be completed in <5 h. PMID:20134434

Differential Interaction of Platelet-Derived Extracellular Vesicles with Leukocyte Subsets in Human Whole Blood.

PubMed

Weiss, René; Gröger, Marion; Rauscher, Sabine; Fendl, Birgit; Eichhorn, Tanja; Fischer, Michael B; Spittler, Andreas; Weber, Viktoria

2018-04-26

Secretion and exchange of biomolecules via extracellular vesicles (EVs) are crucial mechanisms in intercellular communication, and the roles of EVs in infection, inflammation, or thrombosis have been increasingly recognized. EVs have emerged as central players in immune regulation and can enhance or suppress the immune response, depending on the state of donor and recipient cells. We investigated the interaction of blood cell-derived EVs with leukocyte subpopulations (monocytes and their subsets, granulocytes, B cells, T cells, and NK cells) directly in whole blood using a combination of flow cytometry, imaging flow cytometry, cell sorting, and high resolution confocal microscopy. Platelet-derived EVs constituted the majority of circulating EVs and were preferentially associated with granulocytes and monocytes, while they scarcely interacted with lymphocytes. Further flow cytometric differentiation of monocyte subsets provided clear indications for a preferential association of platelet-derived EVs with intermediate (CD14 ++ CD16 + ) monocytes in whole blood.

Innovative application of classic and newer techniques for the characterization of haemocytes in the New Zealand black-footed abalone (Haliotis iris).

PubMed

Grandiosa, Roffi; Mérien, Fabrice; Pillay, Krish; Alfaro, Andrea

2016-01-01

Haemocytes play an important role in innate immune responses within invertebrate organisms. However, identification and quantification of different types of haemocytes can be extremely challenging, and has led to numerous inconsistencies and misinterpretations within the literature. As a step to rectify this issue, we present a comprehensive and detailed approach to characterize haemocytes using a combination of classical (cytochemical and phagocytosis assays with optical microscopy) and novel (flow cytometry with Sysmex XN-1000 and Muse(®) Cell analyser) techniques. The Sysmex XN-1000 is an innovative fluorescent flow cytometric analyser that can effectively detect, identify and count haemocytes, while the Muse(®) Cell analyser provides accurate and rapid haemocyte cell counts and viability. To illustrate this approach, we present the first report on morphological and functional features of New Zealand black-footed abalone (Haliotis iris) haemocyte cells. Two types of haemocytes were identified in this study, including type I (monocyte-like) and type II (lymphocyte-like) cells. Granular cells, which have been reported in other molluscan species, were not detected in H. iris. Cell types were categorized based on shape, size, internal structures and function. The lymphocyte-like haemocytes were the most abundant hemocytes in the haemolymph samples, and they had large nuclei and basic cytoplasms. Monocyte-like cells generally were larger cells compared to lymphocyte-like cells, and had low nucleus-cytoplasm ratios. Monocyte-like cells showed higher phagocytic activity when encountering Zymosan A particles compared to lymphocyte-like cells. The present study provides a comprehensive and accurate new approach to identify and quantify haemocyte cells for future comparative studies on the immune system of abalone and other molluscan species. Copyright © 2015 Elsevier Ltd. All rights reserved.

Detection of early changes in lung cell cytology by flow-systems analysis techniques. Progress report, January 1-June 30, 1979

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steinkamp, J.A.; Wilson, J.S.; Svitra, Z.V.

1979-08-01

This report summarizes results of ongoing experiments designed to develop automated flow-analysis assay methods for discerning damage to exfoliated respiratory tract cells in model test animals exposed by inhalation to physical and chemical agents associated with the production of synthetic fuels from oil shale and coal, the specific goal being the determination of atypical changes in exposed alveolar macrophages and epithelial cells. Animals were exposed to oil shale particles (raw and spent), silica, and polystyrene latex spheres via intratracheal instillation. Respiratory tract cells were obtained by lavaging the lungs with normal saline, stained with mithramycin for DNA content, and analyzedmore » using flow cytometric analysis methods. In addition to measuring DNA content, differential and total cell counts were made on all samples analyzed. DNA content frequency distribution histograms and cytology showed definite atypical changes resulting from exposure to shale and silica particulates when compared to the controls. To continue development of fluorescence staining methods for measuring intracellular enzymes in alveolar macrophages, studies were initiated for determining ..beta..-glucuronidase using naphthol AS-BI-..beta..-d-glucuronic acid as a fluorogenic substrate. As this new technology becomes adapted to analyzing pulmonary macrophages and epithelial cells, the measurement of physical and biochemical properties as a function of exposure to particulate and gaseous toxic agents related to the production of synthetic fuels will be increased. This analytical approach is designed to assist in the establishment of future guideline for estimating the risks to exposed humans.« less

Detection of Legionella pneumophila on clinical samples and susceptibility assessment by flow cytometry.

PubMed

Faria-Ramos, I; Costa-de-Oliveira, S; Barbosa, J; Cardoso, A; Santos-Antunes, J; Rodrigues, A G; Pina-Vaz, C

2012-12-01

Culture in selective media represents the standard diagnostic method to confirm Legionella pneumophila infection, despite requiring a prolonged incubation period; antigen detection by immunofluorescence (IFS) and molecular techniques are also available, but they do not allow antimicrobial susceptibility evaluation. Our objective was to optimise flow cytometry (FC) protocols for the detection of L. pneumophila in respiratory samples and for susceptibility evaluation to first-line drugs. In order to optimise the FC protocol, a specific monoclonal antibody, conjugated with fluorescein isothiocyanate (FITC), was incubated with type strain L. pneumophila ATCC 33152. The limit of detection was established by analysing serial dilutions of bacterial suspension; specificity was assayed using mixtures of prokaryotic and eukaryotic microorganisms. The optimised FC protocol was used to assess 50 respiratory samples and compared with IFS evaluation. The susceptibility profile to erythromycin, ciprofloxacin and levofloxacin was evaluated by FC using propidium iodide and SYBR Green fluorescent dyes; the results were compared with the Etest afterwards. The optimal specific antibody concentration was 20 μg/ml; 10(2)/ml Legionella organisms were detected by this protocol and no cross-reactions with other microorganisms were detected. The five positive respiratory samples (10 %) determined by IFS were also detected by FC, showing 100 % correlation. After 1 h of incubation at 37 °C with different antimicrobials, SYBR Green staining could discriminate between treated and non-treated cells. A novel flow cytometric approach for the detection of L. pneumophila from clinical samples and susceptibility evaluation is now available, representing an important step forward for the diagnosis of this very relevant agent.

Flow cytometry shows added value in diagnosing lymphoma in brain biopsies.

PubMed

van der Meulen, Matthijs; Bromberg, Jacoline E C; Lam, King H; Dammers, Ruben; Langerak, Anton W; Doorduijn, Jeanette K; Kros, Johan M; van den Bent, Martin J; van der Velden, Vincent H J

2018-05-10

To assess the sensitivity, specificity and turnaround time of flow cytometric analysis on brain biopsies compared to histology plus immunohistochemistry analysis in tumors with clinical suspicion of lymphoma. All brain biopsies performed between 2010 and 2015 at our institution and analyzed by both immunohistochemistry and flow cytometry were included in this retrospective study. Immunohistochemistry was considered the gold standard. In a total of 77 biopsies from 71 patients, 49 lymphomas were diagnosed by immunohistochemistry, flow cytometry results were concordant in 71 biopsies (92,2%). We found a specificity and sensitivity of flow cytometry of 100% and 87,8%, respectively. The time between the biopsy and reporting the result (turnaround time) was significantly shorter for flow cytometry, compared to immunohistochemistry (median: 1 versus 5 days). Flow cytometry has a high specificity and can confirm the diagnosis of a lymphoma significantly faster than immunohistochemistry. This allows for rapid initiation of treatment in this highly aggressive tumor. However, since its sensitivity is less than 100%, we recommend to perform histology plus immunohistochemistry in parallel to flow cytometry. This article is protected by copyright. All rights reserved. © 2018 International Clinical Cytometry Society.

Assessment of Telomere Length, Phenotype, and DNA Content

PubMed Central

Kelesidis, Theodoros; Schmid, Ingrid

2017-01-01

Telomere sequences at the end of chromosomes control somatic cell division; therefore, telomere length in a given cell population provides information about its replication potential. This unit describes a method for flow cytometric measurement of telomere length in subpopulations using fluorescence in situ hybridization of fluorescently-labeled probes (Flow-FISH) without prior cell separation. After cells are stained for surface immunofluorescence, antigen-antibody complexes are covalently cross-linked onto cell membranes before FISH with a telomere-specific probe. Cells with long telomeres are included as internal standards. Addition of a DNA dye permits exclusion of proliferating cells during data analysis. DNA ploidy measurements of cells of interest and internal standard are performed on separate aliquots in parallel to Flow-FISH. Telomere fluorescence of G0/1 cells of subpopulations and internal standards obtained from Flow-FISH are normalized for DNA ploidy, and telomere length in subsets of interest is expressed as a fraction of the internal standard telomere length. PMID:28055113

Assessment of Telomere Length, Phenotype, and DNA Content.

PubMed

Kelesidis, Theodoros; Schmid, Ingrid

2017-01-05

Telomere sequences at the end of chromosomes control somatic cell division; therefore, telomere length in a given cell population provides information about its replication potential. This unit describes a method for flow cytometric measurement of telomere length in subpopulations using fluorescence in situ hybridization of fluorescently-labeled probes (Flow-FISH) without prior cell separation. After cells are stained for surface immunofluorescence, antigen-antibody complexes are covalently cross-linked onto cell membranes before FISH with a telomere-specific probe. Cells with long telomeres are included as internal standards. Addition of a DNA dye permits exclusion of proliferating cells during data analysis. DNA ploidy measurements of cells of interest and internal standard are performed on separate aliquots in parallel to Flow-FISH. Telomere fluorescence of G 0/1 cells of subpopulations and internal standards obtained from Flow-FISH are normalized for DNA ploidy, and telomere length in subsets of interest is expressed as a fraction of the internal standard telomere length. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Macroecological patterns of phytoplankton in the northwestern North Atlantic Ocean.

PubMed

Li, W K W

2002-09-12

Many issues in biological oceanography are regional or global in scope; however, there are not many data sets of extensive areal coverage for marine plankton. In microbial ecology, a fruitful approach to large-scale questions is comparative analysis wherein statistical data patterns are sought from different ecosystems, frequently assembled from unrelated studies. A more recent approach termed macroecology characterizes phenomena emerging from large numbers of biological units by emphasizing the shapes and boundaries of statistical distributions, because these reflect the constraints on variation. Here, I use a set of flow cytometric measurements to provide macroecological perspectives on North Atlantic phytoplankton communities. Distinct trends of abundance in picophytoplankton and both small and large nanophytoplankton underlaid two patterns. First, total abundance of the three groups was related to assemblage mean-cell size according to the 3/4 power law of allometric scaling in biology. Second, cytometric diversity (an ataxonomic measure of assemblage entropy) was maximal at intermediate levels of water column stratification. Here, intermediate disturbance shapes diversity through an equitable distribution of cells in size classes, from which arises a high overall biomass. By subsuming local fluctuations, macroecology reveals meaningful patterns of phytoplankton at large scales.

The Isolation and Enrichment of Large Numbers of Highly Purified Mouse Spleen Dendritic Cell Populations and Their In Vitro Equivalents.

PubMed

Vremec, David

2016-01-01

Dendritic cells (DCs) form a complex network of cells that initiate and orchestrate immune responses against a vast array of pathogenic challenges. Developmentally and functionally distinct DC subtypes differentially regulate T-cell function. Importantly it is the ability of DC to capture and process antigen, whether from pathogens, vaccines, or self-components, and present it to naive T cells that is the key to their ability to initiate an immune response. Our typical isolation procedure for DC from murine spleen was designed to efficiently extract all DC subtypes, without bias and without alteration to their in vivo phenotype, and involves a short collagenase digestion of the tissue, followed by selection for cells of light density and finally negative selection for DC. The isolation procedure can accommodate DC numbers that have been artificially increased via administration of fms-like tyrosine kinase 3 ligand (Flt3L), either directly through a series of subcutaneous injections or by seeding with an Flt3L secreting murine melanoma. Flt3L may also be added to bone marrow cultures to produce large numbers of in vitro equivalents of the spleen DC subsets. Total DC, or their subsets, may be further purified using immunofluorescent labeling and flow cytometric cell sorting. Cell sorting may be completely bypassed by separating DC subsets using a combination of fluorescent antibody labeling and anti-fluorochrome magnetic beads. Our procedure enables efficient separation of the distinct DC subsets, even in cases where mouse numbers or flow cytometric cell sorting time is limiting.

Relevance of Flow Cytometric Auto-Crossmatch to the Post-transplant Course of Kidney Transplant Recipients.

PubMed

Demir, E; Yeğit, O; Erol, A; Akgül, S U; Çalışkan, B; Bayraktar, A; Çalışkan, Y; Türkmen, A; Savran, F O; Sever, M S

2017-04-01

The crossmatch test is essential prior to kidney transplantation (tx) to confirm compatibility between the donor and the recipient. However, its results can be misleading due to "undetectable antibodies" in the recipient's serum. To establish if undetectable autoantibodies are responsible for a positive result, an auto-crossmatch test can be performed. In this study, we aim to determine the long-term prognostic value of auto-flow cytometric auto-crossmatch (FCXM) test on kidney survival in kidney tx recipients. The primary outcome variable was reduced renal function. Secondary endpoints were incidence of biopsy-confirmed chronic antibody-mediated rejection (CAMR) and recurrent glomerulonephritis (GN). There were no differences regarding initial serum creatinine levels between the study and control groups (P = .441). Patients who had positive auto-B FCXM had a significantly reduced renal function compared with the control group (P = .016). Four patients developed biopsy-confirmed CAMR in the study group and 1 patient in the control group (P = .047). Five patients had biopsy-confirmed recurrent GN in the GN study group, and only 1 patient had recurrent GN in the GN control group (P = .026). Kidney transplant recipients with positive auto-FCXM test had significantly reduced renal function and a higher incidence of recurrent GN and CAMR compared with the control group. The findings of this study suggest a potential role of auto-antibody causing positive auto-FCXM test result, meanwhile increasing the risk of CAMR, recurrent GN, and new-onset diabetes after tx. Copyright © 2017 Elsevier Inc. All rights reserved.

Antiproliferative and apoptosis inducing effects of citral via p53 and ROS-induced mitochondrial-mediated apoptosis in human colorectal HCT116 and HT29 cell lines.

PubMed

Sheikh, Bassem Y; Sarker, Md Moklesur Rahman; Kamarudin, Muhamad Noor Alfarizal; Mohan, Gokula

2017-12-01

Despite various anticancer reports, antiproliferative and apoptosis inducing activity of citral in HCT116 and HT29 cells have never been reported. This study aimed to evaluate the cytotoxic and apoptosis inducing effects of citral in colorectal cancer cell lines. The citral-treated cells were subjected to MTT assay followed by flow cytometric Annexin V-FITC/PI, mitochondrial membrane potential and intracellular reactive oxygen species (ROS) determination. The apoptotic proteins expression was investigated by Western blot analysis. Citral inhibited the growth of HCT116 and HT29 cells by dose- and time-dependent manner without inducing cytotoxicity in CCD841-CoN normal colon cells. Flow cytometric analysis showed that citral (50-200μM; 24-48h) induced the externalization of phoshpotidylserine and reduced the mitochondrial membrane potential in HCT116 and HT29 cells. Citral elevated intracellular ROS level while attenuating GSH levels in HCT116 and HT29 cells which were reversed with N-acetycysteine (2mM) pre-treatment indicating that citral induced mitochondrial-mediated apoptosis via augmentation of intracellular ROS. Citral induced the phosphorylation of p53 protein and the expression of Bax while decreasing Bc-2 and Bcl-xL expression which promoted the cleavage of caspase-3. Collectively, our data suggest that citral induced p53 and ROS-mediated mitochondrial-mediated apoptosis in human colorectal cancer HCT116 and HT29 cells. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

MicroRNA expression profiling of human breast cancer identifies new markers of tumor subtype.

PubMed

Blenkiron, Cherie; Goldstein, Leonard D; Thorne, Natalie P; Spiteri, Inmaculada; Chin, Suet-Feung; Dunning, Mark J; Barbosa-Morais, Nuno L; Teschendorff, Andrew E; Green, Andrew R; Ellis, Ian O; Tavaré, Simon; Caldas, Carlos; Miska, Eric A

2007-01-01

MicroRNAs (miRNAs), a class of short non-coding RNAs found in many plants and animals, often act post-transcriptionally to inhibit gene expression. Here we report the analysis of miRNA expression in 93 primary human breast tumors, using a bead-based flow cytometric miRNA expression profiling method. Of 309 human miRNAs assayed, we identify 133 miRNAs expressed in human breast and breast tumors. We used mRNA expression profiling to classify the breast tumors as luminal A, luminal B, basal-like, HER2+ and normal-like. A number of miRNAs are differentially expressed between these molecular tumor subtypes and individual miRNAs are associated with clinicopathological factors. Furthermore, we find that miRNAs could classify basal versus luminal tumor subtypes in an independent data set. In some cases, changes in miRNA expression correlate with genomic loss or gain; in others, changes in miRNA expression are likely due to changes in primary transcription and or miRNA biogenesis. Finally, the expression of DICER1 and AGO2 is correlated with tumor subtype and may explain some of the changes in miRNA expression observed. This study represents the first integrated analysis of miRNA expression, mRNA expression and genomic changes in human breast cancer and may serve as a basis for functional studies of the role of miRNAs in the etiology of breast cancer. Furthermore, we demonstrate that bead-based flow cytometric miRNA expression profiling might be a suitable platform to classify breast cancer into prognostic molecular subtypes.

Kidney stone matrix proteins ameliorate calcium oxalate monohydrate induced apoptotic injury to renal epithelial cells.

PubMed

Narula, Shifa; Tandon, Simran; Singh, Shrawan Kumar; Tandon, Chanderdeep

2016-11-01

Kidney stone formation is a highly prevalent disease, affecting 8-10% of the human population worldwide. Proteins are the major constituents of human kidney stone's organic matrix and considered to play critical role in the pathogenesis of disease but their mechanism of modulation still needs to be explicated. Therefore, in this study we investigated the effect of human kidney stone matrix proteins on the calcium oxalate monohydrate (COM) mediated cellular injury. The renal epithelial cells (MDCK) were exposed to 200μg/ml COM crystals to induce injury. The effect of proteins isolated from human kidney stone was studied on COM injured cells. The alterations in cell-crystal interactions were examined by phase contrast, polarizing, fluorescence and scanning electron microscopy. Moreover, its effect on the extent of COM induced cell injury, was quantified by flow cytometric analysis. Our study indicated the antilithiatic potential of human kidney stone proteins on COM injured MDCK cells. Flow cytometric analysis and fluorescence imaging ascertained that matrix proteins decreased the extent of apoptotic injury caused by COM crystals on MDCK cells. Moreover, the electron microscopic studies of MDCK cells revealed that matrix proteins caused significant dissolution of COM crystals, indicating cytoprotection against the impact of calcium oxalate injury. The present study gives insights into the mechanism implied by urinary proteins to restrain the pathogenesis of kidney stone disease. This will provide a better understanding of the formation of kidney stones which can be useful for the proper management of the disease. Copyright © 2016 Elsevier Inc. All rights reserved.

Flow cytometric chemosensitivity assay using JC‑1, a sensor of mitochondrial transmembrane potential, in acute leukemia.

PubMed

Yokosuka, Tomoko; Goto, Hiroaki; Fujii, Hisaki; Naruto, Takuya; Takeuchi, Masanobu; Tanoshima, Reo; Kato, Hiromi; Yanagimachi, Masakatsu; Kajiwara, Ryosuke; Yokota, Shumpei

2013-12-01

The purpose of the study is to establish a simple and relatively inexpensive flow cytometric chemosensitivity assay (FCCA) for leukemia to distinguish leukemic blasts from normal leukocytes in clinical samples. We first examined whether the FCCA with the mitochondrial membrane depolarization sensor, 5, 50, 6, 60-tetrachloro-1, 10, 3, 30 tetraethyl benzimidazolo carbocyanine iodide (JC-1), could detect drug-induced apoptosis as the conventional FCCA by annexin V/7-AAD detection did and whether it was applicable in the clinical samples. Second, we compared the results of the FCCA for prednisolone (PSL) with clinical PSL response in 18 acute lymphoblastic leukemia (ALL) patients to evaluate the reliability of the JC-1 FCCA. Finally, we performed the JC-1 FCCA for bortezomib (Bor) in 25 ALL or 11 acute myeloid leukemia (AML) samples as the example of the clinical application of the FCCA. In ALL cells, the results of the JC-1 FCCA for nine anticancer drugs were well correlated with those of the conventional FCCA using anti-annexin V antibody (P < 0.001). In the clinical samples from 18 children with ALL, the results of the JC-1 FCCA for PSL were significantly correlated with the clinical PSL response (P = 0.005). In ALL samples, the sensitivity for Bor was found to be significantly correlated with the sensitivity for PSL (P = 0.005). In AML samples, the Bor sensitivity was strongly correlated with the cytarabine sensitivity (P = 0.0003). This study showed the reliability of a relatively simple and the FCCA using JC-1, and the possibility for the further clinical application.

A novel tumor-activated ALA fusion protein for specific inhibition on the growth and invasion of breast cancer cells MDA-MB-231.

PubMed

Liu, Xiufeng; Liu, Xintong; Sunchen, Suwen; Liu, Meixia; Shen, Chen; Wu, Juanjuan; Zhao, Wanli; Yu, Boyang; Liu, Jihua

2017-11-01

The aim of this research was to develop a novel ALA fusion protein for target to the malignant cells surface with high uPAR expression and locally release of the scorpion toxin AGAP in an uPA-cleavable manner. It will provide an effective approach for controlled release of the peptide toxins to treat cancerous cells. The ALA fusion proteins were expressed in pichia pastoris, and the recombinant proteins were purified by Ni-NTA affinity chromatography. The proteins were added to human breast cancer cells (MDA-MB-231) and human embryonic kidney cells (HEK-293) in order to investigate the characteristic of selective targeting and releasing of scorpion toxin AGAP in cancer cells with high uPAR expression. The inhibitory effect of ALA on MDA-MB-231, MCF7, LO2 and HEK-293 was evaluated by MTT assay. Moreover, the antiproliferation mechanism of ALA was determined by flow cytometric and western blot analysis. The results showed that ALA could target MDA-MB-231 cells and the scorpion toxin AGAP could be released with high efficiency and selectivity. ALA inhibited the growth and invasion of breast cancer cells MDA-MB231. Also, cell apoptosis pathway was found to be associated with the inhibition mechanism of ALA according to the data of flow cytometric and western blot analysis. Therefore, ALA could be a novel antitumor candidate for targeting treatment of malignant cell. This study successfully demonstrated that fusion of biotoxins with tumor target domain could provide a simple yet effective way to delivery of peptide or protein drugs.

CFTR RECRUITMENT TO PHAGOSOMES IN NEUTROPHILS

PubMed Central

Zhou, Yun; Song, Kejing; Painter, Richard G.; Aiken, Martha; Reiser, Jakob; Stanton, Bruce A.; Nauseef, William M.; Wang, Guoshun

2013-01-01

Optimal microbicidal activity of human polymorphonuclear leukocytes (PMN) relies on generation of toxic agents such as hypochlorous acid (HOCl) in phagosomes. HOCl formation requires H2O2 produced by the NADPH oxidase, myeloperoxidase derived from azurophilic granules, and chloride ion. Chloride transport from cytoplasm into phagosomes requires chloride channels which include cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel. However, the phagosomal targeting of CFTR in PMN has not been defined. Using human peripheral blood PMN, we determined that ~95–99% of LAMP-1 positive mature phagosomes were CFTR-positive, as judged by immunostaining and flow cytometric analysis. To establish a model cell system to evaluate CFTR phagosomal recruitment, we stably expressed EGFP alone, EGFP-wt-CFTR and EGFP-ΔF508-CFTR fusion proteins in promyelocytic PLB-985 cells, respectively. After differentiation into neutrophil-like cells, CFTR presentation to phagosomes was examined. EGFP-wt-CFTR was observed to associate with phagosomes and co-localize with LAMP-1. Flow cytometric analysis of the isolated phagosomes indicated that such a phagosomal targeting was determined by the CFTR portion of the fusion protein. In contrast, significantly less EGFP-ΔF508-CFTR was found in phagosomes, indicating a defective targeting of the molecule to the organelle. Importantly, CFTR corrector compound VRT-325 facilitated the recruitment of ΔF508-CFTR to phagosomes. These data demonstrate the possibility of pharmacologic correction of impaired recruitment of mutant CFTR, thereby providing a potential means to augment chloride supply to the phagosomes of PMN in patients with cystic fibrosis to enhance their microbicidal function. PMID:23486169

EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols

PubMed Central

Kalina, T; Flores-Montero, J; van der Velden, V H J; Martin-Ayuso, M; Böttcher, S; Ritgen, M; Almeida, J; Lhermitte, L; Asnafi, V; Mendonça, A; de Tute, R; Cullen, M; Sedek, L; Vidriales, M B; Pérez, J J; te Marvelde, J G; Mejstrikova, E; Hrusak, O; Szczepański, T; van Dongen, J J M; Orfao, A

2012-01-01

The EU-supported EuroFlow Consortium aimed at innovation and standardization of immunophenotyping for diagnosis and classification of hematological malignancies by introducing 8-color flow cytometry with fully standardized laboratory procedures and antibody panels in order to achieve maximally comparable results among different laboratories. This required the selection of optimal combinations of compatible fluorochromes and the design and evaluation of adequate standard operating procedures (SOPs) for instrument setup, fluorescence compensation and sample preparation. Additionally, we developed software tools for the evaluation of individual antibody reagents and antibody panels. Each section describes what has been evaluated experimentally versus adopted based on existing data and experience. Multicentric evaluation demonstrated high levels of reproducibility based on strict implementation of the EuroFlow SOPs and antibody panels. Overall, the 6 years of extensive collaborative experiments and the analysis of hundreds of cell samples of patients and healthy controls in the EuroFlow centers have provided for the first time laboratory protocols and software tools for fully standardized 8-color flow cytometric immunophenotyping of normal and malignant leukocytes in bone marrow and blood; this has yielded highly comparable data sets, which can be integrated in a single database. PMID:22948490

The life-cycle of Emiliania huxleyi: A brief review and a study of relative ploidy levels analysed by flow cytometry

NASA Astrophysics Data System (ADS)

Green, J. C.; Course, P. A.; Tarran, G. A.

1996-10-01

Emiliania huxleyi exists in several principal forms including the familiar coccolith-bearing C-cell, non-motile naked N-cells, and scale-bearing swarmers (S-cells), but the relationships between these cells are unclear. Flow cytometric analyses have been undertaken on whole cells using fluorochrome staining of the DNA in order to determine the relative DNA content and the relative GC content of the S- and C-cells of selected clones. Results showed that the DNA complement of the S-cells was half that of the C-cells and the two cell types are, therefore, haploid and diploid relative to each other. The S-cells may, therefore, represent a gametic stage, though processes such as sexual fusion and meiosis have not been observed.

Scalable clustering algorithms for continuous environmental flow cytometry.

PubMed

Hyrkas, Jeremy; Clayton, Sophie; Ribalet, Francois; Halperin, Daniel; Armbrust, E Virginia; Howe, Bill

2016-02-01

Recent technological innovations in flow cytometry now allow oceanographers to collect high-frequency flow cytometry data from particles in aquatic environments on a scale far surpassing conventional flow cytometers. The SeaFlow cytometer continuously profiles microbial phytoplankton populations across thousands of kilometers of the surface ocean. The data streams produced by instruments such as SeaFlow challenge the traditional sample-by-sample approach in cytometric analysis and highlight the need for scalable clustering algorithms to extract population information from these large-scale, high-frequency flow cytometers. We explore how available algorithms commonly used for medical applications perform at classification of such a large-scale, environmental flow cytometry data. We apply large-scale Gaussian mixture models to massive datasets using Hadoop. This approach outperforms current state-of-the-art cytometry classification algorithms in accuracy and can be coupled with manual or automatic partitioning of data into homogeneous sections for further classification gains. We propose the Gaussian mixture model with partitioning approach for classification of large-scale, high-frequency flow cytometry data. Source code available for download at https://github.com/jhyrkas/seaflow_cluster, implemented in Java for use with Hadoop. hyrkas@cs.washington.edu Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Evaluation of bone marrow specimens with acute myelogenous leukemia for CD34, CD15, CD117, and myeloperoxidase.

PubMed

Dunphy, C H; Polski, J M; Evans, H L; Gardner, L J

2001-08-01

Immunophenotyping of bone marrow (BM) specimens with acute myelogenous leukemia (AML) may be performed by flow cytometric (FC) or immunohistochemical (IH) techniques. Some markers (CD34, CD15, and CD117) are available for both techniques. Myeloperoxidase (MPO) analysis may be performed by enzyme cytochemical (EC) or IH techniques. To determine the reliability of these markers and MPO by these techniques, we designed a study to compare the results of analyses of these markers and MPO by FC (CD34, CD15, and CD117), EC (MPO), and IH (CD34, CD15, CD117, and MPO) techniques. Twenty-nine AMLs formed the basis of the study. These AMLs all had been immunophenotyped previously by FC analysis; 27 also had had EC analysis performed. Of the AMLs, 29 had BM core biopsies and 26 had BM clots that could be evaluated. The paraffin blocks of the 29 BM core biopsies and 26 BM clots were stained for CD34, CD117, MPO, and CD15. These results were compared with results by FC analysis (CD34, CD15, and CD117) and EC analysis (MPO). Immunodetection of CD34 expression in AML had a similar sensitivity by FC and IH techniques. Immunodetection of CD15 and CD117 had a higher sensitivity by FC analysis than by IH analysis. Detection of MPO by IH analysis was more sensitive than by EC analysis. There was no correlation of French-American-British (FAB) subtype of AML with CD34 or CD117 expression. Expression of CD15 was associated with AMLs with a monocytic component. Myeloperoxidase reactivity by IH analysis was observed in AMLs originally FAB subtyped as M0. CD34 can be equally detected by FC and IH techniques. CD15 and CD117 are better detected by FC analysis and MPO is better detected by IH analysis.

Analytical validation of a flow cytometric protocol for quantification of platelet microparticles in dogs.

PubMed

Cremer, Signe E; Krogh, Anne K H; Hedström, Matilda E K; Christiansen, Liselotte B; Tarnow, Inge; Kristensen, Annemarie T

2018-06-01

Platelet microparticles (PMPs) are subcellular procoagulant vesicles released upon platelet activation. In people with clinical diseases, alterations in PMP concentrations have been extensively investigated, but few canine studies exist. This study aims to validate a canine flow cytometric protocol for PMP quantification and to assess the influence of calcium on PMP concentrations. Microparticles (MP) were quantified in citrated whole blood (WB) and platelet-poor plasma (PPP) using flow cytometry. Anti-CD61 antibody and Annexin V (AnV) were used to detect platelets and phosphatidylserine, respectively. In 13 healthy dogs, CD61 + /AnV - concentrations were analyzed with/without a calcium buffer. CD61 + /AnV - , CD61 + /AnV + , and CD61 - /AnV + MP quantification were validated in 10 healthy dogs. The coefficient of variation (CV) for duplicate (intra-assay) and parallel (inter-assay) analyses and detection limits (DLs) were calculated. CD61 + /AnV - concentrations were higher in calcium buffer; 841,800 MP/μL (526,000-1,666,200) vs without; 474,200 MP/μL (278,800-997,500), P < .05. In WB, PMP were above DLs and demonstrated acceptable (<20%) intra-assay and inter-assay CVs in 9/10 dogs: 1.7% (0.5-8.9) and 9.0% (0.9-11.9), respectively, for CD61 + /AnV - and 2.4% (0.2-8.7) and 7.8% (0.0-12.8), respectively, for CD61 + /AnV + . Acceptable CVs were not seen for the CD61 - /AnV + MP. In PPP, quantifications were challenged by high inter-assay CV, overlapping DLs and hemolysis and lipemia interfered with quantification in 5/10 dogs. Calcium induced higher in vitro PMP concentrations, likely due to platelet activation. PMP concentrations were reliably quantified in WB, indicating the potential for clinical applications. PPP analyses were unreliable due to high inter-CV and DL overlap, and not obtainable due to hemolysis and lipemia interference. © 2018 American Society for Veterinary Clinical Pathology.

Pulsating electromagnetic field stimulation of urothelial cells induces apoptosis and diminishes necrosis: new insight to magnetic therapy in urology.

PubMed

Juszczak, K; Kaszuba-Zwoinska, J; Thor, P J

2012-08-01

The evidence of electromagnetic therapy (EMT) efficacy in stress and/or urge urinary incontinence, as well as in detrusor overactivity is generally lacking in the literature. The potential EMT action of neuromuscular tissue depolarization has been described. Because there is no data on the influence of pulsating electromagnetic fields (PEMF) on the urothelium, we evaluated the effect of PEMF stimulation on rat urothelial cultured cells (RUCC). In our study 15 Wistar rats were used for RUCC preparation. RUCC were exposed to PEMF (50 Hz, 45±5 mT) three times for 4 hours each with 24-hour intervals. The unexposed RUCC was in the same incubator, but in a distance of 35 cm from the PEMF generator. Annexin V-APC (AnV+) labelled was used to determine the percentage of apoptotic cells and propidium iodide (PI+), as standard flow cytometric viability probe to distinguish necrotic cells from viable ones. The results are presented in percentage values. The flow cytometric analysis was carried out on a FACS calibur flow cytometer using Cell-Quest software. In PEMF-unstimulated RUCC, the percentage of AnV+, PI+, and AnV+PI+ positive cells were 1.24±0.34%, 11.03±1.55%, and 12.43±1.96%, respectively. The percentages of AnV+, PI+, and AnV+PI+ positive cells obtained after PEMF stimulation were 1.45±0.16% (p=0.027), 7.03±1.76% (p<0.001), and 9.48±3.40% (p=0.003), respectively. The PEMF stimulation of RUCC induces apoptosis (increase of AnV+ cells) and inhibits necrosis (decrease of PI+ cells) of urothelial cells. This leads us to the conclusion that a low-frequency pulsating electromagnetic field stimulation induces apoptosis and diminishes necrosis of rat urothelial cells in culture.

Flow cytometric detection of Pig-A mutant red blood cells using an erythroid-specific antibody: application of the method for evaluating the in vivo genotoxicity of methylphenidate in adolescent rats.

PubMed

Dobrovolsky, Vasily N; Boctor, Sherin Y; Twaddle, Nathan C; Doerge, Daniel R; Bishop, Michelle E; Manjanatha, Mugimane G; Kimoto, Takafumi; Miura, Daishiro; Heflich, Robert H; Ferguson, Sherry A

2010-03-01

A modified flow cytometry assay for Pig-A mutant rat red blood cells (RBCs) was developed using an antibody that positively identifies rat RBCs (monoclonal antibody HIS49). The assay was used in conjunction with a flow cytometric micronucleus (MN) assay to evaluate gene mutation and clastogenicity/aneugenicity in adolescent male and female rats treated with methylphenidate hydrochloride (MPH). Sprague-Dawley rats were treated orally with 3 mg/kg MPH (70/sex) or water (40/sex) 3 x /day on postnatal days (PNDs) 29-50. Eight additional rats (4/sex) were injected i.p. with N-ethyl-N-nitrosourea (ENU) on PND 28. Blood was collected on PNDs 29, 50, and 90, and used for determining serum MPH levels and/or conducting genotoxicity assays. On the first and last days of MPH treatment (PNDs 29 and 50), serum MPH levels averaged 21 pg/microl, well within the clinical treatment range. Relative to our previously published method (Miura et al. [2008]; Environ Mol Mutagen 49: 614-629), the HIS49 Pig-A mutation assay significantly reduced the background RBC mutant frequency; in the experiments with ENU-treated rats, the modification increased the overall sensitivity of the assay 2-3 fold. Even with the increased assay sensitivity, the 21 consecutive days of MPH treatment produced no evidence of Pig-A mutation induction (measured at PND 90); in addition, MPH treatment did not increase MN frequency (measured at PND 50). These results support the consensus view that the genotoxicity of MPH in pediatric patients reported earlier (El-Zein et al. [2005]: Cancer Lett 230: 284-291) cannot be reproduced in animal models, suggesting that MPH at clinically relevant levels may be nongenotoxic in humans. Published 2009 by Wiley-Liss, Inc.

In-vitro immunosuppression of canine T-lymphocyte-specific proliferation with dexamethasone, cyclosporine, and the active metabolites of azathioprine and leflunomide in a flow-cytometric assay

PubMed Central

Nafe, Laura A.; Dodam, John R.; Reinero, Carol R.

2014-01-01

A high rate of mortality, expense, and complications of immunosuppressive therapy in dogs underscores the need for optimization of drug dosing. The purpose of this study was to determine, using a flow-cytometric assay, the 50% T-cell inhibitory concentration (IC50) of dexamethasone, cyclosporine, and the active metabolites of azathioprine (6-mercaptopurine) and leflunomide (A77 1726) in canine lymphocytes stimulated with concanavalin A (Con A). Whole blood was collected from 5 privately owned, healthy dogs of various ages, genders, and breeds. Peripheral blood mononuclear cells, obtained by density-gradient separation, were cultured for 72 h with Con A, a fluorochrome-tagged cell proliferation dye, and various concentrations of dexamethasone (0.1, 1, 10, 100, 1000, and 10 000 μM), cyclosporine (0.2, 2, 10, 20, 30, 40, 80, and 200 ng/mL), 6-mercaptopurine (0.5, 2.5, 50, 100, 250, and 500 μM), and A77 1726 (1, 5, 10, 25, 50, and 200 μM). After incubation, the lymphocytes were labeled with propidium iodide and an antibody against canine CD5, a pan T-cell surface marker. Flow cytometry determined the percentage of live, proliferating T-lymphocytes incubated with or without immunosuppressants. The mean (± standard error) IC50 was 3460 ± 1900 μM for dexamethasone, 15.8 ± 2.3 ng/mL for cyclosporine, 1.3 ± 0.4 μM for 6-mercaptopurine, and 55.6 ± 22.0 μM for A77 1722. Inhibition of T-cell proliferation by the 4 immunosuppressants was demonstrated in a concentration-dependent manner, with variability between the dogs. These results represent the initial steps to tailor this assay for individual immunosuppressant protocols for dogs with immune-mediated disease. PMID:24982547

Transient changes in bacterioplankton communities induced by the submarine volcanic eruption of El Hierro (Canary Islands).

PubMed

Ferrera, Isabel; Arístegui, Javier; González, José M; Montero, María F; Fraile-Nuez, Eugenio; Gasol, Josep M

2015-01-01

The submarine volcanic eruption occurring near El Hierro (Canary Islands) in October 2011 provided a unique opportunity to determine the effects of such events on the microbial populations of the surrounding waters. The birth of a new underwater volcano produced a large plume of vent material detectable from space that led to abrupt changes in the physical-chemical properties of the water column. We combined flow cytometry and 454-pyrosequencing of 16S rRNA gene amplicons (V1-V3 regions for Bacteria and V3-V5 for Archaea) to monitor the area around the volcano through the eruptive and post-eruptive phases (November 2011 to April 2012). Flow cytometric analyses revealed higher abundance and relative activity (expressed as a percentage of high-nucleic acid content cells) of heterotrophic prokaryotes during the eruptive process as compared to post-eruptive stages. Changes observed in populations detectable by flow cytometry were more evident at depths closer to the volcano (~70-200 m), coinciding also with oxygen depletion. Alpha-diversity analyses revealed that species richness (Chao1 index) decreased during the eruptive phase; however, no dramatic changes in community composition were observed. The most abundant taxa during the eruptive phase were similar to those in the post-eruptive stages and to those typically prevalent in oceanic bacterioplankton communities (i.e. the alphaproteobacterial SAR11 group, the Flavobacteriia class of the Bacteroidetes and certain groups of Gammaproteobacteria). Yet, although at low abundance, we also detected the presence of taxa not typically found in bacterioplankton communities such as the Epsilonproteobacteria and members of the candidate division ZB3, particularly during the eruptive stage. These groups are often associated with deep-sea hydrothermal vents or sulfur-rich springs. Both cytometric and sequence analyses showed that once the eruption ceased, evidences of the volcano-induced changes were no longer observed.

Gallic acid induced apoptotic events in HCT-15 colon cancer cells.

PubMed

Subramanian, Aruna Priyadharshni; Jaganathan, Saravana Kumar; Mandal, Mahitosh; Supriyanto, Eko; Muhamad, Ida Idayu

2016-04-21

To investigate the inhibitory action of diet-derived phenolic compound gallic acid (GA) against HCT-15 colon cancer cells. The antiproliferative effect of GA against colon cancer cells was determined by performing thiazolyl blue tetrazolium bromide (MTT) assay. The colony forming ability of GA treated colon cancer cells was evaluated using the colony forming assay. The cell cycle changes induced by GA in HCT-15 cells were analyzed by propidium iodide staining. Levels of reactive oxygen species (ROS) and mitochondrial membrane potential of HCT-15 exposed to GA was assessed using 2',7'-dichlorfluorescein-diacetate and rhodamine-123 respectively, with the help of flow cytometry. Morphological changes caused by GA treatment in the colon cancer cells were identified by scanning electron microscope and photomicrograph examination. Apoptosis was confirmed using flow cytometric analysis of GA treated HCT-15 cells after staining with Yo-Pro-1. MTT assay results illustrated that GA has an inhibitory effect on HCT-15 cells with IC50 value of 740 μmol/L. A time-dependent inhibition of colony formation was evident with GA treatment. Cell cycle arrest was evident from the accumulation of GA treated HCT-15 cells at sub-G1 phase (0.98 ± 1.03 vs 58.01 ± 2.05) with increasing exposure time. Flow cytometric analysis of GA treated HCT-15 cells depicted early events associated with apoptosis like lipid layer breakage and fall in mitochondrial membrane potential apart from an increase in the generation of ROS which were in a time dependent manner. SEM and photomicrograph images of the GA-treated cells displayed membrane blebbing and cell shrinking characteristics of apoptosis. Further apoptosis confirmation by Yo-Pro-1 staining also showed the time-dependent increase of apoptotic cells after treatment. These results show that GA induced ROS dependent apoptosis and inhibited the growth of colon cancer cells.

Transient Changes in Bacterioplankton Communities Induced by the Submarine Volcanic Eruption of El Hierro (Canary Islands)

PubMed Central

Ferrera, Isabel; Arístegui, Javier; González, José M.; Montero, María F.; Fraile-Nuez, Eugenio; Gasol, Josep M.

2015-01-01

The submarine volcanic eruption occurring near El Hierro (Canary Islands) in October 2011 provided a unique opportunity to determine the effects of such events on the microbial populations of the surrounding waters. The birth of a new underwater volcano produced a large plume of vent material detectable from space that led to abrupt changes in the physical-chemical properties of the water column. We combined flow cytometry and 454-pyrosequencing of 16S rRNA gene amplicons (V1–V3 regions for Bacteria and V3–V5 for Archaea) to monitor the area around the volcano through the eruptive and post-eruptive phases (November 2011 to April 2012). Flow cytometric analyses revealed higher abundance and relative activity (expressed as a percentage of high-nucleic acid content cells) of heterotrophic prokaryotes during the eruptive process as compared to post-eruptive stages. Changes observed in populations detectable by flow cytometry were more evident at depths closer to the volcano (~70–200 m), coinciding also with oxygen depletion. Alpha-diversity analyses revealed that species richness (Chao1 index) decreased during the eruptive phase; however, no dramatic changes in community composition were observed. The most abundant taxa during the eruptive phase were similar to those in the post-eruptive stages and to those typically prevalent in oceanic bacterioplankton communities (i.e. the alphaproteobacterial SAR11 group, the Flavobacteriia class of the Bacteroidetes and certain groups of Gammaproteobacteria). Yet, although at low abundance, we also detected the presence of taxa not typically found in bacterioplankton communities such as the Epsilonproteobacteria and members of the candidate division ZB3, particularly during the eruptive stage. These groups are often associated with deep-sea hydrothermal vents or sulfur-rich springs. Both cytometric and sequence analyses showed that once the eruption ceased, evidences of the volcano-induced changes were no longer observed. PMID:25671714

Clinical CVVH model removes endothelium-derived microparticles from circulation

PubMed Central

Abdelhafeez, Abdelhafeez H.; Jeziorczak, Paul M.; Schaid, Terry R.; Hoefs, Susan L.; Kaul, Sushma; Nanchal, Rahul; Jacobs, Elizabeth R.; Densmore, John C.

2014-01-01

Background Endothelium-derived microparticles (EMPs) are submicron vesicles released from the plasma membrane of endothelial cells in response to injury, apoptosis or activation. We have previously demonstrated EMP-induced acute lung injury (ALI) in animal models and endothelial barrier dysfunction in vitro. Current treatment options for ALI are limited and consist of supportive therapies. We hypothesize that standard clinical continuous venovenous hemofiltration (CVVH) reduces serum EMP levels and may be adapted as a potential therapeutic intervention. Materials and methods EMPs were generated from plasminogen activation inhibitor-1 (PAI-1)-stimulated human umbilical vein endothelial cells (HUVECs). Flow cytometric analysis was used to characterize EMPs as CD31- and annexin V-positive events in a submicron size gate. Enumeration was completed against a known concentration of latex beads. Ultimately, a concentration of ~650,000 EMP/mL perfusate fluid (total 470 mL) was circulated through a standard CVVH filter (pore size 200 μm, flow rate 250 mL/hr) for a period of 70 minutes. 0.5 mL aliquots were removed at 5- to 10-minute intervals for flow cytometric analysis. EMP concentration in the dialysate was measured at the end of 4 hours to better understand the fate of EMPs. Results A progressive decrease in circulating EMP concentration was noted using standard CVVH at 250 mL/hr (a clinical standard rate) from a 470 mL volume modelling a patient's circulation. A 50% reduction was noted within the first 30 minutes. EMPs entering the dialysate after 4 hours were 5.7% of the EMP original concentration. Conclusion These data demonstrate that standard CVVH can remove EMPs from circulation in a circuit modelling a patient. An animal model of hemofiltration with induction of EMP release is required to test the therapeutic potential of this finding and potential of application in early treatment of ALI. PMID:24596654

Gallic acid induced apoptotic events in HCT-15 colon cancer cells

PubMed Central

Subramanian, Aruna Priyadharshni; Jaganathan, Saravana Kumar; Mandal, Mahitosh; Supriyanto, Eko; Muhamad, Ida Idayu

2016-01-01

AIM: To investigate the inhibitory action of diet-derived phenolic compound gallic acid (GA) against HCT-15 colon cancer cells. METHODS: The antiproliferative effect of GA against colon cancer cells was determined by performing thiazolyl blue tetrazolium bromide (MTT) assay. The colony forming ability of GA treated colon cancer cells was evaluated using the colony forming assay. The cell cycle changes induced by GA in HCT-15 cells were analyzed by propidium iodide staining. Levels of reactive oxygen species (ROS) and mitochondrial membrane potential of HCT-15 exposed to GA was assessed using 2’,7’-dichlorfluorescein-diacetate and rhodamine-123 respectively, with the help of flow cytometry. Morphological changes caused by GA treatment in the colon cancer cells were identified by scanning electron microscope and photomicrograph examination. Apoptosis was confirmed using flow cytometric analysis of GA treated HCT-15 cells after staining with Yo-Pro-1. RESULTS: MTT assay results illustrated that GA has an inhibitory effect on HCT-15 cells with IC50 value of 740 μmol/L. A time-dependent inhibition of colony formation was evident with GA treatment. Cell cycle arrest was evident from the accumulation of GA treated HCT-15 cells at sub-G1 phase (0.98 ± 1.03 vs 58.01 ± 2.05) with increasing exposure time. Flow cytometric analysis of GA treated HCT-15 cells depicted early events associated with apoptosis like lipid layer breakage and fall in mitochondrial membrane potential apart from an increase in the generation of ROS which were in a time dependent manner. SEM and photomicrograph images of the GA-treated cells displayed membrane blebbing and cell shrinking characteristics of apoptosis. Further apoptosis confirmation by Yo-Pro-1 staining also showed the time-dependent increase of apoptotic cells after treatment. CONCLUSION: These results show that GA induced ROS dependent apoptosis and inhibited the growth of colon cancer cells. PMID:27099438

IgA and IgG1 reactivities assessed by flow cytometry mirror clinical aspects of infants with ocular congenital toxoplasmosis.

PubMed

de Jesus, Laura Néspoli Nassar Pansini; Tonini, Aline de Castro Zacche; Barros, Geisa Baptista; Coelho-dos-Reis, Jordana Grazziela A; Béla, Samantha Ribeiro; Antonelli, Lis Ribeiro do Valle; Machado, Anderson Silva; Carneiro, Ana Carolina Aguiar Vasconcelos; Andrade, Gláucia Manzan Queiroz; Vasconcelos-Santos, Daniel Vitor; Januário, José Nélio; Teixeira-Carvalho, Andréa; Vitor, Ricardo Wagner Almeida; Ferro, Eloísa A V; Mineo, José Roberto; Bahia-Oliveira, Lilian Maria Garcia; Martins-Filho, Olindo Assis; Lemos, Elenice Moreira

2016-01-01

This study intended to apply the flow cytometric analysis of IgA and IgG reactivity and intracytoplasmic cytokine analysis to understand and decode the clinical aspects of infants with ocular congenital toxoplasmosis. The Toxoplasma gondii-infected infants (TOXO) were subdivided according to their clinical aspects based on the absence (NRL), presence of active (ARL), active/cicatricial (ACRL) or cicatricial retinochoroidal lesions (CRL) and compared to non-infected controls (NI). The reactivity of anti-T. gondii IgG subclasses resembles the clinical aspects of ocular lesions. IgG and IgG1 discriminate infants with cicatricial lesions (ACRL and CRL) from both ARL and NLR. IgG2 and IgG3 are particularly higher in ACRL and CRL as compared to NLR. No differences were observed when IgG4 reactivity was evaluated. Thus, the results indicated that the reactivity patterns of IgA, IgG and IgG subclasses are able to discriminate ARL, ACRL and CRL from NLR or NI. IgA and IgG subclasses are relevant serological biomarkers with diagnostic and prognostic applicability, respectively. Moreover, IgA and IgG1 were closely related to cytokine production by innate/adaptive immunity cells. IgA reactivity was directly associated to TNF-α-derived from neutrophils, monocytes and CD8(+) T-cells, while IgG1 was inversely correlated with IFN-γ-producing CD4(+) and CD8(+) T-cells but positively correlated with IL-10(+) B-cells. These findings provide insights on the relationship between the cytokine production by innate/adaptive immunity and the antibody pattern of infants with ocular congenital toxoplasmosis. In addition, the present study supports the use of flow cytometric serology as a potential tool for the diagnosis and monitoring of ocular lesions in T. gondii-infected infants in the clinical setting. Copyright © 2015 Elsevier B.V. All rights reserved.

Flow cytometric sorting of fresh and frozen-thawed spermatozoa in the western lowland gorilla (Gorilla gorilla gorilla).

PubMed

O'Brien, J K; Stojanov, T; Crichton, E G; Evans, K M; Leigh, D; Maxwell, W M C; Evans, G; Loskutoff, N M

2005-08-01

We adapted flow cytometry technology for high-purity sorting of X chromosome-bearing spermatozoa in the western lowland gorilla (Gorilla gorilla gorilla). Our objectives were to develop methodologies for liquid storage of semen prior to sorting, sorting of liquid-stored and frozen-thawed spermatozoa, and assessment of sorting accuracy. In study 1, the in vitro sperm characteristics of gorilla ejaculates from one male were unchanged (P > 0.05) after 8 hr of liquid storage at 15 degrees C in a non-egg yolk diluent (HEPES-buffered modified Tyrode's medium). In study 2, we examined the efficacy of sorting fresh and frozen-thawed spermatozoa using human spermatozoa as a model for gorilla spermatozoa. Ejaculates from one male were split into fresh and frozen aliquots. X-enriched samples derived from both fresh and frozen-thawed human semen were of high purity, as determined by fluorescence in situ hybridization (FISH; 90.7%+/-2.3%, overall), and contained a high proportion of morphologically normal spermatozoa (86.0%+/-1.0%, overall). In study 3, we processed liquid-stored semen from two gorillas for sorting using a modification of methods for human spermatozoa. The sort rate for enrichment of X-bearing spermatozoa was 7.3+/-2.5 spermatozoa per second. The X-enriched samples were of high purity (single-sperm PCR: 83.7%) and normal morphology (79.0%+/-3.9%). In study 4 we examined frozen-thawed gorilla semen, and the sort rate (8.3+/-2.9 X-bearing sperm/sec), purity (89.7%), and normal morphology (81.4%+/-3.4%) were comparable to those of liquid-stored semen. Depending on the male and the type of sample used (fresh or frozen-thawed), 0.8-2.2% of gorilla spermatozoa in the processed ejaculate were present in the X-enriched sample. These results demonstrate that fresh or frozen-thawed gorilla spermatozoa can be flow cytometrically sorted into samples enriched for X-bearing spermatozoa. Copyright 2005 Wiley-Liss, Inc.

Development of a Cell Sheet Transportation Technique for Regenerative Medicine

PubMed Central

Oie, Yoshinori; Nozaki, Takayuki; Takayanagi, Hiroshi; Hara, Susumu; Hayashi, Ryuhei; Takeda, Shizu; Mori, Keisuke; Moriya, Noboru; Soma, Takeshi; Tsujikawa, Motokazu; Saito, Kazuo

2014-01-01

Purpose: A transportation technique for cell sheets is necessary to standardize regenerative medicine. The aim of this article is to develop and evaluate a new transportation technique for cell sheets. Material and Methods: We developed a transportation container with three basic functions: the maintenance of interior temperature, air pressure, and sterility. The interior temperature and air pressure were monitored by a recorder. Human oral mucosal epithelial cells obtained from two healthy volunteers were cultured on temperature-responsive culture dishes. The epithelial cell sheets were transported via an airplane between the Osaka University and Tohoku University using the developed cell transportation container. Histological and immunohistochemical analyses and flow cytometric analyses for cell viability and cell purity were performed for the cell sheets before and 12 h after transportation to assess the influence of transportation on the cell sheets. Sterility tests and screening for endotoxin and mycoplasma in the cell sheets were performed before and after transportation. Results: During transportation via an airplane, the temperature inside the container was maintained above 32°C, and the changes in air pressure remained within 10 hPa. The cell sheets were well stratified and successfully harvested before and after transportation. The expression patterns of keratin 3/76, p63, and MUC16 were equivalent before and after transportation. However, the expression of ZO-1 in the cell sheet after transportation was slightly weaker than that before transportation. The cell viability was 72.0% before transportation and 77.3% after transportation. The epithelial purity was 94.6% before transportation and 87.9% after transportation. Sterility tests and screening for endotoxin and mycoplasma were negative for all cell sheets. Conclusion: The newly developed transportation technique for air travel is essential technology for regenerative medicine and promotes the standardization and spread of regenerative therapies. PMID:24044382

Development of a cell sheet transportation technique for regenerative medicine.

PubMed

Oie, Yoshinori; Nozaki, Takayuki; Takayanagi, Hiroshi; Hara, Susumu; Hayashi, Ryuhei; Takeda, Shizu; Mori, Keisuke; Moriya, Noboru; Soma, Takeshi; Tsujikawa, Motokazu; Saito, Kazuo; Nishida, Kohji

2014-05-01

A transportation technique for cell sheets is necessary to standardize regenerative medicine. The aim of this article is to develop and evaluate a new transportation technique for cell sheets. We developed a transportation container with three basic functions: the maintenance of interior temperature, air pressure, and sterility. The interior temperature and air pressure were monitored by a recorder. Human oral mucosal epithelial cells obtained from two healthy volunteers were cultured on temperature-responsive culture dishes. The epithelial cell sheets were transported via an airplane between the Osaka University and Tohoku University using the developed cell transportation container. Histological and immunohistochemical analyses and flow cytometric analyses for cell viability and cell purity were performed for the cell sheets before and 12 h after transportation to assess the influence of transportation on the cell sheets. Sterility tests and screening for endotoxin and mycoplasma in the cell sheets were performed before and after transportation. During transportation via an airplane, the temperature inside the container was maintained above 32°C, and the changes in air pressure remained within 10 hPa. The cell sheets were well stratified and successfully harvested before and after transportation. The expression patterns of keratin 3/76, p63, and MUC16 were equivalent before and after transportation. However, the expression of ZO-1 in the cell sheet after transportation was slightly weaker than that before transportation. The cell viability was 72.0% before transportation and 77.3% after transportation. The epithelial purity was 94.6% before transportation and 87.9% after transportation. Sterility tests and screening for endotoxin and mycoplasma were negative for all cell sheets. The newly developed transportation technique for air travel is essential technology for regenerative medicine and promotes the standardization and spread of regenerative therapies.

Photoactive platinum diimine complexes showing induced cancer cell death by apoptosis.

PubMed

Zhang, Zhigang; Dai, Ruihui

2017-02-01

Photoinduced cytotoxicity mediated by a triphenylenamine-modified platinum diimine complex in human breast adenocarcinoma cells has been studied by cell viability assay. The triphenylenamine-modified platinum diimine complex showed more potent cytotoxicity in light than its carboxylate-modified analogue. To gain insights into the mechanism of photodynamic activity of this class of platinum diimine complexes, flow cytometric analyses were performed. The results suggest that upon irradiation the two platinum diimine complexes studied could induce cell cycle arrest in G 2 /M or S phase, and both of them could induce cancer cell death by apoptosis.

Optimization of Dendritic Cell-Mediated Cytotoxic T-Cell Activation by Tracking of Dendritic Cell Migration Using Reporter Gene Imaging.

PubMed

Lee, Hongje; Lee, Ho Won; La Lee, You; Jeon, Yong Hyun; Jeong, Shin Young; Lee, Sang-Woo; Lee, Jaetae; Ahn, Byeong-Cheol

2018-06-01

The aim of this study is to optimize the dendritic cell (DC)-mediated T-cell activation using reporter gene imaging and flow cytometric analysis in living mice. A murine dendritic cell line (DC2.4) co-expressing effluc and Thy1.1 genes were established by transfection with retroviral vectors. Thy1.1 positive cells were sorted by magnetic bead separation system (DC2.4/effluc). Cell proliferation assay and phenotype analysis to determine the effects of gene transduction on the function of dendritic cells between parental DC2.4 and DC2.4/effluc were performed. To optimize the DC-mediated immune response by cell number or frequency, different cell numbers (5 × 10 5 , 1 × 10 6 , and 2 × 10 6  DC2.4/effluc) or different frequencies of DC2.4/effluc (first, second, and third injections) were injected in the right footpad of mice. The migration of the DC2.4/effluc into the draining popliteal lymph node of mice was monitored by bioluminescence imaging (BLI). Flow cytometric analysis was performed with splenocytes to determine the cytotoxic T-cell population after injection of DC2.4/effluc. Parental DC2.4 and DC2.4/effluc exhibit no significant differences in their proliferation and phenotype. BLI signals were observed in the draining popliteal lymph node at day 1 after injection of DC2.4/effluc in 1 × 10 6 and 2 × 10 6 cells-injected groups. The highest BLI signal intensity was detected in 2 × 10 6 cells-injected mice. On day 11, the BLI signal was detected in only 2 × 10 6 cell-injected group but not in other groups. Optimized cell numbers (2 × 10 6 ) were injected in three animal groups with a different frequency (first, second, and third injection groups). The BLI signal was detected at day 1 and maintained until day 7 in the first injection group, but there is low signal intensity in the second and the third injection groups. Although the expression levels of Thy1.1 gene in the first injection group were very high, there reveals no expression of Thy1.1 gene in the second and the third injection groups. The number of tumor-specific CD8 + T-cells in the spleen significantly increased, as the number of DC injections increases. Successful optimization of DC-mediated cytotoxic T-cell activation in living mice using reporter gene imaging and flow cytometric analysis was achieved. The optimization of DC-mediated cytotoxic T-cell activation could be applied for the future DC-based immunotherapy.

Microfluidic Preparation of Polymer-Nucleic Acid Nanocomplexes Improves Nonviral Gene Transfer

NASA Astrophysics Data System (ADS)

Grigsby, Christopher L.; Ho, Yi-Ping; Lin, Chao; Engbersen, Johan F. J.; Leong, Kam W.

2013-11-01

As the designs of polymer systems used to deliver nucleic acids continue to evolve, it is becoming increasingly apparent that the basic bulk manufacturing techniques of the past will be insufficient to produce polymer-nucleic acid nanocomplexes that possess the uniformity, stability, and potency required for their successful clinical translation and widespread commercialization. Traditional bulk-prepared products are often physicochemically heterogeneous and may vary significantly from one batch to the next. Here we show that preparation of bioreducible nanocomplexes with an emulsion-based droplet microfluidic system produces significantly improved nanoparticles that are up to fifty percent smaller, more uniform, and are less prone to aggregation. The intracellular integrity of nanocomplexes prepared with this microfluidic method is significantly prolonged, as detected using a high-throughput flow cytometric quantum dot Förster resonance energy transfer nanosensor system. These physical attributes conspire to consistently enhance the delivery of both plasmid DNA and messenger RNA payloads in stem cells, primary cells, and human cell lines. Innovation in processing is necessary to move the field toward the broader clinical implementation of safe and effective nonviral nucleic acid therapeutics, and preparation with droplet microfluidics represents a step forward in addressing the critical barrier of robust and reproducible nanocomplex production.

Mannich bases of 1,2,4-triazole-3-thione containing adamantane moiety: Synthesis, preliminary anticancer evaluation, and molecular modeling studies.

PubMed

Milošev, Milorad Z; Jakovljević, Katarina; Joksović, Milan D; Stanojković, Tatjana; Matić, Ivana Z; Perović, Milka; Tešić, Vesna; Kanazir, Selma; Mladenović, Milan; Rodić, Marko V; Leovac, Vukadin M; Trifunović, Snežana; Marković, Violeta

2017-06-01

A series of 18 novel N-Mannich bases derived from 5-adamantyl-1,2,4-triazole-3-thione was synthesized and characterized using NMR spectroscopy and X-ray diffraction technique. All derivatives were evaluated for their anticancer potential against four human cancer cell lines. Several tested compounds exerted good cytotoxic activities on K562 and HL-60 cell lines, along with pronounced selectivity, showing lower cytotoxicity against normal fibroblasts MRC-5 compared to cancer cells. The effects of compounds 5b, 5e, and 5j on the cell cycle were investigated by flow cytometric analysis. It was found that these compounds cause the accumulation of cells in the subG1 and G1 phases of the cell cycle and induce caspase-dependent apoptosis, while the anti-angiogenic effects of 5b, 5e, and 5j have been confirmed in EA.hy926 cells using a tube formation assay. Further, the interaction of Bax protein with compound 5b was investigated by means of molecular modeling, applying the combined molecular docking/molecular dynamics approach. © 2016 John Wiley & Sons A/S.

Meroterpenoids with Antitumor Activities from Guava (Psidium guajava).

PubMed

Qin, Xu-Jie; Yu, Qian; Yan, Huan; Khan, Afsar; Feng, Mi-Yan; Li, Pan-Pan; Hao, Xiao-Jiang; An, Lin-Kun; Liu, Hai-Yang

2017-06-21

Psidium guajava L., a species native to South America, has been widely cultivated in the tropical and subtropical areas of China for its popular fruits. The preliminary analysis by liquid chromatography-ultraviolet (LC-UV) indicated the presence of meroterpenoids in the fruits of P. guajava (guava). Subsequent fractionation of the petroleum ether extract resulted in the identification of two new meroterpenoids, psiguajavadials A (1) and B (2), together with 14 previously described meroterpenoids (3-16). Their structures were fully elucidated by comprehensive spectroscopic techniques and theoretical calculations. All of the meroterpenoids showed cytotoxicities against five human cancer cell lines, with guajadial B (12) being the most effective having an IC 50 value of 150 nM toward A549 cells. Furthermore, biochemical topoisomerase I (Top1) assay revealed that psiguajavadial A (1), psiguajavadial B (2), guajadial B (12), guajadial C (14), and guajadial F (16) acted as Top1 catalytic inhibitors and delayed Top1 poison-mediated DNA damage. The flow cytometric analysis indicated that the new meroterpenoids psiguajavadials A (1) and B (2) could induce apoptosis of HCT116 cells. These data suggest that meroterpenoids from guava fruit could be used for the development of antitumor agents.

Synthesis of 1,2,4-triazole-linked urea/thiourea conjugates as cytotoxic and apoptosis inducing agents.

PubMed

Tokala, Ramya; Bale, Swarna; Janrao, Ingle Pavan; Vennela, Aluri; Kumar, Niggula Praveen; Senwar, Kishna Ram; Godugu, Chandraiah; Shankaraiah, Nagula

2018-06-01

A new series of 1,2,4-triazole-linked urea and thiourea conjugates have been synthesized and evaluated for their in vitro cytotoxicity against selected human cancer cell lines namely, breast (MCF-7, MDA-MB-231), lung (A549) prostate (DU145) and one mouse melanoma (B16-F10) cell line and compared with reference drug. The compound 5t showed significant cytotoxicity on MCF-7 breast cancer cell line with a IC 50 value of 7.22 ± 0.47 µM among all the tested compounds. Notably, induction of apoptosis by compound 5t on MCF-7 cells was evaluated using different staining techniques such as acridine orange/ethidium bromide (AO/EB), annexin V-FITC/PI, and DAPI. Further, clonogenic assay indicates the inhibition of colony formation on MCF-7 cells by compound 5t. Moreover, the flow-cytometric analysis also revealed that compound 5t caused the arrest of cells at G0/G1 phase of cell cycle. In addition, the compounds when tested on normal human cells (L-132) were found to be safer with low cytotoxicity profile. Copyright © 2018 Elsevier Ltd. All rights reserved.

Development of a monoclonal anitbody to immuno-cytochemical analysis of the cellular localization of the peripheral benzodiazepine receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dussossoy, D.; Carayon, P.; Feraut, D.

1996-05-01

Based on the amino acid sequence deduced from the cloned human peripheral benzodiazepine receptor (PBR) gene, monoclonal antibody (Mab 8D7) was produced against the C-terminal fragment of the receptor. Immunoblot experiments, performed against purified PBR, indicated that the antipeptide antibody recognized, under denaturing conditions, the corresponding amino acid sequence of the PBR. When mitochondrial membranes form PBR transfected yeast or from THP1 and U937 cells were used on immunoblot analysis, a high level of immunoreactivity was observed at 18 kDa, the PBR molecular mass deduced from cDNA, establishing the specificity of the antibody for the receptor. Moreover, binding experiments realizedmore » with intact mitochondria demonstrated that the immunogenic sequence was accessible to the antibody indicating that the C-terminal fragment of the PBR faces the cytosol. Using this Mab we developed a technique which allowed precise quantification of PBR density per cell. Furthermore, cellular localization studies by flow cytometric analysis and confocal microscopy on cell lines displaying different levels of PBR showed that Mab 8D7 was entirely colocalized with an antimitochondria Mab. 34 refs., 7 figs.« less

Comparison of quantitative flow cytometric data provided by panels with lower and increased color number

NASA Astrophysics Data System (ADS)

Bocsi, József; Mittag, Anja; Pierzchalski, Arkadiusz; Baumgartner, Adolf; Dähnert, Ingo; Tárnok, Attila

2012-03-01

To date the flow cytometry (FCM) industry is booming with new generations of commercial clinical instruments. Long-term clinical studies have the dilemma that moving to new instruments being capable of more complex cell-analysis makes it difficult to compare new data with those obtained on older instruments with less complex analysis panels. Since 15 years we conduct follow-up studies on children with congenital heart diseases. In this period we moved from 2- to 3- and now to 10-color FCM immunophenotyping panels. Questions arise how to compare and transfer data from lower to higher level of complexity. Two comparable antibody panels for leukocyte immunophenotyping (12-tube 2-colors, and 9-tube 4-colors) were measured on a BD FACScalibur FCM (calibration: Spherotech beads) in 19 blood samples from children with congenital heart disease. This increase of colors was accompanied by moving antibodies that were in the 2-color panel either FITC or PE labeled to red dyes such as PerCP or APC. Algorithms were developed for bridging data for quantitative characterization of antigen expression (mean fluorescence intensity) and frequency of different cell subpopulations in combination with rainbow bead standard data. This approach worked for the most relevant antibodies (CD3, CD4, CD8 etc.) well, but rendered substantial uncertainty for activation markers (CD69 etc.). Our techniques are particularly well suited to the analysis in long-term studies and have the potential to compare older and recent results in a standardized way.

Detection of early changes in lung cell cytology by flow-systems analysis techniques. Progress report, July 1--December 31, 1977

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steinkamp, J.A.; Hansen, K.M.; Wilson, J.S.

1978-04-01

This report summarizes ongoing experiments to develop cytological and biochemical indicators for measuring damage to respiratory tract cells exposed by inhalation of environmental toxic agents. The specific goal of this project is to apply flow cytometric methods to analyze and detect changes in lung epithelium as a function of exposure to toxic agents such as those associated with the production of synthetic fuels from oil shale and coal. The objectives during the past 6 months were to complete modifications to the multiparameter cell separator by adding a krypton laser with an output capability of specific wavelengths ranging from the uvmore » to the ir; analyze and separate lung cells based on their DNA content; evaluate some new fluorescent DNA and protein stains; and treat hamster lung cells with proteolytic enzymes for increasing cell yield. Future experiments will involve the continued analysis and characterization of exfoliated lung cells based primarily on cellular DNA content, protein, morphological features, and specific enzyme activities; quantitation of macrophage activity; exposure of hamsters to toxic agents such as oil shale particulates and ozone; and continued analysis of cells based on DNA content. As this new technology becomes adapted to analyzing respiratory tract cells, the measurement of physical and biochemical cell properties as a function of exposure to toxic agents will be increased. This analytical approach is designed to assist in the establishment of guidelines for estimating risks to exposed humans.« less

Evidence of progenitor cells in the adult human cochlea: sphere formation and identification of ABCG2.

PubMed

Massucci-Bissoli, Milene; Lezirovitz, Karina; Oiticica, Jeanne; Bento, Ricardo Ferreira

2017-11-01

The aim of this study was to search for evidence of stem or progenitor cells in the adult human cochlea by testing for sphere formation capacity and the presence of the stem cell marker ABCG2. Cochleas removed from patients undergoing vestibular schwannoma resection (n=2) and from brain-dead organ donors (n=4) were dissociated for either flow cytometry analysis for the stem cell marker ABCG2 or a sphere formation assay that is widely used to test the sphere-forming capacity of cells from mouse inner ear tissue. Spheres were identified after 2-5 days in vitro, and the stem cell marker ABCG2 was detected using flow cytometric analysis after cochlear dissociation. Evidence suggests that there may be progenitor cells in the adult human cochlea, although further studies are required.

Flow cytometric single cell analysis reveals heterogeneity between adipose depots

PubMed Central

Boumelhem, Badwi B.; Assinder, Stephen J.; Bell-Anderson, Kim S.; Fraser, Stuart T.

2017-01-01

ABSTRACT Understanding adipose tissue heterogeneity is hindered by the paucity of methods to analyze mature adipocytes at the single cell level. Here, we report a system for analyzing live adipocytes from different adipose depots in the adult mouse. Single cell suspensions of buoyant adipocytes were separated from the stromal vascular fraction and analyzed by flow cytometry. Compared to other lipophilic dyes, Nile Red uptake effectively distinguished adipocyte populations. Nile Red fluorescence increased with adipocyte size and granularity and could be combined with MitoTracker® Deep Red or fluorescent antibody labeling to further dissect adipose populations. Epicardial adipocytes exhibited the least mitochondrial membrane depolarization and highest fatty-acid translocase CD36 surface expression. In contrast, brown adipocytes showed low surface CD36 expression. Pregnancy resulted in reduced mitochondrial membrane depolarisation and increased CD36 surface expression in brown and epicardial adipocyte populations respectively. Our protocol revealed unreported heterogeneity between adipose depots and highlights the utility of flow cytometry for screening adipocytes at the single cell level. PMID:28453382

Evaluation of antibacterial properties of novel phthalocyanines against Escherichia coli--comparison of analytical methods.

PubMed

Mikula, Premysl; Kalhotka, Libor; Jancula, Daniel; Zezulka, Stepan; Korinkova, Radka; Cerny, Jiri; Marsalek, Blahoslav; Toman, Petr

2014-09-05

We analyzed antibacterial effects of several novel phthalocyanines against Escherichia coli and evaluated the suitability of flow cytometry for the detection of antibacterial effects of phthalocyanines in comparison with routinely used cultivation. After 3h of exposure under cool white light eight cationic phthalocyanines showed very high antibacterial activity in the concentration of 2.00 mg L(-1) and four of them were even efficient in the concentration of 0.20 mg L(-1). Antibacterial activity of neutral and anionic compounds was considerably lower or even negligible. No antibacterial effect was detected when bacteria were exposed without illumination. Binding affinity to bacterial cells was found to represent an important parameter influencing phthalocyanine antibacterial activity that can be modified by total charge of peripheral substituents and by the presence of suitable functional groups inside them. Agglomeration of cells observed in suspensions treated with a higher concentration of certain cationic phthalocyanines (the strongest binders to bacterial membrane) affected cytometric measurements of total cell counts, thus without appropriate pretreatment of the sample before analysis this parameter seems not to be fully valid in the evaluation of phthalocyanine antibacterial activity. Cytometric measurement of cell membrane integrity appears to be a suitable and even more sensitive parameter than cultivation. Copyright © 2014 Elsevier B.V. All rights reserved.

Diploid endosperm formation in Tulipa spp. and identification of a 1:1 maternal-to-paternal genome ratio in endosperms of T. gesneriana L.

PubMed

Mizuochi, Hitoshi; Matsuzaki, Hironori; Moue, Takehiko; Okazaki, Keiichi

2009-03-01

Most Liliaceae plants have the tetrasporic Fritillaria-type embryo sac and normally form diploid embryos and pentaploid endosperms derived from a 4:1 maternal-to-paternal genome ratio (4m:1p) after double fertilization. Here we characterize embryo sac and endosperm formation in Tulipa spp. of Liliaceae. Chromosome analysis using seeds derived from 2x x 2x crosses of Tulipa gesneriana (2n = 2x = 24) identified diploid chromosome number in the endosperm. Similarly, flow cytometric analysis confirmed diploid endosperm formation in T. gesneriana, T. fosteriana (2n = 2x = 24) and T. greigii (2n = 2x = 24). To further study the possible mechanism of diploid endosperm formation, we made interploidy crosses of triploid (2n = 3x = 36) x diploid in which aneuploid seeds with various chromosome numbers (2n = 25-36) were produced. Again, flow cytometric analysis confirmed the same ploidy level in both embryos and endosperms at all aneuploidy levels, suggesting that only a single haploid polar nucleus contributes to endosperm formation at fertilization. Histological observation further confirmed the physical separation of two polar nuclei by a large vacuole in the Fritillaria-type embryo sac of T. gesneriana that appeared to prevent the fusion of the two polar nuclei that originated at the micropylar and chalazal ends before fertilization. Taken together, these results indicate that diploid endosperms (1m:1p) are normally formed in Tulipa spp. by fusion of the micropylar polar nucleus (n) and a spermatid (n) but not by normal triple fusion. We also show that tulip endosperm partially overcomes the triploid block mechanism that occurs in interploidy crosses. Based on these observations, the possible role of triple nuclear fusion in double fertilization is discussed.

Ductal carcinoma of breast: nuclear grade as a predictor of S-phase fraction.

PubMed

Dabbs, D J

1993-06-01

Nuclear grade (NG) and S-phase fraction (SPF) are established independent prognostic variables for ductal breast carcinomas. Nuclear grade can be assigned by a pathologist in a simple fashion during histopathologic evaluation of the tumor, while SPF requires flow cytometric evaluation of tumor samples. This prospective study was undertaken to determine whether elevated SPF could be predicted from NG alone and how NG and SPF correlate with c-erbB-2 expression. Eighty-two breast carcinomas of ductal type were assigned an NG of low (grade 1 or grade 2) or high (grade 3). S-phase fraction was recorded initially from fresh-frozen tissue samples and was designated as either low SPF (below the value designated as the cutoff for elevated SPF) or high SPF (a value at or greater than the cutoff value). On fresh tissue the NG predicted the range of SPF (low or high) in 89% of cases. Four percent of the cases that did not correlate could definitely be attributed to sample error. The remaining 7% that did not correlate could have been due to sample error, specimen quality, or tumor heterogeneity, as demonstrated by reversal of SPF range as performed on paraffin blocks of tumor. Eighty-eight percent of the tumors positive for c-erbB-2 were NG 3 and 12% were NG 2. All c-erbB-2 tumors were aneuploid. This study demonstrates the importance of carefully assigning NGs on tissue and indicates the importance of reviewing flow cytometric data side by side with histopathologic parameters to detect discrepancies between these two modalities. Careful nuclear grading assignment can accurately predict the range of SPF.

Optimized multiparametric flow cytometric analysis of circulating endothelial cells and their subpopulations in peripheral blood of patients with solid tumors: a technical analysis.

PubMed

Zhou, Fangbin; Zhou, Yaying; Yang, Ming; Wen, Jinli; Dong, Jun; Tan, Wenyong

2018-01-01

Circulating endothelial cells (CECs) and their subpopulations could be potential novel biomarkers for various malignancies. However, reliable enumerable methods are warranted to further improve their clinical utility. This study aimed to optimize a flow cytometric method (FCM) assay for CECs and subpopulations in peripheral blood for patients with solid cancers. An FCM assay was used to detect and identify CECs. A panel of 60 blood samples, including 44 metastatic cancer patients and 16 healthy controls, were used in this study. Some key issues of CEC enumeration, including sample material and anticoagulant selection, optimal titration of antibodies, lysis/wash procedures of blood sample preparation, conditions of sample storage, sufficient cell events to enhance the signal, fluorescence-minus-one controls instead of isotype controls to reduce background noise, optimal selection of cell surface markers, and evaluating the reproducibility of our method, were integrated and investigated. Wilcoxon and Mann-Whitney U tests were used to determine statistically significant differences. In this validation study, we refined a five-color FCM method to detect CECs and their subpopulations in peripheral blood of patients with solid tumors. Several key technical issues regarding preanalytical elements, FCM data acquisition, and analysis were addressed. Furthermore, we clinically validated the utility of our method. The baseline levels of mature CECs, endothelial progenitor cells, and activated CECs were higher in cancer patients than healthy subjects ( P <0.01). However, there was no significant difference in resting CEC levels between healthy subjects and cancer patients ( P =0.193). We integrated and comprehensively addressed significant technical issues found in previously published assays and validated the reproducibility and sensitivity of our proposed method. Future work is required to explore the potential of our optimized method in clinical oncologic applications.

Interleukin-15 receptor blockade in non-human primate kidney transplantation.

PubMed

Haustein, Silke; Kwun, Jean; Fechner, John; Kayaoglu, Ayhan; Faure, Jean-Pierre; Roenneburg, Drew; Torrealba, Jose; Knechtle, Stuart J

2010-04-27

Interleukin (IL)-15 is a chemotactic factor to T cells. It induces proliferation and promotes survival of activated T cells. IL-15 receptor blockade in mouse cardiac and islet allotransplant models has led to long-term engraftment and a regulatory T-cell environment. This study investigated the efficacy of IL-15 receptor blockade using Mut-IL-15/Fc in an outbred non-human primate model of renal allotransplantation. Male cynomolgus macaque donor-recipient pairs were selected based on ABO typing, major histocompatibility complex class I typing, and carboxy-fluorescein diacetate succinimidyl ester-based mixed lymphocyte responses. Once animals were assigned to one of six treatment groups, they underwent renal transplantation and bilateral native nephrectomy. Serum creatinine level was monitored twice weekly and as indicated, and protocol biopsies were performed. Rejection was defined as a increase in serum creatinine to 1.5 mg/dL or higher and was confirmed histologically. Complete blood counts and flow cytometric analyses were performed periodically posttransplant; pharmacokinetic parameters of Mut-IL-15/Fc were assessed. Compared with control animals, Mut-IL-15/Fc-treated animals did not demonstrate increased graft survival despite adequate serum levels of Mut-IL-15/Fc. Flow cytometric analysis of white blood cell subgroups demonstrated a decrease in CD8 T-cell and natural killer cell numbers, although this did not reach statistical significance. Interestingly, two animals receiving Mut-IL-15/Fc developed infectious complications, but no infection was seen in control animals. Renal pathology varied widely. Peritransplant IL-15 receptor blockade does not prolong allograft survival in non-human primate renal transplantation; however, it reduces the number of CD8 T cells and natural killer cells in the peripheral blood.

Protective effects of melatonin against 12C6+ beam irradiation-induced oxidative stress and DNA injury in the mouse brain

NASA Astrophysics Data System (ADS)

Wu, Z. H.; Zhang, H.; Wang, X. Y.; Yang, R.; Liu, B.; Liu, Y.; Zhao, W. P.; Feng, H. Y.; Xue, L. G.; Hao, J. F.; Niu, B. T.; Wang, Z. H.

2012-01-01

The purpose of this experiment was to estimate the protective effects of melatonin against radiation-induced brain damages in mice induced by heavy ion beams. Kun-Ming mice were randomly divided into five groups: normal control group, irradiation control group, and three different doses of melatonin (5, 10, and 20 mg/kg, i.p.) treated groups. Apart from the normal control group, the other four groups were exposed to whole-body 4.0 Gy carbon ion beam irradiation (approximately 0.5 Gy/min) after i.p. administration of normal saline or melatonin 1 h before irradiation. The oxidative redox status of brain tissue was assessed by measurement of malondiadehyde (MDA) levels, total superoxide dismutase (T-SOD), cytosolic superoxide dismutase (Cu/ZnSOD, SOD1) and mitochondrial superoxide dismutase (MnSOD, SOD2) activities at 8 h after irradiation. DNA damages were determined using the Comet assay and apoptosis and cell cycle distribution were detected by flow cytometric analyses. A dramatic dose-dependent decrease in MDA levels, tail moment, rates of tailing cells, and apoptosis, and a dose-dependent increase in T-SOD and SOD2 activities, in brain tissues in the melatonin-treated groups were detected compared with the irradiation only group. Furthermore, flow cytometric analysis demonstrated that the percentage of brain cells in the G0/G1 phase decreased significantly, while those in the S and G2/M stage increased dramatically, with mice pretreated with melatonin compared to the irradiation control group. These data indicate that melatonin has protective effects against irradiation-induced brain injury, and that its underlying protective mechanisms may relate to modulation of oxidative stress induced by heavy ionirradiation.

Highly sensitive assays are mandatory for the differential diagnosis of patients presenting with symptoms of mast cell activation: diagnostic work-up of 38 patients.

PubMed

Van den Poel, Bea; Kochuyt, Anne-Marie; Del Biondo, Elke; Dewaele, Barbara; Lierman, Els; Tousseyn, Thomas; de Hertogh, Gert; Vandenberghe, Peter; Boeckx, Nancy

2017-04-01

Mastocytosis is a heterogeneous disease caused by excessive mast cell (MC) proliferation. Diagnosis of systemic mastocytosis (SM) is based on the presence of major and minor criteria defined by the World Health Organization. Symptoms of MC activation can also occur in patients without SM or without allergic or inflammatory disease. These MC activation syndromes (MCAS) can be divided into primary (monoclonal) MCAS (MMAS) vs. secondary and idiopathic MCAS. In this single center study, the diagnostic work-up of 38 patients with a clinical suspicion of SM and/or with elevated basic tryptase levels is presented. Clinical symptoms, biochemical parameters, results of bone marrow investigation, flow cytometric immunophenotyping, and molecular analysis were retrospectively reviewed. Twenty-three patients were found to have a monoclonal MC disorder of which 19 were diagnosed with SM and 4 with MMAS. In 13/19 SM patients, multifocal MC infiltrates in the bone marrow were found (major criterion), while in 6 the diagnosis was based on the presence of ≥3 minor criteria. Flow cytometric analysis of bone marrow showed CD25 expression of MCs in all patients with SM and MMAS (range: 0.002-0.3% of cells). In bone marrow, the KIT D816V mutation was detected in all SM patients but in only 2 patients with MMAS (range: 0.007-9% mutated cells). Basic tryptase elevation was demonstrated in 16/19 patients with SM but also in 9/19 patients without SM. Our study reveals the heterogeneity of primary MC disorders and the importance of sensitive assays in patients suspected of having SM.

Inositol-requiring enzyme 1-mediated endoplasmic reticulum stress triggers apoptosis and fibrosis formation in liver cirrhosis rat models.

PubMed

Jiang, Tianpeng; Wang, Lizhou; Li, Xing; Song, Jie; Wu, Xiaoping; Zhou, Shi

2015-04-01

Long‑term and advanced cirrhosis is usually irreversible and often coincides with variceal hemorrhage or development of hepatocellular carcinoma; therefore, liver cirrhosis is a major cause of morbidity and mortality globally. The aim of the present study was to investigate the specific mechanism behind the formation of fibrosis or cirrhosis using rat models of hepatic fibrosis. The cirrhosis model was established by intraperitoneally administering dimethylnitrosamine to the rats. Hematoxylin and eosin staining was performed on the hepatic tissues of the rats to observe the fibrosis or cirrhosis, and western blot analysis was employed to detect α‑smooth muscle actin and desmin protein expression. Flow cytometric analysis was used to examine early and late apoptosis, and the protein and mRNA expression of endoplasmic reticulum (ER) stress-associated unfolded protein response (UPR) pathway proteins and apoptotic proteins [C/EBP homologous protein (CHOP) and caspase‑12] was detected by western blotting and the reverse-transcription polymerase chain reaction, respectively. The results indicated that the cirrhosis model was established successfully and that fibrosis was significantly increased in the cirrhosis model group compared with that in the normal control group. Flow cytometric analysis showed that early and late apoptosis in the cirrhosis model was significantly higher compared with that in the control group. The expression of the UPR pathway protein inositol-requiring enzyme (IRE) 1, as well as the expression of CHOP, was increased significantly in the cirrhotic rat tissues compared with that in the control group tissues (P<0.05). In conclusion, apoptosis was clearly observed in the hepatic tissue of cirrhotic rats, and the apoptosis was caused by activation of the ER stress-mediated IRE1 and CHOP.

Challenges of flow-cytometric estimation of nuclear genome size in orchids, a plant group with both whole-genome and progressively partial endoreplication.

PubMed

Trávníček, Pavel; Ponert, Jan; Urfus, Tomáš; Jersáková, Jana; Vrána, Jan; Hřibová, Eva; Doležel, Jaroslav; Suda, Jan

2015-10-01

Nuclear genome size is an inherited quantitative trait of eukaryotic organisms with both practical and biological consequences. A detailed analysis of major families is a promising approach to fully understand the biological meaning of the extensive variation in genome size in plants. Although Orchidaceae accounts for ∼10% of the angiosperm diversity, the knowledge of patterns and dynamics of their genome size is limited, in part due to difficulties in flow cytometric analyses. Cells in various somatic tissues of orchids undergo extensive endoreplication, either whole-genome or partial, and the G1-phase nuclei with 2C DNA amounts may be lacking, resulting in overestimated genome size values. Interpretation of DNA content histograms is particularly challenging in species with progressively partial endoreplication, in which the ratios between the positions of two neighboring DNA peaks are lower than two. In order to assess distributions of nuclear DNA amounts and identify tissue suitable for reliable estimation of nuclear DNA content, we analyzed six different tissue types in 48 orchid species belonging to all recognized subfamilies. Although traditionally used leaves may provide incorrect C-values, particularly in species with progressively partial endoreplication, young ovaries and pollinaria consistently yield 2C and 1C peaks of their G1-phase nuclei, respectively, and are, therefore, the most suitable parts for genome size studies in orchids. We also provide new DNA C-values for 22 orchid genera and 42 species. Adhering to the proposed methodology would allow for reliable genome size estimates in this largest plant family. Although our research was limited to orchids, the need to find a suitable tissue with dominant 2C peak of G1-phase nuclei applies to all endopolyploid species. © 2015 International Society for Advancement of Cytometry.

A retrospective review of acute myeloid leukaemia in 35 dogs diagnosed by a combination of morphologic findings, flow cytometric immunophenotyping and cytochemical staining results (2007-2015).

PubMed

Davis, L L; Hume, K R; Stokol, T

2018-06-01

Acute myeloid leukaemia (AML) is an uncommon, rapidly progressive neoplasm in dogs. The aim of this retrospective study was to characterize the clinical presentation, haematologic findings, diagnostic imaging results, treatment and survival time of a contemporary cohort of dogs with AML. Diagnosis was based on >20% blasts in bone marrow or blood identified as myeloid based on morphologic findings, flow cytometric immunophenotyping and cytochemical staining. Medical records of 35 dogs diagnosed with AML from 2007 to 2015 were included. Most dogs presented with inappetence (66%) and lethargy (57%) and physical examination findings of peripheral lymphadenopathy (74%) and tachypnea (62%). Common haematologic findings were quantifiable circulating blasts (85%; median blast count 35 700/μL; range: 300-276 500/μL), anaemia (median haematocrit 34%; range: 11%-52%) and thrombocytopenia (median 57 000/μL; range: 9000-252 000/μL). Bicytopenia and pancytopenia were each found in 44% of dogs. Follow-up information was available for 34 dogs. The overall median survival time from diagnosis was 19 days (range: 1-121 days). Clinical progression in some dogs was not as rapid as previously reported. Haematologic responses to various chemotherapeutics were documented in 3 dogs, with associated survival times of 62, 103 and 121 days. Dogs treated with prednisone or a combination of chemotherapy and prednisone had improved survival compared to dogs that received symptomatic care only (P < .0001). Our results show canine AML has an overlapping clinical presentation with lymphoma. The prognosis for canine AML remains extremely guarded. Further studies are needed to optimize therapeutic regimens for dogs with AML. © 2017 John Wiley & Sons Ltd.

Skeletal Cell Differentiation Is Enhanced by Atmospheric Dielectric Barrier Discharge Plasma Treatment

PubMed Central

Zhang, Jun; Kurpad, Deepa S.; Fridman, Gregory; Fridman, Alexander; Freeman, Theresa A.

2013-01-01

Enhancing chondrogenic and osteogenic differentiation is of paramount importance in providing effective regenerative therapies and improving the rate of fracture healing. This study investigated the potential of non-thermal atmospheric dielectric barrier discharge plasma (NT-plasma) to enhance chondrocyte and osteoblast proliferation and differentiation. Although the exact mechanism by which NT-plasma interacts with cells is undefined, it is known that during treatment the atmosphere is ionized generating extracellular reactive oxygen and nitrogen species (ROS and RNS) and an electric field. Appropriate NT-plasma conditions were determined using lactate-dehydrogenase release, flow cytometric live/dead assay, flow cytometric cell cycle analysis, and Western blots to evaluate DNA damage and mitochondrial integrity. We observed that specific NT-plasma conditions were required to prevent cell death, and that loss of pre-osteoblastic cell viability was dependent on intracellular ROS and RNS production. To further investigate the involvement of intracellular ROS, fluorescent intracellular dyes Mitosox (superoxide) and dihydrorhodamine (peroxide) were used to assess onset and duration after NT-plasma treatment. Both intracellular superoxide and peroxide were found to increase immediately post NT-plasma treatment. These increases were sustained for one hour but returned to control levels by 24 hr. Using the same treatment conditions, osteogenic differentiation by NT-plasma was assessed and compared to peroxide or osteogenic media containing β-glycerolphosphate. Although both NT-plasma and peroxide induced differentiation-specific gene expression, neither was as effective as the osteogenic media. However, treatment of cells with NT-plasma after 24 hr in osteogenic or chondrogenic media significantly enhanced differentiation as compared to differentiation media alone. The results of this study show that NT-plasma can selectively initiate and amplify ROS signaling to enhance differentiation, and suggest this technology could be used to enhance bone fusion and improve healing after skeletal injury. PMID:24349203

Effects of Granulocyte-Macrophage Colony-Stimulating (GM-CSF) Factor on Corneal Epithelial Cells in Corneal Wound Healing Model

PubMed Central

Rho, Chang Rae; Park, Mi-young; Kang, Seungbum

2015-01-01

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that activates granulocyte and macrophage cell lineages. It is also known to have an important function in wound healing. This study investigated the effect of GM-CSF in wound healing of human corneal epithelial cells (HCECs). We used human GM-CSF derived from rice cells (rice cell-derived recombinant human GM-CSF; rhGM-CSF). An in vitro migration assay was performed to investigate the migration rate of HCECs treated with various concentrations of rhGM-CSF (0.1, 1.0, and 10.0 μg/ml). MTT assay and flow cytometric analysis were used to evaluate the proliferative effect of rhGM-CSF. The protein level of p38MAPK was analyzed by western blotting. For in vivo analysis, 100 golden Syrian hamsters were divided into four groups, and their corneas were de-epithelialized with alcohol and a blade. The experimental groups were treated with 10, 20, or 50 μg/ml rhGM-CSF four times daily, and the control group was treated with phosphate-buffered saline. The corneal wound-healing rate was evaluated by fluorescein staining at the initial wounding and 12, 24, 36, and 48 hours after epithelial debridement. rhGM-CSF accelerated corneal epithelial wound healing both in vitro and in vivo. MTT assay and flow cytometric analysis revealed that rhGM-CSF treatment had no effects on HCEC proliferation. Western blot analysis demonstrated that the expression level of phosphorylated p38MAPK increased with rhGM-CSF treatment. These findings indicate that rhGM-CSF enhances corneal wound healing by accelerating cell migration. PMID:26376304

Apomixis is not prevalent in subnival to nival plants of the European Alps

PubMed Central

Hörandl, Elvira; Dobeš, Christoph; Suda, Jan; Vít, Petr; Urfus, Tomáš; Temsch, Eva M.; Cosendai, Anne-Caroline; Wagner, Johanna; Ladinig, Ursula

2011-01-01

Background and Aims High alpine environments are characterized by short growing seasons, stochastic climatic conditions and fluctuating pollinator visits. These conditions are rather unfavourable for sexual reproduction of flowering plants. Apomixis, asexual reproduction via seed, provides reproductive assurance without the need of pollinators and potentially accelerates seed development. Therefore, apomixis is expected to provide selective advantages in high-alpine biota. Indeed, apomictic species occur frequently in the subalpine to alpine grassland zone of the European Alps, but the mode of reproduction of the subnival to nival flora was largely unknown. Methods The mode of reproduction in 14 species belonging to seven families was investigated via flow cytometric seed screen. The sampling comprised 12 species typical for nival to subnival plant communities of the European Alps without any previous information on apomixis (Achillea atrata, Androsace alpina, Arabis caerulea, Erigeron uniflorus, Gnaphalium hoppeanum, Leucanthemopsis alpina, Oxyria digyna, Potentilla frigida, Ranunculus alpestris, R. glacialis, R. pygmaeus and Saxifraga bryoides), and two high-alpine species with apomixis reported from other geographical areas (Leontopodium alpinum and Potentilla crantzii). Key Results Flow cytometric data were clearly interpretable for all 46 population samples, confirming the utility of the method for broad screenings on non-model organisms. Formation of endosperm in all species of Asteraceae was documented. Ratios of endosperm : embryo showed pseudogamous apomixis for Potentilla crantzii (ratio approx. 3), but sexual reproduction for all other species (ratios approx. 1·5). Conclusions The occurrence of apomixis is not correlated to high altitudes, and cannot be readily explained by selective forces due to environmental conditions. The investigated species have probably other adaptations to high altitudes to maintain reproductive assurance via sexuality. We hypothesize that shifts to apomixis are rather connected to frequencies of polyploidization than to ecological conditions. PMID:21724654

Skeletal cell differentiation is enhanced by atmospheric dielectric barrier discharge plasma treatment.

PubMed

Steinbeck, Marla J; Chernets, Natalie; Zhang, Jun; Kurpad, Deepa S; Fridman, Gregory; Fridman, Alexander; Freeman, Theresa A

2013-01-01

Enhancing chondrogenic and osteogenic differentiation is of paramount importance in providing effective regenerative therapies and improving the rate of fracture healing. This study investigated the potential of non-thermal atmospheric dielectric barrier discharge plasma (NT-plasma) to enhance chondrocyte and osteoblast proliferation and differentiation. Although the exact mechanism by which NT-plasma interacts with cells is undefined, it is known that during treatment the atmosphere is ionized generating extracellular reactive oxygen and nitrogen species (ROS and RNS) and an electric field. Appropriate NT-plasma conditions were determined using lactate-dehydrogenase release, flow cytometric live/dead assay, flow cytometric cell cycle analysis, and Western blots to evaluate DNA damage and mitochondrial integrity. We observed that specific NT-plasma conditions were required to prevent cell death, and that loss of pre-osteoblastic cell viability was dependent on intracellular ROS and RNS production. To further investigate the involvement of intracellular ROS, fluorescent intracellular dyes Mitosox (superoxide) and dihydrorhodamine (peroxide) were used to assess onset and duration after NT-plasma treatment. Both intracellular superoxide and peroxide were found to increase immediately post NT-plasma treatment. These increases were sustained for one hour but returned to control levels by 24 hr. Using the same treatment conditions, osteogenic differentiation by NT-plasma was assessed and compared to peroxide or osteogenic media containing β-glycerolphosphate. Although both NT-plasma and peroxide induced differentiation-specific gene expression, neither was as effective as the osteogenic media. However, treatment of cells with NT-plasma after 24 hr in osteogenic or chondrogenic media significantly enhanced differentiation as compared to differentiation media alone. The results of this study show that NT-plasma can selectively initiate and amplify ROS signaling to enhance differentiation, and suggest this technology could be used to enhance bone fusion and improve healing after skeletal injury.

Effects of Granulocyte-Macrophage Colony-Stimulating (GM-CSF) Factor on Corneal Epithelial Cells in Corneal Wound Healing Model.

PubMed

Rho, Chang Rae; Park, Mi-young; Kang, Seungbum

2015-01-01

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that activates granulocyte and macrophage cell lineages. It is also known to have an important function in wound healing. This study investigated the effect of GM-CSF in wound healing of human corneal epithelial cells (HCECs). We used human GM-CSF derived from rice cells (rice cell-derived recombinant human GM-CSF; rhGM-CSF). An in vitro migration assay was performed to investigate the migration rate of HCECs treated with various concentrations of rhGM-CSF (0.1, 1.0, and 10.0 μg/ml). MTT assay and flow cytometric analysis were used to evaluate the proliferative effect of rhGM-CSF. The protein level of p38MAPK was analyzed by western blotting. For in vivo analysis, 100 golden Syrian hamsters were divided into four groups, and their corneas were de-epithelialized with alcohol and a blade. The experimental groups were treated with 10, 20, or 50 μg/ml rhGM-CSF four times daily, and the control group was treated with phosphate-buffered saline. The corneal wound-healing rate was evaluated by fluorescein staining at the initial wounding and 12, 24, 36, and 48 hours after epithelial debridement. rhGM-CSF accelerated corneal epithelial wound healing both in vitro and in vivo. MTT assay and flow cytometric analysis revealed that rhGM-CSF treatment had no effects on HCEC proliferation. Western blot analysis demonstrated that the expression level of phosphorylated p38MAPK increased with rhGM-CSF treatment. These findings indicate that rhGM-CSF enhances corneal wound healing by accelerating cell migration.

Neutrophil chemotaxis in cord blood of term and preterm neonates is reduced in preterm neonates and influenced by the mode of delivery and anaesthesia.

PubMed

Birle, Alexandra; Nebe, C Thomas; Hill, Sandra; Hartmann, Karin; Poeschl, Johannes; Koch, Lutz

2015-01-01

Bacterial infections, even without any perinatal risk factors, are common in newborns, especially in preterm neonates. The aim of this study was to evaluate possible impairment of neutrophil chemotaxis in term and preterm neonates compared with adults as well as neonates with different modes of delivery and anaesthesia. We analysed the expression of the adhesion molecule L-Selectin as well as shape change, spontaneous and N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced transmigration of neutrophils in a flow cytometric assay of chemotaxis after spontaneous delivery with Cesarian Section (CS) under spinal anaesthesia (mepivacaine, sufentanil), epidural anaesthesia (ropivacaine or bupivacaine, sufentanil) or general anaesthesia (ketamine, thiopental, succinylcholine). Chemokinesis was higher (p=0.008) in cord blood neutrophils than in the adult ones, whereas those could be more stimulated by fMLP (p=0.02). After vaginal delivery neutrophils showed a higher spontaneous and fMLP-stimulated chemotactic response compared to neonates after CS without labor. Comparing different types of anaesthesia for CS, spinal anaesthesia resulted in less impairment on chemotaxis than general anaesthesia or epidural anaesthesia. The new flow cytometric assay of neutrophil chemotaxis is an appropriate and objective method to analyse functional differences even in very small volumes of blood, essential in neonatology. Term neonates do not show reduced chemotaxis compared to adults. Preterm neonates present with reduced chemotaxis and chemokinesis, confirming the well known deficits in their neutrophil function. The side effects of maternal drugs on the neonatal immune system have to be considered especially when the immune response is already impaired, as in preterm infants.

Identifying the Presence of Prostate Cancer in Individuals with PSA Levels <20 ng ml-1 Using Computational Data Extraction Analysis of High Dimensional Peripheral Blood Flow Cytometric Phenotyping Data.

PubMed

Cosma, Georgina; McArdle, Stéphanie E; Reeder, Stephen; Foulds, Gemma A; Hood, Simon; Khan, Masood; Pockley, A Graham

2017-01-01

Determining whether an asymptomatic individual with Prostate-Specific Antigen (PSA) levels below 20 ng ml -1 has prostate cancer in the absence of definitive, biopsy-based evidence continues to present a significant challenge to clinicians who must decide whether such individuals with low PSA values have prostate cancer. Herein, we present an advanced computational data extraction approach which can identify the presence of prostate cancer in men with PSA levels <20 ng ml -1 on the basis of peripheral blood immune cell profiles that have been generated using multi-parameter flow cytometry. Statistical analysis of immune phenotyping datasets relating to the presence and prevalence of key leukocyte populations in the peripheral blood, as generated from individuals undergoing routine tests for prostate cancer (including tissue biopsy) using multi-parametric flow cytometric analysis, was unable to identify significant relationships between leukocyte population profiles and the presence of benign disease (no prostate cancer) or prostate cancer. By contrast, a Genetic Algorithm computational approach identified a subset of five flow cytometry features ( CD 8 + CD 45 RA - CD 27 - CD 28 - ( CD 8 + Effector Memory cells); CD 4 + CD 45 RA - CD 27 - CD 28 - ( CD 4 + Terminally Differentiated Effector Memory Cells re-expressing CD45RA); CD 3 - CD 19 + (B cells); CD 3 + CD 56 + CD 8 + CD 4 + (NKT cells)) from a set of twenty features, which could potentially discriminate between benign disease and prostate cancer. These features were used to construct a prostate cancer prediction model using the k-Nearest-Neighbor classification algorithm. The proposed model, which takes as input the set of flow cytometry features, outperformed the predictive model which takes PSA values as input. Specifically, the flow cytometry-based model achieved Accuracy = 83.33%, AUC = 83.40%, and optimal ROC points of FPR = 16.13%, TPR = 82.93%, whereas the PSA-based model achieved Accuracy = 77.78%, AUC = 76.95%, and optimal ROC points of FPR = 29.03%, TPR = 82.93%. Combining PSA and flow cytometry predictors achieved Accuracy = 79.17%, AUC = 78.17% and optimal ROC points of FPR = 29.03%, TPR = 85.37%. The results demonstrate the value of computational intelligence-based approaches for interrogating immunophenotyping datasets and that combining peripheral blood phenotypic profiling with PSA levels improves diagnostic accuracy compared to using PSA test alone. These studies also demonstrate that the presence of cancer is reflected in changes in the peripheral blood immune phenotype profile which can be identified using computational analysis and interpretation of complex flow cytometry datasets.

Flow cytometric characterization of phenotype, DNA indices and p53 gene expression in 55 cases of acute leukemia.

PubMed

Powari, Manish; Varma, Neelam; Varma, Subhash; Marwaha, Ram Kumar; Sandhu, Harpreet; Ganguly, Nirmal Kumar

2002-06-01

To characterize the phenotype of acute leukemia cases using flow cytometry, to detect mixed lineage cases and to use DNA index determination, including S-phase fraction (SPF) and p53 detection, to find if there was any correlation of SPF and p53 expression with outcome. Fifty-five cases of acute leukemia were enrolled in this study. A complete hemogram and routine bone marrow examination, including cytochemistry, was done. Mycloperoxidase-negative cases were evaluated on a flow cytometer using monoclonal antibodies. DNA indices were determined by flow cytometry in all cases, and p53 was detected immunohistochemically using the alkaline phosphatase/antialkaline phosphatase technique. Acute myeloblastic leukemia (AML) was diagnosed in 32 cases; acute lymphoblastic leukemia (ALL) was diagnosed in 18 (14 B lineage and 4 T line age). Four cases showed mixed lineage leukemia, and undifferentiated acute leukemia was diagnosed in one case. The mean/range of SPF for these groups were 3.76/0.33-6.91, 6.25/0.15-21.4, 2.89/0.35-10.64, 2.60/0.72-6.94 and 7.34, respectively. Aneuploidy was detected in two cases of B-lineage ALL and tetraploidy in a case of AML-M7, while all others were diploid p53. Was detected in 6 of 55 cases (10.90%). Follow-up was available for 24 patients. Five patients relapsed, and four had B-cell type ALL and were diploid and expressed no p53 gene. SPF% did not show any correlation with outcome. These data suggest that within acute leukemia subtypes, there is a wide variation in SPF. SPF does not seem to correlate with outcome. Immunophenotyping is essential to determine the lineage in myeloperoxidase-negative cases. It is perhaps the only way to diagnose mixed lineage leukemia and aberrant expression of markers presently. The p53 gene was detected less frequently. However, more studies are required from different centers with longer follow-up to evaluate prognostic significance.

Contribution of flow cytometry to the diagnosis of gastric lymphomas in endoscopic biopsy specimens.

PubMed

Almasri, N M; Zaer, F S; Iturraspe, J A; Braylan, R C

1997-07-01

Gastric lymphomas seem to have unique clinical, pathologic, and immunophenotypic features that set them apart from nodal lymphomas. Microscopic examination of endoscopic biopsy specimens is the most frequent procedure used to diagnose gastric tumors, but it is very difficult, and sometimes impossible, to recognize lymphomas in endoscopic samples by histologic or even immunohistologic methods. Because most gastric lymphomas are of B-cell origin, we used flow cytometry to assess B-cell clonality in gastric biopsy specimens containing dense lymphocytic infiltrates thought to represent lymphoma. We prepared viable cell suspensions from unfixed specimens obtained from 29 consecutive patients who had a previous microscopic diagnosis of suspicious gastric lymphoid infiltrates. We performed immunophenotypic studies with multicolor flow cytometry, and we assessed clonality by examination of immunoglobulin (Ig) light-chain expression analyzed exclusively on B cells identified by anti-CD20 or CD19 antibodies. The mean number of cells recovered was 1.04 x 10(6), from an average of 5.5 gastric biopsy fragments per patient. In 26 of the 29 patients, the number of cells was adequate for analysis. We detected B-cell monoclonality in 16 cases, including 5 in which the percentage of clonal B cells was less than 5%. Of the 16 cases, only 8 could be diagnosed as lymphomas on morphologic grounds alone; the remaining 8 patients had either suspicious lymphoid infiltrates or chronic gastritis. The three cases with an insufficient number of cells were considered non-neoplastic either on histologic grounds alone or in conjunction with Southern analysis of Ig genes. We conclude that flow cytometric immunophenotypic analysis of freshly prepared cell suspensions obtained from endoscopic biopsy specimens can be used to evaluate gastric lymphocytic infiltrates. Specifically, the analysis of surface Ig light-chain expression on B cells distinguishes between monoclonal (lymphoma) and polyclonal (nonlymphoma) infiltrates. The rapidity, ease, quantitative properties, and sensitivity of this technique make it a supplement to the morphologic assessment of gastric lymphoid infiltrates.

Systems biology and clinical cytomics: The 10th Leipziger Workshop and the 3rd International Workshop on Slide-Based Cytometry, Leipzig, Germany, April 2005.

PubMed

Tárnok, Attila; Valet, Günther K; Emmrich, Frank

2006-01-01

Despite very significant technical and software improvements in flow cytometry (FCM) since the 1980's, the demand for a cytometric technology combining both quantitative cell analysis and morphological documentation in Cytomics became evident. Improvements in microtechnology and computing permit nowadays similar quantitative and stoichiometric single cell-based high-throughput analyses by microscopic instruments, like Slide-Based Cytometry (SBC). SBC and related techniques offer unique tools to perform complex immunophenotyping, thereby enabling diagnostic procedures during early disease stages. Multicolor or polychromatic analysis of cells by SBC is of special importance not only as a cytomics technology platform but also because of low quantities of required reagents and biological material. The exact knowledge of the location of each cell on the slide permits repetitive restaining and reanalysis of specimens. Various separate measurements of the same specimen can be ultimately fused to one database increasing the information obtained per cell. Relocation and optical evaluation of cells as typical SBC feature, can be of integral importance for cytometric analysis, since artifacts can be excluded and morphology of measured cells can be documented. Progress in cell analytic: In the SBC, new horizons can be opened by the new techniques of structural and functional analysis with the high resolution from intracellular and membrane (confocal microscopy, nanoscopy, total internal fluorescence microscopy (TIRFM), and tissue level (tissomics), to organ and organism level (in vivo cytometry, optical whole body imaging). Predictive medicine aims at the detection of changes in patient's state prior to the manifestation of the disease or the complication. Such instances concern immune consequences of surgeries or noninfectious posttraumatic shock in intensive care patients or the pretherapeutic identification of high risk patients in cancer cytostatic therapy. Preventive anti-infectious or anti-shock therapy as well as curative chemotherapy in combination with stem cell transplantation may provide better survival chances for patient at concomitant cost containment. Predictive medicine-guided optimization of therapy could lead to individualized medicine that gives significant therapeutic effect and may lower or abrogate potential therapeutic side effects. The 10th Leipziger Workshop combined with the 3rd International Workshop on SBC aimed to offer new methods in Image- and Slide-Based Cytometry for solutions in clinical research. It moved towards practical applications in the clinics and the clinical laboratory. This development will be continued in 2006 at the upcoming Leipziger Workshop and the International Workshop on Slide-Based Cytometry.

Application of advanced cytometric and molecular technologies to minimal residual disease monitoring

NASA Astrophysics Data System (ADS)

Leary, James F.; He, Feng; Reece, Lisa M.

2000-04-01

Minimal residual disease monitoring presents a number of theoretical and practical challenges. Recently it has been possible to meet some of these challenges by combining a number of new advanced biotechnologies. To monitor the number of residual tumor cells requires complex cocktails of molecular probes that collectively provide sensitivities of detection on the order of one residual tumor cell per million total cells. Ultra-high-speed, multi parameter flow cytometry is capable of analyzing cells at rates in excess of 100,000 cells/sec. Residual tumor selection marker cocktails can be optimized by use of receiver operating characteristic analysis. New data minimizing techniques when combined with multi variate statistical or neural network classifications of tumor cells can more accurately predict residual tumor cell frequencies. The combination of these techniques can, under at least some circumstances, detect frequencies of tumor cells as low as one cell in a million with an accuracy of over 98 percent correct classification. Detection of mutations in tumor suppressor genes requires insolation of these rare tumor cells and single-cell DNA sequencing. Rare residual tumor cells can be isolated at single cell level by high-resolution single-cell cell sorting. Molecular characterization of tumor suppressor gene mutations can be accomplished using a combination of single- cell polymerase chain reaction amplification of specific gene sequences followed by TA cloning techniques and DNA sequencing. Mutations as small as a single base pair in a tumor suppressor gene of a single sorted tumor cell have been detected using these methods. Using new amplification procedures and DNA micro arrays it should be possible to extend the capabilities shown in this paper to screening of multiple DNA mutations in tumor suppressor and other genes on small numbers of sorted metastatic tumor cells.

Flow cytometry for immediate follow-up of drinking water networks after maintenance.

PubMed

Van Nevel, Sam; Buysschaert, Benjamin; De Roy, Karen; De Gusseme, Bart; Clement, Lieven; Boon, Nico

2017-03-15

Drinking water networks need maintenance every once in a while, either planned interventions or emergency repairs. When this involves opening of the water pipes, precautionary measures need to be taken to avoid contamination of the drinking water at all time. Drinking water suppliers routinely apply plating for faecal indicator organisms as quality control in such a situation. However, this takes at least 21 h of waiting time, which can be crucial when dealing with major supply pipes. A combination of flow cytometric (FCM) bacterial cell counts with FCM fingerprinting techniques is proposed in this study as a fast and sensitive additional technique. In three full scale situations, major supply pipes with 400-1050 mm diameter were emptied for maintenance, shock-chlorinated and flushed with large amounts of clean drinking water before taking back in operation. FCM measurements of the discharged flushing water revealed fast lowering and stabilizing bacterial concentrations once flushing is initiated. Immediate comparison with clean reference drinking water used for flushing was done, and the moment when both waters had similar bacterial concentrations was considered as the endpoint of the necessary flushing works. This was usually after 2-4 h of flushing. FCM fingerprinting, based on both bacteria and FCM background, was used as additional method to verify how similar flushing and reference samples were and yielded similar results. The FCM approved samples were several hours later approved as well by the drinking water supplier after plating and incubation for total Coliforms and Enterococci. These were used as decisive control to set the pipes back in operation. FCM proved to be a more conservative test than plating, yet it yielded immediate results. Application of these FCM methods can therefore avoid long unnecessary waiting times and large drinking water losses. Copyright © 2016 Elsevier Ltd. All rights reserved.

Development of wide-angle 2D light scattering static cytometry

NASA Astrophysics Data System (ADS)

Xie, Linyan; Liu, Qiao; Shao, Changshun; Su, Xuantao

2016-10-01

We have recently developed a 2D light scattering static cytometer for cellular analysis in a label-free manner, which measures side scatter (SSC) light in the polar angular range from 79 to 101 degrees. Compared with conventional flow cytometry, our cytometric technique requires no fluorescent labeling of the cells, and static cytometry measurements can be performed without flow control. In this paper we present an improved label-free static cytometer that can obtain 2D light scattering patterns in a wider angular range. By illuminating the static microspheres on chip with a scanning optical fiber, wide-angle 2D light scattering patterns of single standard microspheres with a mean diameter of 3.87 μm are obtained. The 2D patterns of 3.87 μm microspheres contain both large-angle forward scatter (FSC) and SSC light in the polar angular range from 40 to 100 degrees, approximately. Experimental 2D patterns of 3.87 μm microspheres are in good agreement with Mie theory simulated ones. The wide-angle light scattering measurements may provide a better resolution for particle analysis as compared with the SSC measurements. Two dimensional light scattering patterns of HL-60 human acute leukemia cells are obtained by using our static cytometer. Compared with SSC 2D light scattering patterns, wide-angle 2D patterns contain richer information of the HL-60 cells. The obtaining of 2D light scattering patterns in a wide angular range could help to enhance the capabilities of our label-free static cytometry for cell analysis.

The HLA-A2 Restricted T Cell Epitope HCV Core35–44 Stabilizes HLA-E Expression and Inhibits Cytolysis Mediated by Natural Killer Cells

PubMed Central

Nattermann, Jacob; Nischalke, Hans Dieter; Hofmeister, Valeska; Ahlenstiel, Golo; Zimmermann, Henning; Leifeld, Ludger; Weiss, Elisabeth H.; Sauerbruch, Tilman; Spengler, Ulrich

2005-01-01

Impaired activity of natural killer cells has been proposed as a mechanism contributing to viral persistence in hepatitis C virus (HCV) infection. Natural cytotoxicity is regulated by interactions of HLA-E with inhibitory CD94/NKG2A receptors on natural killer (NK) cells. Here, we studied whether HCV core encodes peptides that bind to HLA-E and inhibit natural cytotoxicity. We analyzed 30 HCV core-derived peptides. Peptide-induced stabilization of HLA-E expression was measured flow cytometrically after incubating HLA-E-transfected cells with peptides. NK cell function was studied with a 51chromium-release-assay. Intrahepatic HLA-E expression was analyzed by an indirect immunoperoxidase technique and flow cytometry of isolated cells using a HLA-E-specific antibody. We identified peptide aa35–44, a well-characterized HLA-A2 restricted T cell epitope, as a peptide stabilizing HLA-E expression and thereby inhibiting NK cell-mediated lysis. Blocking experiments confirmed that this inhibitory effect of peptide aa35–44 on natural cytotoxicity was mediated via interactions between CD94/NKG2A receptors and enhanced HLA-E expression. In line with these in vitro data we found enhanced intrahepatic HLA-E expression on antigen-presenting cells in HCV-infected patients. Our data indicate the existence of T cell epitopes that can be recognized by HLA-A2 and HLA-E. This dual recognition may contribute to viral persistence in hepatitis C. PMID:15681828

Investigation of functional and morphological changes in Pseudomonas aeruginosa and Staphylococcus aureus cells induced by Origanum compactum essential oil.

PubMed

Bouhdid, S; Abrini, J; Zhiri, A; Espuny, M J; Manresa, A

2009-05-01

Evaluation of the cellular effects of Origanum compactum essential oil on Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213. The damage induced by O. compactum essential oil on these two strains has been studied using different techniques: plate count, potassium leakage, flow cytometry (FC) and transmission electron microscopy (TEM). The results showed that oil treatment led to reduction of cells viability and dissipated potassium ion gradients. Flow cytometric analysis showed that oil treatment promoted the accumulation of bis-oxonol and the membrane-impermeable nucleic acid stain propidium iodide (PI), indicating the loss of membrane potential and permeability. The ability to reduce 5-cyano-2,3-ditolyl tetrazolium chloride was inhibited. Unlike in Ps. aeruginosa, membrane potential and membrane permeability in Staph. aureus cells were affected by oil concentration and contact time. Finally, TEM showed various structural effects. Mesosome-like structures were seen in oil-treated Staph. aureus cells whereas in Ps. aeruginosa, coagulated cytoplasmic material and liberation of membrane vesicles were observed, and intracellular material was seen in the surrounding environment. Both FC and TEM revealed that the effects in Ps. aeruginosa were greater than in Staph. aureus. Oregano essential oil induces membrane damage showed by the leakage of potassium and uptake of PI and bis-oxonol. Ultrastructural alterations and the loss of cell viability were observed. Understanding the mode of antibacterial effect of the oil studied is of a great interest in it further application as natural preservative in food or pharmaceutical industries.

Elevated Plasma Soluble CD14 and Skewed CD16+ Monocyte Distribution Persist despite Normalisation of Soluble CD163 and CXCL10 by Effective HIV Therapy: A Changing Paradigm for Routine HIV Laboratory Monitoring?

PubMed Central

Castley, Alison; Berry, Cassandra; French, Martyn; Fernandez, Sonia; Krueger, Romano; Nolan, David

2014-01-01

Objective We investigated plasma and flow cytometric biomarkers of monocyte status that have been associated with prognostic utility in HIV infection and other chronic inflammatory diseases, comparing 81 HIV+ individuals with a range of treatment outcomes to a group of 21 healthy control blood donors. Our aim is to develop and optimise monocyte assays that combine biological relevance, clinical utility, and ease of adoption into routine HIV laboratory practice. Design Cross-sectional evaluation of concurrent plasma and whole blood samples. Methods A flow cytometry protocol was developed comprising single-tube CD45, CD14, CD16, CD64, CD163, CD143 analysis with appropriately matched isotype controls. Plasma levels of soluble CD14 (sCD14), soluble CD163 (sCD163) and CXCL10 were measured by ELISA. Results HIV status was associated with significantly increased expression of CD64, CD143 and CD163 on CD16+ monocytes, irrespective of the virological response to HIV therapy. Plasma levels of sCD14, sCD163 and CXCL10 were also significantly elevated in association with viremic HIV infection. Plasma sCD163 and CXCL10 levels were restored to healthy control levels by effective antiretroviral therapy while sCD14 levels remained elevated despite virological suppression (p<0.001). Conclusions Flow cytometric and plasma biomarkers of monocyte activation indicate an ongoing systemic inflammatory response to HIV infection, characterised by persistent alterations of CD16+ monocyte expression profiles and elevated sCD14 levels, that are not corrected by antiretroviral therapy and likely to be prognostically significant. In contrast, sCD163 and CXCL10 levels declined on antiretroviral therapy, suggesting multiple activation pathways revealed by these biomarkers. Incorporation of these assays into routine clinical care is feasible and warrants further consideration, particularly in light of emerging therapeutic strategies that specifically target innate immune activation in HIV infection. PMID:25544986

The relative value of cytometry and cytology in the management of bladder cancer: the Memorial Sloan-Kettering Cancer Center experience.

PubMed

Badalament, R A; Fair, W R; Whitmore, W F; Melamed, M R

1988-02-01

The flow cytometric studies presented herein are based almost entirely on DNA measurements and represent an early application of this diagnostic test. Nevertheless, the MSKCC experience with FCM has demonstrated that it is technically feasible and clinically useful. The sensitivity of FCM is in the range of 80% to 85% overall, and is superior to that of conventional voided or bladder wash cytology. In the absence of inflammation secondary to infection, calculi, or intravesical agents such as BCG, the specificity is greater than 90%. In the presence of inflammation, FCM appears to be less specific than conventional cytology. A potential advantage of FCM over cytology is the quantitative nature of the examination permitting comparisons of sequential examinations. Also, there are refinements in technique that hold promise of increasing the accuracy or clinical usefulness of FCM, eg, the joint measurements of DNA and differentiation antigens defined by monoclonal antibodies. Yet, despite the current and potential advantages of FCM, voided urinary cytology continues to be the procedure of choice for detection and monitoring urothelial carcinoma, not only because of our long experience and better understanding of this test, but because of its proven high specificity, because it is noninvasive, widely available, and may help to detect upper tract or urethral tumors.

The Sperm Chromatin Structure Assay (SCSA(®)) and other sperm DNA fragmentation tests for evaluation of sperm nuclear DNA integrity as related to fertility.

PubMed

Evenson, Donald P

2016-06-01

Thirty-five years ago the pioneering paper in Science (240:1131) on the relationship between sperm DNA integrity and pregnancy outcome was featured as the cover issue showing a fluorescence photomicrograph of red and green stained sperm. The flow cytometry data showed a very significant difference in sperm DNA integrity between fertile and subfertile bulls and men. This study utilized heat (100°C, 5min) to denature DNA at sites of DNA strand breaks followed by staining with acridine orange (AO) and measurements of 5000 individual sperm of green double strand (ds) DNA and red single strand (ss) DNA fluorescence. Later, the heat protocol was changed to a low pH protocol to denature the DNA at sites of strand breaks; the heat and acid procedures produced the same results. SCSA data are very advantageously dual parameter with 1024 channels (degrees) of both red and green fluorescence. Hundreds of publications on the use of the SCSA test in animals and humans have validated the SCSA as a highly useful test for determining male breeding soundness. The SCSA test is a rapid, non-biased flow cytometer machine measurement providing robust statistical data with exceptional precision and repeatability. Many genotoxic experiments showed excellent dose response data with very low coefficient of variation that further validated the SCSA as being a highly powerful assay for sperm DNA integrity. Twelve years following the introduction of the SCSA test, the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labelling (TUNEL) test (1993) for sperm was introduced as the only other flow cytometric assay for sperm DNA fragmentation. However, the TUNEL test can also be done by light microscopy with much less statistical robustness. The COMET (1998) and Sperm Chromatin Dispersion (SCD; HALO) (2003) tests were introduced as light microscope tests that don't require a flow cytometer. Since these tests measure only 50-200 sperm per sample, they suffer from the lack of the statistical robustness of flow cytometric measurements. Only the SCSA test has an exact standardization of a fixed protocol. The many variations of the other tests make it very difficult to compare data and thresholds for risk of male factor infertility. Data from these four sperm DNA fragmentation tests plus the light microscope acridine orange test (AOT) are correlated to various degrees. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

FLOCK cluster analysis of mast cell event clustering by high-sensitivity flow cytometry predicts systemic mastocytosis.

PubMed

Dorfman, David M; LaPlante, Charlotte D; Pozdnyakova, Olga; Li, Betty

2015-11-01

In our high-sensitivity flow cytometric approach for systemic mastocytosis (SM), we identified mast cell event clustering as a new diagnostic criterion for the disease. To objectively characterize mast cell gated event distributions, we performed cluster analysis using FLOCK, a computational approach to identify cell subsets in multidimensional flow cytometry data in an unbiased, automated fashion. FLOCK identified discrete mast cell populations in most cases of SM (56/75 [75%]) but only a minority of non-SM cases (17/124 [14%]). FLOCK-identified mast cell populations accounted for 2.46% of total cells on average in SM cases and 0.09% of total cells on average in non-SM cases (P < .0001) and were predictive of SM, with a sensitivity of 75%, a specificity of 86%, a positive predictive value of 76%, and a negative predictive value of 85%. FLOCK analysis provides useful diagnostic information for evaluating patients with suspected SM, and may be useful for the analysis of other hematopoietic neoplasms. Copyright© by the American Society for Clinical Pathology.

Biology and flow cytometry of proangiogenic hematopoietic progenitors cells.

PubMed

Rose, Jonathan A; Erzurum, Serpil; Asosingh, Kewal

2015-01-01

During development, hematopoiesis and neovascularization are closely linked to each other via a common bipotent stem cell called the hemangioblast that gives rise to both hematopoietic cells and endothelial cells. In postnatal life, this functional connection between the vasculature and hematopoiesis is maintained by a subset of hematopoietic progenitor cells endowed with the capacity to differentiate into potent proangiogenic cells. These proangiogenic hematopoietic progenitors comprise a specific subset of bone marrow (BM)-derived cells that homes to sites of neovascularization and possess potent paracrine angiogenic activity. There is emerging evidence that this subpopulation of hematopoietic progenitors plays a critical role in vascular health and disease. Their angiogenic activity is distinct from putative "endothelial progenitor cells" that become structural cells of the endothelium by differentiation into endothelial cells. Proangiogenic hematopoietic progenitor cell research requires multidisciplinary expertise in flow cytometry, hematology, and vascular biology. This review provides a comprehensive overview of proangiogenic hematopoietic progenitor cell biology and flow cytometric methods to detect these cells in the peripheral blood circulation and BM. © 2014 International Society for Advancement of Cytometry.

Alkamides from Echinacea angustifolia Interact with P-glycoprotein of primary brain capillary endothelial cells isolated from porcine brain blood vessels.

PubMed

Mahringer, Anne; Ardjomand-Woelkart, Karin; Bauer, Rudolf; Fricker, Gert; Efferth, Thomas

2013-03-01

The blood-brain barrier prevents the passage of toxic compounds from blood circulation into brain tissue. Unfortunately, drugs for the treatment of neurodegenerative diseases, brain tumors, and other diseases also do not cross the blood-brain barrier. In the present investigation, we used isolated porcine brain capillary endothelial cells and a flow cytometric calcein-AM assay to analyze inhibition of P-glycoprotein, a major constituent of the blood-brain barrier. We tested 8 alkamides isolated from Echinacea angustifolia and found that four of them inhibited P-glycoprotein-mediated calcein transport in porcine brain capillary endothelial cells. Georg Thieme Verlag KG Stuttgart · New York.

Utilization of Ancillary Studies in the Cytologic Diagnosis of Respiratory Lesions

PubMed Central

Layfield, Lester J.; Roy-Chowdhuri, Sinchita; Baloch, Zubair; Ehya, Hormoz; Geisinger, Kim; Hsiao, Susan J.; Lin, Oscar; Lindeman, Neal I.; Roh, Michael; Schmitt, Fernando; Sidiropoulos, Nikoletta; VanderLaan, Paul A.

2017-01-01

The Papanicolaou Society of Cytopathology has developed a set of guidelines for respiratory cytology including indications for sputum examination, bronchial washings and brushings, CT-guided FNA and endobronchial ultrasound guided fine needle aspiration (EBUS-FNA), as well as recommendations for classification and criteria, ancillary testing and post-cytologic diagnosis management and follow-up. All recommendation documents are based on the expertise of committee members, an extensive literature review, and feedback from presentations at national and international conferences. The guideline documents selectively present the results of these discussions. The present document summarizes recommendations for ancillary testing of cytologic samples. Ancillary testing including microbiologic, immunocytochemical, flow cytometric, and molecular testing, including next-generation sequencing are discussed. PMID:27561242

Novel Confocal Microscopic and Flow Cytometric Based Assays to Visualize and Detect the (Beta)2-Adrenergic Receptor in Human Lymphocyte and Mononuclear Cell Populations

NASA Technical Reports Server (NTRS)

Salicru, A. N.; Crucian, B. E.; Nelman, M. A.; Sams, C. F.; Actor, J. K.; Marshall, G. D.

2006-01-01

The data show that immunophenotyping of leukocyte populations with (beta)2AR is possible with the commercially available Ab, although the FC assay is limited to the IST as a result of the Ab binding site to the intracellular C-terminus of the 2AR. The FC assay has applications for measuring alterations in total (beta)2AR in human leukocyte populations as changes in fluorescence. In addition, CM confirms that both surface and intracellular compartments stain positively for the (beta)2AR and can be used for qualitative assays that screen for changes in receptor compartmentalization and localization.

[A new method of in vitro chemosensitivity test using multicellular spheroids of cholangiocarcinoma cell line cocultured with fibroblasts].

PubMed

Kubota, S; Takezawa, T; Mori, Y; Takakuwa, T

1992-09-01

We applied the multicellular spheroids which consist of cholangiocarcinoma cell line (MEC) and human dermal fibroblasts (HDF) to in vitro chemosensitivity test. Five-day multicellular spheroids were incubated with 1.5 micrograms/ml of mitomycin C (MMC) for 24 hrs. Then, cell kinetics of MEC and HDF in a spheroid was determined by flow cytometric analysis. Twenty four hrs after treatment with MMC, both MEC and HDF were accumulated on S phase. Seven-day after treatment, DNA histogram in MEC returned to normal, but that of HDF was disappeared. These results showed that the multicellular assay could be more like on in vivo like chemosensitivity test.

New sesquiterpene lactones from Ambrosia cumanensis Kunth.

PubMed

Jimenez-Usuga, Nora Del Socorro; Malafronte, Nicola; Cotugno, Roberta; De Leo, Marinella; Osorio, Edison; De Tommasi, Nunziatina

2016-09-01

Eleven sesquiterpene lactones, including three new natural products (1-3), were isolated from the n-butanolic extract of Ambrosia cumanensis Kunth. aerial parts. The structure of all isolated compounds was elucidated by 1D- and 2D-NMR, and MS analyses. All compounds were tested for their antiproliferative activity on HeLa, Jurkat, and U937 cell lines. Compound 3, 2,3-dehydropsilostachyn C, showed cytotoxic activity with different potency in all cell lines. By means of flow cytometric studies, compound 3 was demonstrated to induce in Jurkat cells a G2/M cell cycle block, while in U937 elicited both cytostatic and cytotoxic responses. Copyright © 2016 Elsevier B.V. All rights reserved.

Gene cloning, expression and functional characterization of a proliferation-inducing ligand (APRIL) from hedgehog (Erinaceus europaeus).

PubMed

Cui, Xian-Wei; Xiao, Wen; Ji, Chen-Bo; Tian, Ai-Ying; Zhang, Jie; Zhang, Shuang-Quan

2012-05-01

Here we describe the identification of the hedgehog Erinaceus europaeus homologue of a proliferation-inducing ligand (APRIL) of the TNF family (designated heAPRIL). Hedgehog APRIL contains two cysteine residues (Cys(196) and Cys(211)), a furin protease cleavage site and a conserved putative N-glycosylation site (Asn(124)). Real-time quantitative PCR (qPCR) analysis revealed that heAPRIL could be detected in various tissues. MTT assays and flow cytometric analysis revealed that Nus-hesAPRIL and hesAPRIL could promote the survival/proliferation of splenic B cells. Laser scanning confocal microscopy analysis showed GFP-hesAPRIL could successfully bind to the APRIL receptors of lymphocytes.

Flow cytometry beads rather than the antihuman globulin method should be used to detect HLA Class I IgG antibody (PRA) in cadaveric renal regraft candidates.

PubMed

Bryan, Christopher F; McDonald, Scott B; Baier, Karen A; Luger, Alan M; Aeder, Mark I; Murillo, Daniel; Muruve, Nicolas A; Nelson, Paul W; Shield, Charles F; Warady, Bradley A

2002-01-01

HLA Class I antibody screening can be performed by flow cytometry using a mixture of 30 distinct bead populations each coated with the Class I antigen phenotype derived from different cell lines. In this study we compared the efficacy of Class I antibody screens done by flow cytometry beads with the antihuman globulin (AHG) method for patients awaiting cadaveric renal retransplantation. Class I panel reactive antibody (PRA) screening by flow cytometric beads of 21 regraft serum samples that had all been found to be negative by AHG DTT Class I PRA, revealed that 57.1% (12 of 21) had a flow Class I PRA of > or = 10%. Furthermore, when five regraft sera with an intermediate PRA were screened (mean AHG DTT PRA = 33.2 +/- 13%) the mean flow Class I PRA almost doubled (mean flow PRA = 72.4 +/- 10.2%) (p < 0.01). When active UNOS waiting list regraft candidates, after several months of screening the Class I PRA by flow beads, were divided into the three PRA categories based on their peak flow Class I PRA value (0-20%, 21-79% and > or = 80%), the incidence of a positive flow cross-match was 0%, 72% and 85% and the incidence of retransplantation was 60%, 22% and 10%, in each of these groups, respectively. These data provided our histocompatibility laboratory with the rationale to stop performing the AHG PRA and perform only the flow Class I PRA method for regraft candidates.

Three-dimensional cell organization leads to almost immediate HRE activity as demonstrated by molecular imaging of MG-63 spheroids using two-photon excitation microscopy.

PubMed

Indovina, Paola; Collini, Maddalena; Chirico, Giuseppe; Santini, Maria Teresa

2007-02-20

Hypoxia through HRE (hypoxia-responsive element) activity in MG-63 human osteosarcoma cells grown in monolayer and as very small, three-dimensional tumor spheroids was investigated using molecular imaging techniques. MG-63 cells were stably transfected with a vector constructed with multiple copies of the HRE sequence of the human vascular endothelial growth factor (VEGF) gene and with the enhanced green fluorescent protein (EGFP) coding sequence. During hypoxia when HIF-1alpha (hypoxia-inducible factor-1alpha) is stabilized, the binding of HIF-1 to the HRE sequences of the vector allows the transcription of EGFP and the appearance of fluorescence. Transfected monolayer cells were characterized by flow cytometric analysis in response to various hypoxic conditions and HIF-1alpha expression in these cells was assessed by Western blotting. Two-photon excitation (TPE) microscopy was then used to examine both MG-63-transfected monolayer cells and spheroids at 2 and 5 days of growth in normoxic conditions. Monolayer cells reveal almost no fluorescence, whereas even very small spheroids (<100 microm) after 2 days of growth contain regions of high fluorescence. For the first time in the literature, at least to our knowledge, it is demonstrated, using highly sensitive and non-perturbing molecular imaging techniques, that three-dimensional cell organization leads to almost immediate HRE activation. This activation of the HRE sequences, which control a wide variety of genes, suggests that monolayer cells and spheroids of the MG-63 cell line have different genes activated and thus diverse functional activities.

Means and methods for cytometric therapies

DOEpatents

Gillies, George T.; Fillmore, Helen; Broaddus, William C.; Evans, III, Boyd M.; Allison, Stephen W.

2013-03-26

A functionalized tip is incorporated into catheters for the cytometric delivery of cells into the brain and other body parts. For use in the brain, the tip forms part of a neurosurgical probe having a proximal end and a distal end. In addition to the functionalized tip, the probe has at least one cell slurry delivery lumen and a plurality of optical fibers configured along the probe, terminating in the tip to provide the photo-optical capability needed to monitor the viability and physiological behavior of the grafted cells as well as certain characteristics of the cellular environment. Details are also presented of the use of a neurocatheter having a cytometric tip of the type disclosed in the invention, as employed within the context of a feedback and control system for regulating the number of cells delivered to the brain of a patient.

Expansion of blood IgG4+ B, TH2, and regulatory T cells in patients with IgG4-related disease.

PubMed

Heeringa, Jorn J; Karim, A Faiz; van Laar, Jan A M; Verdijk, Robert M; Paridaens, Dion; van Hagen, P Martin; van Zelm, Menno C

2018-05-01

IgG 4 -related disease (IgG 4 -RD) is a systemic fibroinflammatory condition affecting various organs and has a diverse clinical presentation. Fibrosis and accumulation of IgG 4 + plasma cells in tissue are hallmarks of the disease, and IgG 4 -RD is associated with increased IgG 4 serum levels. However, disease pathogenesis is still unclear, and these cellular and molecular parameters are neither sensitive nor specific for the diagnosis of IgG 4 -RD. Here we sought to develop a flow cytometric gating strategy to reliably identify blood IgG 4 + B cells to study their cellular and molecular characteristics and investigate their contribution in disease pathogenesis. Sixteen patients with histologically confirmed IgG 4 -RD, 11 patients with sarcoidosis, and 30 healthy subjects were included for 11-color flow cytometric analysis of peripheral blood for IgG 4 -expressing B cells and T H subsets. In addition, detailed analysis of activation markers and chemokine receptors was performed on IgG 4 -expressing B cells, and IgG 4 transcripts were analyzed for somatic hypermutations. Cellular and molecular analyses revealed increased numbers of blood IgG 4 + memory B cells in patients with IgG 4 -RD. These cells showed reduced expression of CD27 and CXCR5 and increased signs of antibody maturation. Furthermore, patients with IgG 4 -RD, but not patients with sarcoidosis, had increased numbers of circulating plasmablasts and CD21 low B cells, as well as T H 2 and regulatory T cells, indicating a common disease pathogenesis in patients with IgG 4 -RD. These results provide new insights into the dysregulated IgG 4 response in patients with IgG 4 -RD. A specific "peripheral lymphocyte signature" observed in patients with IgG 4 -RD, could support diagnosis and treatment monitoring. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Vinpocetine attenuates neointimal hyperplasia in diabetic rat carotid arteries after balloon injury.

PubMed

Wang, Ke; Wen, Li; Peng, Wenhui; Li, Hailing; Zhuang, Jianhui; Lu, Yuyan; Liu, Baoxin; Li, Xiankai; Li, Weiming; Xu, Yawei

2014-01-01

Diabetes exacerbates abnormal vascular smooth muscle cell (VSMC) accumulation in response to arterial wall injury. Vinpocetine has been shown to improve vascular remolding; however, little is known about the direct effects of vinpocetine on vascular complications mediated by diabetes. The objective of this study was to determine the effects of vinpocetine on hyperglycemia-facilitated neointimal hyperplasia and explore its possible mechanism. Nondiabetic and diabetic rats were subjected to balloon injury of the carotid artery followed by 3-week treatment with either vinpocetine (10 mg/kg/day) or saline. Morphological analysis and proliferating cell nuclear antigen (PCNA) immunostaining were performed on day 21. Rat VSMCs proliferation was determined with 5-ethynyl-20-deoxyuridine cell proliferation assays. Chemokinesis was monitored with scratch assays, and production of reactive oxygen species (ROS) was assessed using a 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) flow cytometric assay. Apoptosis was detected by annexin V-FITC/PI flow cytometric assay. Cell signaling was assessed by immunblotting. Vinpocetine prevented intimal hyperplasia in carotid arteries in both normal (I/M ratio: 93.83 ± 26.45% versus 143.2 ± 38.18%, P<0.05) and diabetic animals (I/M ratio: 120.5 ± 42.55% versus 233.46 ± 33.98%, P<0.05) when compared to saline. The in vitro study demonstrated that vinpocetine significantly inhibited VSMCs proliferation and chemokinesis as well as ROS generation and apoptotic resistance, which was induced by high glucose (HG) treatment. Vinpocetine significantly abolished HG-induced phosphorylation of Akt and JNK1/2 without affecting their total levels. For downstream targets, HG-induced phosphorylation of IκBα was significantly inhibited by vinpocetine. Vinpocetine also attenuated HG-enhanced expression of PCNA, cyclin D1 and Bcl-2. Vinpocetine attenuated neointimal formation in diabetic rats and inhibited HG-induced VSMCs proliferation, chemokinesis and apoptotic resistance by preventing ROS activation and affecting MAPK, PI3K/Akt, and NF-κB signaling.

Vinpocetine Attenuates Neointimal Hyperplasia in Diabetic Rat Carotid Arteries after Balloon Injury

PubMed Central

Peng, Wenhui; Li, Hailing; Zhuang, Jianhui; Lu, Yuyan; Liu, Baoxin; Li, Xiankai; Li, Weiming; Xu, Yawei

2014-01-01

Background Diabetes exacerbates abnormal vascular smooth muscle cell (VSMC) accumulation in response to arterial wall injury. Vinpocetine has been shown to improve vascular remolding; however, little is known about the direct effects of vinpocetine on vascular complications mediated by diabetes. The objective of this study was to determine the effects of vinpocetine on hyperglycemia-facilitated neointimal hyperplasia and explore its possible mechanism. Materials and Methods Nondiabetic and diabetic rats were subjected to balloon injury of the carotid artery followed by 3-week treatment with either vinpocetine (10 mg/kg/day) or saline. Morphological analysis and proliferating cell nuclear antigen (PCNA) immunostaining were performed on day 21. Rat VSMCs proliferation was determined with 5-ethynyl-20-deoxyuridine cell proliferation assays. Chemokinesis was monitored with scratch assays, and production of reactive oxygen species (ROS) was assessed using a 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) flow cytometric assay. Apoptosis was detected by annexin V-FITC/PI flow cytometric assay. Cell signaling was assessed by immunblotting. Results Vinpocetine prevented intimal hyperplasia in carotid arteries in both normal (I/M ratio: 93.83 ± 26.45% versus 143.2 ± 38.18%, P<0.05) and diabetic animals (I/M ratio: 120.5 ± 42.55% versus 233.46 ± 33.98%, P<0.05) when compared to saline. The in vitro study demonstrated that vinpocetine significantly inhibited VSMCs proliferation and chemokinesis as well as ROS generation and apoptotic resistance, which was induced by high glucose (HG) treatment. Vinpocetine significantly abolished HG-induced phosphorylation of Akt and JNK1/2 without affecting their total levels. For downstream targets, HG-induced phosphorylation of IκBα was significantly inhibited by vinpocetine. Vinpocetine also attenuated HG-enhanced expression of PCNA, cyclin D1 and Bcl-2. Conclusions Vinpocetine attenuated neointimal formation in diabetic rats and inhibited HG-induced VSMCs proliferation, chemokinesis and apoptotic resistance by preventing ROS activation and affecting MAPK, PI3K/Akt, and NF-κB signaling. PMID:24819198

Cobra venom cytotoxins; apoptotic or necrotic agents?

PubMed

Ebrahim, Karim; Shirazi, Farshad H; Mirakabadi, Abbas Zare; Vatanpour, Hossein

2015-12-15

Organs homeostasis is controlled by a dynamic balance between cell proliferation and apoptosis. Failure to induction of apoptosis has been implicated in tumor development. Cytotoxin-I (CTX-I) and cytotoxin-II (CTX-II) are two physiologically active polypeptides found in Caspian cobra venom. Anticancer activity and mechanism of cell death induced by these toxins have been studied. The toxins were purified by different chromatographic steps and their cytotoxicity and pattern of cell death were determined by MTT, LDH release, acridine orange/ethidium bromide (AO/EtBr) double staining, flow cytometric analysis, caspase-3 activity and neutral red assays. The IC50 of CTX-II in MCF-7, HepG2, DU-145 and HL-60 was 4.1 ± 1.3, 21.2 ± 4.4, 9.4 ± 1.8 μg/mL and 16.3 ± 1.9 respectively while the IC50 of this toxin in normal MDCK cell line was 54.5 ± 3.9 μg/mL. LDH release suddenly increase after a specific toxins concentrations in all cell lines. AO/EtBr double staining, flow cytometric analysis and caspase-3 activity assay confirm dose and time-dependent induction of apoptosis by both toxins. CTX-I and CTX-II treated cells lost their lysosomal membrane integrity and couldn't uptake neutral red day. CTX-I and CTX-II showed significant anticancer activity with minimum effects on normal cells and better IC50 compared to current anticancer drug; cisplatin. They induce their apoptotic effect via lysosomal pathways and release of cathepsins to cytosol. These effects were seen in limited rage of toxins concentrations and pattern of cell death rapidly changes to necrosis by increase in toxin's concentration. In conclusion, significant apoptogenic effects of these toxins candidate them as a possible anticancer agent. Copyright © 2015 Elsevier Ltd. All rights reserved.

Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting: Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4{sup +} T cells compared

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ford, A.L.; Goodsall, A.L.; Sedgwick, J.D.

1995-05-01

Ramified microglia in the adult central nervous system (CNS) are the principal glial element up-regulating MHC class I and II expression in response to inflammatory events or neuronal damage. A proportion of these cells also express MHC class II constitutively in the normal CNS. The role of microglia as APCs for CD4{sup +} cells extravasating into the CNS remains undefined. In this study, using irradiation bone marrow chimeras in CD45-congenic rats, the phenotype CD45{sup low}CD11b/c{sup +} is shown to identify microglial cells specifically within the CNS. Highly purified populations of microglia and nonmicroglial but CNS-associated macrophages (CD45{sup high}CD11b/c{sup +}) havemore » been obtained directly from the adult CNS, by using flow cytometric sorting. Morphologically, freshly isolated microglia vs other CNS macrophages are quite distinct. Of the two populations recovered from the normal CNS, it is the minority CD45{sup high}CD11 b/c{sup +} transitional macrophage population, and not microglia, that is the effective APC for experimental autoimmune encephalomyelitis-inducing CD4{sup +} myelin basic protein (MBP)-reactive T cells. CD45{sup high}CD11b/c{sup +} CNS macrophages also stimulate MBP-reactive T cells without addition of MBP to culture suggesting presentation of endogenous Ag. This is the first study in which microglia vs other CNS macrophages have been analyzed for APC ability directly from the CNS, with substantial cross-contamination between the two populations eliminated. The heterogeneity of these populations in terms of APC function is clearly demonstrated. Evidence is still lacking that adult CNS microglia have the capacity to interact with and stimulate CD4{sup +} T cells to proliferate or secrete IL-2. 60 refs., 6 figs., 1 tab.« less

Overexpression of adrenomedullin protects mesenchymal stem cells against hypoxia and serum deprivation-induced apoptosis via the Akt/GSK3β and Bcl-2 signaling pathways

PubMed Central

Song, Yuqing; Li, Lili

2018-01-01

The poor survival rate of transplanted mesenchymal stem cells (MSCs) within the ischemic heart limits their therapeutic potential for cardiac repair. Adrenomedullin (ADM) has been identified as a potent apoptotic inhibitor. The present study aimed to investigate the protective effects of ADM on MSCs against hypoxia and serum deprivation (H/SD)-induced apoptosis, and to determine the potential underlying mechanisms. In the present study, a recombinant adenovirus expressing the ADM gene was established and was infected into MSCs. The infection rate was determined via microscopic detection of green fluorescence and flow cytometric analysis. The mRNA expression levels of ADM were detected by reverse transcription-polymerase chain reaction. In addition, a model of H/SD was generated. The MSCs were randomly separated into six groups: Control, enhanced green fluorescent protein (EGFP)-Adv, EGFP-ADM, H/SD, EGFP-Adv + H/SD and EGFP-ADM + H/SD. Cell viability and proliferation were determined using the Cell Counting kit-8 assay. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated-dUTP nick-end labeling assay and flow cytometric analysis using Annexin V-phycoerythrin/7-aminoactinomycin D staining. The protein expression levels of total protein kinase B (Akt), phosphorylated (p)-Akt, total glycogen synthase kinase (GSK)3β, p-GSK3β, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), caspase-3 and cleaved caspase-3 were detected by western blot analysis. The results indicated that ADM overexpression could improve MSC proliferation and viability, and protect MSCs against H/SD-induced apoptosis. In addition, ADM overexpression increased Akt and GSK3β phosphorylation, and Bcl-2/Bax ratio, and decreased the activation of caspase-3. These results suggested that ADM protects MSCs against H/SD-induced apoptosis, which may be mediated via the Akt/GSK3β and Bcl-2 signaling pathways. PMID:29512737

Evaluation of the effect of storage condition on cell extraction and flow cytometric analysis from intestinal biopsies.

PubMed

Wildenberg, Manon E; Duijvestein, Marjolijn; Westera, Liset; van Viegen, Tanja; Buskens, Christianne J; van der Bilt, Jarmila D W; Stitt, Larry; Jairath, Vipul; Feagan, Brian G; Vande Casteele, Niels

2018-06-01

Flow cytometric (FC) analysis of intestinal tissue biopsies requires prompt cell isolation and processing to prevent cell death and generate valid data. We examined the effect of storage conditions prior to cell isolation and FC on viable cell yield and the proportions of immune cell phenotypes from intestinal biopsies. Biopsies (N = 224) from inflamed or non-inflamed ileal and/or colonic tissue from three patients with Crohn's disease were processed and analyzed immediately in duplicate, or stored under different conditions. Cells were isolated and stained for specific markers, followed by FC. Decreased mean live CD45+ cell counts were observed after storage of biopsies at -80 °C dimethyl sulfoxide (DMSO)/citrate buffer compared with immediate processing (1794.3 vs. 19,672.7; p = 0.006]). A non-significant decrease in CD45+ live cell count occurred after storage at -20 °C in DMSO/citrate buffer and cell yield was adequate for subsequent analysis. CD3+ cell proportions were significantly lower after storage at 4 °C in complete medium for 48 h compared with immediate analysis. Mean CD14+ cell proportions were significantly higher after storage of biopsies at -80 °C in DMSO/citrate buffer compared with immediate analysis (2.61% vs. 1.31%, p = 0.007). CD4+, CD8+ and CD4+/CD8+ cell proportions were unaffected by storage condition. Storage of intestinal tissue biopsies at -20 °C in DMSO/citrate buffer for up to 48 h resulted in sufficient viable cell yield for FC analysis without affecting subsequent marker-positive cell proportions. These findings support the potential shipping and storage of intestinal biopsies for centralized FC analysis in multicenter clinical trials. Copyright © 2018 Elsevier B.V. All rights reserved.

ZAP-70 expression in B-cell chronic lymphocytic leukemia: evaluation by external (isotypic) or internal (T/NK cells) controls and correlation with IgV(H) mutations.

PubMed

Zucchetto, Antonella; Bomben, Riccardo; Bo, Michele Dal; Nanni, Paola; Bulian, Pietro; Rossi, Francesca Maria; Del Principe, Maria Ilaria; Santini, Simone; Del Poeta, Giovanni; Degan, Massimo; Gattei, Valter

2006-07-15

Expression of T cell specific zeta-associated protein 70 (ZAP-70) by B-cell chronic lymphocytic leukemia (B-CLL) cells, as investigated by flow cytometry, has both prognostic relevance and predictive power as surrogate for immunoglobulin heavy chain variable region (IgV(H)) mutations, although a standardization of the cytometric protocol is still lacking. Flow cytometric analyses for ZAP-70 were performed in peripheral blood samples from 145 B-CLL (124 with IgV(H) mutations) by a standard three-color protocol. Identification of ZAP-70(+) cell population was based on an external negative control, i.e., the isotypic control (ISO method) or an internal positive control, i.e., the population of residual normal T/NK cells (TNK method). A comparison between these two approaches was performed. While 86/145 cases were concordant as for ZAP-70 expression according to the two methods (ISO(+)TNK(+) or ISO(-)TNK(-)), 59/145 cases had discordant ZAP-70 expression, mainly (56/59) showing a ISO(+)TNK(-) profile. These latter cases express higher levels of ZAP-70 in their normal T cell component. Moreover, discordant ISO(+)TNK(-) cases had a IgV(H) gene mutation profile similar to that of concordantly positive cases and different from ZAP-70 concordantly negative B-CLL. Analysis of ZAP-70 expression by B-CLL cells by using the ISO method allows to overcome the variability in the expression of ZAP-70 by residual T cells and yields a better correlation with IgV(H) gene mutations. A receiver operating characteristic analysis suggests to employ a higher cut-off than the commonly used 20%. A parallel evaluation of the prognostic value of ZAP-70 expression, as determined according to the ISO and TNK methods, is still needed. (c) 2006 International Society for Analytical Cytology.

Cloning and expression of canine CD25 for validation of an anti-human CD25 antibody to compare T regulatory lymphocytes in healthy dogs and dogs with osteosarcoma.

PubMed

Rissetto, K C; Rindt, H; Selting, K A; Villamil, J A; Henry, C J; Reinero, C R

2010-05-15

T regulatory cells (Tregs) are a unique subset of T helper cells that serve to modify/inhibit effector cells of the immune system and thus are essential to prevent autoimmunity. Overzealous Treg activity may contribute to impaired immune responses to cancer. Tregs can be phenotypically identified by proteins expressed on the cell surface (CD4 and CD25) and inside the cell (forkhead box3 (FoxP3)), although in dogs, no anti-canine CD25 antibody exists. We hypothesized that a mouse anti-human CD25 antibody definitively recognizes the canine protein and can be used to identify Tregs in dogs. We describe cloning and transfection of the canine CD25 gene into human HeLa cells with subsequent expression of the canine protein on the cell surface detected using an anti-human CD25 antibody in a flow cytometric assay. Validation of this antibody was used to identify CD4+CD25+FoxP3+ Tregs in 39 healthy dogs and 16 dogs with osteosarcoma (OSA). Results were expressed in five different ways and showed significantly fewer %CD4+CD25+ T lymphocytes expressing FoxP3 in blood of older dogs (>/=7 years) compared with the other two age groups (<2 and 2-6 years) (p<0.001) and fewer %CD4+CD25+FoxP3+ Tregs in the tumor draining lymph nodes of OSA patients compared to the unrelated lymph node (p=0.049). However, there was no significant difference in % Tregs in the peripheral blood or lymph nodes between the control dogs and those with OSA. While the CD25 antibody can be successfully used in a flow cytometric assay to identify Tregs, this study does not support clinical utility of phenotypic recognition of Tregs in dogs with OSA. Copyright 2010 Elsevier B.V. All rights reserved.

Foxn1 Transcription Factor Regulates Wound Healing of Skin through Promoting Epithelial-Mesenchymal Transition

PubMed Central

Gawronska-Kozak, Barbara; Grabowska, Anna; Kur-Piotrowska, Anna; Kopcewicz, Marta

2016-01-01

Transcription factors are key molecules that finely tune gene expression in response to injury. We focused on the role of a transcription factor, Foxn1, whose expression is limited to the skin and thymus epithelium. Our previous studies showed that Foxn1 inactivity in nude mice creates a pro-regenerative environment during skin wound healing. To explore the mechanistic role of Foxn1 in the skin wound healing process, we analyzed post-injured skin tissues from Foxn1::Egfp transgenic and C57BL/6 mice with Western Blotting, qRT-PCR, immunofluorescence and flow cytometric assays. Foxn1 expression in non-injured skin localized to the epidermis and hair follicles. Post-injured skin tissues showed an intense Foxn1-eGFP signal at the wound margin and in leading epithelial tongue, where it co-localized with keratin 16, a marker of activated keratinocytes. This data support the concept that suprabasal keratinocytes, expressing Foxn1, are key cells in the process of re-epithelialization. The occurrence of an epithelial-mesenchymal transition (EMT) was confirmed by high levels of Snail1 and Mmp-9 expression as well as through co-localization of vimentin/E-cadherin-positive cells in dermis tissue at four days post-wounding. Involvement of Foxn1 in the EMT process was verified by co-localization of Foxn1-eGFP cells with Snail1 in histological sections. Flow cytometric analysis showed the increase of double positive E-cadherin/N-cadherin cells within Foxn1-eGFP population of post-wounded skin cells isolates, which corroborated histological and gene expression analyses. Together, our findings indicate that Foxn1 acts as regulator of the skin wound healing process through engagement in re-epithelization and possible involvement in scar formation due to Foxn1 activity during the EMT process. PMID:26938103

A New Submersible Imaging-in-flow Instrument to Monitor Nano- and Microplankton: Imaging FlowCytobot

NASA Astrophysics Data System (ADS)

Olson, R. J.; Sosik, H. M.; Shalapyonok, A.

2004-12-01

Understanding of how coastal plankton communities are regulated has traditionally been limited by undersampling, but cabled observatories now provide opportunities to deploy submersible sensors that have high power and data transmission requirements. We have developed an in situ instrument to carry out high-resolution, long term monitoring of phytoplankton and microzooplankton in the size range 10 to100 micrometers, to be deployed at cabled research facilities such as the Martha's Vineyard Coastal Observatory (MVCO). The new instrument is designed to complement FlowCytobot, a submersible flow cytometer currently deployed at MVCO that uses fluorescence and light scattering signals from a laser beam to characterize the smallest phytoplankton cells (less than 10 micrometers). Imaging FlowCytobot uses a combination of flow cytometric and video technology to capture images of organisms for identification and to measure chlorophyll fluorescence associated with each image. Images will be classified using neural net software, while the measurements of chlorophyll fluorescence will allow us to discriminate heterotrophic from phototrophic cells. The new instrument, like the original FlowCytobot is autonomous but remotely programmable. It utilizes a computer controlled syringe pump and distribution valve that allows periodic anti-fouling treatment and analysis of standard beads. Samples are analyzed continuously (0.25 to 2.5 ml per min) and data is sent over a fiber optic link to a remote computer for analysis. Preliminary results indicate that we can detect cells as small as 5 micrometers and discriminate several taxa of diatoms and dinoflagellates.

Morphologic, cytometric and functional characterization of the common octopus (Octopus vulgaris) hemocytes.

PubMed

Castellanos-Martínez, S; Prado-Alvarez, M; Lobo-da-Cunha, A; Azevedo, C; Gestal, C

2014-05-01

The hemocytes of Octopus vulgaris were morphologically and functionally characterized. Light and electron microscopy (TEM and SEM), and flow cytometry analyses revealed the existence of two hemocyte populations. Large granulocytes showed U-shaped nucleus, a mean of 11.6 μm±1.2 in diameter with basophilic granules, polysaccharide and lysosomic deposits in the cytoplasm. Small granulocytes measured a mean of 8.1 μm±0.7 in diameter, and have a round nucleus occupying almost the entire cell and few or not granules in the cytoplasm. Flow cytometry analysis showed that large granulocytes are the principal cells that develop phagocytosis of latex beads (rising up to 56%) and ROS after zymosan stimulation. Zymosan induced the highest production of both ROS and NO. This study is the first tread towards understanding the O. vulgaris immune system by applying new tools to provide a most comprehensive morpho-functional study of their hemocytes. Copyright © 2013 Elsevier Ltd. All rights reserved.

Flow Cytometric Methods for Circulating Tumor Cell Isolation and Molecular Analysis.

PubMed

Bhagwat, Neha; Carpenter, Erica L

2017-01-01

Circulating tumor cells provide a non-invasive source of tumor material that can be valuable at all stages of disease management, including screening and early diagnosis, monitoring response to therapy, identifying therapeutic targets, and assessing development of drug resistance. Cells isolated from the blood of cancer patients can be used for phenotypic analysis, tumor genotyping, transcriptional profiling, as well as for ex vivo culture of isolated cells. There are a variety of novel technologies currently being developed for the detection and analysis of rare cells in circulation of cancer patients. Flow cytometry is a powerful cell analysis platform that is increasingly being used in this field of study due to its relatively high throughput and versatility with respect to the large number of commercially available antibodies and fluorescent probes available to translational and clinical researchers. More importantly, it offers the ability to easily recover viable cells with high purity that are suitable for downstream molecular analysis, thus making it an attractive technology for cancer research and as a diagnostic tool.

Flow cytometric minimal residual disease assessment of peripheral blood in acute lymphoblastic leukaemia patients has potential for early detection of relapsed extramedullary disease.

PubMed

Keegan, Alissa; Charest, Karry; Schmidt, Ryan; Briggs, Debra; Deangelo, Daniel J; Li, Betty; Morgan, Elizabeth A; Pozdnyakova, Olga

2018-03-27

To evaluate peripheral blood (PB) for minimal residual disease (MRD) assessment in adults with acute lymphoblastic leukaemia (ALL). We analysed 76 matched bone marrow (BM) aspirate and PB specimens independently for the presence of ALL MRD by six-colour flow cytometry (FC). The overall rate of BM MRD-positivity was 24% (18/76) and PB was also MRD-positive in 22% (4/18) of BM-positive cases. We identified two cases with evidence of leukaemic cells in PB at the time of the extramedullary relapse that were interpreted as MRD-negative in BM. The use of PB MRD as a non-invasive method for monitoring of systemic relapse may have added clinical and diagnostic value in patients with high risk of extramedullary disease. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Effect of long-term clopidogrel treatment on platelet function and inflammation in patients undergoing coronary arterial stenting.

PubMed

Antonino, Mark J; Mahla, Elisabeth; Bliden, Kevin P; Tantry, Udaya S; Gurbel, Paul A

2009-06-01

A clopidogrel loading dose administered during stenting attenuates inflammation marker release. However, less is known of the anti-inflammatory effect of clopidogrel maintenance therapy. Platelet reactivity to adenosine diphosphate and inflammation markers were measured in 110 consecutive patients (69 clopidogrel-naive patients and 41 patients receiving long-term clopidogrel therapy for >6 months) before nonemergent stenting by turbidimetric aggregometry and flow cytometry and multianalyte profiling, respectively. All patients were treated with aspirin. Prestenting adenosine diphosphate-induced platelet aggregation, P-selectin, and activated glycoprotein IIb/IIIa expression were lower in patients receiving long-term clopidogrel therapy compared with the clopidogrel-naive group (p <0.001), accompanied by lower levels of selected inflammation markers (p < or = 0.05). Additionally, there were strong correlations between platelet aggregation and flow cytometric measurements (p < or = 0.04) and between specific inflammation markers (p < or = 0.02). In conclusion, in addition to markedly lowering platelet reactivity to adenosine diphosphate, long-term clopidogrel therapy is associated with an anti-inflammatory effect.

Preparation of highly fluorescent magnetic nanoparticles for analytes-enrichment and subsequent biodetection.

PubMed

Zhang, Bingbo; Chen, Bingdi; Wang, Yilong; Guo, Fangfang; Li, Zhuoquan; Shi, Donglu

2011-01-15

Bifunctional nanoparticles with highly fluorescence and decent magnetic properties have been widely used in biomedical application. In this study, highly fluorescent magnetic nanoparticles (FMNPs) with uniform size of ca. 40 nm are prepared by encapsulation of both magnetic nanoparticles (MNPs) and shell/core quantum dots (QDs) with well-designed shell structure/compositions into silica matrix via a one-pot reverse microemulsion approach. The spectral analysis shows that the FMNPs hold high fluorescent quantum yield (QY). The QYs and saturation magnetization of the FMNPs can be regulated by varying the ratio of the encapsulated QDs to MNPs. Moreover, the surface of the FMNPs can be modified to offer chemical groups for antibody conjugation for following use in target-enrichment and subsequent fluorescent detection. The in vitro immunofluorescence assay and flow cytometric analysis indicate that the bifunctional FMNPs-antibody bioconjugates are capable of target-enrichment, magnetic separation and can also be used as alternative fluorescent probes on flow cytometry for biodetection. Copyright © 2010 Elsevier Inc. All rights reserved.

Flow analysis of individual blood extracellular vesicles in acute coronary syndrome.

PubMed

Vagida, Murad; Arakelyan, Anush; Lebedeva, Anna; Grivel, Jean-Charles; Shpektor, Alexander; Vasilieva, Elena; Margolis, Leonid

2017-03-01

A diverse population of small extracellular vesicles (EVs) that are released by various cells has been characterized predominantly in bulk, a procedure whereby the individual characteristics of EVs are lost. Here, we used a new nanotechnology-based flow cytometric analysis to characterize the antigenic composition of individual EVs in patients with acute coronary syndrome (ACS). Plasma EVs were captured with 15-nm magnetic nanoparticles coupled to antibodies against CD31 (predominantly an endothelial marker), CD41a (a marker for platelets), and CD63 or MHC class I (common EV markers). The total amounts of EVs were higher in the ACS patients than in the controls, predominantly due to the contribution of patients with acute myocardial infarction. For all captured fractions, the differences in the EV amounts were restricted to CD41a + EVs. The increase in the numbers of EVs in the ACS patients, predominantly of platelet origin, probably reflects platelet activation and may indicate disease progression.

Flow cytometric monitoring of hormone receptor expression in human solid tumors

NASA Astrophysics Data System (ADS)

Krishan, Awtar

2002-05-01

Hormone receptor expression in human breast and prostate tumors is of diagnostic and therapeutic importance. With the availability of anti-estrogen, androgen and progesterone antibodies, immunohistochemistry has become a standard tool for determination of receptor expression in human tumor biopsies. However, this method is dependent on examination of a small number of cells under a microscope and the data obtained in most cases is not quantitative. As most of the commercially used anti-hormone antibodies have nuclear specificity, we have developed methods for isolation and antigen unmasking of nuclei from formalin fixed/paraffin embedded archival human tumors. After immunostaining with the antibodies and propidium iodide (for DNA content and cell cycle analysis), nuclei are analyzed by multiparametric laser flow cytometry for hormone receptor expression, DNA content, aneuploidy and cell cycle determination. These multiparametric methods are especially important for retrospective studies seeking to correlate hormone receptor expression with clinical response to anti-hormonal therapy of human breast and prostate tumors.

Genetic stock assessment of spawning arctic cisco (Coregonus autumnalis) populations by flow cytometric determination of DNA content.

PubMed

Lockwood, S F; Bickham, J W

1991-01-01

Intraspecific variation in cellular DNA content was measured in five Coregonus autumnalis spawning populations from the Mackenzie River drainage, Canada, using flow cytometry. The rivers assayed were the Peel, Arctic Red, Mountain, Carcajou, and Liard rivers. DNA content was determined from whole blood preparations of fish from all rivers except the Carcajou, for which kidney tissue was used. DNA content measurements of kidney and blood preparations of the same fish from the Mountain River revealed statistically indistinguishable results. Mosaicism was found in blood preparations from the Peel, Arctic Red, Mountain, and Liard rivers, but was not observed in kidney tissue preparations from the Mountain or Carcajou rivers. The Liard River sample had significantly elevated mean DNA content relative to the other four samples; all other samples were statistically indistinguishable. Significant differences in mean DNA content among spawning stocks of a single species reinforces the need for adequate sample sizes of both individuals and populations when reporting "C" values for a particular species.

Effects of pneumonia and malnutrition on the frequency of micronuclei in peripheral blood of pediatric patients

PubMed Central

Elsayh, Khalid I; Sayed, Douaa M; Zahran, Asmaa M; Saad, Khaled; Badr, Gamal

2013-01-01

The aim of this study was to evaluate the effects of bacterial pneumonia and malnutrition on the frequency of micronuclei (MN) in peripheral blood of pediatric patients through flow cytometric analysis. The study was an analytical case-control study carried out on 35 malnourished children with bacterial pneumonia and 20 well-nourished children with bacterial pneumonia, in addition to 20 healthy children as controls. Complete physical examination including; anthropometric measurement, Chest roentgenograms were done for all cases. Assessment of MN was done by FACSCalibur flow cytometry. The frequency of micronucleated reticulocytes (MN-RETs) was higher both in the malnourished children with pneumonia and well-nourished children with pneumonia than the controls. Within the malnourished children with pneumonia, patients with kwashiorkor had more micronucleated mature erythrocytes (MN-RBCs) and MN-RETs than patients with marasmus. In conclusion: Pneumonia is associated with an increased frequency of MN and this increment is more pronounced in children with severe malnutrition especially kwashiorkor group. PMID:24260601

Physics of a rapid CD4 lymphocyte count with colloidal gold.

PubMed

Hansen, P; Barry, D; Restell, A; Sylvia, D; Magnin, O; Dombkowski, D; Preffer, F

2012-03-01

The inherent surface charges and small diameters that confer colloidal stability to gold particle conjugates (immunogold) are detrimental to rapid cell surface labeling and distinct cluster definition in flow cytometric light scatter assays. Although the inherent immunogold surface charge prevents self aggregation when stored in liquid suspension, it also slows binding to cells to timeframes of hours and inhibits cell surface coverage. Although the small diameter of immunogold particles prevents settling when in liquid suspension, small particles have small light scattering cross sections and weak light scatter signals. We report a new, small particle lyophilized immunogold reagent that maintains activity after 42°C storage for a year and can be rapidly dissolved into stable liquid suspension for use in labelling cells with larger particle aggregates that have enhanced scattering cross section. Labeling requires less than 1 min at 20°C, which is ∼30 times faster than customary fluorescent antibody labeling. The labeling step involves neutralizing the surface charge of immunogold and creating specifically bound aggregates of gold on the cell surface. This process provides distinct side-scatter cluster separation with blue laser light at 488 nm, which is further improved by using red laser light at 640 nm. Similar comparisons using LED light sources showed less improvement with red light, thereby indicating that coherent light scatter is of significance in enhancing side-scatter cluster separation. The physical principles elucidated here for this technique are compatible with most flow cytometers; however, future studies of its clinical efficacy should be of primary interest in point-of-care applications where robust reagents and rapid results are important. Copyright © 2011 International Society for Advancement of Cytometry.

Development of a flow cytometric method to analyze subpopulations of bacteria in probiotic products and dairy starters.

PubMed

Bunthof, Christine J; Abee, Tjakko

2002-06-01

Flow cytometry (FCM) is a rapid and sensitive technique that can determine cell numbers and measure various physiological characteristics of individual cells by using appropriate fluorescent probes. Previously, we developed an FCM assay with the viability probes carboxyfluorescein diacetate (cFDA) and TOTO-1 [1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3dihydro(benzo-1,3-oxazole)-2-methylidene]-1-(3'-trimethylammoniumpropyl)-pyridinium tetraiodide] for (stressed) lactic acid bacteria (C. J. Bunthof, K. Bloemen, P. Breeuwer, F. M. Rombouts, and T. Abee, Appl. Environ. Microbiol. 67:2326-2335, 2001). cFDA stains intact cells with enzymatic activity, and TOTO-1 stains membrane-permeabilized cells. Here we used this assay to study the viability of bacterial suspensions in milk, dairy fermentation starters, and probiotic products. To facilitate FCM analysis of bacteria in milk, a commercially available milk-clearing solution was used. The procedure was optimized to increase the signal-to-noise ratio. FCM enumerations were accurate down to a concentration of 10(5) cells ml(-1). The level of retrieval of Lactobacillus plantarum WCFS 1 suspended in milk was high, and viability was not affected by the procedure. The plate counts for cleared samples of untreated cell suspensions were nearly as high as the total FCM counts, and the correlation was strong (r > 0.99). In dairy fermentation starters and in probiotic products the FCM total cell counts were substantially higher than the numbers of CFU. Three functional populations could be distinguished: culturable cells, cells that are intact and metabolically active but not culturable, and permeabilized cells. The proportions of the populations differed in the products tested. This FCM method provides tools to assess the functionality of different populations in fermentation starters and probiotic products.

Development of a Flow Cytometric Method To Analyze Subpopulations of Bacteria in Probiotic Products and Dairy Starters

PubMed Central

Bunthof, Christine J.; Abee, Tjakko

2002-01-01

Flow cytometry (FCM) is a rapid and sensitive technique that can determine cell numbers and measure various physiological characteristics of individual cells by using appropriate fluorescent probes. Previously, we developed an FCM assay with the viability probes carboxyfluorescein diacetate (cFDA) and TOTO-1 {1′-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3dihydro(benzo-1,3-oxazole)-2-methylidene]-1-(3′-trimethylammoniumpropyl)-pyridinium tetraiodide} for (stressed) lactic acid bacteria (C. J. Bunthof, K. Bloemen, P. Breeuwer, F. M. Rombouts, and T. Abee, Appl. Environ. Microbiol. 67:2326-2335, 2001). cFDA stains intact cells with enzymatic activity, and TOTO-1 stains membrane-permeabilized cells. Here we used this assay to study the viability of bacterial suspensions in milk, dairy fermentation starters, and probiotic products. To facilitate FCM analysis of bacteria in milk, a commercially available milk-clearing solution was used. The procedure was optimized to increase the signal-to-noise ratio. FCM enumerations were accurate down to a concentration of 105 cells ml−1. The level of retrieval of Lactobacillus plantarum WCFS 1 suspended in milk was high, and viability was not affected by the procedure. The plate counts for cleared samples of untreated cell suspensions were nearly as high as the total FCM counts, and the correlation was strong (r > 0.99). In dairy fermentation starters and in probiotic products the FCM total cell counts were substantially higher than the numbers of CFU. Three functional populations could be distinguished: culturable cells, cells that are intact and metabolically active but not culturable, and permeabilized cells. The proportions of the populations differed in the products tested. This FCM method provides tools to assess the functionality of different populations in fermentation starters and probiotic products. PMID:12039752

Flow cytometric sex sorting affects CD4 membrane distribution and binding of exogenous DNA on bovine sperm cells.

PubMed

Domingues, William Borges; da Silveira, Tony Leandro Rezende; Komninou, Eliza Rossi; Monte, Leonardo Garcia; Remião, Mariana Härter; Dellagostin, Odir Antônio; Corcini, Carine Dahl; Varela Junior, Antônio Sergio; Seixas, Fabiana Kömmling; Collares, Tiago; Campos, Vinicius Farias

2017-08-01

Bovine sex-sorted sperm have been commercialized and successfully used for the production of transgenic embryos of the desired sex through the sperm-mediated gene transfer (SMGT) technique. However, sex-sorted sperm show a reduced ability to internalize exogenous DNA. The interaction between sperm cells and the exogenous DNA has been reported in other species to be a CD4-like molecule-dependent process. The flow cytometry-based sex-sorting process subjects the spermatozoa to different stresses causing changes in the cell membrane. The aim of this study was to elucidate the relationship between the redistribution of CD4-like molecules and binding of exogenous DNA to sex-sorted bovine sperm. In the first set of experiments, the membrane phospholipid disorder and the redistribution of the CD4 were evaluated. The second set of experiments was conducted to investigate the effect of CD4 redistribution on the mechanism of binding of exogenous DNA to sperm cells and the efficiency of lipofection in sex-sorted bovine sperm. Sex-sorting procedure increased the membrane phospholipid disorder and induced the redistribution of CD4-like molecules. Both X-sorted and Y-sorted sperm had decreased DNA bound to membrane in comparison with the unsorted sperm; however, the binding of the exogenous DNA was significantly increased with the addition of liposomes. Moreover, we demonstrated that the number of sperm-bound exogenous DNA was decreased when these cells were preincubated with anti-bovine CD4 monoclonal antibody, supporting our hypothesis that CD4-like molecules indeed play a crucial role in the process of exogenous DNA/bovine sperm cells interaction.

NOTCH1 Is Aberrantly Activated in Chronic Lymphocytic Leukemia Hematopoietic Stem Cells.

PubMed

Di Ianni, Mauro; Baldoni, Stefano; Del Papa, Beatrice; Aureli, Patrizia; Dorillo, Erica; De Falco, Filomena; Albi, Elisa; Varasano, Emanuela; Di Tommaso, Ambra; Giancola, Raffaella; Accorsi, Patrizia; Rotta, Gianluca; Rompietti, Chiara; Silva Barcelos, Estevão Carlos; Campese, Antonio Francesco; Di Bartolomeo, Paolo; Screpanti, Isabella; Rosati, Emanuela; Falzetti, Franca; Sportoletti, Paolo

2018-01-01

To investigate chronic lymphocytic leukemia (CLL)-initiating cells, we assessed NOTCH1 mutation/expression in hematopoietic stem cells (HSCs). In NOTCH1- mutated CLL, we detected subclonal mutations in 57% CD34+/CD38- HSCs. NOTCH1 mutation was present in 66% CD34+/CD38+ progenitor cells displaying an increased mutational burden compared to HSCs. Flow cytometric analysis revealed significantly higher NOTCH1 activation in CD34+/CD38- and CD34+/CD38+ cells from CLL patients, regardless NOTCH1 mutation compared to healthy donors. Activated NOTCH1 resulted in overexpression of the NOTCH1 target c-MYC. We conclude that activated NOTCH1 is an early event in CLL that may contribute to aberrant HSCs in this disease.

Assessment of cell death mechanisms triggered by 177Lu-anti-CD20 in lymphoma cells.

PubMed

Azorín-Vega, E; Rojas-Calderón, E; Martínez-Ventura, B; Ramos-Bernal, J; Serrano-Espinoza, L; Jiménez-Mancilla, N; Ordaz-Rosado, D; Ferro-Flores, G

2018-08-01

The aim of this research was to evaluate the cell cycle redistribution and activation of early and late apoptotic pathways in lymphoma cells after treatment with 177 Lu-anti-CD20. Experimental and computer models were used to calculate the radiation absorbed dose to cancer cell nuclei. The computer model (Monte Carlo, PENELOPE) consisted of twenty spheres representing cells with an inner sphere (cell nucleus) embedded in culture media. Radiation emissions of the radiopharmaceutical located in cell membranes and in culture media were considered for nuclei dose calculations. Flow cytometric analyses demonstrated that doses as low as 4.8Gy are enough to induce cell cycle arrest and activate late apoptotic pathways. Copyright © 2017 Elsevier Ltd. All rights reserved.

Acute B cell lymphoblastic leukaemia in a 12-week-old greyhound.

PubMed

Adams, J; Mellanby, R J; Villiers, E; Baines, S; Woodger, N

2004-11-01

A 12-week-old greyhound had a two-day history of lethargy, inappetence and shifting lameness. Clinical examination revealed pyrexia and hepatosplenomegaly. Haematological examination showed anaemia, thrombocytopenla, neutropenla and large numbers of atypical mononuclear leucocytes. A diagnosis of acute B cell lymphoblastic leukaemia was made following flow cytometric immunophenotyping of the leucocytes. The owner declined further evaluation and the dog was treated symptomatically with antibiotics. After a brief improvement, the dog's condition deteriorated and it was euthanased four days after initial presentation. The case was unusual because acute lymphoid leukaemia in the dog is most frequently reported in mature animals. This is in contrast to humans, where acute leukaemia is one of the most common childhood cancers.

Role of receptor occupancy assays by flow cytometry in drug development.

PubMed

Stewart, Jennifer J; Green, Cherie L; Jones, Nicholas; Liang, Meina; Xu, Yuanxin; Wilkins, Danice E C; Moulard, Maxime; Czechowska, Kamila; Lanham, David; McCloskey, Thomas W; Ferbas, John; van der Strate, Barry W A; Högerkorp, Carl-Magnus; Wyant, Timothy; Lackey, Alan; Litwin, Virginia

2016-03-01

The measurement of the binding of a biotherapeutic to its cellular target, receptor occupancy (RO), is increasingly important in development of biologically-based therapeutic agents. Receptor occupancy (RO) assays by flow cytometry describe the qualitative and/or quantitative assessment of the binding of a therapeutic agent to its cell surface target. Such RO assays can be as simple as measuring the number of cell surface receptors bound by an antireceptor therapeutic agent or can be designed to address more complicated scenarios such as internalization or shedding events once a receptor engages the administered therapeutic agent. Data generated from RO assays can also be used to model whether given doses of an experimental therapeutic agent and their administration schedules lead to predicted levels of receptor occupancy and whether the receptor is modulated (up or down) on cells engaged by the therapeutic agent. There are a variety of approaches that can be used when undertaking RO assays and with the ability to measure distinct subsets in heterogeneous populations, flow cytometry is ideally suited to RO measurements. This article highlights the importance of RO assays on the flow cytometric platform in the development of biotherapeutic agents. © 2016 The Authors Cytometry Part B: Clinical Cytometry Published by Wiley Periodicals, Inc.

Liposuction Preserves the Morphological Integrity of the Microvascular Network: Flow Cytometry and Confocal Microscopy Evidence in a Controlled Study.

PubMed

Bertheuil, Nicolas; Chaput, Benoit; Berger-Müller, Sandra; Ménard, Cédric; Mourcin, Frédéric; Watier, Eric; Grolleau, Jean-Louis; Garrido, Ignacio; Tarte, Karin; Sensébé, Luc; Varin, Audrey

2016-05-01

Liposuction is a very popular technique in plastic surgery that allows for the taking adipose tissue (AT) on large surfaces with little risk of morbidity. Although liposuction was previously shown to preserve large perforator vessels, little is known about the effects of liposuction on the microvasculature network. The aim of this study was to analyze the effect of liposuction on the preservation of microvessels at tissue and cellular levels by flow cytometry and confocal microscopy following abdominoplasty procedure. Percentage of endothelial cells in AT from liposuction and en bloc AT was determined by multicolor flow cytometry. Moreover, vessel density and adipocyte content were analyzed in situ in 3 different types of AT (en bloc, from liposuction, and residual AT after liposuction) by confocal microscopy. Flow cytometric analysis showed that en bloc AT contained 30.6% ± 12.9% and AT from liposuction 21.6% ± 9.9% of endothelial cells (CD31(pos)/CD45(neg)/CD235a(neg)/CD11b(neg)) (P = .009). Moreover, analysis of paired AT from the same patients (n = 5) confirmed a lower percentage of endothelial cells in AT from liposuction compared to en bloc AT (17.7% ± 4.5% vs 21.9% ± 3.3%, P = .031). Likewise, confocal microscopy showed that en bloc AT contained 8.2% ± 6.3%, AT from liposuction only 1.6% ± 1.0% (P < .0001), and AT after liposuction 8.9% ± 4.1% (P = .111) of CD31(pos) vessels. Conversely, adipocyte content was 39.5% ± 14.5% in the en bloc AT, 45% ± 18.4% in AT from liposuction (P = .390), and 18.8 ± 14.8% in AT after liposuction (P = .011). For the first time, we demonstrate that liposuction preserves the microvascular network. Indeed, a low percentage of endothelial cells was found in AT from liposuction and we confirm the persistence of microvessels in the tissue after liposuction. © 2015 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com.

Flow cytometry of sputum: assessing inflammation and immune response elements in the bronchial airways

PubMed Central

Lay, John C.; Peden, David B.; Alexis, Neil E.

2012-01-01

Background The evaluation of sputum leukocytes by flow cytometry is an opportunity to assess characteristics of cells residing in the central airways, yet it is hampered by certain inherent properties of sputum including mucus and large amounts of contaminating cells and debris. Objective To develop a gating strategy based on specific antibody panels in combination with light scatter properties for flow cytometric evaluation of sputum cells. Methods Healthy and mild asthmatic volunteers underwent sputum induction. Manually selected mucus “plug” material was treated with dithiothrietol, filtered and total leukocytes acquired. Multicolor flow cytometry was performed using specific gating strategies based on light scatter properties, differential expression of CD45 and cell lineage markers to discriminate leukocytes from squamous epithelial cells and debris. Results The combination of forward scatter and CD45 expression reliably segregated sputum leukocytes from contaminating squamous epithelial cells and debris. Overlap of major leukocyte populations (neutrophils, macrophages/monocytes) required the use of specific antibodies (e.g. CD16, CD64, CD14, HLA-DR) that differentiated granulocytes from monocytes and macrophages. These gating strategies allowed identification of small populations of eosinophils, CD11c+ myeloid dendritic cells, B cells and NK cells. Conclusions Multicolor flow cytometry can be successfully applied to sputum samples to identify and characterize leukocyte populations residing on the surfaces of the central airways. PMID:21639708

Flow cytometry for receptor analysis from ex-vivo brain tissue in adult rat.

PubMed

Benoit, A; Guillamin, M; Aitken, P; Smith, P F; Philoxene, B; Sola, B; Poulain, L; Coquerel, A; Besnard, S

2018-07-01

Flow cytometry allows single-cell analysis of peripheral biological samples and is useful in many fields of research and clinical applications, mainly in hematology, immunology, and oncology. In the neurosciences, the flow cytometry separation method was first applied to stem cell extraction from healthy or cerebral tumour tissue and was more recently tested in order to phenotype brain cells, hippocampal neurogenesis, and to detect prion proteins. However, it remains sparsely applied in quantifying membrane receptors in relation to synaptic plasticity. We aimed to optimize a flow cytometric procedure for receptor quantification in neurons and non-neurons. A neural dissociation process, myelin separation, fixation, and membrane permeability procedures were optimized to maximize cell survival and analysis in hippocampal tissue obtained from adult rodents. We then aimed to quantify membrane muscarinic acetylcholine receptors (mAChRs) in rats with and without bilateral vestibular loss (BVL). mAChR's were quantified for neuronal and non-neuronal cells in the hippocampus and striatum following BVL. At day 30 but not at day 7 following BVL, there was a significant increase (P ≤ 0.05) in the percentage of neurons expressing M 2/4 mAChRs in both the hippocampus and the striatum. Here, we showed that flow cytometry appears to be a reliable method of membrane receptor quantification in ex-vivo brain tissue. Copyright © 2018 Elsevier B.V. All rights reserved.

Immunophenotyping of posttraumatic neutrophils on a routine haematology analyser.

PubMed

Groeneveld, Kathelijne Maaike; Heeres, Marjolein; Leenen, Loek Petrus Hendrikus; Huisman, Albert; Koenderman, Leo

2012-01-01

Flow cytometry markers have been proposed as useful predictors for the occurrence of posttraumatic inflammatory complications. However, currently the need for a dedicated laboratory and the labour-intensive analytical procedures make these markers less suitable for clinical practice. We tested an approach to overcome these limitations. Neutrophils of healthy donors were incubated with antibodies commonly used in trauma research: CD11b (MAC-1), L-selectin (CD62L), FcγRIII (CD16), and FcγRII (CD32) in active form (MoPhab A27). Flow cytometric analysis was performed both on a FACSCalibur, a standard flow cytometer, and on a Cell-Dyn Sapphire, a routine haematology analyser. There was a high level of agreement between the two types of analysers, with 41% for FcγRIII, 80% for L-selectin, 98% for CD11b, and even a 100% agreement for active FcγRII. Moreover, analysis on the routine haematology analyser was possible in less than a quarter of the time in comparison to the flow cytometer. Analysis of neutrophil phenotype on the Cell-Dyn Sapphire leads to the same conclusion compared to a standard flow cytometer. The markedly reduced time necessary for analysis and reduced labour intensity constitutes a step forward in implementation of this type of analysis in clinical diagnostics in trauma research. Copyright © 2012 Kathelijne Maaike Groeneveld et al.

EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes

PubMed Central

van Dongen, J J M; Lhermitte, L; Böttcher, S; Almeida, J; van der Velden, V H J; Flores-Montero, J; Rawstron, A; Asnafi, V; Lécrevisse, Q; Lucio, P; Mejstrikova, E; Szczepański, T; Kalina, T; de Tute, R; Brüggemann, M; Sedek, L; Cullen, M; Langerak, A W; Mendonça, A; Macintyre, E; Martin-Ayuso, M; Hrusak, O; Vidriales, M B; Orfao, A

2012-01-01

Most consensus leukemia & lymphoma antibody panels consist of lists of markers based on expert opinions, but they have not been validated. Here we present the validated EuroFlow 8-color antibody panels for immunophenotyping of hematological malignancies. The single-tube screening panels and multi-tube classification panels fit into the EuroFlow diagnostic algorithm with entries defined by clinical and laboratory parameters. The panels were constructed in 2–7 sequential design–evaluation–redesign rounds, using novel Infinicyt software tools for multivariate data analysis. Two groups of markers are combined in each 8-color tube: (i) backbone markers to identify distinct cell populations in a sample, and (ii) markers for characterization of specific cell populations. In multi-tube panels, the backbone markers were optimally placed at the same fluorochrome position in every tube, to provide identical multidimensional localization of the target cell population(s). The characterization markers were positioned according to the diagnostic utility of the combined markers. Each proposed antibody combination was tested against reference databases of normal and malignant cells from healthy subjects and WHO-based disease entities, respectively. The EuroFlow studies resulted in validated and flexible 8-color antibody panels for multidimensional identification and characterization of normal and aberrant cells, optimally suited for immunophenotypic screening and classification of hematological malignancies. PMID:22552007

Advances in Automated Plankton Imaging: Enhanced Throughput, Automated Staining, and Extended Deployment Modes for Imaging FlowCytobot

NASA Astrophysics Data System (ADS)

Sosik, H. M.; Olson, R. J.; Brownlee, E.; Brosnahan, M.; Crockford, E. T.; Peacock, E.; Shalapyonok, A.

2016-12-01

Imaging FlowCytobot (IFCB) was developed to fill a need for automated identification and monitoring of nano- and microplankton, especially phytoplankton in the size range 10 200 micrometer, which are important in coastal blooms (including harmful algal blooms). IFCB uses a combination of flow cytometric and video technology to capture high resolution (1 micrometer) images of suspended particles. This proven, now commercially available, submersible instrument technology has been deployed in fixed time series locations for extended periods (months to years) and in shipboard laboratories where underway water is automatically analyzed during surveys. Building from these successes, we have now constructed and evaluated three new prototype IFCB designs that extend measurement and deployment capabilities. To improve cell counting statistics without degrading image quality, a high throughput version (IFCB-HT) incorporates in-flow acoustic focusing to non-disruptively pre-concentrate cells before the measurement area of the flow cell. To extend imaging to all heterotrophic cells (even those that do not exhibit chlorophyll fluorescence), Staining IFCB (IFCB-S) incorporates automated addition of a live-cell fluorescent stain (fluorescein diacetate) to samples before analysis. A horizontally-oriented IFCB-AV design addresses the need for spatial surveying from surface autonomous vehicles, including design features that reliably eliminate air bubbles and mitigate wave motion impacts. Laboratory evaluation and test deployments in waters near Woods Hole show the efficacy of each of these enhanced IFCB designs.

Low-Coherence Reflectometry for Refractive Index Measurements of Cells in Micro-Capillaries.

PubMed

Carpignano, Francesca; Rigamonti, Giulia; Mazzini, Giuliano; Merlo, Sabina

2016-10-11

The refractive index of cells provides insights into their composition, organization and function. Moreover, a good knowledge of the cell refractive index would allow an improvement of optical cytometric and diagnostic systems. Although interferometric techniques undoubtedly represent a good solution for quantifying optical path variation, obtaining the refractive index of a population of cells non-invasively remains challenging because of the variability in the geometrical thickness of the sample. In this paper, we demonstrate the use of infrared low-coherence reflectometry for non-invasively quantifying the average refractive index of cell populations gently confined in rectangular glass micro-capillaries. A suspension of human red blood cells in plasma is tested as a reference. As a use example, we apply this technique to estimate the average refractive index of cell populations belonging to epithelial and hematological families.

Low-Coherence Reflectometry for Refractive Index Measurements of Cells in Micro-Capillaries

PubMed Central

Carpignano, Francesca; Rigamonti, Giulia; Mazzini, Giuliano; Merlo, Sabina

2016-01-01

The refractive index of cells provides insights into their composition, organization and function. Moreover, a good knowledge of the cell refractive index would allow an improvement of optical cytometric and diagnostic systems. Although interferometric techniques undoubtedly represent a good solution for quantifying optical path variation, obtaining the refractive index of a population of cells non-invasively remains challenging because of the variability in the geometrical thickness of the sample. In this paper, we demonstrate the use of infrared low-coherence reflectometry for non-invasively quantifying the average refractive index of cell populations gently confined in rectangular glass micro-capillaries. A suspension of human red blood cells in plasma is tested as a reference. As a use example, we apply this technique to estimate the average refractive index of cell populations belonging to epithelial and hematological families. PMID:27727172

A critical review of published methods for analysis of red cell antigen-antibody reactions by flow cytometry, and approaches for resolving problems with red cell agglutination.

PubMed

Arndt, Patricia A; Garratty, George

2010-07-01

Flow cytometry operators often apply familiar white blood cell (WBC) methods when studying red blood cell (RBC) antigens and antibodies. Some WBC methods are not appropriate for RBCs, as the analysis of RBCs requires special considerations, for example, avoidance of agglutination. One hundred seventy-six published articles from 88 groups studying RBC interactions were reviewed. Three fourths of groups used at least one unnecessary WBC procedure for RBCs, and about one fourth did not use any method to prevent/disperse RBC agglutination. Flow cytometric studies were performed to determine the effect of RBC agglutination on results and compare different methods of preventing and/or dispersing agglutination. The presence of RBC agglutinates have been shown to be affected by the type of pipette tip used for mixing RBC suspensions, the number of antigen sites/RBC, the type and concentration of primary antibody, and the type of secondary antibody. For quantitation methods, for example, fetal maternal hemorrhage, the presence of agglutinates have been shown to adversely affect results (fewer fetal D+ RBCs detected). Copyright 2010 Elsevier Inc. All rights reserved.

Measuring T cell-mediated cytotoxicity using fluorogenic caspase substrates.

PubMed

Chahroudi, A; Silvestri, G; Feinberg, M B

2003-10-01

Cytotoxic T lymphocytes (CTLs) play a major role in the immune response against viruses and other intracellular pathogens. In addition, CTLs are implicated in the control of tumor cells in certain settings. Accurate measures of CTL function are of critical importance to study the pathogenesis of infectious diseases and to evaluate the efficacy of new vaccines and immunotherapies. To this end, we have recently developed a flow cytometry-based CTL (FCC) assay that measures the CTL-induced caspase activation within target cells using cell permeable fluorogenic caspase substrates. This novel assay reliably detects, by flow cytometry or fluorescence/confocal microscopy, antigen-specific CTLs in a wide variety of human and murine systems, and is safer and more informative than the standard 51Cr-release assay. In addition, the flow cytometric CTL (FCC) assay provides an alternative method that is often more sensitive and physiologically informative when compared to previously described FCC assays, as it measures a biological indicator of apoptosis within the target cell. The FCC assay may thus represent a useful tool to further understand the molecular and cellular mechanisms that underlie CTL-mediated killing during tumorigenesis or following infection with viruses or other intracellular pathogens.

Interlaboratory assessment of mitotic index by flow cytometry confirms superior reproducibility relative to microscopic scoring.

PubMed

Roberts, D J; Spellman, R A; Sanok, K; Chen, H; Chan, M; Yurt, P; Thakur, A K; DeVito, G L; Murli, H; Stankowski, L F

2012-05-01

A flow cytometric procedure for determining mitotic index (MI) as part of the metaphase chromosome aberrations assay, developed and utilized routinely at Pfizer as part of their standard assay design, has been adopted successfully by Covance laboratories. This method, using antibodies against phosphorylated histone tails (H3PS10) and nucleic acid stain, has been evaluated by the two independent test sites and compared to manual scoring. Primary human lymphocytes were treated with cyclophosphamide, mitomycin C, benzo(a)pyrene, and etoposide at concentrations inducing dose-dependent cytotoxicity. Deming regression analysis indicates that the results generated via flow cytometry (FCM) were more consistent between sites than those generated via microscopy. Further analysis using the Bland-Altman modification of the Tukey mean difference method supports this finding, as the standard deviations (SDs) of differences in MI generated by FCM were less than half of those generated manually. Decreases in scoring variability owing to the objective nature of FCM, and the greater number of cells analyzed, make FCM a superior method for MI determination. In addition, the FCM method has proven to be transferable and easily integrated into standard genetic toxicology laboratory operations. Copyright © 2012 Wiley Periodicals, Inc.

Determination by flow cytometry polyploidy inducing-capacity of colchicine in Cajanus cajan (L.) Mill sp.

PubMed

Udensi, O U; Ontui, V

2013-07-01

The need to optimize flow cytometric analysis for the determination of ploidy level is a worthwhile venture to precisely know at what concentration of a mutagen and at what time of exposure polyploidy could be induced. Flow cytometry was used to determine the polyploidy inducing-capacity of colchicine in pigeon pea (Cajanus cajan (L.) Mill sp). Seeds of pigeon pea were soaked in three different concentrations of colchicine-5 mg, 10 and 15 mg L(-1) for 24, 48 and 72 h, respectively, while the control group was soaked in water. Treated seeds and those from the control were planted in a greenhouse using a Completely Randomized Design (CRD). Results show that colchicine induced tetraploids (4n) and mixoploids (2n+ 4n) as the concentration of colchicine increased and soaking duration. Days to seedling emergence increased as concentration of colchicine and duration of soaking increased while germination rate decreased proportionately with the increase in colchicine concentration and soaking duration but did not significantly affect percentage seedling survival. Explicitly, colchicine has the capacity of inducing polyploidy; especially tetraploids on the seeds of pigeon pea, which obviously could be harnessed for further breeding and improvement of the pigeon pea.

[Changes of the immune cells, cytokines and growth hormone in teenager drug addicts].

PubMed

Kuang, Ying-min; Zhu, Yue-chun; Kuang, Ying; Sun, Yuan; Hua, Chen; He, Wen-yi

2007-09-01

To investigate the effect of heroin on the immune function, growth and development in the teenager heroin addicts by measuring their T-lymphocyte subsets, Th1/Th2 cytokines and serum growth hormone. Tlymphocyte subsets of peripheral blood from the teenager heroin addicts were measured by direct microvolume whole blood immunofluorescent staining technique by flow cytometer (FCM). Thl / Th2 cytokines were measured by BD cytometric bead array and serum growth hormone was assayed using the chemiluminescence method in the 20 teenager heroin addicts and 23 healthy teenagers. The levels of CD3(+), CD3(+) + CD4(+), CD3(+) + CD4(+)/CD3(+)+ CD8(+), Th1 cytokines(IL-2, TNF-alpha and IFN-gamma) and Th2 cytokines(IL-4 and IL-10) reduced significantly in the teenager heroin addicts compared with the healthy control group (P < 0.01 or P < 0.05). The level of Th1 cytokines(IL-2 + TNF-alpha+IFN-gamma) decreased more than that of Th2 cytokines(IL-4 + IL-5 + IL-10)(P < 0.05). The level of serum growth hormone from the teenager heroin addicts was remarkably higher than that in control group (P<0.01). Heroin can inhibit the immunofunction especially the celluar immunity of the teenager heroin addicts. Besides, it can increase the level of serum growth hormone of the teenager heroin addicts.

Aberrant Production of Th1/Th2/Th17-Related Cytokines in Serum of C57BL/6 Mice after Short-Term Formaldehyde Exposure

PubMed Central

Wei, Haiyan; Tan, Kehong; Sun, Rongli; Yin, Lihong; Zhang, Juan; Pu, Yuepu

2014-01-01

Previous studies have shown that formaldehyde (FA) could cause immunotoxicity by changing the number of T lymphocytes and that cytokines play a pivotal role in the regulation of T lymphocytes. However, the previously used cytokine detection methods are difficult to use in the measurement of several cytokines in a small amount of sample for one test. Therefore, the cytometric bead array (CBA) technique was used. CBA showed better analytical efficiency and sensitivity than the previous methods. C57BL/6 mice were exposed to the control (normal saline), low FA concentration (0.5 mg/kg), and high FA concentration (2 mg/kg) for 1 week or 1 month. The contents of cytokines, including Th1-related cytokines (IL-2, IFN-γ, and tumor necrosis factor), Th2-related cytokines (IL-4, IL-6, and IL-10), and Th17-related cytokines (IL-17A), were measured by using the BD FACS Canto II Flow Cytometer and analyzed by FCAP ArrayTM Software. Th1/Th2/Th17-related cytokines showed a slightly decreasing trend after low FA exposure. Conversely, a significantly increasing trend was found after high FA exposure. Th1/Th2/Th17-related cytokines all serve important functions in the immune reactions in mice after FA exposure. PMID:25264680

Peptidoglycan and lipoteichoic acid, components of the streptococcal cell wall, have marked and differential effects on adhesion molecule expression and the production of reactive oxygen species in human whole blood leukocytes.

PubMed

Saetre, T; Kähler, H; Foster, S J; Lyberg, T

2000-07-01

To elucidate the pathophysiology of infections with Streptococcus pyogenes we applied flow cytometric techniques to study dose-response and time-related effects of the streptococcal cell-wall-derived components lipoteichoic acid (LTA 0.005 to 50 microg/ml) and peptidoglycan (10 and 100 microg/ml) on the expression of leukocyte adhesion molecules, the CD14 receptor, and the production of leukocyte reactive oxygen species (ROS). LTA (50 microg/ml, 1-2 h) markedly increased the expression of CD11b (approximately 5-fold), CD11c (approximately 2-fold) and CD11a. Concomitantly, CD62L was downregulated (60%). Peptidoglycan alone or in combination with LTA had little effect on adhesion molecules, except for an amplification of the downregulation of CD62L to 90%. Monocyte CD14 expression was doubled by LTA. Leukocyte ROS production was 10-fold and 5-fold increased by peptidoglycan in granulocytes and monocytes, respectively. LTA alone had no effect, while the combination of peptidoglycan with LTA doubled the increase in ROS caused by peptidoglycan. LTA and peptidoglycan had marked and differential effects: LTA caused mainly adhesion molecule modulation, whereas peptidoglycan mainly increased ROS production. These changes are important in inflammatory cell activation and recruitment, intracellular microbial killing and adverse tissue injury.

Design of a multi-center immunophenotyping analysis of peripheral blood, sputum and bronchoalveolar lavage fluid in the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS).

PubMed

Freeman, Christine M; Crudgington, Sean; Stolberg, Valerie R; Brown, Jeanette P; Sonstein, Joanne; Alexis, Neil E; Doerschuk, Claire M; Basta, Patricia V; Carretta, Elizabeth E; Couper, David J; Hastie, Annette T; Kaner, Robert J; O'Neal, Wanda K; Paine, Robert; Rennard, Stephen I; Shimbo, Daichi; Woodruff, Prescott G; Zeidler, Michelle; Curtis, Jeffrey L

2015-01-27

Subpopulations and Intermediate Outcomes in COPD Study (SPIROMICS) is a multi-center longitudinal, observational study to identify novel phenotypes and biomarkers of chronic obstructive pulmonary disease (COPD). In a subset of 300 subjects enrolled at six clinical centers, we are performing flow cytometric analyses of leukocytes from induced sputum, bronchoalveolar lavage (BAL) and peripheral blood. To minimize several sources of variability, we use a "just-in-time" design that permits immediate staining without pre-fixation of samples, followed by centralized analysis on a single instrument. The Immunophenotyping Core prepares 12-color antibody panels, which are shipped to the six Clinical Centers shortly before study visits. Sputum induction occurs at least two weeks before a bronchoscopy visit, at which time peripheral blood and bronchoalveolar lavage are collected. Immunostaining is performed at each clinical site on the day that the samples are collected. Samples are fixed and express shipped to the Immunophenotyping Core for data acquisition on a single modified LSR II flow cytometer. Results are analyzed using FACS Diva and FloJo software and cross-checked by Core scientists who are blinded to subject data. Thus far, a total of 152 sputum samples and 117 samples of blood and BAL have been returned to the Immunophenotyping Core. Initial quality checks indicate useable data from 126 sputum samples (83%), 106 blood samples (91%) and 91 BAL samples (78%). In all three sample types, we are able to identify and characterize the activation state or subset of multiple leukocyte cell populations (including CD4+ and CD8+ T cells, B cells, monocytes, macrophages, neutrophils and eosinophils), thereby demonstrating the validity of the antibody panel. Our study design, which relies on bi-directional communication between clinical centers and the Core according to a pre-specified protocol, appears to reduce several sources of variability often seen in flow cytometric studies involving multiple clinical sites. Because leukocytes contribute to lung pathology in COPD, these analyses will help achieve SPIROMICS aims of identifying subgroups of patients with specific COPD phenotypes. Future analyses will correlate cell-surface markers on a given cell type with smoking history, spirometry, airway measurements, and other parameters. This study was registered with ClinicalTrials.gov as NCT01969344 .

Method for flow cytometric monitoring of Renibacterium salmoninarum inactivation

USGS Publications Warehouse

Pascho, R.J.; Ongerth, J.E.

2000-01-01

The slow growth of Renibacterium salmoninarum limits the usefulness of culture as a research tool. Development of a 2-color flow cytometric assay to quantify the proportions of live and dead R. salmoninarum in a test population is described. Bacteria were simultaneously stained with fluorescein isothiocyanate-conjugated immunoglobulin and exposed to the exclusion dye propidium iodide. Propidium iodide red fluorescence profiles of control groups of untreated and killed R. salmoninarum were compared with those for bacteria exposed to chlorine. Bacterial inactivation was based on mean red fluorescence intensity, and analyzed by high-red fluorescence intensity (HRFI) and curve subtraction (CS) analyses. When the concentration of R. salmoninarum was 8.65 x 106 bacteria ml-1 and the bacteria exposed to chlorine at 1 mg l-1 for periods from 1 to 20 min (high-Rs assessment), the mean red fluorescence intensity of the profile for each chlorine-exposure group was higher than that for the untreated control (p < 0.0001). When the concentration of R. salmoninarum was reduced to 1.76 x 106 bacteria ml-1 and exposed to 0.8 mg l-1 free chlorine level for periods from 20 s to 5 min (reduced-Rs assessment), the mean red fluorescence intensities of the exposure groups were higher than that for the untreated control only when the R. salmoninarum was exposed to chlorine for at least 1 min (p ??? 0.01). On the basis of red fluorescence intensity, the proportion of dead cells generally increased with the duration of chlorine exposure. Whereas the rates of inactivation derived from the HRFI and CS analyses did not correlate with the duration of exposure in the high-Rs assessment (r2 ??? 0.27), there was a correlation between these estimates and the duration of exposure in the reduced-Rs assessment (r2 ??? 0.92). Because of the rapid loss of culturable R. salmoninarum in both assessments following chlorine exposure, neither the duration of exposure nor the inactivation estimates correlated with bacteriological culture (r2 ??? 0.22). In both assessments, there was a correlation between the estimates of inactivation based upon HRFI and CS analyses (r2 > 0.99). These results suggest that flow cytometry can be used as a supplementary or alternative method to bacteriological culture for monitoring the inactivation of R. salmoninarum.

Mechanism of antiproliferative action of a new d-secoestrone-triazole derivative in cervical cancer cells and its effect on cancer cell motility.

PubMed

Bózsity, Noémi; Minorics, Renáta; Szabó, Johanna; Mernyák, Erzsébet; Schneider, Gyula; Wölfling, János; Wang, Hui-Chun; Wu, Chin-Chung; Ocsovszki, Imre; Zupkó, István

2017-01-01

Cervical cancer is the fourth most frequently diagnosed tumor and the fourth leading cause of cancer death in females worldwide. Cervical cancer is predominantly related with human papilloma virus (HPV) infection, with the most oncogenic types being HPV-18 and -16. Our previous studies demonstrated that some d-secoestrone derivatives exert pronounced antiproliferative activity. The aim of the current investigation was to characterize the mechanism of action of d-secoestrone-triazole (D-SET) on three cervical cancer cell lines with different pathological backgrounds. The growth-inhibitory effects of D-SET were determined by a standard MTT assay. We have found that D-SET exerts a pronounced growth-inhibitory effect on HPV 18-positive HeLa and HPV-negative C-33 A cells, but it has no substantial inhibitory activity on HPV 16-positive SiHa or on intact fibroblast MRC-5 cell lines. After 24h incubation, cells showed the morphological and biochemical signs of apoptosis determined by fluorescent double staining, flow cytometry and caspase-3 activity assay. Besides the elevation of the ratio of cells in the subG1 phase, flow cytometric analysis revealed a cell cycle arrest at G2/M in both HeLa and C-33 A cell lines. To distinguish the G2/M cell population immunocytochemical flow cytometric analysis was performed on HeLa cells. The results show that D-SET significantly increases the ratio of phosphorylated histone H3, indicating cell accumulation in the M phase. Additionally, D-SET significantly increased the maximum rate of microtube formation measured by an in vitro tubulin polymerization assay. Besides its direct antiproliferative activity, the antimigratory property of D-SET has been investigated. Our results demonstrate that D-SET significantly inhibits the migration and invasion of HeLa cells after 24h incubation. These results suggests that D-SET is a potent antiproliferative agent against HPV 16+ and HPV-negative cervical cancer cell lines, with an efficacious motility-inhibiting activity against HPV 16+ cells. Accordingly D-SET can be regarded as a potential drug candidate with a promising new mechanism of action among the antiproliferative steroids, potentially allowing for the design of novel anticancer agents. Copyright © 2016 Elsevier Ltd. All rights reserved.

Chromosome banding in Amphibia. XXVIII. Homomorphic XY sex chromosomes and a derived Y-autosome translocation in Eleutherodactylus riveroi (Anura, Leptodactylidae).

PubMed

Schmid, M; Feichtinger, W; Steinlein, C; Visbal García, R; Fernández Badillo, A

2003-01-01

Extensive cytogenetic analyses on a population of the leptodactylid frog Eleutherodactylus riveroi in northern Venezuela revealed the existence of multiple XXAA male/XYAA female/XAA(Y) female sex chromosomes. The XAA(Y) karyotype originated by a centric (Robertsonian) fusion between the original, free Y chromosome and an autosome. 46.2% of the male individuals in this population are carriers of this Y-autosome fusion. In male meiosis the XAA(Y) sex chromosomes pair in the expected trivalent configuration. In the same population 53.8% of the male animals still possess the original, free XY sex chromosomes. E. riveroi is only the second vertebrate species discovered in which a derived Y-autosome fusion coexists with the ancestral free XY sex chromosomes. The free XY sex chromosomes, as well as the multiple XA(Y) sex chromosomes are still in a very primitive (homomorphic) stage of differentiation. With no banding technique applied it is possible to distinguish the Y from the X. Various banding techniques and in situ hybridizations have been carried out to characterize the karyotypes. DNA flow cytometric measurements show that the genome size of E. riveroi resembles that of other Eleutherodactylus species. The cytogenetic data obtained in E. riveroi are compared with those of the sole other vertebrate known to possess the extremely rare, multiple XXAA male/XYAA female/XAA(Y) female sex chromosomes. Surprisingly enough, this vertebrate again is a frog belonging to the genus Eleutherodactylus [E. ((maussi) biporcatus] which lives exactly in the same habitat in northern Venezuela as does E. riveroi. Copyright 2003 S. Karger AG, Basel

Immobilization and detection of platelet-derived extracellular vesicles on functionalized silicon substrate: cytometric and spectrometric approach.

PubMed

Gajos, Katarzyna; Kamińska, Agnieszka; Awsiuk, Kamil; Bajor, Adrianna; Gruszczyński, Krzysztof; Pawlak, Anna; Żądło, Andrzej; Kowalik, Artur; Budkowski, Andrzej; Stępień, Ewa

2017-02-01

Among the various biomarkers that are used to diagnose or monitor disease, extracellular vesicles (EVs) represent one of the most promising targets in the development of new therapeutic strategies and the application of new diagnostic methods. The detection of circulating platelet-derived microvesicles (PMVs) is a considerable challenge for laboratory diagnostics, especially in the preliminary phase of a disease. In this study, we present a multistep approach to immobilizing and detecting PMVs in biological samples (microvesicles generated from activated platelets and human platelet-poor plasma) on functionalized silicon substrate. We describe the application of time-of-flight secondary ion mass spectrometry (TOF-SIMS) and spectroscopic ellipsometry methods to the detection of immobilized PMVs in the context of a novel imaging flow cytometry (ISX) technique and atomic force microscopy (AFM). This novel approach allowed us to confirm the presence of the abundant microvesicle phospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) on a surface with immobilized PMVs. Phosphatidylcholine groups (C 5 H 12 N + ; C 5 H 15 PNO 4 + ) were also detected. Moreover, we were able to show that ellipsometry permitted the immobilization of PMVs on a functionalized surface to be evaluated. The sensitivity of the ISX technique depends on the size and refractive index of the analyzed microvesicles. Graphical abstract Human platelets activated with thrombin (in concentration 1IU/mL) generate population of PMVs (platelet derived microvesicles), which can be detected and enumerated with fluorescent-label method (imaging cytometry). Alternatively, PMVs can be immobilized on the modified silicon substrate which is functionalized with a specific IgM murine monoclonal antibody against human glycoprotein IIb/IIIa complex (PAC-1). Immobilized PMVs can be subjected to label-free analyses by means ellipsometry, atomic force microscopy (AFM) and time-of-flight secondary ion mass spectrometry (TOF-SIMS).

Measurement of Lipid Accumulation in Chlorella vulgaris via Flow Cytometry and Liquid-State ¹H NMR Spectroscopy for Development of an NMR-Traceable Flow Cytometry Protocol

PubMed Central

Bono Jr., Michael S.; Garcia, Ravi D.; Sri-Jayantha, Dylan V.; Ahner, Beth A.; Kirby, Brian J.

2015-01-01

In this study, we cultured Chlorella vulgaris cells with a range of lipid contents, induced via nitrogen starvation, and characterized them via flow cytometry, with BODIPY 505/515 as a fluorescent lipid label, and liquid-state 1H NMR spectroscopy. In doing so, we demonstrate the utility of calibrating flow cytometric measurements of algal lipid content using triacylglyceride (TAG, also known as triacylglycerol or triglyceride) content per cell as measured via quantitative 1H NMR. Ensemble-averaged fluorescence of BODIPY-labeled cells was highly correlated with average TAG content per cell measured by bulk NMR, with a linear regression yielding a linear fit with r 2 = 0.9974. This correlation compares favorably to previous calibrations of flow cytometry protocols to lipid content measured via extraction, and calibration by NMR avoids the time and complexity that is generally required for lipid quantitation via extraction. Flow cytometry calibrated to a direct measurement of TAG content can be used to investigate the distribution of lipid contents for cells within a culture. Our flow cytometry measurements showed that Chlorella vulgaris cells subjected to nitrogen limitation exhibited higher mean lipid content but a wider distribution of lipid content that overlapped the relatively narrow distribution of lipid content for replete cells, suggesting that nitrogen limitation induces lipid accumulation in only a subset of cells. Calibration of flow cytometry protocols using direct in situ measurement of TAG content via NMR will facilitate rapid development of more precise flow cytometry protocols, enabling investigation of algal lipid accumulation for development of more productive algal biofuel feedstocks and cultivation protocols. PMID:26267664

Measurement of lipid accumulation in Chlorella vulgaris via flow cytometry and liquid-state ¹H NMR spectroscopy for development of an NMR-traceable flow cytometry protocol.

PubMed

Bono, Michael S; Garcia, Ravi D; Sri-Jayantha, Dylan V; Ahner, Beth A; Kirby, Brian J

2015-01-01

In this study, we cultured Chlorella vulgaris cells with a range of lipid contents, induced via nitrogen starvation, and characterized them via flow cytometry, with BODIPY 505/515 as a fluorescent lipid label, and liquid-state 1H NMR spectroscopy. In doing so, we demonstrate the utility of calibrating flow cytometric measurements of algal lipid content using triacylglyceride (TAG, also known as triacylglycerol or triglyceride) content per cell as measured via quantitative 1H NMR. Ensemble-averaged fluorescence of BODIPY-labeled cells was highly correlated with average TAG content per cell measured by bulk NMR, with a linear regression yielding a linear fit with r2 = 0.9974. This correlation compares favorably to previous calibrations of flow cytometry protocols to lipid content measured via extraction, and calibration by NMR avoids the time and complexity that is generally required for lipid quantitation via extraction. Flow cytometry calibrated to a direct measurement of TAG content can be used to investigate the distribution of lipid contents for cells within a culture. Our flow cytometry measurements showed that Chlorella vulgaris cells subjected to nitrogen limitation exhibited higher mean lipid content but a wider distribution of lipid content that overlapped the relatively narrow distribution of lipid content for replete cells, suggesting that nitrogen limitation induces lipid accumulation in only a subset of cells. Calibration of flow cytometry protocols using direct in situ measurement of TAG content via NMR will facilitate rapid development of more precise flow cytometry protocols, enabling investigation of algal lipid accumulation for development of more productive algal biofuel feedstocks and cultivation protocols.

Building an Ontology-driven Database for Clinical Immune Research

PubMed Central

Ma, Jingming

2006-01-01

The clinical researches of immune response usually generate a huge amount of biomedical testing data over a certain period of time. The user-friendly data management systems based on the relational database will help immunologists/clinicians to fully manage the data. On the other hand, the same biological assays such as ELISPOT and flow cytometric assays are involved in immunological experiments no matter of different study purposes. The reuse of biological knowledge is one of driving forces behind this ontology-driven data management. Therefore, an ontology-driven database will help to handle different clinical immune researches and help immunologists/clinicians easily understand the immunological data from each other. We will discuss some outlines for building an ontology-driven data management for clinical immune researches (ODMim). PMID:17238637

Gynogenic plant regeneration from unpollinated flower explants of Eragrostis tef (Zuccagni) Trotter.

PubMed

Gugsa, Likyelesh; Sarial, Ashok K; Lörz, Horst; Kumlehn, Jochen

2006-12-01

Tef [Eragrostis tef (Zucc.) Trotter] is the most important cereal in Ethiopia. In its wild relative E. mexicana, regeneration of six green plants resulted from culture of 121 non-pollinated immature pistils. In the allotetraploid crop species tef, however, only callus and root formation was obtained by this method. By contrast, immature spikelets and panicle segments of E. tef proved amenable to gynogenic plant regeneration. Upon step-wise optimization of the protocol, efficient plant formation was achieved in all three cultivars tested. In cv. DZ-01-196, culture of 1305 immature spikelets resulted in formation of 159 green plants. Flow cytometric analysis revealed (di)haploid, triploid, tetraploid and octoploid regenerants, from which the vast majority was tetraploid. Tef-breeding programs will likely benefit substantially from efficient generation of true-breeding plants.

Parthenogenesis in a whitetip reef shark Triaenodon obesus involves a reduction in ploidy.

PubMed

Portnoy, D S; Hollenbeck, C M; Johnston, J S; Casman, H M; Gold, J R

2014-08-01

Genetic analysis of a female whitetip reef shark Triaenodon obesus and her stillborn pup, assumed to be of parthenogenetic origin, revealed that the pup was homozygous at all 24 nuclear-encoded microsatellites assayed, consistent with the idea that diploidy in the pup had been restored via terminal fusion. Flow cytometric analysis, however, indicated that the genome size of the pup was no more than half that of the mother, and microscopy revealed that nuclear volume was c. 1.73 times larger in the mother than in the pup. Together these data suggest that the pup was genetically haploid, developing directly from an unfertilized egg; as far as is known, this is the first observation of a spontaneously produced haploid vertebrate. © 2014 The Fisheries Society of the British Isles.

Dielectric inspection of erythrocyte morphology.

PubMed

Hayashi, Yoshihito; Oshige, Ikuya; Katsumoto, Yoichi; Omori, Shinji; Yasuda, Akio; Asami, Koji

2008-05-21

We performed a systematic study of the sensitivity of dielectric spectroscopy to erythrocyte morphology. Namely, rabbit erythrocytes of four different shapes were prepared by precisely controlling the pH of the suspending medium, and their complex permittivities over the frequency range from 0.1 to 110 MHz were measured and analyzed. Their quantitative analysis shows that the characteristic frequency and the broadening parameter of the dielectric relaxation of interfacial polarization are highly specific to the erythrocyte shape, while they are insensitive to the cell volume fraction. Therefore, these two dielectric parameters can be used to differentiate erythrocytes of different shapes, if dielectric spectroscopy is applied to flow-cytometric inspection of single blood cells. In addition, we revealed the applicability and limitations of the analytical theory of interfacial polarization to explain the experimental permittivities of non-spherical erythrocytes.

Flow cytometric analysis of leukocytes and reticulocytes stained with proflavine.

PubMed

Sagawa, H; Tatsumi, N

1997-12-01

Proflavine, an acridine analog for industrial use, was used to stain blood cells. A drop of blood treated with ethylenediaminetetraacetic acid-2K was mixed with a 0.00001% solution of the dye and observed immediately by fluorescence microscopy with a green filter. Leukocytes, platelets, and reticulocytes were stained but mature red blood cells were not. Chromatin in the nuclei of all leukocytes and nucleoli of lymphocytes and monocytes had greenish-yellow fluorescence, and the kind of cell could be identified by the tone and intensity of this color. Granules in granulocytes were in green. Reticular fine-granular or granulofibrous structures in the reticulocytes were brownish. The proflavine could be used routinely in clinical laboratories because this single stain makes possible simultaneous differentiation of leukocytes and counting of reticulocytes.

2',4'-Dihydroxychalcone-induced apoptosis of human gastric cancer MGC-803 cells via down-regulation of survivin mRNA.

PubMed

Lou, Chenghua; Yang, Guangming; Cai, Hao; Zou, Mingchang; Xu, Zisheng; Li, Yu; Zhao, Fengming; Li, Weidong; Tong, Li; Wang, Mingyan; Cai, Baochang

2010-08-01

2',4'-Dihydroxychalcone (TFC), a main component in Herba Oxytropis, is grouped under flavonoids, which are well known to have antitumor activities in vitro. In this study, the possible antitumor mechanism of TFC in human gastric cancer MGC-803 cells is examined. Hoechst 33258 staining analysis indicates that TFC causes MGC-803 cell shrinkage and apoptotic body formation, typical characteristics of apoptosis. Flow cytometric analysis demonstrates that TFC causes cell cycle arrest in the G2/M phase. Furthermore, TFC significantly increases caspase-3 activity but decreases survivin mRNA expression. Therefore, TFC can induce the apoptosis of MGC-803 cells via down-regulation of survivin mRNA expression. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Host cell recruitment patterns by bone morphogenetic protein-2 releasing hyaluronic acid hydrogels in a mouse subcutaneous environment.

PubMed

Todeschi, Maria R; El Backly, Rania M; Varghese, Oommen P; Hilborn, Jöns; Cancedda, Ranieri; Mastrogiacomo, Maddalena

2017-07-01

This study aimed to identify host cell recruitment patterns in a mouse model in response to rhBMP-2 releasing hyaluronic acid hydrogels and influence of added nano-hydroxyapatite particles on rhBMP-2 release and pattern of bone formation. Implanted gels were retrieved after implantation and cells were enzymatically dissociated for flow cytometric analysis. Percentages of macrophages, progenitor endothelial cells and putative mesenchymal stem cells were measured. Implants were evaluated for BMP-2 release by ELISA and by histology to monitor tissue formation. Hyaluronic acid+BMP-2 gels influenced the inflammatory response in the bone healing microenvironment. Host-derived putative mesenchymal stem cells were major contributors. Addition of hydroxyapatite nanoparticles modified the release pattern of rhBMP-2, resulting in enhanced bone formation.

Polystyrene Core-Silica Shell Particles with Defined Nanoarchitectures as a Versatile Platform for Suspension Array Technology.

PubMed

Sarma, Dominik; Gawlitza, Kornelia; Rurack, Knut

2016-04-19

The need for rapid and high-throughput screening in analytical laboratories has led to significant growth in interest in suspension array technologies (SATs), especially with regard to cytometric assays targeting a low to medium number of analytes. Such SAT or bead-based assays rely on spherical objects that constitute the analytical platform. Usually, functionalized polymer or silica (SiO2) microbeads are used which each have distinct advantages and drawbacks. In this paper, we present a straightforward synthetic route to highly monodisperse SiO2-coated polystyrene core-shell (CS) beads for SAT with controllable architectures from smooth to raspberry- and multilayer-like shells by varying the molecular weight of poly(vinylpyrrolidone) (PVP), which was used as the stabilizer of the cores. The combination of both organic polymer core and a structurally controlled inorganic SiO2 shell in one hybrid particle holds great promises for flexible next-generation design of the spherical platform. The particles were characterized by electron microscopy (SEM, T-SEM, and TEM), thermogravimetry, flow cytometry, and nitrogen adsorption/desorption, offering comprehensive information on the composition, size, structure, and surface area. All particles show ideal cytometric detection patterns and facile handling due to the hybrid structure. The beads are endowed with straightforward modification possibilities through the defined SiO2 shells. We successfully implemented the particles in fluorometric SAT model assays, illustrating the benefits of tailored surface area which is readily available for small-molecule anchoring. Very promising assay performance was shown for DNA hybridization assays with quantification limits down to 8 fmol.

Sperm DNA fragmentation induced by cryopreservation: new insights and effect of a natural extract from Opuntia ficus-indica.

PubMed

Meamar, Mehrdad; Zribi, Nassira; Cambi, Marta; Tamburrino, Lara; Marchiani, Sara; Filimberti, Erminio; Fino, Maria Grazia; Biggeri, Annibale; Menezo, Yves; Forti, Gianni; Baldi, Elisabetta; Muratori, Monica

2012-08-01

To analyze the effect of cryopreservation on sperm DNA fragmentation (SDF) in two cytometric sperm populations, PI(brighter) and PI(dimmer), and to test the effects of Opuntia ficus-indica (OFI) extracts, which contain antioxidants and flavanoids, and of resveratrol on cryopreservation of human semen. In vitro prospective study. Institutional study. Twenty-one normozoospermic men undergoing semen analysis for couple infertility. Cryopreservation using the routine method in the presence of OFI extracts or resveratrol. Measurement of SDF by TUNEL/PI flow cytometric method to evaluate sperm motility (by automated motion analysis, CASA system) and viability (by eosin/nigrosin staining) in the two populations of sperm PI(br) and PI(dim). Cryopreservation induced an increase of SDF only in the PI(br) sperm population. The increase was negatively dependent on the basal values of SDF in the same population. Addition of OFI extracts and resveratrol to the cryopreservation medium slightly but statistically significantly reduced SDF in the PI(br) population without affecting the deleterious effect of cryopreservation on sperm motion parameters or viability. The increase of SDF in the PI(br) population, which is unrelated to semen quality, suggests that caution must be taken in using cryopreserved semen, as morphologically normal and motile sperm may be damaged. The addition of substances with multifunctional properties such as OFI extracts to cryopreservation medium is only slightly effective in preventing the dramatic effects on SDF. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Cytometric Approach for Detection of Encephalitozoon intestinalis, an Emergent Agent▿

PubMed Central

Barbosa, Joana; Rodrigues, Acácio Gonçalves; Pina-Vaz, Cidália

2009-01-01

Encephalitozoon intestinalis is responsible for intestinal disease in patients with AIDS and immunocompetent patients. The infectious form is a small spore that is resistant to water treatment procedures. Its detection is very important, but detection is very cumbersome and time-consuming. Our main objective was to develop and optimize a specific flow cytometric (FC) protocol for the detection of E. intestinalis in hospital tap water and human feces. To determine the optimal specific antibody (Microspor-FA) concentration, a known concentration of E. intestinalis spores (Waterborne, Inc.) was suspended in hospital tap water and stool specimens with different concentrations of Microspor-FA, and the tap water and stool specimens were incubated under different conditions. The sensitivity limit and specificity were also evaluated. To study spore infectivity, double staining with propidium iodide (PI) and Microspor-FA was undertaken. Distinct approaches for filtration and centrifugation of the stool specimens were used. E. intestinalis spores stained with 10 μg/ml of Microspor-FA at 25°C overnight provided the best results. The detection limit was 5 × 104 spores/ml, and good specificity was demonstrated. Simultaneous staining with Microspor-FA and PI ensured that the E. intestinalis spores were dead and therefore noninfectious. With the stool specimens, better spore recovery was observed with a saturated solution of NaCl and centrifugation at 1,500 × g for 15 min. A new approach for the detection of E. intestinalis from tap water or human feces that ensures that the spores are not viable is now available and represents an important step for the prevention of this threat to public health. PMID:19439525

α2β1 integrin affects metastatic potential of ovarian carcinoma spheroids by supporting disaggregation and proteolysis

PubMed Central

Shield, Kristy; Riley, Clyde; Quinn, Michael A; Rice, Gregory E; Ackland, Margaret L; Ahmed, Nuzhat

2007-01-01

Background Ovarian cancer is characterized by a wide-spread intra-abdominal metastases which represents a major clinical hurdle in the prognosis and management of the disease. A significant proportion of ovarian cancer cells in peritoneal ascites exist as multicellular aggregates or spheroids. We hypothesize that these cellular aggregates or spheroids are invasive with the capacity to survive and implant on the peritoneal surface. This study was designed to elucidate early inherent mechanism(s) of spheroid survival, growth and disaggregation required for peritoneal metastases Methods In this study, we determined the growth pattern and adhesive capacity of ovarian cancer cell lines (HEY and OVHS1) grown as spheroids, using the well established liquid overlay technique, and compared them to a normal ovarian cell line (IOSE29) and cancer cells grown as a monolayer. The proteolytic capacity of these spheroids was compared with cells grown as a monolayer using a gelatin zymography assay to analyze secreted MMP-2/9 in conditioned serum-free medium. The disaggregation of cancer cell line spheroids was determined on extracellular matrices (ECM) such as laminin (LM), fibronectin (FN) and collagen (CI) and the expression of α2, α3, αv, α6 and β1 interin was determined by flow cytometric analysis. Neutralizing antibodies against α2, β1 subunits and α2β1 integrin was used to inhibit disaggregation as well as activation of MMPs in spheroids. Results We demonstrate that ovarian cancer cell lines grown as spheroids can sustain growth for 10 days while the normal ovarian cell line failed to grow beyond 2 days. Compared to cells grown as a monolayer, cancer cells grown as spheroids demonstrated no change in adhesion for up to 4 days, while IOSE29 cells had a 2–4-fold loss of adhesion within 2 days. Cancer cell spheroids disaggregated on extracellular matrices (ECM) and demonstrated enhanced expression of secreted pro-MMP2 as well as activated MMP2/MMP9 with no such activation of MMP's observed in monolayer cells. Flow cytometric analysis demonstrated enhanced expression of α2 and diminution of α6 integrin subunits in spheroids versus monolayer cells. No change in the expression of α3, αv and β1 subunits was evident. Conversely, except for αv integrin, a 1.5–7.5-fold decrease in α2, α3, α6 and β1 integrin subunit expression was observed in IOSE29 cells within 2 days. Neutralizing antibodies against α2, β1 subunits and α2β1 integrin inhibited disaggregation as well as activation of MMPs in spheroids. Conclusion Our results suggest that enhanced expression of α2β1 integrin may influence spheroid disaggregation and proteolysis responsible for the peritoneal dissemination of ovarian carcinoma. This may indicate a new therapeutic target for the suppression of the peritoneal metastasis associated with advanced ovarian carcinomas. PMID:17567918

Novel full-spectral flow cytometry with multiple spectrally-adjacent fluorescent proteins and fluorochromes and visualization of in vivo cellular movement.

PubMed

Futamura, Koji; Sekino, Masashi; Hata, Akihiro; Ikebuchi, Ryoyo; Nakanishi, Yasutaka; Egawa, Gyohei; Kabashima, Kenji; Watanabe, Takeshi; Furuki, Motohiro; Tomura, Michio

2015-09-01

Flow cytometric analysis with multicolor fluoroprobes is an essential method for detecting biological signatures of cells. Here, we present a new full-spectral flow cytometer (spectral-FCM). Unlike conventional flow cytometer, this spectral-FCM acquires the emitted fluorescence for all probes across the full-spectrum from each cell with 32 channels sequential PMT unit after dispersion with prism, and extracts the signals of each fluoroprobe based on the spectral shape of each fluoroprobe using unique algorithm in high speed, high sensitive, accurate, automatic and real-time. The spectral-FCM detects the continuous changes in emission spectra from green to red of the photoconvertible protein, KikGR with high-spectral resolution and separates spectrally-adjacent fluoroprobes, such as FITC (Emission peak (Em) 519 nm) and EGFP (Em 507 nm). Moreover, the spectral-FCM can measure and subtract autofluorescence of each cell providing increased signal-to-noise ratios and improved resolution of dim samples, which leads to a transformative technology for investigation of single cell state and function. These advances make it possible to perform 11-color fluorescence analysis to visualize movement of multilinage immune cells by using KikGR-expressing mice. Thus, the novel spectral flow cytometry improves the combinational use of spectrally-adjacent various FPs and multicolor fluorochromes in metabolically active cell for the investigation of not only the immune system but also other research and clinical fields of use. © 2015 International Society for Advancement of Cytometry.

Children with postsurgical capillary leak syndrome can be distinguished by antigen expression on neutrophils and monocytes

NASA Astrophysics Data System (ADS)

Tarnok, Attila; Pipek, Michal; Valet, Guenter; Richter, Jacqueline; Hambsch, Joerg; Schneider, Peter

1999-04-01

Our initial studies indicate that children who develop post- operative capillary leak syndrome (CLS) following cardiac surgery with cardiopulmonary bypass (CPB) can be distinguished based on their pre-operative level of circulating cytokines an adhesion molecules. We tested flow cytometric analysis of surface antigen expression as a potential assay for risk assessment of CLS. 24th preoperative blood samples were stained with monoclonal antibodies for the adhesion molecules ICAM-1, LFA1, MAC1, (beta) -integrin, activation markers CD25, CD54, CD69, HLA- DR, CD14 or CD4. Cells were measured on a dual-laser flow cytometer calibrated with microbeads. Antigen expression was detected as mean fluorescence intensity. The data indicate, that neutrophils of CLS patients express preoperatively higher levels of LFA1 and monocytes higher levels of HLA-DR and activation markers thus are in a state of activation. This could in combination with surgical trauma and CPB lead to their additional stimulation and migration into sites of inflammation and induce postoperative CLS. It is planned to set up a Flow-Classification program for individual risk assessment. By discriminate analysis over 80 percent of the patients were correctly classified. Our preliminary study indicates that flow cytometry with its low samples requirements and rapid access of the results could be a powerful tool to perform risk assessment prior to pediatric open heart surgery.

Quantitative analysis of gold and carbon nanoparticles in mammalian cells by flow cytometry light scattering

NASA Astrophysics Data System (ADS)

Zhou, Gang; Liu, Naicheng; Wang, Zhenheng; Shi, Tongguo; Gan, Jingjing; Wang, Zhenzhen; Zhang, Junfeng

2017-02-01

Nanoparticle-based applications for diagnostics and therapeutics have been extensively studied. These applications require a profound understanding of the fate of nanoparticles (NPs) in cellular environments. However, until now, few analytical methods are available and most of them rely on fluorescent properties or special elements of NPs; therefore, for NPs without observable optical properties or special elements, the existing methods are hardly applicable. In this study, we introduce a flow cytometry light scattering (FCLS)-based approach that quantifies in situ NPs accurately in mammalian cells. Continuous cells of heterogeneous human epithelial colorectal adenocarcinoma (Caco-2 cells), mouse peritoneal macrophages (MPM), and human adenocarcinomic alveolar basal epithelia (A549 cells) were cultured with NPs with certain concentrations and size. The intensity of the flow cytometric side scattered light, which indicates the quantity of NPs in the cells, was analyzed. The result shows an accurate size- and dose-dependent uptake of Au NPs (5, 30, 250 nm) in Caco-2 cells. The size- and dose- dependence of Au NPs (5, 30, 250 nm) and carbon NPs (50, 500 nm) in cells was validated by transmission electron microscope (TEM). This paper demonstrates the great potential of flow cytometry light scattering in the quantitative study of the size and dose effect on in situ metallic or non-metallic NPs in mammalian cells.

Quantitation of nanoparticle accumulation in flow using optimized microfluidic chambers

PubMed Central

Kusunose, J.; Gagnon, M. K. J.; Seo, J. W.; Ferrara, K. W.

2014-01-01

Background The vascular cell adhesion molecule-1 (VCAM-1) targeting peptide sequence, VHPKQHR, is a promising moiety for targeting atherosclerosis through incorporation into nanoparticles such as dendrimers and liposomes. Purpose We aim to develop VCAM-1-targeted nanoparticles that effectively accumulate on the endothelium under shear conditions and to develop robust microfluidic chambers able to house sufficient cells for flow cytometric measurements. Methods Carboxyfluorescein-labeled monomeric VHP-peptide, tetrameric VHP-dendrimers (bisbidentate or radial architecture, with or without N-terminal acetylation) and VHP-peptide liposomes were prepared. Human umbilical vein endothelial cells were treated with nano-particles under 0 or 2.9 dyne/cm2 shear, and particle binding was quantified. Flow chambers cured at various temperatures, with or without glass backings were fabricated, characterized for deformation and applied in experiments. Results Although liposomes accumulated with highest efficiency, dendrimers also demonstrated specific binding. N-terminal acetylation significantly reduced dendrimer binding, and despite shorter movement range, bisbidentate dendrimers outperformed radial dendrimers, suggesting multiple epitope presence within its estimated arm-span of 57 Å. Under shear, while liposome binding increased 300%, dendrimer binding to cells decreased 65%. Through higher temperature curing and glass backing insertion, polydimethylsiloxane flow chambers maintaining rectangular cross-section with aspect-ratio as low as 1:111 were achieved. Conclusion Optimized dendrimers and liposomal nanocarriers specifically accumulated onto cells within microfluidic chambers. PMID:24079404

Interaction study between synthetic glycoconjugate ligands and endocytic receptors using flow cytometry.

PubMed

Yura, Hirofumi; Ishihara, Masayuki; Kanatani, Yasuhiro; Takase, Bonpei; Hattori, Hidemi; Suzuki, Shinya; Kawakami, Mitsuyuki; Matsui, Takemi

2006-04-01

Flow cytometric analysis of synthetic galactosyl polymers, asialofetuin and LDL derivatives labeled with FITC (Fluorescein Isothiocyanate) was carried out to determine the phenotypes of endocytic receptors, such as asialoglycoprotein (ASPG) and the LDL receptor, on various types of cells. When FITC-labeled galactosyl polystyrene (GalCPS), being a synthetic ligand of ASPG, was applied to rat hepatocytes and human cancer cells (Hep G2 and Chang Liver), surface fluorescence intensities varied according to receptor expression on the cells. The fluorescence intensity originates from the calcium-dependent binding of the FITC-labeled GalCPS. Although unaltered by pre-treatment with glucosyl polystyrene (GluCPS), fetuin and LDL, the fluorescence intensity was suppressed by pre-treatment with (non-labeled) GalCPS and asialofetuin. Flow cytometry allowed us to demonstrate that the calcium-dependent binding of FITC-labeled LDL (prepared from rabbits) upon the addition of 17alpha-ethinyl estradiol enhances LDL receptor expression, and the expression is suppressed upon the addition of a monoclonal antibody to the LDL receptor. The binding efficiency based on the combination of FITC-labeled ligands suggests a possible application for the classification of cell types and conditions corresponding to endocytic receptor expression without the need for immuno-active antibodies or radiolabeled substances. Furthermore, the synthetic glycoconjugate (GalCPS) is shown to be a sensitive and useful marker for classification based on cell phenotype using flow cytometry.

Maturation of sperm volume regulation in the rat epididymis

PubMed Central

Damm, Oliver S.; Cooper, Trevor G.

2010-01-01

Sperm maturation in the epididymis may involve differences between mature and immature spermatozoa in their volume regulatory osmolyte response. Spermatozoa obtained from the rat caput and cauda epididymidis were examined for their ability to regulate volume after transfer from in situ epididymal osmolality (measured to be 343 ± 13 and 365 ± 19 mmol kg−1, respectively) to that of the female tract in single- and multiple-step protocols. Cells withstood the single-step treatment better than the multistep protocol. Sperm volume estimates by flow cytometric measurements of forward scatter of cells with intact head membranes was more sensitive than those by assessing cell coiling microscopically. At osmolalites below 210 mmol kg−1 both caput and cauda cells ruptured, limiting the use of flow cytometry. Above this critical value, the use of quinine showed that both caput and cauda cells could regulate volume, but cauda cells were the more effective. Of several organic osmolytes studied, myo-inositol, glutamate and KCl caused only temporary and slight swelling of spermatozoa cells in hypotonic medium. Spermatozoa of both maturities seemed to use potassium as the preferred osmolyte for regulating volume. PMID:20531277

The immunomodulator AS101 suppresses production of inflammatory cytokines and ameliorates the pathogenesis of experimental autoimmune encephalomyelitis.

PubMed

Xie, Li; Chen, Jing; McMickle, Anthony; Awar, Nadia; Nady, Soad; Sredni, Benjamin; Drew, Paul D; Yu, Shiguang

2014-08-15

We reported that AS101 (organotellurium compound, trichloro(dioxoethylene-O,O') tellurate) inhibited the differentiation of Th17 cells and reduced the production of IL-17 and GM-CSF. In addition, AS101 promoted the production of IL-2 in activated T cells. Flow cytometric analysis showed that AS101 inhibited Th17 cell proliferation. AS101 blocked the activation of transcriptional factor NFAT, Stat3, and RORγt, and increased activation of Erk1/2, suggesting a mechanism of action of AS101. We further demonstrated that AS101 was effective in amelioration of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Finally, by real-time PCR analysis we showed that AS101 reduces the IL-17, IFN-γ, GM-CSF, and IL-6 mRNA expression in inflammatory cells of spinal cords. Additionally, flow cytometry analysis also indicated that the CD4+ T cells and IL-17 and GM-CSF-producing cells were reduced in the spinal cords of AS101 treated mice compared to those treated with PBS. Copyright © 2014 Elsevier B.V. All rights reserved.

The immunomodulator AS101suppresses production of inflammatory cytokines and ameliorates the pathogenesis of experimental autoimmune encephalomyelitis

PubMed Central

Xie, Li; Chen, Jing; McMickle, Anthony; Awar, Nadia; Nady, Soad; Sredni, Benjamin; Drew, Paul D.; Yu, Shiguang

2014-01-01

We reported that AS101 (organotellurium compound, trichloro(dioxoethylene-O,O′) tellurate) inhibited the differentiation of Th17 cells and reduced the production of IL-17 and GM-CSF. In addition, AS101 promoted the production of IL-2 in activated T cells. Flow cytometric analysis showed that AS101 inhibited Th17 cell proliferation. AS101 blocked the activation of transcriptional factor NFAT, Stat3, and RORγt, and increased activation of Erk1/2, suggesting a mechanism of action of AS101. We further demonstrated that AS101 was effective in amelioration of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Finally, by real-time PCR analysis we showed that AS101 reduces the IL-17, IFN-γ, GM-CSF, and IL-6 mRNA expression in inflammatory cells of spinal cords. Additionally, flow cytometry analysis also indicated that the CD4+ T cells and IL-17 and GM-CSF-producing cells were reduced in the spinal cords of AS101 treated mice compared to those treated with PBS. PMID:24975323

Flow cytometric analysis of red-eared slider turtles (Trachemys scripta) from Tar Creek Superfund Site.

PubMed

Hays, Kimberly A; McBee, Karen

2007-05-01

Tar Creek Superfund Site (TCSFS) was heavily mined from the 1890s to 1970 and currently is contaminated with lead, zinc, and cadmium. Flow cytometry (FCM) was used to measure variation in nuclear DNA content of red blood cells collected from Trachemys scripta living within TCSFS and reference sites, Lake Carl Blackwell (LCB) and Sequoyah National Wildlife Refuge (SNWR). We also used atomic absorption spectrometry to measure Pb in blood and carapace and Cd in blood samples of turtles from TCSFS and SNWR. Mean coefficients of variation around the G(1) peak ranged from 5.33 to 5.48 and showed no significant difference between contaminated and reference populations; however, there was a significantly higher frequency of aneuploidy at TCSFS when compared with both reference populations. Blood Pb levels were not significantly different between TCSFS and SNWR populations. Pb levels in carapace samples did not differ significantly between sites; however, Pb levels were higher in carapace than blood for both populations. Blood Cd was significantly higher in animals at TCSFS than SNWR.

Minimal disease detection of B-cell lymphoproliferative disorders by flow cytometry: multidimensional cluster analysis.

PubMed

Duque, Ricardo E

2012-04-01

Flow cytometric analysis of cell suspensions involves the sequential 'registration' of intrinsic and extrinsic parameters of thousands of cells in list mode files. Thus, it is almost irresistible to describe phenomena in numerical terms or by 'ratios' that have the appearance of 'accuracy' due to the presence of numbers obtained from thousands of cells. The concepts involved in the detection and characterization of B cell lymphoproliferative processes are revisited in this paper by identifying parameters that, when analyzed appropriately, are both necessary and sufficient. The neoplastic process (cluster) can be visualized easily because the parameters that distinguish it form a cluster in multidimensional space that is unique and distinguishable from neighboring clusters that are not of diagnostic interest but serve to provide a background. For B cell neoplasia it is operationally necessary to identify the multidimensional space occupied by a cluster whose kappa:lambda ratio is 100:0 or 0:100. Thus, the concept of kappa:lambda ratio is without meaning and would not detect B cell neoplasia in an unacceptably high number of cases.

Induction of micronuclei and apoptosis in natural killer cells compared to T lymphocytes after gamma-irradiation.

PubMed

Louagie, H; Philippé, J; Vral, A; Cornelissen, M; Thierens, H; De Ridder, L

1998-02-01

To investigate the chromosomal damage caused by gamma-irradiation in T lymphocytes and natural killer (NK) cells and compare this with apoptosis induction in both lymphocyte subsets. Apoptosis induction by gamma-irradiation in T lymphocytes and NK cells was quantified using the annexin V flow cytometric assay. The cytokinesis-block micronucleus (MN) assay was used to evaluate the induced cytogenetic damage. For the MN assays on NK cells, gamma-irradiated peripheral blood mononuclear cells were cultured and stimulated with interleukin 15 (IL-15). Afterwards the NK cells (characterized by the CD3-/CD56+ phenotype) were separated with the FACSort flow cytometer and the number of MN in the sorted binuclear cells was scored. Doses of 1 and 2 Gy gamma-irradiation were applied. Higher numbers of MN in NK cells were found compared with the MN yield in T lymphocytes. In contrast, NK cells were less than T lymphocytes prone to apoptosis after gamma-irradiation. The results support the view that cytogenetic damage and apoptosis after gamma-irradiation are not necessarily correlated.

Effect of feeding soybean meal and differently processed peas on the gut mucosal immune system of broilers.

PubMed

Röhe, I; Göbel, T W; Goodarzi Boroojeni, F; Zentek, J

2017-07-01

Peas are traditionally used as a protein source for poultry. However, peas contain antinutritional factors (ANF), which are associated with the initiation of local and systemic immune reactions. The current study examined the effect of feeding raw or differently processed peas in comparison with feeding a soybean meal (SBM) based control diet (C) on the gut mucosal immune system of broilers in a 35 day feeding trial. In six replicates, a total of 360 one-day-old male broilers were randomly allocated to four different groups receiving C, or three treatment diets containing raw, fermented, and enzymatically pre-digested peas, each supplying 30% of required crude protein. After slaughtering, jejunal samples were taken for immunohistochemical, flow cytometric, and gene expression analyses. Investigations were focused on the topological distribution of intraepithelial leukocytes (villus tip, villus mid, and crypt region) as well as on the further characterization of the different intraepithelial lymphocytes (IEL) and concomitant pro- and anti-inflammatory cytokines. Broilers receiving the raw or processed pea diets had higher numbers of intraepithelial CD45+ leukocytes in the tip (P = 0.004) and mid region (P < 0.001) of villi than birds fed C. Higher numbers of intraepithelial CD3+ lymphocytes were found in the villus tip (P = 0.002) and mid region (P = 0.003) of birds fed raw or processed pea containing diets in comparison with those fed C. The flow cytometric phenotyping showed a similar relative distribution of IEL among the feeding groups. The expression of intestinal pro- and anti-inflammatory cytokines was affected by feeding the different diets only to a minor extent. To conclude, feeding of diets formulated with raw and processed peas in comparison with feeding a SBM control diet initiated mucosal immune responses in the jejunum of broilers indicated by a quantitative increase of intraepithelial T cells. Further research is needed in order to ascertain the specific factors which are responsible for observed local immune reactions and how these local reactions might affect the immune status and health of broilers. © 2017 Poultry Science Association Inc.

Flow cytometric cell cycle analysis of muscle precursor cells cultured within 3D scaffolds in a perfusion bioreactor.

PubMed

Flaibani, Marina; Luni, Camilla; Sbalchiero, Elisa; Elvassore, Nicola

2009-01-01

It has been widely demonstrated that perfusion bioreactors improve in vitro three-dimensional (3D) cultures in terms of high cell density and uniformity of cell distribution; however, the studies reported in literature were primarily based on qualitative analysis (histology, immunofluorescent staining) or on quantitative data averaged on the whole population (DNA assay, PCR). Studies on the behavior, in terms of cell cycle, of a cell population growing in 3D scaffolds in static or dynamic conditions are still absent. In this work, a perfusion bioreactor suitable to culture C(2)C(12) muscle precursor cells within 3D porous collagen scaffolds was designed and developed and a method based on flowcytometric analyses for analyzing the cell cycle in the cell population was established. Cells were extracted by enzymatic digestion of the collagen scaffolds after 4, 7, and 10 days of culture, and flow cytometric live/dead and cell cycle analyses were performed with Propidium Iodide. A live/dead assay was used for validating the method for cell extraction and staining. Moreover, to investigate spatial heterogeneity of the cell population under perfusion conditions, two stacked scaffolds in the 3D domain, of which only the upstream layer was seeded, were analyzed separately. All results were compared with those obtained from static 3D cultures. The live/dead assay revealed the presence of less than 20% of dead cells, which did not affect the cell cycle analysis. Cell cycle analyses highlighted the increment of cell fractions in proliferating phases (S/G(2)/M) owing to medium perfusion in long-term cultures. After 7-10 days, the percentage of proliferating cells was 8-12% for dynamic cultures and 3-5% for the static controls. A higher fraction of proliferating cells was detected in the downstream scaffold. From a general perspective, this method provided data with a small standard deviation and detected the differences between static and dynamic cultures and between upper and lower scaffolds. Our methodology can be extended to other cell types to investigate the influence of 3D culture conditions on the expression of other relevant cell markers.

A novel and easy FxCycle™ violet based flow cytometric method for simultaneous assessment of DNA ploidy and six-color immunophenotyping.

PubMed

Tembhare, Prashant; Badrinath, Yajamanam; Ghogale, Sitaram; Patkar, Nikhil; Dhole, Nilesh; Dalavi, Pooja; Kunder, Nikesh; Kumar, Ashok; Gujral, Sumeet; Subramanian, P G

2016-03-01

Abnormal DNA ploidy is a valuable prognostic factor in many neoplasms, especially in hematological neoplasms like B-cell acute lymphoblastic leukemia (B-ALL) and multiple myeloma (MM). Current methods of flow-cytometric (FC) DNA-ploidy evaluation are either technically difficult or limited to three- to four-color immunophenotyping and hence, challenging to evaluate DNA-ploidy in minute tumor population with background rich of its normal counterpart cells and other hematopoietic cells. We standardized a novel sensitive and easy method of simultaneous evaluation of six- to seven-color immunophenotyping and DNA-ploidy using a dye-FxCycle Violet (FCV). Linearity, resolution, and coefficient of variation (CV) for FCV were studied using chicken erythrocyte nuclei. Ploidy results of FCV were compared with Propidium iodide (PI) in 20 samples and intra-assay variation for FCV was studied. Using this six-color immunophenotyping & FCV-protocol DNA-ploidy was determined in bone-marrow samples from 124 B-ALL & 50 MM patients. Dilution experiment was also conducted to determine the sensitivity in detection of aneuploidy in minute tumor population. FCV revealed high linearity and resolution in 450/50 channel. On comparison with PI, CV of Go/G1-peak with FCV (mean-CV 4.1%) was slightly higher than PI (mean-CV 2.9%) but had complete agreement in ploidy results. Dilution experiment showed that aneuploidy could be accurately detected up to the limit of 0.01% tumor cells. Intra-assay variation was very low with CV of 0.005%. In B-ALL, hypodiploidy was noted in 4%, hyperdiploidy in 24%, near-hyperdiploidy in 13% and remaining 59% were diploid. In MM, hypodiploidy was in 2%, hyperdiploidy in 58%, near-hyperdiploidy in 8% and remaining 30% were diploid. FCV-based DNA-ploidy method is a sensitive and easy method for simultaneous evaluation of six-color immunophenotyping and DNA analysis. It is useful in DNA-ploidy evaluation of minute tumor population in cases like minimal residual disease and MM precursor conditions. © 2015 International Society for Advancement of Cytometry.

Evaluation of Macaca mulatta as a model for genotoxicity studies.

PubMed

Dobrovolsky, Vasily N; Shaddock, Joseph G; Mittelstaedt, Roberta A; Manjanatha, Mugimane G; Miura, Daishiro; Uchikawa, Makoto; Mattison, Donald R; Morris, Suzanne M

2009-02-19

We have investigated the use of peripheral blood from the nonhuman primate (NHP) rhesus monkey (Macaca mulatta) as a model system for mutation detection. The rhesus monkey is metabolically closer to humans than most common laboratory animals, and therefore may be a relevant model for hazard identification and human risk assessment. To validate the model, conditions were determined for in vitro selection and expansion of 6-thioguanine-resistant (6-TGr) HPRT mutant and proaerolysin-resistant (ProAERr) PIG-A mutant lymphocytes from peripheral blood obtained by routine venipuncture. Also, flow cytometric methods were developed for the rapid detection of PIG-A mutant erythrocytes. The flow cytometric analysis of PIG-A mutant erythrocytes was based on enumerating cells deficient in surface markers attached to the cellular membrane via glycosylphosphatidyl inositol (GPI) anchors. Mutant cells were enumerated over an extended period of time in peripheral blood of male monkeys receiving daily doses of the electrolyte replenisher Prangtrade mark (a common carrier for oral delivery of drugs in NHPs), and in the blood of one male monkey treated with a single i.p. dose of 50mg/kg of N-ethyl-N-nitrosourea at approximately 2 years of age and another similar injection at approximately 3.5 years of age. The spontaneous PIG-A and HPRT T-cell mutant frequency (MF) was low in animals receiving Prang (0-8x10(-6)), and treatment with ENU resulted in a clearly detectable increase in the frequency of ProAERr and 6-TGr lymphocytes (up to approximately 28x10(-6) and approximately 30x10(-6), respectively). Also, the ENU-treated animal had higher frequency of GPI-deficient erythrocytes (46.5x10(-6) in the treated animal vs. 7.8+/-4.2x10(-6) in control animals). Our results indicate that the rhesus monkey can be a valuable model for the identification of agents that may impact upon human health as mutagens and that the PIG-A gene can be a useful target for detection of mutation in both white and red blood cells.

Heat shock 70-kDa protein 8 isoform 1 is expressed on the surface of human embryonic stem cells and downregulated upon differentiation.

PubMed

Son, Yeon Sung; Park, Jae Hyun; Kang, Young Kook; Park, Jin-Sung; Choi, Hong Seo; Lim, Ji Young; Lee, Jeoung Eun; Lee, Jung Bok; Ko, Myoung Seok; Kim, Yong-Sam; Ko, Jeong-Heon; Yoon, Hyun Soo; Lee, Kwang-Woong; Seong, Rho Hyun; Moon, Shin Yong; Ryu, Chun Jeih; Hong, Hyo Jeong

2005-01-01

The cell-surface markers used routinely to define the undifferentiated state and pluripotency of human embryonic stem cells (hESCs) are those used in mouse embryonic stem cells (mESCs) because of a lack of markers directly originated from hESC itself. To identify more hESC-specific cell-surface markers, we generated a panel of monoclonal antibodies (MAbs) by immunizing the irradiated cell clumps of hESC line Miz-hES1, and selected 26 MAbs that were able to bind to Miz-hES1 cells but not to mESCs, mouse embryonic fibroblast cells, and STO cells. Most antibodies did not bind to human neural progenitor cells derived from the Miz-hES1 cells, either. Of these, MAb 20-202S (IgG1, kappa) immunoprecipitated a cell-surface protein of 72-kDa from the lysate of biotin-labeled Miz-hES1 cells, which was identified to be heat shock 70-kDa protein 8 isoform 1 (HSPA8) by quadrupole time-of-flight tandem mass spectrometry. Immunocytochemical analyses proved that the HSPA8 protein was also present on the surface of hESC lines Miz-hES4, Miz-hES6, and HSF6. Two-color flow cytometric analysis of Miz-hES1 and HSF6 showed the coexpression of the HSPA8 protein with other hESC markers such as stage-specific embryonic antigen 3 (SSEA3), SSEA4, TRA-1-60, and TRA-1-81. Flow cytometric and Western blot analyses using various cells showed that MAb 20-202S specifically bound to the HSPA8 protein on the surface of Miz-hES1, contrary to other anti-HSP70 antibodies examined. Furthermore, the surface expression of the HSPA8 protein on Miz-hES1 was markedly downregulated upon differentiation. These data indicate that a novel MAb 20-202S recognizes the HSPA8 protein on the surface of hESCs and suggest that the HSPA8 protein is a putative cell-surface marker for undifferentiated hESCs.

Genotoxic Mode of Action Predictions from a Multiplexed Flow Cytometric Assay and a Machine Learning Approach

PubMed Central

Bryce, Steven M.; Bernacki, Derek T.; Bemis, Jeffrey C.; Dertinger, Stephen D.

2015-01-01

Several endpoints associated with cellular responses to DNA damage as well as overt cytotoxicity were multiplexed into a miniaturized, “add and read” type flow cytometric assay. Reagents included a detergent to liberate nuclei, propidium iodide and RNase to serve as a pan-DNA dye, fluorescent antibodies against γH2AX, phospho-histone H3, and p53, and fluorescent microspheres for absolute nuclei counts. The assay was applied to TK6 cells and 67 diverse reference chemicals that served as a training set. Exposure was for 24 hrs in 96 well plates, and unless precipitation or foreknowledge about cytotoxicity suggested otherwise, the highest concentration was 1 mM. At 4 and 24 hrs aliquots were removed and added to microtiter plates containing the reagent mix. Following a brief incubation period robotic sampling facilitated walk-away data acquisition. Univariate analyses identified biomarkers and time points that were valuable for classifying agents into one of three groups: clastogenic, aneugenic, or non-genotoxic. These mode of action predictions were optimized with a forward-stepping process that considered Wald test p-values, receiver operator characteristic curves, and pseudo R2 values, among others. A particularly high performing multinomial logistic regression model was comprised of four factors: 4hr γH2AX and phospho-histone H3 values, and 24 hr p53 and polyploidy values. For the training set chemicals, the four-factor model resulted in 94% concordance with our a priori classifications. Cross validation occurred via a leave-one-out approach, and in this case 91% concordance was observed. A test set of 17 chemicals that were not used to construct the model were evaluated, some of which utilized a short-term treatment in the presence of a metabolic activation system, and in 16 cases mode of action was correctly predicted. These initial results are encouraging as they suggest a machine learning strategy can be used to rapidly and reliably predict new chemicals’ genotoxic mode of action based on data from an efficient and highly scalable multiplexed assay. PMID:26764165

Genotoxic mode of action predictions from a multiplexed flow cytometric assay and a machine learning approach.

PubMed

Bryce, Steven M; Bernacki, Derek T; Bemis, Jeffrey C; Dertinger, Stephen D

2016-04-01

Several endpoints associated with cellular responses to DNA damage as well as overt cytotoxicity were multiplexed into a miniaturized, "add and read" type flow cytometric assay. Reagents included a detergent to liberate nuclei, RNase and propidium iodide to serve as a pan-DNA dye, fluorescent antibodies against γH2AX, phospho-histone H3, and p53, and fluorescent microspheres for absolute nuclei counts. The assay was applied to TK6 cells and 67 diverse reference chemicals that served as a training set. Exposure was for 24 hrs in 96-well plates, and unless precipitation or foreknowledge about cytotoxicity suggested otherwise, the highest concentration was 1 mM. At 4- and 24-hrs aliquots were removed and added to microtiter plates containing the reagent mix. Following a brief incubation period robotic sampling facilitated walk-away data acquisition. Univariate analyses identified biomarkers and time points that were valuable for classifying agents into one of three groups: clastogenic, aneugenic, or non-genotoxic. These mode of action predictions were optimized with a forward-stepping process that considered Wald test p-values, receiver operator characteristic curves, and pseudo R(2) values, among others. A particularly high performing multinomial logistic regression model was comprised of four factors: 4 hr γH2AX and phospho-histone H3 values, and 24 hr p53 and polyploidy values. For the training set chemicals, the four-factor model resulted in 94% concordance with our a priori classifications. Cross validation occurred via a leave-one-out approach, and in this case 91% concordance was observed. A test set of 17 chemicals that were not used to construct the model were evaluated, some of which utilized a short-term treatment in the presence of a metabolic activation system, and in 16 cases mode of action was correctly predicted. These initial results are encouraging as they suggest a machine learning strategy can be used to rapidly and reliably predict new chemicals' genotoxic mode of action based on data from an efficient and highly scalable multiplexed assay. © 2016 Wiley Periodicals, Inc.

Binding of fluoresceinated epidermal growth factor to A431 cell sub-populations studied using a model-independent analysis of flow cytometric fluorescence data.

PubMed Central

Chatelier, R C; Ashcroft, R G; Lloyd, C J; Nice, E C; Whitehead, R H; Sawyer, W H; Burgess, A W

1986-01-01

A method is developed for determining ligand-cell association parameters from a model-free analysis of data obtained with a flow cytometer. The method requires measurement of the average fluorescence per cell as a function of ligand and cell concentration. The analysis is applied to data obtained for the binding of fluoresceinated epidermal growth factor to a human epidermoid carcinoma cell line, A431. The results indicate that the growth factor binds to two classes of sites on A431 cells: 4 X 10(4) sites with a dissociation constant (KD) of less than or equal to 20 pM, and 1.5 X 10(6) sites with a KD of 3.7 nM. A derived plot of the average fluorescence per cell versus the average number of bound ligands per cell is used to construct binding isotherms for four sub-populations of A431 cells fractionated on the basis of low-angle light scatter. The four sub-populations bind the ligand with equal affinity but differ substantially in terms of the number of binding sites per cell. We also use this new analysis to critically evaluate the use of 'Fluorotrol' as a calibration standard in flow cytometry. PMID:3015587

Effects of pre-analytical variables on flow cytometric diagnosis of canine lymphoma: A retrospective study (2009-2015).

PubMed

Comazzi, S; Cozzi, M; Bernardi, S; Zanella, D R; Aresu, L; Stefanello, D; Marconato, L; Martini, V

2018-02-01

Flow cytometry (FC) is increasingly being used for immunophenotyping and staging of canine lymphoma. The aim of this retrospective study was to assess pre-analytical variables that might influence the diagnostic utility of FC of lymph node (LN) fine needle aspirate (FNA) specimens from dogs with lymphoproliferative diseases. The study included 987 cases with LN FNA specimens sent for immunophenotyping that were submitted to a diagnostic laboratory in Italy from 2009 to 2015. Cases were grouped into 'diagnostic' and 'non-diagnostic'. Pre-analytical factors analysed by univariate and multivariate analyses were animal-related factors (breed, age, sex, size), operator-related factors (year, season, shipping method, submitting veterinarian) and sample-related factors (type of sample material, cellular concentration, cytological smears, artefacts). The submitting veterinarian, sample material, sample cellularity and artefacts affected the likelihood of having a diagnostic sample. The availability of specimens from different sites and of cytological smears increased the odds of obtaining a diagnostic result. Major artefacts affecting diagnostic utility included poor cellularity and the presence of dead cells. Flow cytometry on LN FNA samples yielded conclusive results in more than 90% of cases with adequate sample quality and sampling conditions. Copyright © 2018 Elsevier Ltd. All rights reserved.

Flow Cytometry Enables Multiplexed Measurements of Genetically Encoded Intramolecular FRET Sensors Suitable for Screening.

PubMed

Doucette, Jaimee; Zhao, Ziyan; Geyer, Rory J; Barra, Melanie M; Balunas, Marcy J; Zweifach, Adam

2016-07-01

Genetically encoded sensors based on intramolecular FRET between CFP and YFP are used extensively in cell biology research. Flow cytometry has been shown to offer a means to measure CFP-YFP FRET; we suspected it would provide a unique way to conduct multiplexed measurements from cells expressing different FRET sensors, which is difficult to do with microscopy, and that this could be used for screening. We confirmed that flow cytometry accurately measures FRET signals using cells transiently transfected with an ERK activity reporter, comparing responses measured with imaging and cytometry. We created polyclonal long-term transfectant lines, each expressing a different intramolecular FRET sensor, and devised a way to bar-code four distinct populations of cells. We demonstrated the feasibility of multiplexed measurements and determined that robust multiplexed measurements can be conducted in plate format. To validate the suitability of the method for screening, we measured responses from a plate of bacterial extracts that in unrelated experiments we had determined contained the protein kinase C (PKC)-activating compound teleocidin A-1. The multiplexed assay correctly identifying the teleocidin A-1-containing well. We propose that multiplexed cytometric FRET measurements will be useful for analyzing cellular function and for screening compound collections. © 2016 Society for Laboratory Automation and Screening.

Laboratory Tests of Multiplex Detection of PCR Amplicons Using the Luminex 100 Flow Analyzer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Venkateswaran, K.S.; Nasarabadi, S.; Langlois, R.G.

2000-05-05

Lawrence Livermore National Laboratory (LLNL) demonstrated the power of flow cytometry in detecting the biological agents simulants at JFT III. LLNL pioneered in the development of advanced nucleic acid analyzer (ANM) for portable real time identification. Recent advances in flow cytometry provide a means for multiplexed nucleic acid detection and immunoassay of pathogenic microorganisms. We are presently developing multiplexed immunoassays for the simultaneous detection of different simulants. Our goal is to build an integrated instrument for both nucleic acid analysis and immuno detection. In this study we evaluated the Luminex LX 100 for concurrent identification of more than one PCRmore » amplified product. ANAA has real-time Taqman fluorescent detection capability for rapid identification of field samples. However, its multiplexing ability is limited by the combination of available fluorescent labels. Hence integration of ANAA with flow cytometry can give the rapidity of ANAA amplification and the multiplex capability of flow cytometry. Multiplexed flow cytometric analysis is made possible using a set of fluorescent latex microsphere that are individually identified by their red and infrared fluorescence. A green fluorochrome is used as the assay signal. Methods were developed for the identification of specific nucleic acid sequences from Bacillus globigii (Bg), Bacillus thuringensis (Bt) and Erwinia herbicola (Eh). Detection sensitivity using different reporter fluorochromes was tested with the LX 100, and also different assay formats were evaluated for their suitability for rapid testing. A blind laboratory trial was carried out December 22-27, 1999 to evaluate bead assays for multiplex identification of Bg and Bt PCR products. This report summarizes the assay development, fluorochrome comparisons, and the results of the blind trial conducted at LLNL for the laboratory evaluation of the LX 100 flow analyzer.« less

GRP78 mediates the therapeutic efficacy of curcumin on colon cancer.

PubMed

Chang, Yu-Jia; Huang, Chien-Yu; Hung, Chin-Sheng; Chen, Wei-Yu; Wei, Po-Li

2015-02-01

Glucose-regulated protein 78 (GRP78) is the key regulator of endoplasmic reticular (ER) function. Expression of GRP78 was correlated with malignancy in different cancers. However, the role of GRP78 in the cytotoxic effect of curcumin on colon cancer cells is still unclear. A silencing RNA (siRNA) technique was used to knock down GRP78 expression. The anticancer effects of curcumin were assessed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, a flow cytometric cell cycle analysis, and a terminal dexynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. HT-29 cells expressed lower GRP78 compared with DLD-1 cells. The MTT assay revealed that HT-29 cells were more resistant to curcumin treatment than DLD-1 cells. GRP78KD cells showed more resistance to curcumin treatment compared with scrambled control cells. Overexpressed GRP78 in HT-29 cells increased the sensitivity to curcumin treatment. According to the cell cycle analysis and TUNEL assay, we found that apoptosis dramatically increased in scrambled control cells compared to GRP78KD DLD-1 cells after curcumin treatment. Finally, we evaluated levels of Bcl-2, BAX, and Bad and found that an increase of Bcl-2 level was observed in GRP78KD cells treated with curcumin. Those results were consistent with the increasing of resistance to curcumin after silencing of GRP78. The levels of GRP78 expression might determine the therapeutic efficacy of curcumin against colon cancer cells.

Chromosome banding in Amphibia. XXVI. Coexistence of homomorphic XY sex chromosomes and a derived Y-autosome translocation in Eleutherodactylus maussi (Anura, Leptodactylidae).

PubMed

Schmid, M; Feichtinger, W; Steinlein, C; Haaf, T; Schartl, M; Visbal García, R; Manzanilla Pupo, J; Fernández Badillo, A

2002-01-01

A 15-year cytogenetic survey on one population of the leaf litter frog Eleutherodactylus maussi in northern Venezuela confirmed the existence of multiple XXAA male symbol /XAA(Y) female symbol sex chromosomes which originated by a centric (Robertsonian) fusion between the original Y chromosome and an autosome. 95% of the male individuals in this population are carriers of this Y-autosome fusion. In male meiosis the XAA(Y) sex chromosomes pair in the expected trivalent configuration. In the same population, 5% of the male animals still possess the original, free XY sex chromosomes. In a second population of E. maussi analyzed, all male specimens are characterized by these ancestral XY chromosomes which form normal bivalents in meiosis. E. maussi apparently represents the first vertebrate species discovered in which a derived Y-autosome fusion still coexists with the ancestral free XY sex chromosomes. The free XY sex chromosomes, as well as the multiple XA(Y) sex chromosomes are still in a very primitive (homomorphic) stage of differentiation. With no banding technique applied it is possible to distinguish the Y from the X. DNA flow cytometric measurements show that the genome of E. maussi is among the largest in the anuran family Leptodactylidae. The present study also supplies further data on differential chromosome banding and fluorescence in situ hybridization experiments in this amphibian species. Copyright 2003 S. Karger AG, Basel

Homeostatic 'bystander' proliferation of human peripheral blood B cells in response to polyclonal T-cell stimulation in vitro.

PubMed

Jasiulewicz, Aleksandra; Lisowska, Katarzyna A; Pietruczuk, Krzysztof; Frąckowiak, Joanna; Fulop, Tamas; Witkowski, Jacek M

2015-11-01

The mechanisms of maintenance of adequate numbers of B lymphocytes and of protective levels of immunoglobulins in the absence of antigenic (re)stimulation remain not fully understood. Meanwhile, our results presented here show that both peripheral blood naive and memory B cells can be activated strongly and non-specifically (in a mitogen-like fashion) in 5-day in vitro cultures of anti-CD3- or concanavalin A (Con A)-stimulated peripheral blood mononuclear cells of healthy people. This polyclonal, bystander activation of the B cells includes multiple divisions of most of them (assessed here by the flow cytometric technique of dividing cell tracking) and significant antibody [immunoglobulin M (IgM) and IgG] secretion. Observed proliferation of the CD19(+) B cells depends on contact with stimulated T helper (Th) cells (via CD40-CD40L interaction) and on the response of B cells to secreted interleukins IL-5, IL-10 and IL-4, and is correlated with the levels of these Th-derived molecules, while it does not involve the ligation of the BCR/CD19 complex. We suggest that the effect might reflect the situation occurring in vivo as the homeostatic proliferation of otherwise non-stimulated, peripheral B lymphocytes, providing an always ready pool for efficient antibody production to any new (or cognate) antigen challenge. © The Japanese Society for Immunology. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Hyperchromatic laser scanning cytometry

NASA Astrophysics Data System (ADS)

Tárnok, Attila; Mittag, Anja

2007-02-01

In the emerging fields of high-content and high-throughput single cell analysis for Systems Biology and Cytomics multi- and polychromatic analysis of biological specimens has become increasingly important. Combining different technologies and staining methods polychromatic analysis (i.e. using 8 or more fluorescent colors at a time) can be pushed forward to measure anything stainable in a cell, an approach termed hyperchromatic cytometry. For cytometric cell analysis microscope based Slide Based Cytometry (SBC) technologies are ideal as, unlike flow cytometry, they are non-consumptive, i.e. the analyzed sample is fixed on the slide. Based on the feature of relocation identical cells can be subsequently reanalyzed. In this manner data on the single cell level after manipulation steps can be collected. In this overview various components for hyperchromatic cytometry are demonstrated for a SBC instrument, the Laser Scanning Cytometer (Compucyte Corp., Cambridge, MA): 1) polychromatic cytometry, 2) iterative restaining (using the same fluorochrome for restaining and subsequent reanalysis), 3) differential photobleaching (differentiating fluorochromes by their different photostability), 4) photoactivation (activating fluorescent nanoparticles or photocaged dyes), and 5) photodestruction (destruction of FRET dyes). With the intelligent combination of several of these techniques hyperchromatic cytometry allows to quantify and analyze virtually all components of relevance on the identical cell. The combination of high-throughput and high-content SBC analysis with high-resolution confocal imaging allows clear verification of phenotypically distinct subpopulations of cells with structural information. The information gained per specimen is only limited by the number of available antibodies and by sterical hindrance.

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy.

PubMed

Lemcke, Heiko; Voronina, Natalia; Steinhoff, Gustav; David, Robert

2017-06-19

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Hypereosinophilia with abnormal T cells, trisomy 7 and elevated TARC serum level.

PubMed

Roumier, A S; Grardel, N; Laï, J L; Becqueriaux, I; Ghomari, K; de Lavareille, A; Roufosse, F; Prin, L; Capron, M

2003-07-01

The idiopathic hypereosinophilic syndrome (HES) is a rare heterogeneous disorder, characterized by persistent blood eosinophilia with possible organ involvement. We describe here the case of a 20-year-old atopic male presenting chronic hypereosinophilia and eczema since childhood. Biological findings included hypereosinophilia (9.5 x 10(9)/L), hyperlymphocytosis (10.9 x 10(9)/L), polyclonal hypergammaglobulinemia and elevated IgE serum level. Flow cytometric analysis of blood lymphoid cells showed a population of CD2+CD3-CD4+TCRab-TCRgd- lymphocytes. These cells displayed a Th0/Th2 cytokine profile, and a clonal TCR rearrangement pattern. A high serum TARC level was observed. Karyotype studies on blood stimulated culture or lymph nodes revealed a cellular hyperdiploïd clone 47, XY, +7. To our knowledge, this chromosomal aberration has never been reported in such case.

Monoclonal antibodies to human hemoglobin S and cell lines for the production thereof

DOEpatents

Jensen, Ronald H.; Vanderlaan, Martin; Bigbee, William L.; Stanker, Larry H.; Branscomb, Elbert W.; Grabske, Robert J.

1988-01-01

The present invention provides monoclonal antibodies specific to and distinguish between hemoglobin S and hemoglobin A and methods for their production and use. These antibodies are capable of distinguishing between two hemoglobin types which differ from each other by only a single amino acid residue. The antibodies produced according to the present method are useful as immunofluorescent markers to enumerate circulating red blood cells which have the property of altered expression of the hemoglobin gene due to somatic mutation in stem cells. Such a measurement is contemplated as an assay for in vivo cellular somatic mutations in humans. Since the monoclonal antibodies produced in accordance with the instant invention exhibit a high degree of specificity to and greater affinity for hemoglobin S, they are suitable for labeling human red blood cells for flow cytometric detection of hemoglobin genotype.

Apoptosis in liver cancer (HepG2) cells induced by functionalized gold nanoparticles.

PubMed

Ashokkumar, Thirunavukkarasu; Prabhu, Durai; Geetha, Ravi; Govindaraju, Kasivelu; Manikandan, Ramar; Arulvasu, Chinnasamy; Singaravelu, Ganesan

2014-11-01

An ethnopharmacological approach for biosynthesis of gold nanoparticles is being demonstrated using seed coat of Cajanus cajan. Medicinal value of capping molecule investigated for anticancer activity and results disclose its greater potential. The active principle of the seed coat [3-butoxy-2-hydroxypropyl 2-(2,4-dihydroxyphenyl) acetate] is elucidated. Rapid one-step synthesis yields highly stable, monodisperse (spherical) gold nanoparticles in the size ranging from 9 to 41 nm. Anticancer activity has been studied using liver cancer cells and cytotoxic mechanism has been evaluated using MTT, Annexin-V/PI Double-Staining Assay, Cell cycle, Comet assay and Flow cytometric analysis for apoptosis. The present investigation will open up a new possibility of functionalizing gold nanoparticles for apoptosis studies in liver cancer cells. Copyright © 2014 Elsevier B.V. All rights reserved.

Tributyltin induces cell cycle arrest at G1 phase in the yeast Saccharomyces cerevisiae.

PubMed

Sekito, Takayuki; Sugimoto, Naoko; Ishimoto, Masaya; Kawano-Kawada, Miyuki; Akiyama, Koichi; Nishimoto, Sogo; Sugahara, Takuya; Kakinuma, Yoshimi

2014-04-01

Tributyltin (TBT) has long been recognized as a major environmental pollutant that can cause significant damage to the cellular functions as well as disruption of endocrine homeostasis. TBT induces apoptosis accompanied by production of reactive oxygen species (ROS) in mammalian and yeast cells. We observed that the budding yeast cells exposed to this compound at low concentrations exhibited cell growth arrest, but not cell death. Flow cytometric analysis of yeast cells without synchronization and morphological assessment of cells synchronized at M phase by nocodazole treatment indicated that TBT-exposed Saccharomyces cerevisiae cells were arrested at G1 phase of the cell cycle. This arrest was recovered by the addition of N-acetylcysteine, suggesting the involvement of ROS production by TBT. This is the first study to evaluate the action of TBT on cell cycle events.

Essential roles of caspases and their upstream regulators in rotenone-induced apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee Jihjong; Huang, M.-S.; Yang, I-C.

2008-06-20

In the present study, we examined whether caspases and their upstream regulators are involved in rotenone-induced cytotoxicity. Rotenone significantly inhibited the proliferation of oral cancer cell lines in a dose-dependent manner compared to normal oral mucosal fibroblasts. Flow cytometric analysis of DNA content showed that rotenone treatment induced apoptosis following G2/M arrest. Western blotting showed activation of both the caspase-8 and caspase-9 pathways, which differed from previous studies conducted in other cell types. Furthermore, p53 protein and its downstream pro-apoptotic target, Bax, were induced in SAS cells after treatment with rotenone. Rotenone-induced apoptosis was inhibited by antioxidants (glutathione, N-acetylcysteine, andmore » tiron). In conclusion, our results demonstrate significant involvement of caspases and their upstream regulators in rotenone-induced cytotoxicity.« less

New Cytotoxic Triterpenoid Saponins from the Roots of Albizia gummifera (J.F.Gmel.) C.A.Sm.

PubMed

Simo, Line Made; Noté, Olivier Placide; Mbing, Joséphine Ngo; Aouazou, Sarah Ali; Guillaume, Dominique; Muller, Christian Dominique; Pegnyemb, Dieudonné Emmanuel; Lobstein, Annelise

2017-10-01

As part of our search for new bioactive saponins from Cameroonian medicinal plants, two new oleanane-type saponins, named gummiferaosides D and E (1 and 2), along with one known saponin, julibroside J 8 (3), were isolated from the roots of Albizia gummifera. Their structures were established on the basis of extensive 1D- and 2D-NMR ( 1 H- and 13 C-NMR, DEPT, COSY, TOCSY, NOESY, HSQC, HSQC-TOCSY, and HMBC) and HR-ESI-MS studies, and by chemical evidence. The apoptotic effect of saponins 1 - 3 was evaluated on the A431 human epidermoid cancer cell. Flow cytometric analyses showed that saponins 1 - 3 induced apoptosis of human epidermoid cancer cell (A431) in a dose-dependent manner. © 2017 Wiley-VHCA AG, Zurich, Switzerland.

Synthesis and biological evaluation of new coumarins bearing 2,4-diaminothiazole-5-carbonyl moiety.

PubMed

Ayati, Adileh; Oghabi Bakhshaiesh, Tayebeh; Moghimi, Setareh; Esmaeili, Rezvan; Majidzadeh-A, Keivan; Safavi, Maliheh; Firoozpour, Loghman; Emami, Saeed; Foroumadi, Alireza

2018-06-07

A series of new coumarin-containing compounds 3a-l and 4a-c was designed and synthesized based on the chalcone-type 4-amino-5-cinnamoylthiazole scaffold 2, and screened for their in vitro anticancer and antioxidant activities. Representatively, the 2-thiomorpholinothiazole derivative 3k with IC 50 values of 7.5-16.9 μg/ml demonstrated good cytotoxic effects against tested cell lines MCF-7, HepG2 and SW480. Further investigation by flow cytometric analysis confirmed that this compound induces apoptotic cell death in MCF-7 cells and cause G1-phase arrest in the cell cycle. Moreover, most of compounds had intrinsic potential for radical scavenging activity and ferric-reducing power as investigated by DPPH and FRAP assays. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Bead-based microfluidic immunoassay for diagnosis of Johne's disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wadhwa, Ashutosh; Foote, Robert; Shaw, Robert W

2012-01-01

Microfluidics technology offers a platform for development of point-of-care diagnostic devices for various infectious diseases. In this study, we examined whether serodiagnosis of Johne s disease (JD) can be conducted in a bead-based microfluidic assay system. Magnetic micro-beads were coated with antigens of the causative agent of JD, Mycobacterium avium subsp. paratuberculosis. The antigen-coated beads were incubated with serum samples of JD-positive or negative serum samples and then with a fluorescently-labeled secondary antibody (SAB). To confirm binding of serum antibodies to the antigen, the beads were subjected to flow cytometric analysis. Different conditions (dilutions of serum and SAB, types ofmore » SAB, and types of magnetic beads) were optimized for a great degree of differentiation between the JD-negative and JD-positive samples. Using the optimized conditions, we tested a well-classified set of 155 serum samples from JD negative and JD-positive cattle by using the bead-based flow cytometric assay. Of 105 JD-positive samples, 63 samples (60%) showed higher antibody binding levels than a cut-off value determined by using antibody binding levels of JD-negative samples. In contrast, only 43-49 JD-positive samples showed higher antibody binding levels than the cut-off value when the samples were tested by commercially-available immunoassays. Microfluidic assays were performed by magnetically immobilizing a number of beads within a microchannel of a glass microchip and detecting antibody on the collected beads by laser-induced fluorescence. Antigen-coated magnetic beads treated with bovine serum sample and fluorescently-labeled SAB were loaded into a microchannel to measure the fluorescence (reflecting level of antibody binding) on the beads in the microfluidic system. When the results of five bovine serum samples obtained with the system were compared to those obtained with the flow cytometer, a high level of correlation (linear regression, r2 = 0.994) was observed. In a further experiment, we magnetically immobilized antigen-coated beads in a microchannel, reacted the beads with serum and SAB in the channel, and detected antibody binding to the beads in the microfluidic system. A strong antibody binding in JD-positive serum was detected, whereas there was only negligible binding in negative control experiments. Our data suggest that the bead-based microfluidic system may form a basis for development of an on-site serodiagnosis of JD. Key Words: Mycobacterium avium ssp. paratuberculosis, Johne s disease, microfluidics, lab-on-a-chip.« less

Cellular and Molecular Effect of MEHP Involving LXRα in Human Fetal Testis and Ovary

PubMed Central

Muczynski, Vincent; Lecureuil, Charlotte; Messiaen, Sébastien; Guerquin, Marie-Justine; N’Tumba-Byn, Thierry; Moison, Delphine; Hodroj, Wassim; Benjelloun, Hinde; Baijer, Jan; Livera, Gabriel; Frydman, René; Benachi, Alexandra; Habert, René; Rouiller-Fabre, Virginie

2012-01-01

Background Phthalates have been shown to have reprotoxic effects in rodents and human during fetal life. Previous studies indicate that some members of the nuclear receptor (NR) superfamilly potentially mediate phthalate effects. This study aimed to assess if expression of these nuclear receptors are modulated in the response to MEHP exposure on the human fetal gonads in vitro. Methodology/Principal Findings Testes and ovaries from 7 to 12 gestational weeks human fetuses were exposed to 10−4M MEHP for 72 h in vitro. Transcriptional level of NRs and of downstream genes was then investigated using TLDA (TaqMan Low Density Array) and qPCR approaches. To determine whether somatic or germ cells of the testis are involved in the response to MEHP exposure, we developed a highly efficient cytometric germ cell sorting approach. In vitro exposure of fetal testes and ovaries to MEHP up-regulated the expression of LXRα, SREBP members and of downstream genes involved in the lipid and cholesterol synthesis in the whole gonad. In sorted testicular cells, this effect is only observable in somatic cells but not in the gonocytes. Moreover, the germ cell loss induced by MEHP exposure, that we previously described, is restricted to the male gonad as oogonia density is not affected in vitro. Conclusions/Significance We evidenced for the first time that phthalate increases the levels of mRNA for LXRα, and SREBP members potentially deregulating lipids/cholesterol synthesis in human fetal gonads. Interestingly, this novel effect is observable in both male and female whereas the germ cell apoptosis is restricted to the male gonad. Furthermore, we presented here a novel and potentially very useful flow cytometric cell sorting method to analyse molecular changes in germ cells versus somatic cells. PMID:23118965

Cellular and molecular effect of MEHP Involving LXRα in human fetal testis and ovary.

PubMed

Muczynski, Vincent; Lecureuil, Charlotte; Messiaen, Sébastien; Guerquin, Marie-Justine; N'tumba-Byn, Thierry; Moison, Delphine; Hodroj, Wassim; Benjelloun, Hinde; Baijer, Jan; Livera, Gabriel; Frydman, René; Benachi, Alexandra; Habert, René; Rouiller-Fabre, Virginie

2012-01-01

Phthalates have been shown to have reprotoxic effects in rodents and human during fetal life. Previous studies indicate that some members of the nuclear receptor (NR) superfamilly potentially mediate phthalate effects. This study aimed to assess if expression of these nuclear receptors are modulated in the response to MEHP exposure on the human fetal gonads in vitro. Testes and ovaries from 7 to 12 gestational weeks human fetuses were exposed to 10(-4)M MEHP for 72 h in vitro. Transcriptional level of NRs and of downstream genes was then investigated using TLDA (TaqMan Low Density Array) and qPCR approaches. To determine whether somatic or germ cells of the testis are involved in the response to MEHP exposure, we developed a highly efficient cytometric germ cell sorting approach. In vitro exposure of fetal testes and ovaries to MEHP up-regulated the expression of LXRα, SREBP members and of downstream genes involved in the lipid and cholesterol synthesis in the whole gonad. In sorted testicular cells, this effect is only observable in somatic cells but not in the gonocytes. Moreover, the germ cell loss induced by MEHP exposure, that we previously described, is restricted to the male gonad as oogonia density is not affected in vitro. We evidenced for the first time that phthalate increases the levels of mRNA for LXRα, and SREBP members potentially deregulating lipids/cholesterol synthesis in human fetal gonads. Interestingly, this novel effect is observable in both male and female whereas the germ cell apoptosis is restricted to the male gonad. Furthermore, we presented here a novel and potentially very useful flow cytometric cell sorting method to analyse molecular changes in germ cells versus somatic cells.

Evaluation of optimal DNA staining for triggering by scanning fluorescence microscopy (SFM)

NASA Astrophysics Data System (ADS)

Mittag, Anja; Marecka, Monika; Pierzchalski, Arkadiusz; Malkusch, Wolf; Bocsi, József; Tárnok, Attila

2009-02-01

In imaging and flow cytometry, DNA staining is a common trigger signal for cell identification. Selection of the proper DNA dye is restricted by the hardware configuration of the instrument. The Zeiss Imaging Solutions GmbH (München, Germany) introduced a new automated scanning fluorescence microscope - SFM (Axio Imager.Z1) which combines fluorescence imaging with cytometric parameters measurement. The aim of the study was to select optimal DNA dyes as trigger signal in leukocyte detection and subsequent cytometric analysis of double-labeled leukocytes by SFM. Seven DNA dyes (DAPI, Hoechst 33258, Hoechst 33342, POPO-3, PI, 7-AAD, and TOPRO-3) were tested and found to be suitable for the implemented filtersets (fs) of the SFM (fs: 49, fs: 44, fs: 20). EDTA blood was stained after erythrocyte lysis with DNA dye. Cells were transferred on microscopic slides and embedded in fluorescent mounting medium. Quality of DNA fluorescence signal as well as spillover signals were analyzed by SFM. CD45-APC and CD3-PE as well as CD4-FITC and CD8-APC were selected for immunophenotyping and used in combination with Hoechst. Within the tested DNA dyes DAPI showed relatively low spillover and the best CV value. Due to the low spillover of UV DNA dyes a triple staining of Hoechst and APC and PE (or APC and FITC, respectively) could be analyzed without difficulty. These results were confirmed by FCM measurements. DNA fluorescence is applicable for identifying and triggering leukocytes in SFM analyses. Although some DNA dyes exhibit strong spillover in other fluorescence channels, it was possible to immunophenotype leukocytes. DAPI seems to be best suitable for use in the SFM system and will be used in protocol setups as primary parameter.

Flow cytometric assay to assess short-term effects of personal care products on the marine microalga Tetraselmis suecica.

PubMed

Seoane, Marta; Esperanza, Marta; Rioboo, Carmen; Herrero, Concepción; Cid, Ángeles

2017-03-01

Large quantities of personal care products (PCPs) are used daily and many of their chemical ingredients are subsequently released into marine environments. Cultures of the marine microalga Tetraselmis suecica were exposed for 24 h to three emerging compounds included in the main classes of PCPs: the UV filter benzophenone-3 (BP-3), the disinfectant triclosan (TCS) and the fragrance tonalide (AHTN). Concentrations tested, expressed as cellular quota (pg cell -1 ), ranged from 5 to 40 for BP-3, from 2 to 16 for TCS and from 1.2 to 2.4 for AHTN. A small cytometric panel was carried out to evaluate key cytotoxicity biomarkers including inherent cell properties, growth and metabolic activity and cytoplasmic membrane properties. BP-3 caused a significant increase in growth rate, metabolic activity and chlorophyll a fluorescence from 10 pg cell -1 . However, growth and esterase activity decreased in cells exposed to all TCS and AHTN concentrations, except the lowest ones. Also these two compounds provoked a significant swelling of cells, more pronounced in the case of TCS-exposed cells. Although all treated cells remained viable, changes in membrane potential were observed. BP-3 and AHTN caused a significant depolarization of cells from 10 to 1.6 pg cell -1 , respectively; however all TCS concentrations assayed caused a noticeable hyperpolarization of cells. Metabolic activity and cytoplasmic membrane potential were the most sensitive parameters. It can be concluded that the toxicological model used and the toxicological parameters evaluated are suitable to assess the toxicity of these emerging contaminants. Copyright © 2016 Elsevier Ltd. All rights reserved.

Novel full‐spectral flow cytometry with multiple spectrally‐adjacent fluorescent proteins and fluorochromes and visualization of in vivo cellular movement

PubMed Central

Futamura, Koji; Sekino, Masashi; Hata, Akihiro; Ikebuchi, Ryoyo; Nakanishi, Yasutaka; Egawa, Gyohei; Kabashima, Kenji; Watanabe, Takeshi; Furuki, Motohiro

2015-01-01

Abstract Flow cytometric analysis with multicolor fluoroprobes is an essential method for detecting biological signatures of cells. Here, we present a new full‐spectral flow cytometer (spectral‐FCM). Unlike conventional flow cytometer, this spectral‐FCM acquires the emitted fluorescence for all probes across the full‐spectrum from each cell with 32 channels sequential PMT unit after dispersion with prism, and extracts the signals of each fluoroprobe based on the spectral shape of each fluoroprobe using unique algorithm in high speed, high sensitive, accurate, automatic and real‐time. The spectral‐FCM detects the continuous changes in emission spectra from green to red of the photoconvertible protein, KikGR with high‐spectral resolution and separates spectrally‐adjacent fluoroprobes, such as FITC (Emission peak (Em) 519 nm) and EGFP (Em 507 nm). Moreover, the spectral‐FCM can measure and subtract autofluorescence of each cell providing increased signal‐to‐noise ratios and improved resolution of dim samples, which leads to a transformative technology for investigation of single cell state and function. These advances make it possible to perform 11‐color fluorescence analysis to visualize movement of multilinage immune cells by using KikGR‐expressing mice. Thus, the novel spectral flow cytometry improves the combinational use of spectrally‐adjacent various FPs and multicolor fluorochromes in metabolically active cell for the investigation of not only the immune system but also other research and clinical fields of use. © 2015 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of ISAC PMID:26217952

Generation of anti-porcine CD69 monoclonal antibodies and their usefulness to evaluate early activation of cellular immunity by flow cytometric analysis.

PubMed

Hayashi, Yumiko; Okutani, Mie; Ogawa, Shohei; Tsukahara, Takamitsu; Inoue, Ryo

2018-05-01

T cell-mediated cellular immunity and humoral immunity are equally important for the prevention of diseases. To assess activation of human and mouse cellular immunity, early activation markers of lymphocytes are often used in flow cytometry targeting expression of CD69 molecules. Response of humoral immunity against infection or vaccination has been well investigated in pigs, but that of cellular immunity has been largely neglected due to lack of direct evaluation tools. Thus, in pig research a proper assay of antibody reacted with porcine CD69 is still unavailable. In the present study, two anti-porcine CD69 mAb-producing mouse hybridomas, 01-14-22-51 (IgG2b-κ) and 01-22-44-102 (IgG2a-κ), both showing fine reactivity with phorbol 12-myristate 13-acetate (PMA) and ionomycin-stimulated porcine peripheral blood lymphocytes in flow cytometry, were established. When porcine peripheral blood lymphocytes were activated with PMA and ionomycin and analyzed by flow cytometry, it was found that both mAbs generated in this study stained about 70% of lymphocytes. In contrast, after an identical procedure, only 5% and 13.5% of lymphocytes were stained with anti-interferon-γ mAb and anti-tumor necrosis factor-α mAb, respectively. These results indicate that evaluation of cellular immunity activation turns more sensitive after using our newly generated mAbs. © 2018 Japanese Society of Animal Science.

Absolute counting of neutrophils in whole blood using flow cytometry.

PubMed

Brunck, Marion E G; Andersen, Stacey B; Timmins, Nicholas E; Osborne, Geoffrey W; Nielsen, Lars K

2014-12-01

Absolute neutrophil count (ANC) is used clinically to monitor physiological dysfunctions such as myelosuppression or infection. In the research laboratory, ANC is a valuable measure to monitor the evolution of a wide range of disease states in disease models. Flow cytometry (FCM) is a fast, widely used approach to confidently identify thousands of cells within minutes. FCM can be optimised for absolute counting using spiked-in beads or by measuring the sample volume analysed. Here we combine the 1A8 antibody, specific for the mouse granulocyte protein Ly6G, with flow cytometric counting in straightforward FCM assays for mouse ANC, easily implementable in the research laboratory. Volumetric and Trucount™ bead assays were optimized for mouse neutrophils, and ANC values obtained with these protocols were compared to ANC measured by a dual-platform assay using the Orphee Mythic 18 veterinary haematology analyser. The single platform assays were more precise with decreased intra-assay variability compared with ANC obtained using the dual protocol. Defining ANC based on Ly6G expression produces a 15% higher estimate than the dual protocol. Allowing for this difference in ANC definition, the flow cytometry counting assays using Ly6G can be used reliably in the research laboratory to quantify mouse ANC from a small volume of blood. We demonstrate the utility of the volumetric protocol in a time-course study of chemotherapy induced neutropenia using four drug regimens. © 2014 International Society for Advancement of Cytometry.

Chromosome Numbers and Genome Size Variation in Indian Species of Curcuma (Zingiberaceae)

PubMed Central

Leong-Škorničková, Jana; Šída, Otakar; Jarolímová, Vlasta; Sabu, Mamyil; Fér, Tomáš; Trávníček, Pavel; Suda, Jan

2007-01-01

Background and Aims Genome size and chromosome numbers are important cytological characters that significantly influence various organismal traits. However, geographical representation of these data is seriously unbalanced, with tropical and subtropical regions being largely neglected. In the present study, an investigation was made of chromosomal and genome size variation in the majority of Curcuma species from the Indian subcontinent, and an assessment was made of the value of these data for taxonomic purposes. Methods Genome size of 161 homogeneously cultivated plant samples classified into 51 taxonomic entities was determined by propidium iodide flow cytometry. Chromosome numbers were counted in actively growing root tips using conventional rapid squash techniques. Key Results Six different chromosome counts (2n = 22, 42, 63, >70, 77 and 105) were found, the last two representing new generic records. The 2C-values varied from 1·66 pg in C. vamana to 4·76 pg in C. oligantha, representing a 2·87-fold range. Three groups of taxa with significantly different homoploid genome sizes (Cx-values) and distinct geographical distribution were identified. Five species exhibited intraspecific variation in nuclear DNA content, reaching up to 15·1 % in cultivated C. longa. Chromosome counts and genome sizes of three Curcuma-like species (Hitchenia caulina, Kaempferia scaposa and Paracautleya bhatii) corresponded well with typical hexaploid (2n = 6x = 42) Curcuma spp. Conclusions The basic chromosome number in the majority of Indian taxa (belonging to subgenus Curcuma) is x = 7; published counts correspond to 6x, 9x, 11x, 12x and 15x ploidy levels. Only a few species-specific C-values were found, but karyological and/or flow cytometric data may support taxonomic decisions in some species alliances with morphological similarities. Close evolutionary relationships among some cytotypes are suggested based on the similarity in homoploid genome sizes and geographical grouping. A new species combination, Curcuma scaposa (Nimmo) Škorničk. & M. Sabu, comb. nov., is proposed. PMID:17686760

Evaluation of the immune responses of the brown mussel Perna perna as indicators of fecal pollution.

PubMed

Silva Dos Santos, Fernanda; Neves, Raquel Almeida Ferrando; Carvalho, Wanderson Fernandes de; Krepsky, Natascha; Crapez, Mirian Araújo Carlos

2018-06-01

The mussel Perna perna is an intertidal bivalve that is widely distributed, cultivated and consumed in South Africa, Brazil and Venezuela. Among marine resources, bivalve mollusks are one of the most impacted by anthropogenic pollution, as they can accumulate pathogenic bacteria and water pollutants. Hemocytes are molluscan defense cells, and their abundance and functions can be affected in response to contaminants, such as bacterial load. However, no previous study has investigated the immune response of P. perna hemocytes. The aim of this study was to evaluate several immune parameters in P. perna as indicators of fecal pollution in mussel hemolymph and in seawater. We collected mussels and adjacent seawater from beaches with different levels of fecal contamination in Rio de Janeiro state (Brazil): Vermelha Beach (VB); Icaraí Beach (IB); Urca Beach (UB); and Jurujuba Beach (JB). Hemocyte parameters (density, morphology, phagocytic activity and production of Reactive Oxygen Species - ROS) were evaluated using flow cytometry. We quantified Fecal Indicator Bacteria (FIB) in seawater by the multiple tubes technique for each beach and for hemolymph by the spread-plate technique. In agreement with historical evaluation of fecal contamination levels, UB presented the highest FIB abundance in seawater (thermotolerant coliforms, TEC = 1600 NMP 100 mL -1 ), whereas VB exhibited the lowest (TEC = 17 NMP 100 mL -1 ). UB mussels had six and eight times higher hemocyte density and phagocytic activity, respectively, than mussels from VB. Mussels from VB and IB presented a significantly lower number of total coliforms in hemolymph and a significantly higher relative internal complexity of hemocytes than those from UB and JB (p ≤ 0.01, PERMANOVA). ROS production by hemocytes was significantly lower in mussels from VB compared to those from JB (p = 0.04, ANOVA). Our results indicate a significant relationship between the level of fecal contamination in aquatic environments and the immune response of mussel hemocytes. Immune-related parameters may therefore be useful as indicators of bivalve health and environmental quality. Our flow cytometric analysis of P. perna hemocytes represents a new approach for studying Perna perna biology and might represent a novel tool for measuring organic pollution and water quality. Copyright © 2018 Elsevier Ltd. All rights reserved.

Routine conventional karyotyping of lymphoma staging bone marrow samples does not contribute clinically relevant information.

PubMed

Nardi, Valentina; Pulluqi, Olja; Abramson, Jeremy S; Dal Cin, Paola; Hasserjian, Robert P

2015-06-01

Bone marrow (BM) evaluation is an important part of lymphoma staging, which guides patient management. Although positive staging marrow is defined as morphologically identifiable disease, such samples often also include flow cytometric analysis and conventional karyotyping. Cytogenetic analysis is a labor-intensive and costly procedure and its utility in this setting is uncertain. We retrospectively reviewed pathological reports of 526 staging marrow specimens in which conventional karyotyping had been performed. All samples originated from a single institution from patients with previously untreated Hodgkin and non-Hodgkin lymphomas presenting in an extramedullary site. Cytogenetic analysis revealed clonal abnormalities in only eight marrow samples (1.5%), all of which were positive for lymphoma by morphologic evaluation. Flow cytometry showed a small clonal lymphoid population in three of the 443 morphologically negative marrow samples (0.7%). Conventional karyotyping is rarely positive in lymphoma staging marrow samples and, in our cohort, the BM karyotype did not contribute clinically relevant information in the vast majority of cases. Our findings suggest that karyotyping should not be performed routinely on BM samples taken to stage previously diagnosed extramedullary lymphomas unless there is pathological evidence of BM involvement by lymphoma. © 2015 Wiley Periodicals, Inc.

Multiparameter flow cytometric analysis of a pH sensitive formyl peptide with application to receptor structure and processing kinetics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fay, S.P.; Domalewski, M.D.; Houghton, T.G.

1994-02-01

Environmentally sensitive molecules have many potential cellular applications. The authors have investigated the utility of a pH sensitive ligand for the formyl peptide receptor, CHO-Met-Leu-Phe-Phe-Lys (SNAFL)-OH (SNAFL-seminaphthofluorescein), because in previous studies protonation has been used to explain the quenching when the fluorescinated formyl pentapeptide ligand binds to this receptor. Moreover, acidification in intracellular compartments is a general mechanism occurring in cells during processing of ligand-receptor complexes. Because the protonated form of SNAFL is excited at 488 nm with emission at 530 nm and the unprotonated form is excited at 568 nm with emission at 650 nm, the ratio of protonatedmore » and unprotonated forms can be examined by multiparameter flow cytometry. The authors found that the receptor-bound ligand is sensitive to both the extracellular and intracellular pH. There is a small increase in the pK[sub a] of the ligand upon binding to the receptor consistent with protonation in the binding pocket. Once internalized, spectral changes in the probe consistent with acidification and ligand dissociation from the receptor are observed. 22 refs., 4 figs.« less

In vitro induction of tetraploid plants from diploid Zizyphus jujuba Mill. cv. Zhanhua.

PubMed

Gu, X F; Yang, A F; Meng, H; Zhang, J R

2005-12-01

Tetraploid plants of Zizyphus jujuba Mill. cv. Zhanhua were obtained with in vitro colchicine treatment. Shoot tips from in vitro-grown plants were treated with five different concentrations of colchicine (0.01, 0.03, 0.05, 0.1, 0.3%) in liquid MS medium (Murashige and Skoog 1962), and shaken (100 rpm) at 25 degrees C in darkness for 24, 48, 72 or 96 h, respectively. Tetraploids were obtained at a frequency of over 3% by using 0.05% colchicine (48 h, 72 h) and 0.1% colchicine (24 h, 48 h) treatment as determined by flow cytometry. Cytological and morphological evidence confirmed the results of flow cytometric analysis. The chromosome number of diploid plants was 24 and that of tetraploid plants was 48. The stomata sizes of tetraploid plants were significantly larger than those of diploid plants, while the frequency of stomata were reduced significantly. Similarly, the chloroplast number of guard cells of tetraploid plants increased significantly. The selected tetraploid plants were grafted onto mature trees of Z. jujuba Mill. cv. Zhanhua in the field, resulted in thicker stems, rounder and succulent leaves, larger flowers and a delay in florescence time (3-4 days later) than diploid plants.

In situ associations between marine photosynthetic picoeukaryotes and potential parasites - a role for fungi?

PubMed

Lepère, Cécile; Ostrowski, Martin; Hartmann, Manuela; Zubkov, Mikhail V; Scanlan, David J

2016-08-01

Photosynthetic picoeukaryotes (PPEs) are important components of the marine picophytoplankton community playing a critical role in CO2 fixation but also as bacterivores, particularly in the oligotrophic gyres. Despite an increased interest in these organisms and an improved understanding of the genetic diversity of this group, we still know little of the environmental factors controlling the abundance of these organisms. Here, we investigated the quantitative importance of eukaryotic parasites in the free-living fraction as well as in associations with PPEs along a transect in the South Atlantic. Using tyramide signal amplification-fluorescence in situ hybridization (TSA-FISH), we provide quantitative evidence of the occurrence of free-living fungi in open ocean marine systems, while the Perkinsozoa and Syndiniales parasites were not abundant in these waters. Using flow cytometric cell sorting of different PPE populations followed by a dual-labelled TSA-FISH approach, we also demonstrate fungal associations, potentially parasitic, occurring with both pico-Prymnesiophyceae and pico-Chrysophyceae. These data highlight the necessity for further work investigating the specific role of marine fungi as parasites of phytoplankton to improve understanding of carbon flow in marine ecosystems. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Immune gene expression for diverse haemocytes derived from pacific white shrimp, Litopenaeus vannamei.

PubMed

Yang, Chih-Chiu; Lu, Chung-Lun; Chen, Sherwin; Liao, Wen-Liang; Chen, Shiu-Nan

2015-05-01

In this study, diverse haemocytes from Pacific white shrimp Litopenaeus vannamei were spread by flow cytometer sorting system. Using the two commonly flow cytometric parameters FSC and SSC, the haemocytes could be divided into three populations. Microscopy observation of L. vannamei haemocytes in anticoagulant buffer revealed three morphologically distinct cell types designated as granular cell, hyaline cell and semigranular cell. Immune genes, which includes prophenoloxidase (proPO), lipopolysaccharide-β-glucan binding protein (LGBP), peroxinectin, crustin, lysozyme, penaeid-3a and transglutaminase (TGase), expressed from different haemocyte were analysed by quantitative real time PCR (qPCR). Results from the mRNA expression was estimated by relative level of each gene to β-actin gene. Finally, the seven genes could be grouped by their dominant expression sites. ProPO, LGBP and peroxinectin were highly expressed in granular cells, while LGBP, crustin, lysozyme and P-3a were highly expressed in semigranular cells and TGase was highly expressed in hyaline cells. In this study, L. vannamei haemocytes were firstly grouped into three different types and the immune related genes expression in grouped haemocytes were estimated. Copyright © 2015 Elsevier Ltd. All rights reserved.

Hybrid male sterility between the fresh- and brackish-water types of ninespine stickleback Pungitius pungitius (Pisces, Gasterosteidae).

PubMed

Takahashi, Hiroshi; Nagai, Terumi; Goto, Akira

2005-01-01

Two ecologically distinct forms, fresh- and brackish-water types, of ninespine stickleback co-exist in several freshwater systems on the coast of eastern Hokkaido. Recent genetic analyses of 13 allozyme loci revealed genetic separation between the two types even though their spawning grounds were in close proximity. On the other hand, there is only a small difference in mitochondrial DNA (mtDNA) sequence between the two types suggesting that they diverged quite recently or that mtDNA introgression occurred between them. To test for postzygotic reproductive isolating mechanisms and hybrid mediated gene flow, we examined the viability and reproductive performance of reciprocal F1 hybrids. The hybrids grew to the adult size normally and both sexes expressed secondary sexual characters in the reciprocal crosses. The female hybrids were reciprocally fertile, while the male hybrids were reciprocally sterile. Histological and flow-cytometric analyses of the hybrid testis revealed that the sterility pattern was classified as 'gametic sterility,' with gonads of normal size but abnormal spermatogenesis. To our knowledge, the present finding is a novel example of one sex hybrid sterility in the stickleback family (Gasterosteidae).

Reference method for detection of Pgp mediated multidrug resistance in human hematological malignancies: a method validated by the laboratories of the French Drug Resistance Network.

PubMed

Huet, S; Marie, J P; Gualde, N; Robert, J

1998-12-15

Multidrug resistance (MDR) associated with overexpression of the MDR1 gene and of its product, P-glycoprotein (Pgp), plays an important role in limiting cancer treatment efficacy. Many studies have investigated Pgp expression in clinical samples of hematological malignancies but failed to give definitive conclusion on its usefulness. One convenient method for fluorescent detection of Pgp in malignant cells is flow cytometry which however gives variable results from a laboratory to another one, partly due to the lack of a reference method rigorously tested. The purpose of this technical note is to describe each step of a reference flow cytometric method. The guidelines for sample handling, staining and analysis have been established both for Pgp detection with monoclonal antibodies directed against extracellular epitopes (MRK16, UIC2 and 4E3), and for Pgp functional activity measurement with Rhodamine 123 as a fluorescent probe. Both methods have been validated on cultured cell lines and clinical samples by 12 laboratories of the French Drug Resistance Network. This cross-validated multicentric study points out crucial steps for the accuracy and reproducibility of the results, like cell viability, data analysis and expression.

Mechanisms of the ultrasound-mediated intracellular delivery of liposomes and dextrans.

PubMed

Afadzi, Mercy; Strand, Sabina P; Nilssen, Esben A; Måsøy, Svein-Erik; Johansen, Tonni F; Hansen, Rune; Angelsen, Bjørn A; de L Davies, Catharina

2013-01-01

The mechanism involved in the ultrasoundenhanced intracellular delivery of fluorescein-isothiocyanate (FITC)-dextran (molecular weight 4 to 2000 kDa) and liposomes containing doxorubicin (Dox) was studied using HeLa cells and an ultrasound transducer at 300 kHz, varying the acoustic power. The cellular uptake and cell viability were measured using flow cytometry and confocal microscopy. The role of endocytosis was investigated by inhibiting clathrin- and caveolae-mediated endocytosis, as well as macropinocytosis. Microbubbles were found to be required during ultrasound treatment to obtain enhanced cellular uptake. The percentage of cells internalizing Dox and dextran increased with increasing mechanical index. Confocal images and flow cytometric analysis indicated that the liposomes were disrupted extracellularly and that released Dox was taken up by the cells. The percentage of cells internalizing dextran was independent of the molecular weight of dextrans, but the amount of the small 4-kDa dextran molecules internalized per cell was higher than for the other dextrans. The inhibition of endocytosis during ultrasound exposure resulted in a significant decrease in cellular uptake of dextrans. Therefore, the improved uptake of Dox and dextrans may be a result of both sonoporation and endocytosis.

Gamma delta T-cell large granular lymphocyte lymphoma in a dog.

PubMed

Ortiz, Ana Liza; Carvalho, Sofia; Leo, Chiara; Riondato, Fulvio; Archer, Joy; Cian, Francesco

2015-09-01

A 2-year and 6-month-old female neutered Labrador Retriever with Horner syndrome, megaesophagus, and a mediastinal mass was referred to the Queen Mother Hospital for Animals of the Royal Veterinary College. A large granular lymphocyte (LGL) lymphoma was diagnosed on cytology; flow cytometric analysis revealed a γδ T-cell phenotype (CD3+, CD5+, CD45+, TCRγδ+, CD4-, CD8-, CD34-, CD21-). Chemotherapy was started with a combination of lomustine, vincristine, procarbazine, and prednisolone, followed by bleyomicin. Euthanasia was elected by the owners, due to progressive deterioration and lack of quality of life, 28 days after diagnosis. This is the first cytologic and immunophenotypic characterization of a canine γδ T-cell lymphoma with LGL morphology and probably of mediastinal origin. The role of chemotherapy in delaying the disease progression remains unknown. © 2015 American Society for Veterinary Clinical Pathology.

Bone marrow mesenchymal stem cells ameliorate inflammatory factor-induced dysfunction of INS-1 cells on chip.

PubMed

Sun, Yu; Yao, Zhina; Lin, Peng; Hou, Xinguo; Chen, Li

2014-05-01

Using a microfluidic chip, we have investigated whether bone marrow mesenchymal stem cells (BM-MSCs) could ameliorate IL-1β/IFN-γ-induced dysfunction of INS-1 cells. BM-MSCs were obtained from diabetes mellitus patients and their cell surface antigen expression profiles were analyzed by flow cytometric. INS-1 cells were cocultured with BM-MSCs on a microfluidic chip with persistent perfusion of medium containing 1 ng/mL IL-1β and 2.5 U/mL IFN-γ for 72 h. BM-MSCs could partially rescue INS-1 cells from cytokine-induced dysfunction and ameliorate the expression of insulin and PDX-1 gene in INS-1 cells. Thus BM-MSCs can be viewed as a promising stem cell source to depress inflammatory factor-induced dysfunction of pancreatic β cells in diabetic patients. © 2014 International Federation for Cell Biology.

Extranodal posttransplant plasmacytic hyperplasia with subsequent posttransplant plasmacytic malignancy: six-year interval case report and review of the literature.

PubMed

Dunphy, Cherie H; Galambos, Csaba; Polski, Jacek M; Evans, H Lance; Gardner, Laura J; Grosso, Leonard E; Montone, Kathleen T

2002-03-01

Posttransplant lymphoproliferative disorders (PTLDs) represent a morphologic, immunophenotypic, and genotypic spectrum of disease. Most recently, Knowles et al divided PTLDs into 3 distinct categories: (1) plasmacytic hyperplasia, (2) polymorphic B-cell hyperplasia and polymorphic B-cell lymphoma, and (3) immunoblastic lymphoma and multiple myeloma. Although one form of PTLD may progress to another form, only 1 previous case has been reported in which multiple myeloma developed 14 months after an original diagnosis of plasmacytic hyperplasia. The type of solid organ transplant was not specified in that case. We report a post--cardiac transplant plasmacytic hyperplasia developing 7 years posttransplant. Six years subsequent to the plasmacytic hyperplasia, the patient developed a posttransplant plasmacytic malignancy, supported by morphology, flow cytometric immunophenotyping, and genotypic studies. Since we have no data to support disseminated bony disease or an abnormal serum protein, we have not used the term "multiple myeloma" for this case.

Acute promyelocytic leukemia, hypogranular variant, with uncharacteristic staining with chloroacetate esterase.

PubMed

Dunphy, C H; Polski, J M; Johns, G; Evans, H L; Gardner, L J

2001-06-01

A diagnosis of the hypogranular variant of acute promyelocytic leukemia (APLv) may be difficult to establish based on cytomorphology alone. However, the great majority of cases have a classical immunophenotype by flow cytometric immunophenotyping (FCI) (CD13+, CD33+, dim CD64+, HLA-DR-, and CD34-) and a classical enzyme cytochemical (EC) staining pattern. [intensely staining with myeloperoxidase, Sudan Black B, and chloroacetate esterase (CAE) and negative with alpha'-naphthyl acetate and butyrate esterases]. Although the immunophenotype of APLv by FCI has varied in the literature (HLA-DR +/- and CD34 +/-), the EC staining pattern has remained constant. We report a case of APLv with characteristic cytomorphology, compatible FCI data (CD13+, CD33+, dim CD64+, HLA-DR +/-, and CD34-), chromosomal detection of t(15; 17), and molecular detection of the PML/RAR alpha fusion gene; however, staining of the leukemic cells with CAE was quite uncharacteristic. We describe our findings.

Monoclonal antibodies to human hemoglobin S and cell lines for the production thereof

DOEpatents

Jensen, R.H.; Vanderlaan, M.; Bigbee, W.L.; Stanker, L.H.; Branscomb, E.W.; Grabske, R.J.

1984-11-29

The present invention provides monoclonal antibodies specific to and distinguishing between hemoglobin S and hemoglobin A and methods for their production and use. These antibodies are capable of distinguishing between two hemoglobin types which differ from each other by only a single amino acid residue. The antibodies produced according to the present method are useful as immunofluorescent markers to enumerate circulating red blood cells which have the property of altered expression of the hemoglobin gene due to somatic mutation in stem cells. Such a measurement is contemplated as an assay for in vivo cellular somatic mutations in humans. Since the monoclonal antibodies produced in accordance with the instant invention exhibit a high degree of specificity to and greater affinity for hemoglobin S, they are suitable for labeling human red blood cells for flow cytometric detection of hemoglobin genotype. 4 figs.

Evidence for specific annexin I-binding proteins on human monocytes.

PubMed Central

Goulding, N J; Pan, L; Wardwell, K; Guyre, V C; Guyre, P M

1996-01-01

Recombinant human annexin I and a monoclonal antibody specific for this protein (mAb 1B) were used to investigate surface binding of this member of the annexin family of proteins to peripheral blood monocytes. Flow cytometric analysis demonstrated trypsin-sensitive, saturable binding of annexin I to human peripheral blood monocytes but not to admixed lymphocytes. A monoclonal antibody that blocks the anti-phospholipase activity of annexin I also blocked its binding to monocytes. These findings suggest the presence of specific binding sites on monocytes. Furthermore, surface iodination, immunoprecipitation and SDS/PAGE analysis were used to identify two annexin I-binding proteins on the surface of monocytes with molecular masses of 15 kDa and 18 kDa respectively. The identification and characterization of these annexin I-binding molecules should help us to better understand the specific interactions of annexin I with monocytes that lead to down-regulation of pro-inflammatory cell functions. PMID:8687405

Unveiling the mode of action of antibacterial labdane diterpenes from Alpinia nigra (Gaertn.) B. L. Burtt seeds.

PubMed

Ghosh, Sudipta; Indukuri, Kiran; Bondalapati, Somasekhar; Saikia, Anil K; Rangan, Latha

2013-08-01

The labdane diterpene, (E)-labda-8(17), 12-diene-15, 16-dial (compound A) and its epoxide analogue, (E)-8β, 17-Epoxylabd-12-ene-15, 16-dial (compound B) were isolated from the seeds of Alpinia nigra for the first time. The antibacterial activities of both compounds were evaluated against three Gram-positive and four Gram-negative bacteria, and flow cytometric analysis revealed that these compounds caused significant damage to the bacterial cell membranes. Further, field emission scanning electron microscope imaging and cell leakage analysis confirmed that the labdane diterpenes were responsible for bacterial cell membrane damage and disintegration. Our findings provide new insight into the broad-spectrum effects of two natural labdane diterpenes that may be useful in the future development of herbal antibiotic products. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Stem/progenitor cells in pituitary organ homeostasis and tumourigenesis

PubMed Central

Manshaei, Saba

2018-01-01

Evidence for the presence of pituitary gland stem cells has been provided over the last decade using a combination of approaches including in vitro clonogenicity assays, flow cytometric side population analysis, immunohistochemical analysis and genetic approaches. These cells have been demonstrated to be able to self-renew and undergo multipotent differentiation to give rise to all hormonal lineages of the anterior pituitary. Furthermore, evidence exists for their contribution to regeneration of the organ and plastic responses to changing physiological demand. Recently, stem-like cells have been isolated from pituitary neoplasms raising the possibility that a cytological hierarchy exists, in keeping with the cancer stem cell paradigm. In this manuscript, we review the evidence for the existence of pituitary stem cells, their role in maintaining organ homeostasis and the regulation of their differentiation. Furthermore, we explore the emerging concept of stem cells in pituitary tumours and their potential roles in these diseases. PMID:28855316

Kefiran protects Caco-2 cells from cytopathic effects induced by Bacillus cereus infection.

PubMed

Medrano, Micaela; Hamet, Maria F; Abraham, Analía G; Pérez, Pablo F

2009-11-01

The aim of this work was to evaluate the ability of kefiran to antagonize cytopathic effects triggered by Bacillus cereus strain B10502 on cultured human enterocytes (Caco-2 cells). Cell damage was evaluated by F-actin labelling, scanning electron microscopy and determination of ratios of necrotic and detached cells. To assess the interaction between kefiran and bacteria or eukaryotic cells, flow cytometric analysis was conducted with FITC-labelled kefiran. Kefiran significantly protected infected cells from cytopathic effects induced by B. cereus such as cell necrosis, F-actin disorganisation and microvilli effacement, although presence of kefiran did not modify the adhesion of microorganisms to cultured human enterocytes. Results could be ascribed to the ability of kefiran to interact with both bacteria and eukaryotic cells thus antagonizing interactions necessary for maximal biological effects. Our findings encourage further research on the use of bacterial exopolysaccharides to antagonize virulence factors associated to direct bacteria-cell interactions.

Type-I interferon receptor expression: its circadian rhythm and downregulation after interferon-alpha administration in peripheral blood cells from renal cancer patients.

PubMed

Shiba, Masahiro; Nonomura, Norio; Nakai, Yasutomo; Nakayama, Masashi; Takayama, Hitoshi; Inoue, Hitoshi; Tsujimura, Akira; Nishimura, Kazuo; Okuyama, Akihiko

2009-04-01

To investigate the regulation of interferon-alpha (IFN-alpha) receptor expression in metastatic renal cell carcinoma (RCC) after IFN-alpha administration. Blood sampling was carried out in eight patients with metastatic RCC and six healthy volunteers. Flow-cytometric analysis using a monoclonal antibody against the active subunit of the type-I IFN-alpha receptor (IFNAR2) was carried out to examine the circadian rhythm of IFNAR2 expression in peripheral blood mononuclear cells (PBMC) as well as its downregulation after IFN-alpha administration. According to its circadian rhythm IFNAR2 in PBMC had a peak expression at night. Once IFN-alpha is administered, IFNAR2 levels in PBMC showed downregulation within 48 h and recovered within another 48 h. Our findings might support the establishment of an optimal schedule for IFN-alpha administration.

INHIBITORY EFFECT OF CHITOSAN OLIGOSACCHARIDE ON HUMAN HEPATOMA CELLS IN VITRO.

PubMed

Liu, Likun; Xin, Yi; Liu, Jia; Zhang, Ershao; Li, Weiling

2017-01-01

Chitosan oligosaccharide, the degradation products of chitin, was reported to have a wide range of physiological functions and biological activities. In this study, we explored the inhibitory effect of Chitosan oligosaccharide on human hepatoma cells. MTT assay was applied to detect cell viability of the human hepatoma cells treated with Chitosan oligosaccharide. Flow cytometric analysis was used to investigate the apoptosis of the human hepatoma cells treated with Chitosan oligosaccharide. We employed western blot to investigate the underlying mechanisms involved in the apoptosis. Our data indicated that chitosan oligosaccharide dose-dependently inhibited the growth of hepatoma cells and induced apoptosis. On the molecular level, chitosan oligosaccharide decreased Bcl-2 and increased Caspase-3 expression which may be related to the apoptosis of hepatoma cells. Our results provide an experimental basis for the clinical development of Chitosan oligosaccharide as a novel anti-hepatoma drug.

Opium induces apoptosis in jurkat cells.

PubMed

Igder, Somayeh; Asadikaram, Gholam Reza; Sheykholeslam, Farzaneh; Sayadi, Ahmad Reza; Mahmoodi, Mehdi; Kazemi Arababadi, Mohammad; Rasaee, Mohammad Javad

2013-01-01

The direct effect of some opioids on immune cells has been demonstrated. The aim of this study was to assess the apoptotic effect of opium on Jurkat T lymphocyte cells. Different concentrations of opium (2.86 × 10-3 to 2.86 × 10-11 g/ml) were added to 24-well plates containing 5 × 105 Jurkat cells. Apoptotic events were assessed after 6, 24, and 72 hours by flow-cytometric detection of surface phosphatidylserine. Significant differences in apoptosis of Jurkat cells were seen at 24 and 72 hours in different concentrations of opium (P < 0.05). After 72 hours, significant increase in necrosis of Jurkat cells was seen in opium concentration of 2.85 × 10-3 g/ml compared to cells without opium (control) (P < 0.05). These results showed that opium directly increases apoptosis and necrosis of T lymphocytes. This effect may play a role in immune dysfunction in opium addicts.

Opium Induces Apoptosis in Jurkat Cells

PubMed Central

Igder, Somayeh; Asadikaram, Gholam Reza; Sheykholeslam, Farzaneh; Sayadi, Ahmad Reza; Mahmoodi, Mehdi; Kazemi Arababadi, Mohammad; Rasaee, Mohammad Javad

2013-01-01

Background The direct effect of some opioids on immune cells has been demonstrated. The aim of this study was to assess the apoptotic effect of opium on Jurkat T lymphocyte cells. Methods Different concentrations of opium (2.86 × 10-3 to 2.86 × 10-11 g/ml) were added to 24-well plates containing 5 × 105 Jurkat cells. Apoptotic events were assessed after 6, 24, and 72 hours by flow-cytometric detection of surface phosphatidylserine. Findings Significant differences in apoptosis of Jurkat cells were seen at 24 and 72 hours in different concentrations of opium (P < 0.05). After 72 hours, significant increase in necrosis of Jurkat cells was seen in opium concentration of 2.85 × 10-3 g/ml compared to cells without opium (control) (P < 0.05). Conclusion These results showed that opium directly increases apoptosis and necrosis of T lymphocytes. This effect may play a role in immune dysfunction in opium addicts. PMID:24494155

Normal T lymphocytes can express two different T cell receptor beta chains: implications for the mechanism of allelic exclusion

PubMed Central

1995-01-01

We have examined the extent of allelic exclusion at the T cell receptor (TCR) beta locus using monoclonal antibodies specific for V beta products. A small proportion (approximately 1%) of human peripheral blood T cells express two V beta as determined by flow cytometric analysis, isolation of representative clones, and sequencing of the corresponding V beta chains. Dual beta T cells are present in both the CD45R0+ and CD45R0- subset. These results indicate that dual beta expression is compatible with both central and peripheral selection. They also suggest that the substantial degree of TCR beta allelic exclusion is dependent only on asynchronous rearrangements at the beta locus, whereas the role of the pre-TCR is limited to signaling the presence of at least one functional beta protein. PMID:7699339

Molecular modeling, simulation and virtual screening of MurD ligase protein from Salmonella typhimurium LT2.

PubMed

Samal, Himanshu Bhusan; Das, Jugal Kishore; Mahapatra, Rajani Kanta; Suar, Mrutyunjay

2015-01-01

The Mur enzymes of the peptidoglycan biosynthesis pathway constitute ideal targets for the design of new classes of antimicrobial inhibitors in Gram-negative bacteria. We built a homology model of MurD of Salmonella typhimurium LT2 using MODELLER (9v12) software. 'The homology model was subjected to energy minimization by molecular dynamics (MD) simulation study with GROMACS software for a simulation time of 20 ns in water environment. The model was subjected for virtual screening study from the Zinc Database using Dockblaster software. Inhibition assay for the best inhibitor, 3-(amino methyl)-n-(4-methoxyphenyl) aniline, by flow cytometric analysis revealed the effective inhibition of peptidoglycan biosynthesis. Results from this study provide new insights for the molecular understanding and development of new antibacterial drugs against the pathogen. Copyright © 2015 Elsevier Inc. All rights reserved.

INHIBITORY EFFECT OF CHITOSAN OLIGOSACCHARIDE ON HUMAN HEPATOMA CELLS IN VITRO

PubMed Central

Liu, Likun; Xin, Yi; Liu, Jia; Zhang, Ershao; Li, Weiling

2017-01-01

Background: Chitosan oligosaccharide, the degradation products of chitin, was reported to have a wide range of physiological functions and biological activities. In this study, we explored the inhibitory effect of Chitosan oligosaccharide on human hepatoma cells Materials and Methods: MTT assay was applied to detect cell viability of the human hepatoma cells treated with Chitosan oligosaccharide. Flow cytometric analysis was used to investigate the apoptosis of the human hepatoma cells treated with Chitosan oligosaccharide. We employed western blot to investigate the underlying mechanisms involved in the apoptosis. Results: Our data indicated that chitosan oligosaccharide dose-dependently inhibited the growth of hepatoma cells and induced apoptosis. On the molecular level, chitosan oligosaccharide decreased Bcl-2 and increased Caspase-3 expression which may be related to the apoptosis of hepatoma cells. Conclusion: Our results provide an experimental basis for the clinical development of Chitosan oligosaccharide as a novel anti-hepatoma drug. PMID:28638890

Lactoferricin treatment decreases the rate of cell proliferation of a human colon cancer cell line.

PubMed

Freiburghaus, C; Janicke, B; Lindmark-Månsson, H; Oredsson, S M; Paulsson, M A

2009-06-01

Food components modify the risk of cancer at a large number of sites but the mechanism of action is unknown. In the present investigation, we studied the effect of the peptide lactoferricin derived from bovine milk lactoferrin on human colon cancer CaCo-2 cells. The cells were either untreated or treated with 2.0, 0.2, or 0.02 microM lactoferricin. Cell cycle kinetics were investigated with a bromodeoxyuridine DNA flow cytometric method. The results show that lactoferricin treatment slightly but significantly prolonged the S phase of the cell cycle. Lactoferricin treatment lowered the level of cyclin E1, a protein involved in the regulation of genes required for G(1)/S transition and consequently for efficient S phase progression. The slight prolongation of the S phase resulted in a reduction of cell proliferation, which became more apparent after a long treatment time.

Longitudinal evaluation of the efficacy of heat treatment procedures against Legionella spp. in hospital water systems by using a flow cytometric assay.

PubMed

Allegra, Severine; Grattard, Florence; Girardot, Françoise; Riffard, Serge; Pozzetto, Bruno; Berthelot, Philippe

2011-02-01

Legionella spp. are frequently isolated in hospital water systems. Heat shock (30 min at 70°C) is recommended by the World Health Organization to control its multiplication. The aim of the study was to evaluate retrospectively the efficacy of heat treatments by using a flow cytometry assay (FCA) able to identify viable but nonculturable (VBNC) cells. The study included Legionella strains (L. pneumophila [3 clusters] and L. anisa [1 cluster]) isolated from four hot water circuits of different hospital buildings in Saint-Etienne, France, during a 20-year prospective surveillance. The strains recovered from the different circuits were not epidemiologically related, but the strains isolated within a same circuit over time exhibited an identical genotypic profile. After an in vitro treatment of 30 min at 70°C, the mean percentage of viable cells and VBNC cells varied from 4.6% to 71.7%. The in vitro differences in heat sensitivity were in agreement with the observed efficacy of preventive and corrective heating measures used to control water contamination. These results suggest that Legionella strains can become heat resistant after heating treatments for a long time and that flow cytometry could be helpful to check the efficacy of heat treatments on Legionella spp. and to optimize the decontamination processes applied to water systems for the control of Legionella proliferation.

Evaluation of droplet digital PCR for quantification of residual leucocytes in red blood cell concentrates.

PubMed

Doescher, A; Loges, U; Petershofen, E K; Müller, T H

2017-11-01

Enumeration of residual white blood cells in leucoreduced blood components is essential part of quality control. Digital PCR has substantially facilitated quantitative PCR and was thus evaluated for measurements of leucocytes. Target for quantification of leucocytes by digital droplet PCR was the blood group gene RHCE. The SPEF1 gene was added as internal control for the entire assay starting with automated DNA extraction. The sensitivity of the method was determined by serial dilutions of standard samples. Quality control samples were analysed within 24 h, 7 days and 6 months after collection. Routine samples from leucodepleted red blood cell concentrates (n = 150) were evaluated in parallel by flow-cytometry (LeucoCount) and by digital PCR. Digital PCR reliably detected at least 0·4 leucocytes per assay. The mean difference between PCR and flow-cytometric results from 150 units was -0·01 (±1·0). DNA samples were stable for up to at least six months. PCR measurement of leucocytes in samples from plasma and platelet concentrates also provided valid results in a pilot study. Droplet digital PCR to enumerate leucocytes offers an alternative for quality control of leucoreduced blood products. Sensitivity, specificity and reproducibility are comparable to flow-cytometry. The option to collect samples over an extended period of time and the automatization introduce attractive features for routine quality control. © 2017 International Society of Blood Transfusion.

Probing Metabolic Activity of Deep Subseafloor Life with NanoSIMS

NASA Astrophysics Data System (ADS)

Morono, Y.; Terada, T.; Itoh, M.; Inagaki, F.

2014-12-01

There are very few natural environments where life is absent in the Earth's surface biosphere. However, uninhabitable region is expected to be exist in the deep subsurface biosphere, of which extent and constraining factor(s) have still remained largly unknown. Scientific ocean drilling have revealed that microbial communities in sediments are generally phylogenetically distinct from known spieces isolated from the Earth's surface biosphere, and hence metabolic functions of the deep subseafloor life remain unknown. In addition, activity of subseafloor microbial cells are thought to be extraordinally slow, as indicated by limited supply of neutrient and energy substrates. To understand the limits of the Earth's subseafloor biosphere and metabolic functions of microbial populations, detection and quantification of the deeply buried microbial cells in geological habitats are fundamentary important. Using newly developed cell separation techniques as well as an discriminative cell detection system, the current quantification limit of sedimentary microbial cells approaches to 102 cells/cm3. These techniques allow not only to assess very small microbial population close to the subsurface biotic fringe, but also to separate and sort the target cells using flow cytometric cell sorter. Once the deep subseafloor microbial cells are detached from mineral grains and sorted, it opens new windows to subsequent molecular ecological and element/isotopic analyses. With a combined use of nano-scale secondary ion masspectrometry (NanoSIMS) and stable isotope-probing techniques, it is possible to detect and measure activity of substrate incorporation into biomass, even for extremely slow metabolic processes such as uncharacteriszed deep subseafloor life. For example, it was evidenced by NanoSIMS that at least over 80% of microbial cells at ~200 meters-deep, 460,000-year-old sedimentary habitat are indeed live, which substrate incooporation was found to be low (10-15 gC/cell/day) even under the lab incubation condition. Also microbial activity in ultraoligotrophic biosphere samples such as the South Pacific Gyre (i.e., IODP Expeditions 329) will be shown. Our results demonstrates metabolic potential of microbes that have been survived for geological timescale in extremely starved condition.

Physiology and phylogeny of the candidate phylum "Atribacteria" (formerly OP9/JS1) inferred from single-cell genomics and metagenomics

NASA Astrophysics Data System (ADS)

Dodsworth, J. A.; Murugapiran, S.; Blainey, P. C.; Nobu, M.; Rinke, C.; Schwientek, P.; Gies, E.; Webster, G.; Kille, P.; Weightman, A.; Liu, W. T.; Hallam, S.; Tsiamis, G.; Swingley, W.; Ross, C.; Tringe, S. G.; Chain, P. S.; Scholz, M. B.; Lo, C. C.; Raymond, J.; Quake, S. R.; Woyke, T.; Hedlund, B. P.

2014-12-01

Single-cell sequencing and metagenomics have extended the genomics revolution to yet-uncultivated microorganisms and provided insights into the coding potential of this so-called "microbial dark matter", including microbes belonging candidate phyla with no cultivated representatives. As more datasets emerge, comparison of individual genomes from different lineages and habitats can provide insight into the phylogeny, conserved features, and potential metabolic diversity of candidate phyla. The candidate bacterial phylum OP9 was originally found in Obsidian Pool, Yellowstone National Park, and it has since been detected in geothermal springs, petroleum reservoirs, and engineered thermal environments worldwide. JS1, another uncultivated bacterial lineage affiliated with OP9, is often abundant in marine sediments associated with methane hydrates, hydrocarbon seeps, and on continental margins and shelves, and is found in other non-thermal marine and subsurface environments. The phylogenetic relationship between OP9, JS1, and other Bacteria has not been fully resolved, and to date no axenic cultures from these lineages have been reported. Recently, 31 single amplified genomes (SAGs) from six distinct OP9 and JS1 lineages have been obtained using flow cytometric and microfluidic techniques. These SAGs were used to inform metagenome binning techniques that identified OP9/JS1 sequences in several metagenomes, extending genomic coverage in three of the OP9 and JS1 lineages. Phylogenomic analyses of these SAG and metagenome bin datasets suggest that OP9 and JS1 constitute a single, deeply branching phylum, for which the name "Atribacteria" has recently been proposed. Overall, members of the "Atribacteria" are predicted to be heterotrophic anaerobes without the capacity for respiration, with some lineages potentially specializing in secondary fermentation of organic acids. A set of signature "Atribacteria" genes was tentatively identified, including components of a bacterial microcompartments gene cluster that may be involved in carbohydrate catabolism. The "Atribacteria" may play important roles in biomass processing in anaerobic geothermal and subsurface environments.

Culture-Independent Techniques for Rapid Detection of Bacteria Associated with Loss of Chloramine Residual in a Drinking Water System

PubMed Central

Hoefel, Daniel; Monis, Paul T.; Grooby, Warwick L.; Andrews, Stuart; Saint, Christopher P.

2005-01-01

Chloramination is often the disinfection regimen of choice for extended drinking water systems. However, this process is prone to instability due to the growth of nitrifying bacteria. This is the first study to use alternative approaches for rapid investigation of chloraminated drinking water system instability in which flow cytometric cell sorting of bacteria with intact membranes (membrane-intact fraction) (BacLight kit) or with active esterases (esterase-active fraction) (carboxyfluorescein diacetate) was combined with 16S rRNA gene-directed PCR and denaturing gradient gel electrophoresis (DGGE). No active bacteria were detected when water left the water treatment plant (WTP), but 12 km downstream the chloramine residual had diminished and the level of active bacteria in the bulk water had increased to more than 1 × 105 bacteria ml−1. The bacterial diversity in the system was represented by six major DGGE bands for the membrane-intact fraction and 10 major DGGE bands for the esterase-active fraction. PCR targeting of the 16S rRNA gene of chemolithotrophic ammonia-oxidizing bacteria (AOB) and subsequent DGGE and DNA sequence analysis revealed the presence of an active Nitrosospira-related species and Nitrosomonas cryotolerans in the system, but no AOB were detected in the associated WTP. The abundance of active AOB was then determined by quantitative real-time PCR (qPCR) targeting the amoA gene; 3.43 × 103 active AOB ml−1 were detected in the membrane-intact fraction, and 1.40 × 104 active AOB ml−1 were detected in the esterase-active fraction. These values were several orders of magnitude greater than the 2.5 AOB ml−1 detected using a routine liquid most-probable-number assay. Culture-independent techniques described here, in combination with existing chemical indicators, should allow the water industry to obtain more comprehensive data with which to make informed decisions regarding remedial action that may be required either prior to or during an instability event. PMID:16269672

Controlled intracellular generation of reactive oxygen species in human mesenchymal stem cells using porphyrin conjugated nanoparticles

NASA Astrophysics Data System (ADS)

Lavado, Andrea S.; Chauhan, Veeren M.; Alhaj Zen, Amer; Giuntini, Francesca; Jones, D. Rhodri E.; Boyle, Ross W.; Beeby, Andrew; Chan, Weng C.; Aylott, Jonathan W.

2015-08-01

Nanoparticles capable of generating controlled amounts of intracellular reactive oxygen species (ROS), that advance the study of oxidative stress and cellular communication, were synthesized by functionalizing polyacrylamide nanoparticles with zinc(ii) porphyrin photosensitisers. Controlled ROS production was demonstrated in human mesenchymal stem cells (hMSCs) through (1) production of nanoparticles functionalized with varying percentages of Zn(ii) porphyrin and (2) modulating the number of doses of excitation light to internalized nanoparticles. hMSCs challenged with nanoparticles functionalized with increasing percentages of Zn(ii) porphyrin and high numbers of irradiations of excitation light were found to generate greater amounts of ROS. A novel dye, which is transformed into fluorescent 7-hydroxy-4-trifluoromethyl-coumarin in the presence of hydrogen peroxide, provided an indirect indicator for cumulative ROS production. The mitochondrial membrane potential was monitored to investigate the destructive effect of increased intracellular ROS production. Flow cytometric analysis of nanoparticle treated hMSCs suggested irradiation with excitation light signalled controlled apoptotic cell death, rather than uncontrolled necrotic cell death. Increased intracellular ROS production did not induce phenotypic changes in hMSC subcultures.Nanoparticles capable of generating controlled amounts of intracellular reactive oxygen species (ROS), that advance the study of oxidative stress and cellular communication, were synthesized by functionalizing polyacrylamide nanoparticles with zinc(ii) porphyrin photosensitisers. Controlled ROS production was demonstrated in human mesenchymal stem cells (hMSCs) through (1) production of nanoparticles functionalized with varying percentages of Zn(ii) porphyrin and (2) modulating the number of doses of excitation light to internalized nanoparticles. hMSCs challenged with nanoparticles functionalized with increasing percentages of Zn(ii) porphyrin and high numbers of irradiations of excitation light were found to generate greater amounts of ROS. A novel dye, which is transformed into fluorescent 7-hydroxy-4-trifluoromethyl-coumarin in the presence of hydrogen peroxide, provided an indirect indicator for cumulative ROS production. The mitochondrial membrane potential was monitored to investigate the destructive effect of increased intracellular ROS production. Flow cytometric analysis of nanoparticle treated hMSCs suggested irradiation with excitation light signalled controlled apoptotic cell death, rather than uncontrolled necrotic cell death. Increased intracellular ROS production did not induce phenotypic changes in hMSC subcultures. Electronic supplementary information (ESI) available: Materials and experimental methods for the synthesis of (1) positively charged alkyne functionalized nanoparticles (2) Zn(ii) and Cu(ii) centred porphyrin (3); conjugating porphyrins to alkyne-functionalized nanoparticles via click chemistry (4) nanoparticle characterisation (size charge and fluorescence), (5) synthesis of BPTFMC (6) hMSC collection, storage and preparation (7) delivery of porphyrin functionalized nanoparticles (8) staining mitochondria, cumulative ROS production and determination of nanoparticles subcellular localisation (9) fluorescence microscopy and controlled irradiation of hMSCs (10) flow cytometry and controlled irradiation using a custom built irradiator. In addition, results highlighting: (1) nanoparticles emission spectra, size and charge, (2) BPTFMC fluorescence response and (3) hMSCs following light irradiation using flow cytometry. See DOI: 10.1039/c5nr00795j

Validation of a HLA-A2 tetramer flow cytometric method, IFNgamma real time RT-PCR, and IFNgamma ELISPOT for detection of immunologic response to gp100 and MelanA/MART-1 in melanoma patients

PubMed Central

Xu, Yuanxin; Theobald, Valerie; Sung, Crystal; DePalma, Kathleen; Atwater, Laura; Seiger, Keirsten; Perricone, Michael A; Richards, Susan M

2008-01-01

Background HLA-A2 tetramer flow cytometry, IFNγ real time RT-PCR and IFNγ ELISPOT assays are commonly used as surrogate immunological endpoints for cancer immunotherapy. While these are often used as research assays to assess patient's immunologic response, assay validation is necessary to ensure reliable and reproducible results and enable more accurate data interpretation. Here we describe a rigorous validation approach for each of these assays prior to their use for clinical sample analysis. Methods Standard operating procedures for each assay were established. HLA-A2 (A*0201) tetramer assay specific for gp100209(210M) and MART-126–35(27L), IFNγ real time RT-PCR and ELISPOT methods were validated using tumor infiltrating lymphocyte cell lines (TIL) isolated from HLA-A2 melanoma patients. TIL cells, specific for gp100 (TIL 1520) or MART-1 (TIL 1143 and TIL1235), were used alone or spiked into cryopreserved HLA-A2 PBMC from healthy subjects. TIL/PBMC were stimulated with peptides (gp100209, gp100pool, MART-127–35, or influenza-M1 and negative control peptide HIV) to further assess assay performance characteristics for real time RT-PCR and ELISPOT methods. Validation parameters included specificity, accuracy, precision, linearity of dilution, limit of detection (LOD) and limit of quantification (LOQ). In addition, distribution was established in normal HLA-A2 PBMC samples. Reference ranges for assay controls were established. Results The validation process demonstrated that the HLA-A2 tetramer, IFNγ real time RT-PCR, and IFNγ ELISPOT were highly specific for each antigen, with minimal cross-reactivity between gp100 and MelanA/MART-1. The assays were sensitive; detection could be achieved at as few as 1/4545–1/6667 cells by tetramer analysis, 1/50,000 cells by real time RT-PCR, and 1/10,000–1/20,000 by ELISPOT. The assays met criteria for precision with %CV < 20% (except ELISPOT using high PBMC numbers with %CV < 25%) although flow cytometric assays and cell based functional assays are known to have high assay variability. Most importantly, assays were demonstrated to be effective for their intended use. A positive IFNγ response (by RT-PCR and ELISPOT) to gp100 was demonstrated in PBMC from 3 melanoma patients. Another patient showed a positive MART-1 response measured by all 3 validated methods. Conclusion Our results demonstrated the tetramer flow cytometry assay, IFNγ real-time RT-PCR, and INFγ ELISPOT met validation criteria. Validation approaches provide a guide for others in the field to validate these and other similar assays for assessment of patient T cell response. These methods can be applied not only to cancer vaccines but to other therapeutic proteins as part of immunogenicity and safety analyses. PMID:18945350

DNA flow cytometric analysis of primary operable breast cancer. Relation of ploidy and S-phase fraction to outcome of patients in NSABP B-04.

PubMed

Fisher, B; Gunduz, N; Costantino, J; Fisher, E R; Redmond, C; Mamounas, E P; Siderits, R

1991-10-01

Between 1971 and 1974, 1665 women with primary operable breast cancer were randomized into a National Surgical Adjuvant Breast and Bowel Project (NSABP) trial (B-04) conducted to evaluate the effectiveness of several different regimens of surgical and radiation therapy. No systemic therapy was given. Cells from archival paraffin-embedded tumor tissue taken from 398 patients were analyzed for ploidy and S-phase fraction (SPF) using flow cytometry. Characteristics and outcome of patients with satisfactory DNA histograms were comparable to those from whom no satisfactory cytometric studies were available. In patients with diploid tumors (43%), the mean SPF was 3.4% +/- 2.3%; in the aneuploid population (57%), the SPF was 7.9% +/- 6.3%. Only 29.9% +/- 17.3% of cells in aneuploid tumors were aneuploid. Diploid tumors were more likely than aneuploid tumors to be of good nuclear grade (P less than 0.001) and smaller size (P equals 0.03). More tumors with high SPF were of poor nuclear grade than were tumors with low SPF (P equals 0.002). No significant difference in 10-year disease-free survival (P equals 0.3) or survival (P equals 0.1) was found between women with diploid or aneuploid tumors. Patients with low SPF tumors had a 13% better disease-free survival (P equals 0.0006) than those with a high SPF and a 14% better survival (P equals 0.007) at 10 years than patients with high SPF tumors. After adjustment for clinical tumor size, the difference in both disease-free survival and survival between patients with high and low SPF tumors was only 10% (P equals 0.04 and 0.08, respectively). Although SPF was found to be of independent prognostic significance for disease-free survival and marginal significance for survival, it did not detect patients with such a good prognosis as to preclude their receiving chemotherapy. The overall survival of patients with low SPF was only 53% at 10 years. These findings and those of others indicate that additional studies are necessary before tumor ploidy and SPF can be used to select patients who should or should not receive systemic therapy.

Reduced-HMGB1 suppresses poly(I:C)-induced inflammation in keratinocytes.

PubMed

Mori, Hideki; Murakami, Masamoto; Tsuda, Teruko; Kameda, Kenji; Utsunomiya, Ryo; Masuda, Kana; Shiraishi, Ken; Dai, Xiuju; Tohyama, Mikiko; Nakaoka, Hiroki; Sayama, Koji

2018-05-01

High mobility group box 1 (HMGB1) is a nuclear protein that stabilizes DNA and facilitates gene transcription. Additionally, cell stress or death induces the release of HMGB1 outside the cell membrane, where HMGB1 functions as an alarmin, causing an inflammatory response in combination with other cytokines, damage-associated molecular patterns (DAMPs), and pathogen-associated molecular patterns (PAMPs). To evaluate the effect of reduced-HMGB1 (previously termed chemoattractive-HMGB1) on polyinosine-polycytidylic acid [poly(I:C)]-induced inflammation in normal human keratinocytes (NHKs). We focused on downstream components of the poly(I:C)-Toll-like receptor 3 (TLR3), retinoic acid-inducible gene-I (RIG-I), and melanoma differentiation-associated protein 5 (MDA5) pathways, including IκBα, nuclear factor (NF)-κB p65, mitogen-activated protein kinase (MAPK), and interferon regulatory factor 3 (IRF3), and assessed whether these pathways are involved in the suppression of poly(I:C)-induced inflammation in NHKs by HMGB1. An immunoprecipitation was performed to know whether HMGB1 could bind to poly(I:C), and immunofluorescence staining and flow cytometric analysis were performed to check whether reduced-HMGB interferes with cellular uptake of poly(I:C) translocation (possibly by endocytosis). Application of exogenous HMGB1 before, but not after, exerted a suppressive effect on poly(I:C)-induced inflammation in NHKs. In addition, reduced-HMGB1, but not disulfide-HMGB1, exerted a suppressive effect on poly(I:C)-induced inflammation in NHKs, suggesting the importance of the redox status of exogenous HMGB1. Pre-treatment with reduced-HMGB1 inhibited the phosphorylation of IκBα, NF-κB p65, and IRF3 induced by poly(I:C) stimulation in NHKs; however, phosphorylation of p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) was unaffected. Disulfide-HMGB1 formed a complex with poly(I:C), as did reduced- and oxidized-HMGB1, albeit to a lesser extent. Immunofluorescence staining and flow cytometric analysis indicated that reduced-HMGB interferes with cellular uptake of poly(I:C) translocation (possibly by endocytosis). These findings suggest that pre-treatment with reduced-HMGB1 ameliorates poly(I:C)-mediated inflammation in NHKs. Copyright © 2018 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Cellular effect of styrene substituted biscoumarin caused cellular apoptosis and cell cycle arrest in human breast cancer cells.

PubMed

Perumalsamy, Haribalan; Sankarapandian, Karuppasamy; Kandaswamy, Narendran; Balusamy, Sri Renukadevi; Periyathambi, Dhaiveegan; Raveendiran, Nanthini

2017-11-01

Coumarins occurs naturally across plant kingdoms exhibits significant pharmacological properties and pharmacokinetic activity. The conventional, therapeutic agents are often associated with poor stability, absorption and increased side effects. Therefore, identification of a drug that has little or no-side effect on humans is consequential. Here, we investigated the antiproliferative activity of styrene substituted biscoumarin against various human breast cancer cell lines, such as MCF-7, (ER-) MDA-MB-231 and (AR+) MDA-MB-453. Styrene substituted biscoumarin induced cell death by apoptosis in MDA-MB-231 cell line was analyzed. Antiproliferative activity of Styrene substituted biscoumarin was performed by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Styrene substituted biscoumarin induced apoptosis was assessed by Hoechst staining, Annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) staining and flow cytometric analysis. Migratory and proliferating characteristic of breast cancer cell line MDA-MB-231 was also analyzed by wound healing and colony formation assay. Furthermore, mRNA expression of BAX and BCL-2 were quantified using qRT-PCR and protein expression level analyzed by Western blot. The inhibition concentration (IC 50 ) of styrene substituted biscoumarin was assayed against three breast cancer cell lines. The inhibition concentration (IC 50 ) value of styrene substituted biscoumarin toward MDA-MB-231, MDA-MB-453 and MCF-7 cell lines was 5.63, 7.30 and 10.84μg/ml respectively. Styrene substituted biscoumarin induced apoptosis was detected by Hoechst staining, DAPI/PI analysis and flow-cytometric analysis. The migration and proliferative efficiency of MDA-MB-231 cells were completely arrested upon styrene substituted biscoumarin treatment. Also, mRNA gene expression and protein expression of pro-apoptotic (BAX) and anti-apoptotic (BCL-2) genes were analyzed by qRT-PCR and western blot analysis upon styrene substituted biscoumarin treatment to MDA-MB-231 cells. Our results showed that styrene substituted biscoumarin downregulated BCL-2 gene expression and upregulated BAX gene expression to trigger apoptotic process. Styrene substituted biscoumarin could induce apoptosis through intrinsic mitochondrial pathway in breast cancer cell lines, particularly in MDA-MB-231. Our data suggest that styrene substituted biscoumarin may act as a potential chemotherapeutic agent against breast cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

[Salubrinal improves cardiac function in rats with heart failure post myocardial infarction through reducing endoplasmic reticulum stress-associated apoptosis].

PubMed

Liu, Y; Qi, S Y; Ru, L S; Ding, C; Wang, H J; Li, A Y; Xu, B Y; Zhang, G H; Wang, D M

2016-06-24

Endoplasmic reticulum (ER) stress plays an important role in ischemia-mediated cell death. The aim of the current study is to investigate the effects of salubrinal (Sal), a selective eIF2a dephosphorylation inhibitor, on heart failure rats and related mechanisms. Heart failure was induced by coronary artery ligation (MI) in adult male Sprague-Dawley rats. To ensure comparable MI sizes post coronary artery ligation on various groups, echocardiography examination was performed before and 30 minutes after ligation in MI groups. Then rats were randomly assigned to 4 groups: Sham group (n=12), MI group (n=10), MI plus vehicle injections group (DMSO group, n=12) and MI plus Sal injection group (Sal group, n=12). Sal (1 mg/kg) or DMSO was injected via the tail vein daily for the first 3 days (starting at 30 minutes after ligation of the left coronary artery), followed by subcutaneous injections twice per week for 8 weeks. Cardiac function was assessed by echocardiography and cell apoptosis assessed by flow cytometric analysis after 8 weeks. Protein and mRNA levels of ER stress markers were evaluated by immunohistochemistry and real time RT-PCR respectively. Eight weeks later, LVEF was significantly higher, while LVESD and LVEDD values were significantly lower in Sal group compared to MI and DMSO groups (all P<0.05); LV/BW ratio was significantly higher in MI group than in Sham group ((2.30±0.40) mg/g vs.(1.78±0.31) mg/g, P<0.05), which was significantly reduced in Sal group ((1.88±0.25) mg/g), but not in DMSO group((2.25±0.36) mg/g, P<0.05 vs. MI). In addition, flow cytometric analysis showed that Sal treatment significantly reduced apoptosis but not necrosis in post MI. Immunohistochemistry and real time PCR analysis showed that the myocardial protein and mRNA expression of ER stress markers were significantly lower in Sal group than in MI group, myocardial caspase-12 expression was significantly upregulated in MI group and significantly reduced by Sal treatment. Our results suggest that reduction of ER stress and myocardial apoptosis through inhibition of eIF2α dephosphorylation may serve as the potential mechanisms for the improved cardiac function and attenuated cardiac remodeling post Sal treatment in this heart failure rat model.

Survival Benefit with Kidney Transplants from HLA-Incompatible Live Donors.

PubMed

Orandi, Babak J; Luo, Xun; Massie, Allan B; Garonzik-Wang, Jacqueline M; Lonze, Bonne E; Ahmed, Rizwan; Van Arendonk, Kyle J; Stegall, Mark D; Jordan, Stanley C; Oberholzer, Jose; Dunn, Ty B; Ratner, Lloyd E; Kapur, Sandip; Pelletier, Ronald P; Roberts, John P; Melcher, Marc L; Singh, Pooja; Sudan, Debra L; Posner, Marc P; El-Amm, Jose M; Shapiro, Ron; Cooper, Matthew; Lipkowitz, George S; Rees, Michael A; Marsh, Christopher L; Sankari, Bashir R; Gerber, David A; Nelson, Paul W; Wellen, Jason; Bozorgzadeh, Adel; Gaber, A Osama; Montgomery, Robert A; Segev, Dorry L

2016-03-10

A report from a high-volume single center indicated a survival benefit of receiving a kidney transplant from an HLA-incompatible live donor as compared with remaining on the waiting list, whether or not a kidney from a deceased donor was received. The generalizability of that finding is unclear. In a 22-center study, we estimated the survival benefit for 1025 recipients of kidney transplants from HLA-incompatible live donors who were matched with controls who remained on the waiting list or received a transplant from a deceased donor (waiting-list-or-transplant control group) and controls who remained on the waiting list but did not receive a transplant (waiting-list-only control group). We analyzed the data with and without patients from the highest-volume center in the study. Recipients of kidney transplants from incompatible live donors had a higher survival rate than either control group at 1 year (95.0%, vs. 94.0% for the waiting-list-or-transplant control group and 89.6% for the waiting-list-only control group), 3 years (91.7% vs. 83.6% and 72.7%, respectively), 5 years (86.0% vs. 74.4% and 59.2%), and 8 years (76.5% vs. 62.9% and 43.9%) (P<0.001 for all comparisons with the two control groups). The survival benefit was significant at 8 years across all levels of donor-specific antibody: 89.2% for recipients of kidney transplants from incompatible live donors who had a positive Luminex assay for anti-HLA antibody but a negative flow-cytometric cross-match versus 65.0% for the waiting-list-or-transplant control group and 47.1% for the waiting-list-only control group; 76.3% for recipients with a positive flow-cytometric cross-match but a negative cytotoxic cross-match versus 63.3% and 43.0% in the two control groups, respectively; and 71.0% for recipients with a positive cytotoxic cross-match versus 61.5% and 43.7%, respectively. The findings did not change when patients from the highest-volume center were excluded. This multicenter study validated single-center evidence that patients who received kidney transplants from HLA-incompatible live donors had a substantial survival benefit as compared with patients who did not undergo transplantation and those who waited for transplants from deceased donors. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases.).

Image Cytometric Analysis of Algal Spores for Evaluation of Antifouling Activities of Biocidal Agents.

PubMed

Il Koo, Bon; Lee, Yun-Soo; Seo, Mintae; Seok Choi, Hyung; Leng Seah, Geok; Nam, Taegu; Nam, Yoon Sung

2017-07-31

Chemical biocides have been widely used as marine antifouling agents, but their environmental toxicity impose regulatory restriction on their use. Although various surrogate antifouling biocides have been introduced, their comparative effectiveness has not been well investigated partly due to the difficulty of quantitative evaluation of their antifouling activity. Here we report an image cytometric method to quantitatively analyze the antifouling activities of seven commercial biocides using Ulva prolifera as a target organism, which is known to be a dominant marine species causing soft fouling. The number of spores settled on a substrate is determined through image analysis using the intrinsic fluorescence of chlorophylls in the spores. Pre-determined sets of size and shape of spores allow for the precise determination of the number of settled spores. The effects of biocide concentration and combination of different biocides on the spore settlement are examined. No significant morphological changes of Ulva spores are observed, but the amount of adhesive pad materials is appreciably decreased in the presence of biocides. It is revealed that the growth rate of Ulva is not directly correlated with the antifouling activities against the settlement of Ulva spores. This work suggests that image cytometric analysis is a very convenient, fast-processable method to directly analyze the antifouling effects of biocides and coating materials.

Recovery of soil unicellular eukaryotes: an efficiency and activity analysis on the single cell level.

PubMed

Lentendu, Guillaume; Hübschmann, Thomas; Müller, Susann; Dunker, Susanne; Buscot, François; Wilhelm, Christian

2013-12-01

Eukaryotic unicellular organisms are an important part of the soil microbial community, but they are often neglected in soil functional microbial diversity analysis, principally due to the absence of specific investigation methods in the special soil environment. In this study we used a method based on high-density centrifugation to specifically isolate intact algal and yeast cells, with the aim to analyze them with flow cytometry and sort them for further molecular analysis such as deep sequencing. Recovery efficiency was tested at low abundance levels that fit those in natural environments (10(4) to 10(6) cells per g soil). Five algae and five yeast morphospecies isolated from soil were used for the testing. Recovery efficiency was between 1.5 to 43.16% and 2 to 30.2%, respectively, and was dependent on soil type for three of the algae. Control treatments without soil showed that the majority of cells were lost due to the method itself (58% and 55.8% respectively). However, the cell extraction technique did not much compromise cell vitality because a fluorescein di-acetate assay indicated high viability percentages (73.3% and 97.2% of cells, respectively). The low abundant algae and yeast morphospecies recovered from soil were cytometrically analyzed and sorted. Following, their DNA was isolated and amplified using specific primers. The developed workflow enables isolation and enrichment of intact autotrophic and heterotrophic soil unicellular eukaryotes from natural environments for subsequent application of deep sequencing technologies. Copyright © 2013 Elsevier B.V. All rights reserved.

Responses of Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus to Simulated Food Processing Treatments, Determined Using Fluorescence-Activated Cell Sorting and Plate Counting▿

PubMed Central

Kennedy, Deirdre; Cronin, Ultan P.; Wilkinson, Martin G.

2011-01-01

Three common food pathogenic microorganisms were exposed to treatments simulating those used in food processing. Treated cell suspensions were then analyzed for reduction in growth by plate counting. Flow cytometry (FCM) and fluorescence-activated cell sorting (FACS) were carried out on treated cells stained for membrane integrity (Syto 9/propidium iodide) or the presence of membrane potential [DiOC2(3)]. For each microbial species, representative cells from various subpopulations detected by FCM were sorted onto selective and nonselective agar and evaluated for growth and recovery rates. In general, treatments giving rise to the highest reductions in counts also had the greatest effects on cell membrane integrity and membrane potential. Overall, treatments that impacted cell membrane permeability did not necessarily have a comparable effect on membrane potential. In addition, some bacterial species with extensively damaged membranes, as detected by FCM, appeared to be able to replicate and grow after sorting. Growth of sorted cells from various subpopulations was not always reflected in plate counts, and in some cases the staining protocol may have rendered cells unculturable. Optimized FCM protocols generated a greater insight into the extent of the heterogeneous bacterial population responses to food control measures than did plate counts. This study underlined the requirement to use FACS to relate various cytometric profiles generated by various staining protocols with the ability of cells to grow on microbial agar plates. Such information is a prerequisite for more-widespread adoption of FCM as a routine microbiological analytical technique. PMID:21602370

Biogenic terbium oxide nanoparticles as the vanguard against osteosarcoma

NASA Astrophysics Data System (ADS)

Iram, Sana; Khan, Salman; Ansary, Abu Ayoobul; Arshad, Mohd; Siddiqui, Sahabjada; Ahmad, Ejaz; Khan, Rizwan H.; Khan, Mohd Sajid

2016-11-01

The synthesis of inner transition metal nanoparticles via an ecofriendly route is quite difficult. This study, for the first time, reports synthesis of terbium oxide nanoparticles using fungus, Fusarium oxysporum. The biocompatible terbium oxide nanoparticles (Tb2O3 NPs) were synthesized by incubating Tb4O7 with the biomass of fungus F. oxysporum. Multiple physical characterization techniques, such as UV-visible and photoluminescence spectroscopy, TEM, SAED, and zeta-potential were used to confirm the synthesis, purity, optical and surface characteristics, crystallinity, size, shape, distribution, and stability of the nanoemulsion of Tb2O3 NPs. The Tb2O3 NPs were found to inhibit the propagation of MG-63 and Saos-2 cell-lines (IC50 value of 0.102 μg/mL) and remained non-toxic up to a concentration of 0.373 μg/mL toward primary osteoblasts. Cell viability decreased in a concentration-dependent manner upon exposure to 10 nm Tb2O3 NPs in the concentration range 0.023-0.373 μg/mL. Cell toxicity was evaluated by observing changes in cell morphology, cell viability, oxidative stress parameters, and FACS analysis. Morphological examinations of cells revealed cell shrinkage, nuclear condensation, and formation of apoptotic bodies. The level of ROS within the cells-an indicator of oxidative stress was significantly increased. The induction of apoptosis at concentrations ≤ IC50 was corroborated by 4‧,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining (DNA damage and nuclear fragmentation). Flow-cytometric studies indicated that the response was dose dependent with a threshold effect.

Regeneration of skin tissue promoted by mesenchymal stem cells seeded in nanostructured membrane.

PubMed

Souza, C M C O; Mesquita, L A F; Souza, D; Irioda, A C; Francisco, J C; Souza, C F; Guarita-Souza, L C; Sierakowski, M-R; Carvalho, K A T

2014-01-01

The mesenchymal stem cell therapy has proven to be an effective option in the treatment of skin injuries. The combination of these cells with nanostructured membranes seems to be the future for tissues recovery. The aim of this project was to use biomolecules of polysaccharides to be incorporated on regenerated cellulose membranes and to prospect the improvement as bioactive wound dressings with mesenchymal stem cells. The biocomposites were obtained after defibrillation with the use of never-dried bacterial cellulose to form a pulp, and, after the films were regenerated, in the presence of gellan gum with or without fluconazole. Membrane atomic force microscopy was performed for comparison of their structures. Adipose-derived mesenchymal stem cells were obtained from human adipose tissue liposuction in accordance with Zuk et al. The flow cytometric analysis and induction tests for adipocytes and osteocytes were performed. In vitro assays were performed on different membranes to evaluate the ability of these cells to adhere at 2 hours and proliferate at 7 days; the results were obtained by use of the MTT cell counting technique. In vivo testing allowed us to observe cell migration and participation in wound-healing by fluorescence labeling of the cells with BrdU. The bioactive curative, seeded with cells, was tested in skin burned in a murine model. The bacterial cellulose with gelan gum membrane incorporated with fluconazole presented the best performance in adhesion and proliferation tests. The cells can be identified in burned host tissue after occurrence of the wound. Copyright © 2014 Elsevier Inc. All rights reserved.

Assessment of the antioxidant activity of an olive oil total polyphenolic fraction and hydroxytyrosol from a Greek Olea europea variety in endothelial cells and myoblasts

PubMed Central

Kouka, Paraskevi; Priftis, Alexandros; Stagos, Dimitrios; Angelis, Apostolis; Stathopoulos, Panagiotis; Xinos, Nikos; Skaltsounis, Alexios-Léandros; Mamoulakis, Charalampos; Tsatsakis, Aristides M.; Spandidos, Demetrios A.; Kouretas, Demetrios

2017-01-01

Olive oil (OO) constitutes the basis of the Mediterranean diet, and it seems that its biophenols, such as hydroxytyrosol (HT) may scavenge free radicals, attracting distinct attention due to their beneficial effects in many pathological conditions, such as cancer. To the best of our knowedge, this is the first study in which the functional properties of an OO total polyphenolic fraction (TPF) and pure HT were examined in order to determine their antioxidant effects at a cellular level in endothelial cells and myoblasts. The test compounds were isolated using a green gradient-elution centrifugal partition chromatography-based method that allows the isolation of large volumes of OO in a continuous extraction procedure and with extremely low solvent consumption. For the isolation of HT, a combination of two chromatographic techniques was used, which is effective for the recovery of pure compounds from complex natural extracts. Moreover, TPF and HT exhibited potent free radical scavenging activity in vitro. The cells were treated with non-cytotoxic concentrations and their redox status [in terms of glutathione (GSH) and reactive oxygen species (ROS) levels] was assessed. TPF extract was less cytotoxic than HT, and the observed differences between the two cell lines used suggest a tissue-specific activity. Finally, flow cytometric analysis revealed that both TPF and HT improved the redox status by increasing the levels of GSH, one of the most important antioxidant molecules, in both endothelial cells and myoblasts, while the ROS levels were not significantly affected. PMID:28731131

Case report and literature review: transient Inab phenotype and an agglutinating anti-IFC in a patient with a gastrointestinal problem.

PubMed

Yazer, Mark H; Judd, W John; Davenport, Robertson D; Dake, Louann R; Lomas-Francis, Christine; Hue-Roye, Kim; Powell, Vivien; Reid, Marion

2006-09-01

The Inab phenotype is a rare deficiency of all Cromer antigens. These antigens are carried on the decay-accelerating factor (DAF, CD55) molecule that is attached to the red blood cell (RBC) membrane by a glycosylphosphatidylinositol (GPI) anchor. Although typically inherited, an acquired and transient form of the Inab phenotype also exists. A patient with the triad of transient Inab phenotype, a direct-agglutinating anti-IFC, and gastrointestinal (GI) abnormalities is reported. An 18-month-old boy with gastroesophageal reflux disease requiring a feeding tube, milk and soy intolerance, and severe growth retardation, as well as vision and hearing deficits from cytomegalovirus infection, was identified when pretransfusion testing revealed a potent panagglutinin (titer > 2000 at 4 degrees C). This antibody did not react with Dr(a-) and IFC RBCs, and the autocontrol was negative. The patient's RBCs lacked CD55 by flow cytometric techniques but had normal levels of CD59 and antigens such as Yt(a) and Emm, carried on GPI-linked proteins, thus excluding paroxysmal nocturnal hemoglobinuria. Several months after initial detection, the anti-IFC was virtually undetectable and his cells reacted weakly with anti-IFC, anti-Dr(a), and anti-CD55. RBCs from the propositus' parents and brother demonstrated normal CD55 and CD59 expression. This is the first example of a direct-agglutinating anti-IFC. The cause of the transient depression in CD55 protein (and thus Cromer system antigens) and appearance of anti-IFC remains unknown, as does the relationship between the patient's GI system abnormalities and these serologic findings.

Characterizing the Phytoplankton Community of the South China Sea

NASA Astrophysics Data System (ADS)

Aleman, M.; Subramaniam, A.; Hay, I.

2016-12-01

The South China Sea (SCS) is a semi-enclosed tropical basin that receives nutrients from two main sources: coastal upwelling and river runoff from the surrounding countries. The southwest (SW) summer monsoon that occurs between March and September drives upwelling off the coast of Vietnam, a rarity compared to most upwelling systems that are driven by eastern boundary current systems. The upwelling in the SCS and the intensity of the SW monsoon are sensitive to climate variability and are affected by phenomenon such as El Niño Southern Oscillation (ENSO) and Indian Ocean Dipole (IOD). The size and location of the Mekong River plume also varies from year to year due to the variation in rainfall and wind strength of the monsoon. The riverine input is also influenced by anthropogenic activity such as agriculture, damming, and land-use changes. Different nutrients are supplied from these two main sources and in different quantities, and affect the structure of the phytoplankton community. Phytoplankton comprise the base of the food web, supporting the growth of higher organisms and ultimately support fisheries. We need to understand how the supply of different nutrients alters the phytoplankton community of the SCS. We sampled 22 stations along the coast of Vietnam aboard the R/V Falkor of the Schmidt Ocean Institute during the pre-monsoon season. High performance liquid chromatography (HPLC) and flow cytometric techniques will be used to characterize the phytoplankton community. The relationship between the different water masses, nutrient sources, and phytoplankton community found in the SCS will be presented.

Assessment of the antioxidant activity of an olive oil total polyphenolic fraction and hydroxytyrosol from a Greek Olea europea variety in endothelial cells and myoblasts.

PubMed

Kouka, Paraskevi; Priftis, Alexandros; Stagos, Dimitrios; Angelis, Apostolis; Stathopoulos, Panagiotis; Xinos, Nikos; Skaltsounis, Alexios-Léandros; Mamoulakis, Charalampos; Tsatsakis, Aristides M; Spandidos, Demetrios A; Kouretas, Demetrios

2017-09-01

Olive oil (OO) constitutes the basis of the Mediterranean diet, and it seems that its biophenols, such as hydroxytyrosol (HT) may scavenge free radicals, attracting distinct attention due to their beneficial effects in many pathological conditions, such as cancer. To the best of our knowedge, this is the first study in which the functional properties of an OO total polyphenolic fraction (TPF) and pure HT were examined in order to determine their antioxidant effects at a cellular level in endothelial cells and myoblasts. The test compounds were isolated using a green gradient‑elution centrifugal partition chromatography‑based method that allows the isolation of large volumes of OO in a continuous extraction procedure and with extremely low solvent consumption. For the isolation of HT, a combination of two chromatographic techniques was used, which is effective for the recovery of pure compounds from complex natural extracts. Moreover, TPF and HT exhibited potent free radical scavenging activity in vitro. The cells were treated with non‑cytotoxic concentrations and their redox status [in terms of glutathione (GSH) and reactive oxygen species (ROS) levels] was assessed. TPF extract was less cytotoxic than HT, and the observed differences between the two cell lines used suggest a tissue‑specific activity. Finally, flow cytometric analysis revealed that both TPF and HT improved the redox status by increasing the levels of GSH, one of the most important antioxidant molecules, in both endothelial cells and myoblasts, while the ROS levels were not significantly affected.

Effect of cigarette smoke extract and nicotine on the expression of thrombomodulin and endothelial protein C receptor in cultured human umbilical vein endothelial cells

PubMed Central

Wei, Yujie; Lai, Bin; Liu, Huiliang; Li, Yi; Zhen, Wang; Fu, Ling

2018-01-01

The present study investigated the influence of cigarette smoke extract (CSE) and nicotine on the expression of thrombomodulin (TM) and endothelial protein C receptor (EPCR) in human umbilical vein endothelial cells (HUVECs). Smoking is associated with intravascular thrombosis. As a vital anticoagulation cofactor, TM is located on the endothelial cell surface and regulates intravascular coagulation by binding to thrombin, hence activating protein C. Activated protein C is a natural anticoagulant that interacts with EPCR to enhance the function of anticoagulation system. The effects of CSE (0.5–5%) and nicotine (10-3-10-9 mol/l) on the expression of TM and EPCR in HUVECs were observed. Reverse transcription-quantitative polymerase chain reaction and flow cytometric analysis techniques were used for detecting TM and EPCR mRNA and protein expression levels, respectively. After 6-h exposure, TM protein and mRNA expression levels decreased in a dose-dependent manner. Stimulation with 5% CSE for 0, 6, 10, 12 and 24 h led to a decrease in the levels of TM mRNA and protein over time, which reached a peak at 12 h. The levels were significantly reduced compared with the control group (P<0.001). However, CSE had no effect on EPCR. Furthermore, nicotine had no influence on TM and EPCR. In conclusion, the present study supports a novel molecular mechanism of cigarette smoking-associated thrombosis by the decreased expression of TM. Further studies are required to identify specific components in CSE responsible for decreasing TM expression and its associated consequences. PMID:29257196

Detection of bacterial contamination in platelet concentrates by a sensitive flow cytometric assay (BactiFlow): a multicentre validation study.

PubMed

Vollmer, T; Dreier, J; Schottstedt, V; Bux, J; Tapernon, K; Sibrowski, W; Kleesiek, K; Knabbe, C

2012-08-01

Bacterial contamination of platelet concentrates (PCs) still represents an ongoing risk. As a result of septic complications, particularly observed with older PCs, the shelf life of PCs has been reduced in Germany to 4 days. In this study, bacterial screening of PCs by BactiFlow (BF) flow cytometry was introduced in three German blood services to evaluate the robustness and applicability of the assay. Results were used to discuss the potential for the extension of PC shelf life to 5 days. A total of 1956 PCs were tested on days 4 or 5+ after PC production using the BF, whereas the BacT/Alert culture system served as reference method. Two PCs were confirmed positive by culture only and were identified as Propionibacterium acnes and Staphylococcus species. Two PCs were confirmed positive for Streptococcus mitis by BF and culture. Additionally, two PCs were culture-positive only in one culture bottle (aerobic: S. mitis and anaerobic: S. hominis). Retrospective analysis of bacterial growth kinetics provide the indication that corresponding bacterial titres were most likely below the BF analytical detection limit (<150 CFU mL(-1) ) and had probably no transfusion relevance. All remaining specimens were tested negative. Testing of PCs by BF was successfully implemented. The BF proved sufficient as a rapid screening method to improve PC safety. This study further provides data supporting the extension of PC shelf life to 5 days after negative BF testing on day 4. © 2012 The Authors. Transfusion Medicine © 2012 British Blood Transfusion Society.

Flow Cytometry Pulse Width Data Enables Rapid and Sensitive Estimation of Biomass Dry Weight in the Microalgae Chlamydomonas reinhardtii and Chlorella vulgaris

PubMed Central

Chioccioli, Maurizio; Hankamer, Ben; Ross, Ian L.

2014-01-01

Dry weight biomass is an important parameter in algaculture. Direct measurement requires weighing milligram quantities of dried biomass, which is problematic for small volume systems containing few cells, such as laboratory studies and high throughput assays in microwell plates. In these cases indirect methods must be used, inducing measurement artefacts which vary in severity with the cell type and conditions employed. Here, we utilise flow cytometry pulse width data for the estimation of cell density and biomass, using Chlorella vulgaris and Chlamydomonas reinhardtii as model algae and compare it to optical density methods. Measurement of cell concentration by flow cytometry was shown to be more sensitive than optical density at 750 nm (OD750) for monitoring culture growth. However, neither cell concentration nor optical density correlates well to biomass when growth conditions vary. Compared to the growth of C. vulgaris in TAP (tris-acetate-phosphate) medium, cells grown in TAP + glucose displayed a slowed cell division rate and a 2-fold increased dry biomass accumulation compared to growth without glucose. This was accompanied by increased cellular volume. Laser scattering characteristics during flow cytometry were used to estimate cell diameters and it was shown that an empirical but nonlinear relationship could be shown between flow cytometric pulse width and dry weight biomass per cell. This relationship could be linearised by the use of hypertonic conditions (1 M NaCl) to dehydrate the cells, as shown by density gradient centrifugation. Flow cytometry for biomass estimation is easy to perform, sensitive and offers more comprehensive information than optical density measurements. In addition, periodic flow cytometry measurements can be used to calibrate OD750 measurements for both convenience and accuracy. This approach is particularly useful for small samples and where cellular characteristics, especially cell size, are expected to vary during growth. PMID:24832156

Flow Cytometric and Cytokine ELISpot Approaches To Characterize the Cell-Mediated Immune Response in Ferrets following Influenza Virus Infection

PubMed Central

DiPiazza, Anthony; Richards, Katherine; Batarse, Frances; Lockard, Laura; Zeng, Hui; García-Sastre, Adolfo; Albrecht, Randy A.

2016-01-01

ABSTRACT Influenza virus infections represent a significant socioeconomic and public health burden worldwide. Although ferrets are considered by many to be ideal for modeling human responses to influenza infection and vaccination, efforts to understand the cellular immune response have been severely hampered by a paucity of standardized procedures and reagents. In this study, we developed flow cytometric and T cell enzyme-linked immunosorbent spot (ELISpot) approaches to characterize the leukocyte composition and antigen-specific T cell response within key lymphoid tissues following influenza virus infection in ferrets. Through a newly designed and implemented set of serological reagents, we used multiparameter flow cytometry to directly quantify the frequency of CD4+ and CD8+ T cells, Ig+ B cells, CD11b+ myeloid-derived cells, and major histocompatibility complex (MHC) class II-positive antigen-presenting cells (APCs) both prior to and after intranasal infection with A/California/04/09 (H1N1). We found that the leukocyte composition was altered at 10 days postinfection, with notable gains in the frequency of T cells and myeloid cells within the draining lymph node. Furthermore, these studies revealed that the antigen specificity of influenza virus-reactive CD4 and CD8 T cells was very broad, with recognition of the viral HA, NA, M1, NS1, and NP proteins, and that total reactivity to influenza virus postinfection represented approximately 0.1% of the circulating peripheral blood mononuclear cells (PBMC). Finally, we observed distinct patterns of reactivity between individual animals, suggesting heterogeneity at the MHC locus in ferrets within commercial populations, a finding of considerable interest in efforts to move the ferret model forward for influenza vaccine and challenge studies. IMPORTANCE Ferrets are an ideal animal model to study transmission, diseases, and vaccine efficacies of respiratory viruses because of their close anatomical and physiological resemblances to humans. However, a lack of reagents has limited our understanding of the cell-mediated immune response following infection and vaccination. In this study, we used cross-reactive and ferret-specific antibodies to study the leukocyte composition and antigen-specific CD4 and CD8 T cell responses following influenza A/California/04/09 (H1N1) virus infection. These studies revealed strikingly distinct patterns of reactivity between CD4 and CD8 T cells, which were overlaid with differences in protein-specific responses between individual animals. Our results provide a first, in-depth look at the T cell repertoire in response to influenza infection and suggest that there is considerable heterogeneity at the MHC locus, which is akin to that in humans and an area of intense research interest. PMID:27356897