Tracking by flow cytometry antigen-specific follicular helper T cells in wild-type animals after protein vaccination.

PubMed

Chakarov, Svetoslav; Fazilleau, Nicolas

2015-01-01

Flow cytometry is a valuable technology used in immunology to characterize and enumerate the different cell subpopulations specific for a nonself-antigen in the context of an ongoing immune response. Among them, follicular helper T cells are the cognate regulators of B cells in secondary lymphoid tissues. Thus, tracking them is of high interest especially in the context of protein vaccination. For this purpose, transgenic antigen-receptor mouse models have been largely used. It is now clear that transgenic models are not always the best means to study the dynamics of the immune response since they can modify the response. In this chapter, we describe how to track endogenous antigen-specific follicular helper T cells by flow cytometry after protein vaccination in nonmodified wild-type animals, which ultimately provides a comprehensive way to enumerate, characterize, and isolate these particular cells in vivo.

In Vivo Myeloperoxidase Imaging and Flow Cytometry Analysis of Intestinal Myeloid Cells.

PubMed

Hülsdünker, Jan; Zeiser, Robert

2016-01-01

Myeloperoxidase (MPO) imaging is a non-invasive method to detect cells that produce the enzyme MPO that is most abundant in neutrophils, macrophages, and inflammatory monocytes. While lacking specificity for any of these three cell types, MPO imaging can provide guidance for further flow cytometry-based analysis of tissues where these cell types reside. Isolation of leukocytes from the intestinal tract is an error-prone procedure. Here, we describe a protocol for intestinal leukocyte isolation that works reliable in our hands and allows for flow cytometry-based analysis, in particular of neutrophils.

Guide to red fluorescent proteins and biosensors for flow cytometry.

PubMed

Piatkevich, Kiryl D; Verkhusha, Vladislav V

2011-01-01

Since the discovery of the first red fluorescent protein (RFP), named DsRed, 12 years ago, a wide pallet of red-shifted fluorescent proteins has been cloned and biotechnologically developed into monomeric fluorescent probes for optical microscopy. Several new types of monomeric RFPs that change the emission wavelength either with time, called fluorescent timers, or after a brief irradiation with violet light, known as photoactivatable proteins, have been also engineered. Moreover, RFPs with a large Stokes shift of fluorescence emission have been recently designed. Because of their distinctive excitation and fluorescence detection conditions developed specifically for microscopy, these fluorescent probes can be suboptimal for flow cytometry. Here, we have selected and summarized the advanced orange, red, and far-red fluorescent proteins with the properties specifically required for the flow cytometry applications. Their effective brightness was calculated for the laser sources available for the commercial flow cytometers and sorters. Compatibility of the fluorescent proteins of different colors in a multiparameter flow cytometry was determined. Novel FRET pairs, utilizing RFPs, RFP-based intracellular biosensors, and their application to a high-throughput screening, are also discussed. Copyright © 2011 Elsevier Inc. All rights reserved.

qFlow Cytometry-Based Receptoromic Screening: A High-Throughput Quantification Approach Informing Biomarker Selection and Nanosensor Development.

PubMed

Chen, Si; Weddell, Jared; Gupta, Pavan; Conard, Grace; Parkin, James; Imoukhuede, Princess I

2017-01-01

Nanosensor-based detection of biomarkers can improve medical diagnosis; however, a critical factor in nanosensor development is deciding which biomarker to target, as most diseases present several biomarkers. Biomarker-targeting decisions can be informed via an understanding of biomarker expression. Currently, immunohistochemistry (IHC) is the accepted standard for profiling biomarker expression. While IHC provides a relative mapping of biomarker expression, it does not provide cell-by-cell readouts of biomarker expression or absolute biomarker quantification. Flow cytometry overcomes both these IHC challenges by offering biomarker expression on a cell-by-cell basis, and when combined with calibration standards, providing quantitation of biomarker concentrations: this is known as qFlow cytometry. Here, we outline the key components for applying qFlow cytometry to detect biomarkers within the angiogenic vascular endothelial growth factor receptor family. The key aspects of the qFlow cytometry methodology include: antibody specificity testing, immunofluorescent cell labeling, saturation analysis, fluorescent microsphere calibration, and quantitative analysis of both ensemble and cell-by-cell data. Together, these methods enable high-throughput quantification of biomarker expression.

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

Rapid Identification of Staphylococcus aureus and Methicillin Resistance by Flow Cytometry Using a Peptide Nucleic Acid Probe ▿

PubMed Central

Shrestha, Nabin K.; Scalera, Nikole M.; Wilson, Deborah A.; Brehm-Stecher, Byron; Procop, Gary W.

2011-01-01

A total of 56 Staphylococcus aureus isolates incubated for 2 h in the presence or absence of oxacillin were analyzed by flow cytometry after labeling with an S. aureus-specific peptide nucleic acid (PNA) probe. Two defined ratios, the paired signal count ratio (PSCR) and the gate signal count ratio (GSCR), differentiated methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) with sensitivities of 100% each and specificities of 96% and 100%, respectively. PMID:21795508

Rapid identification of Staphylococcus aureus and methicillin resistance by flow cytometry using a peptide nucleic acid probe.

PubMed

Shrestha, Nabin K; Scalera, Nikole M; Wilson, Deborah A; Brehm-Stecher, Byron; Procop, Gary W

2011-09-01

A total of 56 Staphylococcus aureus isolates incubated for 2 h in the presence or absence of oxacillin were analyzed by flow cytometry after labeling with an S. aureus-specific peptide nucleic acid (PNA) probe. Two defined ratios, the paired signal count ratio (PSCR) and the gate signal count ratio (GSCR), differentiated methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) with sensitivities of 100% each and specificities of 96% and 100%, respectively.

[Clinical usefulness of urine-formed elements' information obtained from bacteria detection by flow cytometry method that uses nucleic acid staining].

PubMed

Nakagawa, Hiroko; Yuno, Tomoji; Itho, Kiichi

2009-03-01

Recently, specific detection method for Bacteria, by flow cytometry method using nucleic acid staining, was developed as a function of automated urine formed elements analyzer for routine urine testing. Here, we performed a basic study on this bacteria analysis method. In addition, we also have a comparison among urine sediment analysis, urine Gram staining and urine quantitative cultivation, the conventional methods performed up to now. As a result, the bacteria analysis with flow cytometry method that uses nucleic acid staining was excellent in reproducibility, and higher sensitivity compared with microscopic urinary sediment analysis. Based on the ROC curve analysis, which settled urine culture method as standard, cut-off level of 120/microL was defined and its sensitivity = 85.7%, specificity = 88.2%. In the analysis of scattergram, accompanied with urine culture method, among 90% of rod positive samples, 80% of dots were appeared in the area of 30 degrees from axis X. In addition, one case even indicated that analysis of bacteria by flow cytometry and scattergram of time series analysis might be helpful to trace the progress of causative bacteria therefore the information supposed to be clinically significant. Reporting bacteria information with nucleic acid staining flow cytometry method is expected to contribute to a rapid diagnostics and treatment of urinary tract infections. Besides, the contribution to screening examination of microbiology and clinical chemistry, will deliver a more efficient solution to urine analysis.

Data File Standard for Flow Cytometry, version FCS 3.1.

PubMed

Spidlen, Josef; Moore, Wayne; Parks, David; Goldberg, Michael; Bray, Chris; Bierre, Pierre; Gorombey, Peter; Hyun, Bill; Hubbard, Mark; Lange, Simon; Lefebvre, Ray; Leif, Robert; Novo, David; Ostruszka, Leo; Treister, Adam; Wood, James; Murphy, Robert F; Roederer, Mario; Sudar, Damir; Zigon, Robert; Brinkman, Ryan R

2010-01-01

The flow cytometry data file standard provides the specifications needed to completely describe flow cytometry data sets within the confines of the file containing the experimental data. In 1984, the first Flow Cytometry Standard format for data files was adopted as FCS 1.0. This standard was modified in 1990 as FCS 2.0 and again in 1997 as FCS 3.0. We report here on the next generation flow cytometry standard data file format. FCS 3.1 is a minor revision based on suggested improvements from the community. The unchanged goal of the standard is to provide a uniform file format that allows files created by one type of acquisition hardware and software to be analyzed by any other type.The FCS 3.1 standard retains the basic FCS file structure and most features of previous versions of the standard. Changes included in FCS 3.1 address potential ambiguities in the previous versions and provide a more robust standard. The major changes include simplified support for international characters and improved support for storing compensation. The major additions are support for preferred display scale, a standardized way of capturing the sample volume, information about originality of the data file, and support for plate and well identification in high throughput, plate based experiments. Please see the normative version of the FCS 3.1 specification in Supporting Information for this manuscript (or at http://www.isac-net.org/ in the Current standards section) for a complete list of changes.

Data File Standard for Flow Cytometry, Version FCS 3.1

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

Spidlen, Josef; Moore, Wayne; Parks, David

2009-11-10

The flow cytometry data file standard provides the specifications needed to completely describe flow cytometry data sets within the confines of the file containing the experimental data. In 1984, the first Flow Cytometry Standard format for data files was adopted as FCS 1.0. This standard was modified in 1990 as FCS 2.0 and again in 1997 as FCS 3.0. We report here on the next generation flow cytometry standard data file format. FCS 3.1 is a minor revision based on suggested improvements from the community. The unchanged goal of the standard is to provide a uniform file format that allowsmore » files created by one type of acquisition hardware and software to be analyzed by any other type. The FCS 3.1 standard retains the basic FCS file structure and most features of previous versions of the standard. Changes included in FCS 3.1 address potential ambiguities in the previous versions and provide a more robust standard. The major changes include simplified support for international characters and improved support for storing compensation. The major additions are support for preferred display scale, a standardized way of capturing the sample volume, information about originality of the data file, and support for plate and well identification in high throughput, plate based experiments. Please see the normative version of the FCS 3.1 specification in Supporting Information for this manuscript (or at http://www.isac-net.org/ in the Current standards section) for a complete list of changes.« less

flowVS: channel-specific variance stabilization in flow cytometry

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

Azad, Ariful; Rajwa, Bartek; Pothen, Alex

Comparing phenotypes of heterogeneous cell populations from multiple biological conditions is at the heart of scientific discovery based on flow cytometry (FC). When the biological signal is measured by the average expression of a biomarker, standard statistical methods require that variance be approximately stabilized in populations to be compared. Since the mean and variance of a cell population are often correlated in fluorescence-based FC measurements, a preprocessing step is needed to stabilize the within-population variances.

flowVS: channel-specific variance stabilization in flow cytometry

DOE PAGES

Azad, Ariful; Rajwa, Bartek; Pothen, Alex

2016-07-28

Comparing phenotypes of heterogeneous cell populations from multiple biological conditions is at the heart of scientific discovery based on flow cytometry (FC). When the biological signal is measured by the average expression of a biomarker, standard statistical methods require that variance be approximately stabilized in populations to be compared. Since the mean and variance of a cell population are often correlated in fluorescence-based FC measurements, a preprocessing step is needed to stabilize the within-population variances.

Determination of allergenic load and pollen count of Cupressus arizonica pollen by flow cytometry using Cup a1 polyclonal antibody.

PubMed

Benítez, Francisco Moreno; Camacho, Antonio Letrán; del Cuvillo Bernal, Alfonso; de Medina, Pedro Lobatón Sánchez; García Cózar, Francisco J; Romeu, Marisa Espinazo

2014-01-01

There is an increase in the incidence of pollen related allergy, thus information on pollen schedules would be a great asset for physicians to improve the clinical care of patients. Like cypress pollen sensitization shows a high prevalence among the causes of allergic rhinitis, and therefore it is of interest to use it like a model of study, distinguishing cypress pollen, pollen count, and allergenic load level. In this work, we use a flow cytometry based technique to obtain both Cupressus arizonica pollen count and allergenic load, using specific rabbit polyclonal antibody Cup a1 and its comparison with optical microscopy technique measurement. Airborne samples were collected from Burkard Spore-Trap and Burkard Cyclone Cupressus arizonica pollen was studied using specific rabbit polyclonal antibody Cup a1, labeled with AlexaFluor(®) 488 or 750 and analysed by Flow Cytometry in both an EPICS XL and Cyan ADP cytometers (Beckman Coulter(®) ). Optical microscopy study was realized with a Leica optical microscope. Bland and Altman was used to determine agreement between both techniques measured. We can identify three different populations based on rabbit polyclonal antibody Cup a1 staining. The main region (44.5%) had 97.3% recognition, a second region (25%) with 28% and a third region (30.5%) with 68% respectively. Immunofluorescence and confocal microscopy showed that main region corresponds to whole pollen grains, the second region are pollen without exine and the third region is constituted by smaller particles with allergenic properties. Pollen schedule shows a higher correlation measured by optical microscopy and flow cytometry in the pollen count with a P-value: 0.0008 E(-2) and 0.0002 with regard to smaller particles, so the Bland and Altman measurement showed a good correlation between them, P-value: 0.0003. Determination of pollen count and allergenic load by flow cytometry represents an important tool in the determination of airborne respiratory allergens. We showed that not only whole pollen but also smaller particles could induce allergic sensitization. This is the first study where flow cytometry is used for calculating pollen counts and allergenic load. Copyright © 2013 Clinical Cytometry Society.

Targeting Breast Cancer Vasculature

DTIC Science & Technology

2006-03-01

E., and Hanahan, D. Stage-specific vascular markers revealed by phage display in a mouse model of pancreatic islet tumorigenesis. Cancer Cell 4:393...expressing full-length myc-tagged metadherin, myc-vimen- tin, or myc-pCMV were analyzed by flow cytometry . Anti-myc antibodies were applied to the cells...In 4T1 tumor cell extracts, anti-metadherin(378-440) detectedthen stained with anti-myc antibodies. Using flow cytometry , proteins with apparent

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.

Determination of allergenic load and pollen count of Cupressus arizonica pollen by flow cytometry using Cup a1 polyclonal antibody.

PubMed

Benítez, Francisco Moreno; Camacho, Antonio Letrán; Del Cuvillo Bernal, Alfonso; de Medina, Pedro Lobatón Sánchez; Cózar, Francisco J García; Romeu, Ma Luisa Espinazo

2013-07-10

Background: There is an increase in the incidence of pollen related allergy, thus information on pollen schedules would be a great asset for physicians to improve the clinical care of patients. Like cypress pollen sensitization shows a high prevalence among the causes of allergic rhinitis, and therefore it is of interest to use it like a model of study, distinguishing cypress pollen, pollen count and allergenic load level. In this work, we use a flow cytometry based technique to obtain both Cupressus arizonica pollen count and allergenic load, using specific rabbit polyclonal antibody Cup a1 and its comparison with optical microscopy technique measurement. Methods: Airborne samples were collected from Burkard Spore-Trap and Burkard Cyclone Cupressus arizonica pollen was studied using specific rabbit polyclonal antibody Cup a1, labelled with AlexaFluor ® 488 or 750 and analysed by Flow Cytometry in both an EPICS XL and Cyan ADP cytometers (Beckman Coulter ® ). Optical microscopy study was realized with a Leica optical microscope. Bland & Altman was used to determine agreement between both techniques measured. Results: We can identify three different populations based on rabbit polyclonal antibody Cup a1 staining. The main region (44.5%) had 97.3% recognition, a second region (25%) with 28% and a third region (30.5%) with 68% respectively. Immunofluorescence and confocal microscopy showed that main region corresponds to whole pollen grains, the second region are pollen without exine and the third region is constituted by smaller particles with allergenic properties. Pollen schedule shows a higher correlation measured by optical microscopy and flow cytometry in the pollen count with a p-value: 0.0008E -2 and 0.0002 with regard to smaller particles, so the Bland & Altman measurement showed a good correlation between them, p-value: 0,0003. Conclusion: Determination of pollen count and allergenic load by flow cytometry represents an important tool in the determination of airborne respiratory allergens. We showed that not only whole pollen but also smaller particles could induce allergic sensitization. This is the first study where flow cytometry is used for calculating pollen counts and allergenic load. © 2013 Clinical Cytometry Society. Copyright © 2013 Clinical Cytometry Society.

Detection of site specific glycosylation in proteins using flow cytometry†

PubMed Central

Jayakumar, Deepak; Marathe, Dhananjay D.; Neelamegham, Sriram

2009-01-01

We tested the possibility that it is possible to express unique peptide probes on cell surfaces and detect site-specific glycosylation on these peptides using flow cytometry. Such development can enhance the application of flow cytometry to detect and quantify post-translational modifications in proteins. To this end, the N-terminal section of the human leukocyte glycoprotein PSGL-1 (P-selectin glycoprotein ligand-1) was modified to contain a poly-histidine tag followed by a proteolytic cleavage site. Amino acids preceding the cleavage site have a single O-linked glycosylation site. The recombinant protein called PSGL-1 (HT) was expressed on the surface of two mammalian cell lines, CHO and HL-60, using a lentiviral delivery approach. Results demonstrate that the N-terminal portion of PSGL-1 (HT) can be released from these cells by protease, and the resulting peptide can be readily captured and detected using cytometry-bead assays. Using this strategy, the peptide was immunoprecipitated onto beads bearing mAbs against either the poly-histidine sequence or the human PSGL-1. The carbohydrate epitope associated with the released peptide was detected using HECA-452 and CSLEX-1, monoclonal antibodies that recognize the sialyl Lewis-X epitope. Finally, the peptide released from cells could be separated and enriched using nickel chelate beads. Overall, such an approach that combines recombinant protein expression with flow cytometry, may be useful to quantify changes in site-specific glycosylation for basic science and clinical applications. PMID:19735085

Development of Antibodies Against Novel Cell Surface Proteins In Hormone Refractory Prostate Cancer. Addendum

DTIC Science & Technology

2011-07-01

marker of hormone refractory metastatic prostate cancer. Clin Cancer Res, 2005. 15: 2237- 43 . 3. Tomita K, van Bokhoven A, van Leenders GJ, Ruijter...Santa Cruz Biotechnology), vimentin, Ki-67 (DakoCytomation) and caspase-3 (Cell Signaling Technology). Flow cytometry was performed with N...transduced cells were labeled with N-cadherin–specific antibody and sorted by flow cytometry , gating for a GFP-positive, N-cadherinlow population. The

Screening of carcinoma metastasis by flow cytometry: A study of 238 cases.

PubMed

Acosta, Maria; Pereira, José; Arroz, Maria

2016-05-01

Malignant epithelial cells may be detected in different specimens, by immunophenotyping using flow cytometry (FCM). CD326 (epithelial-specific antigen, clone Ber-Ep4) was used to identify epithelial cells, CD45 to discriminate between leucocytes (positive for this antigen) and non-hematological cells (negative for this antigen), and CD33 to identify monocytes/macrophages. This combination is particularly useful in effusions to characterize large cells and distinguish between monocyte/macrophages (CD45+ CD33+ CD326-), mesothelial cells (CD45 ± (dim) CD33 - CD326-) and epithelial cells (CD45 - CD33 - CD326 +). We evaluated the efficiency of flow cytometry to detect malignant epithelial cells in 238 fresh samples, including effusions, lymph node biopsies, fine needle aspirates, bone marrow aspirates, cerebrospinal fluid, among others. These are specimens expected to lack epithelial cells. FCM results were then compared to the results of smear and cell block morphology, as well as immunocytochemistry on paraffin wax embedded cell blocks, when available. Final diagnosis was the gold standard and a very good sensitivity (96.7%) and specificity (99.3%) were obtained. We concluded that the detection of CD326 positive cells using FCM is strongly indicative of the presence of carcinoma cells. © 2015 International Clinical Cytometry Society. © 2015 International Clinical Cytometry Society.

ggCyto: Next Generation Open-Source Visualization Software for Cytometry.

PubMed

Van, Phu; Jiang, Wenxin; Gottardo, Raphael; Finak, Greg

2018-06-01

Open source software for computational cytometry has gained in popularity over the past few years. Efforts such as FlowCAP, the Lyoplate and Euroflow projects have highlighted the importance of efforts to standardize both experimental and computational aspects of cytometry data analysis. The R/BioConductor platform hosts the largest collection of open source cytometry software covering all aspects of data analysis and providing infrastructure to represent and analyze cytometry data with all relevant experimental, gating, and cell population annotations enabling fully reproducible data analysis. Data visualization frameworks to support this infrastructure have lagged behind. ggCyto is a new open-source BioConductor software package for cytometry data visualization built on ggplot2 that enables ggplot-like functionality with the core BioConductor flow cytometry data structures. Amongst its features are the ability to transform data and axes on-the-fly using cytometry-specific transformations, plot faceting by experimental meta-data variables, and partial matching of channel, marker and cell populations names to the contents of the BioConductor cytometry data structures. We demonstrate the salient features of the package using publicly available cytometry data with complete reproducible examples in a supplementary material vignette. https://bioconductor.org/packages/devel/bioc/html/ggcyto.html. gfinak@fredhutch.org. Supplementary data are available at Bioinformatics online and at http://rglab.org/ggcyto/.

Recombinant scFv antibodies against infectious pancreatic necrosis virus isolated by flow cytometry.

PubMed

Xu, Li-Ming; Zhao, Jing-Zhuang; Liu, Miao; Cao, Yong-Sheng; Yin, Jia-Sheng; Liu, Hong-Bai; Lu, Tongyan

2016-11-01

Infectious pancreatic necrosis is a significant disease of farmed salmonids in China. In this study, a single chain variable fragment (scFv) antibody library derived from rainbow trout (Oncorhynchus mykiss) and viral protein VP2 of a Chinese infectious pancreatic necrosis virus (IPNV) isolate ChRtm213 were co-expressed by a bacterial display technology. The library was subjected to three rounds of screening by flow cytometry (FCM) to select IPNV specific antibodies. Six antibody clones with different mean fluorescence intensities (MFI) were obtained by picking colonies at random. The antibody clones were expressed and purified. The purified IPNV-specific scFv antibodies were used successfully in Western blotting, enzyme linked immunosorbent assay (ELISA) and an immunofluorescence antibody test (IFAT). This method provides a high throughput means to screen an antibody library by flow cytometry, and isolate a panel of antibody that can be used as potential reagents for the detection and study of IPNV that are prevalent in China. Copyright © 2016 Elsevier B.V. All rights reserved.

Microfluidic Impedance Flow Cytometry Enabling High-Throughput Single-Cell Electrical Property Characterization

PubMed Central

Chen, Jian; Xue, Chengcheng; Zhao, Yang; Chen, Deyong; Wu, Min-Hsien; Wang, Junbo

2015-01-01

This article reviews recent developments in microfluidic impedance flow cytometry for high-throughput electrical property characterization of single cells. Four major perspectives of microfluidic impedance flow cytometry for single-cell characterization are included in this review: (1) early developments of microfluidic impedance flow cytometry for single-cell electrical property characterization; (2) microfluidic impedance flow cytometry with enhanced sensitivity; (3) microfluidic impedance and optical flow cytometry for single-cell analysis and (4) integrated point of care system based on microfluidic impedance flow cytometry. We examine the advantages and limitations of each technique and discuss future research opportunities from the perspectives of both technical innovation and clinical applications. PMID:25938973

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.

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.

Detection of endogenous alkaline phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF-97 phosphatase substrate

NASA Technical Reports Server (NTRS)

Telford, W. G.; Cox, W. G.; Stiner, D.; Singer, V. L.; Doty, S. B.

1999-01-01

BACKGROUND: The alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF((R))-97 for enzyme-labeled fluorescence) has been found useful for the histochemical detection of endogenous AP activity and AP-tagged proteins and oligonucleotide probes. In this study, we evaluated its effectiveness at detecting endogenous AP activity by flow cytometry. METHODS: The ELF-97 phosphatase substrate was used to detect endogenous AP activity in UMR-106 rat osteosarcoma cells and primary cultures of chick chondrocytes. Cells were labeled with the ELF-97 reagent and analyzed by flow cytometry using an argon ultraviolet (UV) laser. For comparison purposes, cells were also assayed for AP using a Fast Red Violet LB azo dye assay previously described for use in detecting AP activity by flow cytometry. RESULTS: The ELF-97 phosphatase substrate effectively detected endogenous AP activity in UMR-106 cells, with over 95% of the resulting fluorescent signal resulting from AP-specific activity (as determined by levamisole inhibition of AP activity). In contrast, less than 70% of the fluorescent signal from the Fast Red Violet LB (FRV) assay was AP-dependent, reflecting the high intrinsic fluorescence of the unreacted components. The ELF-97 phosphatase assay was also able to detect very low AP activity in chick chondrocytes that was undetectable by the azo dye method. CONCLUSIONS: The ELF-97 phosphatase assay was able to detect endogenous AP activity in fixed mammalian and avian cells by flow cytometry with superior sensitivity to previously described assays. This work also shows the applicability of ELF-97 to flow cytometry, supplementing its previously demonstrated histochemical applications. Copyright 1999 Wiley-Liss, Inc.

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

Srivastava, Nimisha; Singh, Anup K

Microfluidic devices and methods for flow cytometry are described. In described examples, various sample handling and preparation steps may be carried out within a same microfluidic device as flow cytometry steps. A combination of imaging and flow cytometry is described. In some examples, spiral microchannels serve as incubation chambers. Examples of automated sample handling and flow cytometry are described.

Application of flow cytometry to wine microorganisms.

PubMed

Longin, Cédric; Petitgonnet, Clément; Guilloux-Benatier, Michèle; Rousseaux, Sandrine; Alexandre, Hervé

2017-04-01

Flow cytometry (FCM) is a powerful technique allowing detection and enumeration of microbial populations in food and during food process. Thanks to the fluorescent dyes used and specific probes, FCM provides information about cell physiological state and allows enumeration of a microorganism in a mixed culture. Thus, this technique is increasingly used to quantify pathogen, spoilage microorganisms and microorganisms of interest. Since one decade, FCM applications to the wine field increase greatly to determine population and physiological state of microorganisms performing alcoholic and malolactic fermentations. Wine spoilage microorganisms were also studied. In this review we briefly describe FCM principles. Next, a deep revision concerning enumeration of wine microorganisms by FCM is presented including the fluorescent dyes used and techniques allowing a yeast and bacteria species specific enumeration. Then, the last chapter is dedicated to fluorescent dyes which are used to date in fluorescent microscopy but applicable in FCM. This chapter also describes other interesting "future" techniques which could be applied to study the wine microorganisms. Thus, this review seeks to highlight the main advantages of the flow cytometry applied to wine microbiology. Copyright © 2016 Elsevier Ltd. All rights reserved.

Differences in leukocyte differentiation molecule abundances on domestic sheep (Ovis aries) and bighorn sheep (Ovis canadensis) neutrophils identified by flow cytometry.

PubMed

Highland, Margaret A; Schneider, David A; White, Stephen N; Madsen-Bouterse, Sally A; Knowles, Donald P; Davis, William C

2016-06-01

Although both domestic sheep (DS) and bighorn sheep (BHS) are affected by similar respiratory bacterial pathogens, experimental and field data indicate BHS are more susceptible to pneumonia. Cross-reactive monoclonal antibodies (mAbs) for use in flow cytometry (FC) are valuable reagents for interspecies comparative immune system analyses. This study describes cross-reactive mAbs that recognize leukocyte differentiation molecules (LDMs) and major histocompatibility complex antigens on DS and BHS leukocytes. Characterization of multichannel eosinophil autofluorescence in this study permitted cell-type specific gating of granulocytes for evaluating LDMs, specifically on neutrophils, by single-label FC. Evaluation of relative abundances of LDMs by flow cytometry revealed greater CD11a, CD11b, CD18 (β2 integrins) and CD 172a (SIRPα) on DS neutrophils and greater CD14 (lipopolysaccharide receptor) on BHS neutrophils. Greater CD25 (IL-2) was identified on BHS lymphocytes following Concavalin A stimulation. While DS and BHS have similar total peripheral blood leukocyte counts, BHS have proportionately more neutrophils. Published by Elsevier Ltd.

Utility of peripheral blood immunophenotyping by flow cytometry in the diagnosis of pediatric acute leukemia.

PubMed

Metrock, Laura K; Summers, Ryan J; Park, Sunita; Gillespie, Scott; Castellino, Sharon; Lew, Glen; Keller, Frank G

2017-10-01

Childhood acute leukemia is traditionally diagnosed from a bone marrow aspirate (BMA). New-onset acute leukemia patients do not always have visible circulating blasts in the peripheral blood (PB) at diagnosis. While the role of bone marrow flow cytometry for the diagnosis of acute leukemia is well established, the utility of PB flow cytometry (PBFC) is unknown. We performed a single-institution retrospective analysis to compare PBFC versus BMA in establishing or excluding a diagnosis of childhood acute leukemia. We retrospectively identified 485 PBFC samples with concurrent BMA from 2008 to 2013. Results of four-color flow cytometry for immunophenotypic characterization of leukemic versus nonclonal disease were characterized. Sensitivity and specificity were calculated among patients without a known diagnosis or prior therapy. Among 485 samples eligible for analysis, 120 had negative PBFC and BMA, 359 had positive PBFC and BMA, 3 had negative PBFC and positive BMA, and 3 had positive PBFC and negative BMA. There were small but significant differences in sensitivity (100 vs. 93.8%; P = 0.002) and positive predictive value (100 vs. 93.8%; P = 0.002) favoring BMA over PBFC among those demonstrating absence of circulating morphologic blasts. PBFC has high sensitivity and specificity for the diagnosis of childhood acute leukemia. The predictive value of PBFC remains high for patients without visible circulating blasts and may enhance the diagnostic process for determining the indications for marrow testing. © 2017 Wiley Periodicals, Inc.

Flow Cytometry: Evolution of Microbiological Methods for Probiotics Enumeration.

PubMed

Pane, Marco; Allesina, Serena; Amoruso, Angela; Nicola, Stefania; Deidda, Francesca; Mogna, Luca

2018-05-14

The purpose of this trial was to verify that the analytical method ISO 19344:2015 (E)-IDF 232:2015 (E) is valid and reliable for quantifying the concentration of the probiotic Lactobacillus rhamnosus GG (ATCC 53103) in a finished product formulation. Flow cytometry assay is emerging as an alternative rapid method for microbial detection, enumeration, and population profiling. The use of flow cytometry not only permits the determination of viable cell counts but also allows for enumeration of damaged and dead cell subpopulations. Results are expressed as TFU (Total Fluorescent Units) and AFU (Active Fluorescent Units). In December 2015, the International Standard ISO 19344-IDF 232 "Milk and milk products-Starter cultures, probiotics and fermented products-Quantification of lactic acid bacteria by flow cytometry" was published. This particular ISO can be applied universally and regardless of the species of interest. Analytical method validation was conducted on 3 different industrial batches of L. rhamnosus GG according to USP39<1225>/ICH Q2R1 in term of: accuracy, precision (repeatability), intermediate precision (ruggedness), specificity, limit of quantification, linearity, range, robustness. The data obtained on the 3 batches of finished product have significantly demonstrated the validity and robustness of the cytofluorimetric analysis. On the basis of the results obtained, the ISO 19344:2015 (E)-IDF 232:2015 (E) "Quantification of lactic acid bacteria by flow cytometry" can be used for the enumeration of L. rhamnosus GG in a finished product formulation.

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.

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

Time-gated flow cytometry: an ultra-high selectivity method to recover ultra-rare-event μ-targets in high-background biosamples

NASA Astrophysics Data System (ADS)

Jin, Dayong; Piper, James A.; Leif, Robert C.; Yang, Sean; Ferrari, Belinda C.; Yuan, Jingli; Wang, Guilan; Vallarino, Lidia M.; Williams, John W.

2009-03-01

A fundamental problem for rare-event cell analysis is auto-fluorescence from nontarget particles and cells. Time-gated flow cytometry is based on the temporal-domain discrimination of long-lifetime (>1 μs) luminescence-stained cells and can render invisible all nontarget cell and particles. We aim to further evaluate the technique, focusing on detection of ultra-rare-event 5-μm calibration beads in environmental water dirt samples. Europium-labeled 5-μm calibration beads with improved luminescence homogeneity and reduced aggregation were evaluated using the prototype UV LED excited time-gated luminescence (TGL) flow cytometer (FCM). A BD FACSAria flow cytometer was used to sort accurately a very low number of beads (<100 events), which were then spiked into concentrated samples of environmental water. The use of europium-labeled beads permitted the demonstration of specific detection rates of 100%+/-30% and 91%+/-3% with 10 and 100 target beads, respectively, that were mixed with over one million nontarget autofluorescent background particles. Under the same conditions, a conventional FCM was unable to recover rare-event fluorescein isothiocyanate (FITC) calibration beads. Preliminary results on Giardia detection are also reported. We have demonstrated the scientific value of lanthanide-complex biolabels in flow cytometry. This approach may augment the current method that uses multifluorescence-channel flow cytometry gating.

Comparison of methods for in-house screening of HLA-B*57:01 to prevent abacavir hypersensitivity in HIV-1 care.

PubMed

De Spiegelaere, Ward; Philippé, Jan; Vervisch, Karen; Verhofstede, Chris; Malatinkova, Eva; Kiselinova, Maja; Trypsteen, Wim; Bonczkowski, Pawel; Vogelaers, Dirk; Callens, Steven; Ruelle, Jean; Kabeya, Kabamba; De Wit, Stephane; Van Acker, Petra; Van Sandt, Vicky; Emonds, Marie-Paule; Coucke, Paul; Sermijn, Erica; Vandekerckhove, Linos

2015-01-01

Abacavir is a nucleoside reverse transcriptase inhibitor used as part of combination antiretroviral therapy in HIV-1-infected patients. Because this drug can cause a hypersensitivity reaction that is correlated with the presence of the HLA-B*57:01 allotype, screening for the presence of HLA-B*57:01 is recommended before abacavir initiation. Different genetic assays have been developed for HLA-B*57:01 screening, each with specific sensitivity, turnaround time and assay costs. Here, a new real-time PCR (qPCR) based analysis is described and compared to sequence specific primer PCR with capillary electrophoresis (SSP PCR CE) on 149 patient-derived samples, using sequence specific oligonucleotide hybridization combined with high resolution SSP PCR as gold standard. In addition to these PCR based methods, a complementary approach was developed using flow cytometry with an HLA-B17 specific monoclonal antibody as a pre-screening assay to diminish the number of samples for genetic testing. All three assays had a maximum sensitivity of >99. However, differences in specificity were recorded, i.e. 84.3%, 97.2% and >99% for flow cytometry, qPCR and SSP PCR CE respectively. Our data indicate that the most specific and sensitive of the compared methods is the SSP PCR CE. Flow cytometry pre-screening can substantially decrease the number of genetic tests for HLA-B*57:01 typing in a clinical setting.

CytometryML: a data standard which has been designed to interface with other standards

NASA Astrophysics Data System (ADS)

Leif, Robert C.

2007-02-01

Because of the differences in the requirements, needs, and past histories including existing standards of the creating organizations, a single encompassing cytology-pathology standard will not, in the near future, replace the multiple existing or under development standards. Except for DICOM and FCS, these standardization efforts are all based on XML. CytometryML is a collection of XML schemas, which are based on the Digital Imaging and Communications in Medicine (DICOM) and Flow Cytometry Standard (FCS) datatypes. The CytometryML schemas contain attributes that link them to the DICOM standard and FCS. Interoperability with DICOM has been facilitated by, wherever reasonable, limiting the difference between CytometryML and the previous standards to syntax. In order to permit the Resource Description Framework, RDF, to reference the CytometryML datatypes, id attributes have been added to many CytometryML elements. The Laboratory Digital Imaging Project (LDIP) Data Exchange Specification and the Flowcyt standards development effort employ RDF syntax. Documentation from DICOM has been reused in CytometryML. The unity of analytical cytology was demonstrated by deriving a microscope type and a flow cytometer type from a generic cytometry instrument type. The feasibility of incorporating the Flowcyt gating schemas into CytometryML has been demonstrated. CytometryML is being extended to include many of the new DICOM Working Group 26 datatypes, which describe patients, specimens, and analytes. In situations where multiple standards are being created, interoperability can be facilitated by employing datatypes based on a common set of semantics and building in links to standards that employ different syntax.

Technical advances in flow cytometry-based diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria

PubMed Central

Correia, Rodolfo Patussi; Bento, Laiz Cameirão; Bortolucci, Ana Carolina Apelle; Alexandre, Anderson Marega; Vaz, Andressa da Costa; Schimidell, Daniela; Pedro, Eduardo de Carvalho; Perin, Fabricio Simões; Nozawa, Sonia Tsukasa; Mendes, Cláudio Ernesto Albers; Barroso, Rodrigo de Souza; Bacal, Nydia Strachman

2016-01-01

ABSTRACT Objective: To discuss the implementation of technical advances in laboratory diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria for validation of high-sensitivity flow cytometry protocols. Methods: A retrospective study based on analysis of laboratory data from 745 patient samples submitted to flow cytometry for diagnosis and/or monitoring of paroxysmal nocturnal hemoglobinuria. Results: Implementation of technical advances reduced test costs and improved flow cytometry resolution for paroxysmal nocturnal hemoglobinuria clone detection. Conclusion: High-sensitivity flow cytometry allowed more sensitive determination of paroxysmal nocturnal hemoglobinuria clone type and size, particularly in samples with small clones. PMID:27759825

Flow Cytometry of Spinach Chloroplasts 1

PubMed Central

Schröder, Wolfgang P.; Petit, Patrice X.

1992-01-01

Intact spinach (Spinacia oleracea) chloroplasts, thylakoid membranes, and inside-out or right-side-out thylakoid vesicles have been characterized by flow cytometry with respect to forward angle light scatter, right angle light scatter, and chlorophyll fluorescence. Analysis of intact chloroplasts with respect to forward light scatter and the chlorophyll fluorescence parameter revealed the presence of truly “intact” and “disrupted” chloroplasts. The forward light scatter parameter, normally considered to reflect object size, was instead found to reflect the particle density. One essential advantage of flow cytometry is that additional parameters such as Ricinus communis agglutinin (linked to fluorescein isothiocyanate) fluorescence can be determined through logical conditions placed on bit-maps, amounting to an analytical purification procedure. In the present case, chloroplast subpopulations with fully preserved envelopes, thylakoid membrane, and inside-out or right-side-out thylakoid membranes vesicles can be distinguished. Flow cytometry is also a useful tool to address the question of availability of glycosyl moities on the membrane surfaces if one keeps in mind that organelle-to-organelle interactions could be partially mediated through a recognition process. A high specific binding of R. communis agglutinin and peanut lectin to the chloroplast envelope was detected. This showed that galactose residues were exposed and accessible to specific lectins on the chloroplast surface. No exposed glucose, fucose, or mannose residues could be detected by the appropriate lectins. Ricin binding to the intact chloroplasts caused a strong aggregation. Disruption of these aggregates by resuspension or during passage in the flow cytometer induced partial breakage of the chloroplasts. Only minor binding of R. communis agglutinin and peanut lectin to the purified thylakoid membranes was detected; the binding was found to be low for both inside-out and right-side-out vesicles of the thylakoid membranes. Images Figure 1 Figure 1 Figure 1 PMID:16653090

Dissecting the Role of IGFBP-2 in Development of Acute Myeloid Leukemia

DTIC Science & Technology

2011-06-01

surface proteins on freshly isolated and cultured cells, as determined by flow cytometry ... Surface Immune Molecules on Phenotypic HSCs during Culture (A and B) A summary of the result of flow cytometry analysis of surface expression of indicated...from the distant implanted tumor were counted by flow cytometry analysis. The flow cytometry result was confirmed by counting GFP+ surface foci of

Flow cytometry: basic principles and applications.

PubMed

Adan, Aysun; Alizada, Günel; Kiraz, Yağmur; Baran, Yusuf; Nalbant, Ayten

2017-03-01

Flow cytometry is a sophisticated instrument measuring multiple physical characteristics of a single cell such as size and granularity simultaneously as the cell flows in suspension through a measuring device. Its working depends on the light scattering features of the cells under investigation, which may be derived from dyes or monoclonal antibodies targeting either extracellular molecules located on the surface or intracellular molecules inside the cell. This approach makes flow cytometry a powerful tool for detailed analysis of complex populations in a short period of time. This review covers the general principles and selected applications of flow cytometry such as immunophenotyping of peripheral blood cells, analysis of apoptosis and detection of cytokines. Additionally, this report provides a basic understanding of flow cytometry technology essential for all users as well as the methods used to analyze and interpret the data. Moreover, recent progresses in flow cytometry have been discussed in order to give an opinion about the future importance of this technology.

Development of a bead-based multiplexed assay for simultaneous quantification of five bovine cytokines by flow cytometry.

PubMed

Rodrigues, Valérie; Baudier, Jean Baptiste; Chantal, Isabelle

2017-09-01

Quantifying cytokines is extremely important in studies of host-pathogen interactions. Multiplex assays are commercially available but only for human and mouse cytokines. Here a method for the simultaneous quantification of five important bovine cytokines IFNγ, IL-4, IL-10, IL-12, and TNFα in cell culture supernatants, using flow cytometry was reported. Functional beads from BD Biosciences expressing specific APC intensity were used. Commercially available antibodies against bovine cytokines were covalently coupled to beads as capture antibodies. Fixed recombinant cytokines were revealed with a second monoclonal antibody coupled with biotin, then revealed with streptavidin-PE. This complex was analyzed using a standard flow cytometer. Experiments were performed to check no cross reactions had occurred. The limits of detection ranged between 0.08 and 0.4 ng/ml depending on the cytokine, and the linearity between the lower and higher limits was remarkable (R 2  > 99.8%). Finally, native cytokines from cell culture supernatants were tested. Results were compared using the standard ELISA test and showed that concentrations of native cytokine in cell culture supernatants were comparable with the two methods, with a wider dynamic range using beads and flow cytometry than with ELISA assays. Bovine IFNγ, IL-4, IL-10, IL-12, and TNFα in culture supernatants can be now simultaneously detected in a single assay, using a standard flow cytometer for both basic and high-throughput analyses. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Final Report Nucleic Acid System - Hybrid PCR and Multiplex Assay Project Phase 2

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

Koopman, R P; Langlois, R G; Nasarabadi, S

2002-04-17

This report covers phase 2 (year 2) of the Nucleic Acid System--Hybrid PCR and Multiplex Assay project. The objective of the project is to reduce to practice the detection and identification of biological warfare pathogens by the nucleic acid recognition technique of PCR (polymerase chain reaction) in a multiplex mode using flow cytometry. The Hybrid instrument consists of a flow-through PCR module capable of handling a multiplexed PCR assay, a hybridizing module capable of hybridizing multiplexed PCR amplicons and beads, and a flow cytometer module for bead-based identification, all controlled by a single computer. Multiplex immunoassay using bead-based Luminex flowmore » cytometry is available, allowing rapid screening for many agents. PCR is highly specific and complements and verifies immunoassay. It can also be multiplexed and detection provided using the bead-based Luminex flow cytometer. This approach allows full access to the speed and 100-fold multiplex capability of flow cytometry for rapid screening as well as the accuracy and specificity of PCR. This project has two principal activities: (1) Design, build and test a prototype hybrid PCR/flow cytometer with the basic capabilities for rapid, broad spectrum detection and identification, and (2) Develop and evaluate multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products. This project requires not only building operationally functional instrumentation but also developing the chemical assays for detection of priority pathogens. This involves development and evaluation of multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products.« less

RNA Flow Cytometry Using the Branched DNA Technique.

PubMed

Soh, Kah Teong; Wallace, Paul K

2018-01-01

The systematic modulation of mRNA and proteins governs the complicated and intermingled biological functions of our cells. Traditionally, transcriptomic technologies such as DNA microarray and RNA-Seq have been used to identify, characterize, and profile gene expression data. These are, however, considered bulk methods as they are unable to measure gene expression at the single-cell level, unless the cells are pre-sorted. Branched DNA is a flow cytometry-based detection platform that has been developed recently to measure mRNA at the single-cell level. Originally adapted from microscopy, the current system has been modified to achieve compatibility with the detection of surface and intracellular antigens using monoclonal antibodies conjugated to fluorochromes, thus permitting simultaneous detection of mRNAs and proteins. The Branched DNA method offers a variety of advantages when compared to traditional or standard methods used for the quantification of mRNA, such as (a) the detection of specific mRNA on a per cell basis, (b) an alternate detection tool when the measurement of a protein is technically infeasible (i.e., no quality antibody exists) or the epitope is not assessable, and (c) correlate the analysis of mRNA with protein. Compared to earlier attempts at measuring nucleic acid by flow cytometry, the hybridization temperature applied in the Branched DNA assay is much lower, thus preserving the integrity of cellular structures for further characterization. It also has greatly increased specificity and sensitivity. Here, we provide detailed instruction for performing the Branched DNA method using it in a model system to correlate the expression of CD8 mRNA and CD8 protein by flow cytometry.

A critical evaluation of a flow cytometer used for detecting enterococci in recreational waters.

PubMed

King, Dawn N; Brenner, Kristen P; Rodgers, Mark R

2007-06-01

The current U. S. Environmental Protection Agency-approved method for enterococci (Method 1600) in recreational water is a membrane filter (MF) method that takes 24 hours to obtain results. If the recreational water is not in compliance with the standard, the risk of exposure to enteric pathogens may occur before the water is identified as hazardous. Because flow cytometry combined with specific fluorescent antibodies has the potential to be used as a rapid detection method for microorganisms, this technology was evaluated as a rapid, same-day method to detect enterococci in bathing beach waters. The flow cytometer chosen for this study was a laser microbial detection system designed to detect labeled antibodies. A comparison of MF counts with flow cytometry counts of enterococci in phosphate buffer and sterile-filtered recreational water showed good agreement between the two methods. However, when flow cytometry was used, the counts were several orders of magnitude higher than the MF counts with no correlation to Enterococcus spike concentrations. The unspiked sample controls frequently had higher counts than the samples spiked with enterococci. Particles within the spiked water samples were probably counted as target cells by the flow cytometer because of autofluorescence or non-specific adsorption of antibody and carryover to subsequent samples. For these reasons, this technology may not be suitable for enterococci detection in recreational waters. Improvements in research and instrument design that will eliminate high background and carryover may make this a viable technology in the

An active, collaborative approach to learning skills in flow cytometry.

PubMed

Fuller, Kathryn; Linden, Matthew D; Lee-Pullen, Tracey; Fragall, Clayton; Erber, Wendy N; Röhrig, Kimberley J

2016-06-01

Advances in science education research have the potential to improve the way students learn to perform scientific interpretations and understand science concepts. We developed active, collaborative activities to teach skills in manipulating flow cytometry data using FlowJo software. Undergraduate students were given compensated clinical flow cytometry listmode output (FCS) files and asked to design a gating strategy to diagnose patients with different hematological malignancies on the basis of their immunophenotype. A separate cohort of research trainees was given uncompensated data files on which they performed their own compensation, calculated the antibody staining index, designed a sequential gating strategy, and quantified rare immune cell subsets. Student engagement, confidence, and perceptions of flow cytometry were assessed using a survey. Competency against the learning outcomes was assessed by asking students to undertake tasks that required understanding of flow cytometry dot plot data and gating sequences. The active, collaborative approach allowed students to achieve learning outcomes not previously possible with traditional teaching formats, for example, having students design their own gating strategy, without forgoing essential outcomes such as the interpretation of dot plots. In undergraduate students, favorable perceptions of flow cytometry as a field and as a potential career choice were correlated with student confidence but not the ability to perform flow cytometry data analysis. We demonstrate that this new pedagogical approach to teaching flow cytometry is beneficial for student understanding and interpretation of complex concepts. It should be considered as a useful new method for incorporating complex data analysis tasks such as flow cytometry into curricula. Copyright © 2016 The American Physiological Society.

Development of EMab-51, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody in Flow Cytometry, Western Blot, and Immunohistochemistry.

PubMed

Itai, Shunsuke; Kaneko, Mika K; Fujii, Yuki; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Handa, Saori; Chang, Yao-Wen; Suzuki, Hiroyoshi; Harada, Hiroyuki; Kato, Yukinari

2017-10-01

The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases and is involved in cell growth and differentiation. EGFR homodimers or heterodimers with other HER members, such as HER2 and HER3, activate downstream signaling cascades in many cancers. In this study, we developed novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. First, we expressed the full-length or ectodomain of EGFR in LN229 glioblastoma cells and then immunized mice with LN229/EGFR or ectodomain of EGFR, and performed the first screening using enzyme-linked immunosorbent assays. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical (fourth screening) analyses. Among 100 mAbs, only one clone EMab-51 (IgG 1 , kappa) reacted with EGFR in Western blot analysis. Finally, immunohistochemical analyses with EMab-51 showed sensitive and specific reactions against oral cancer cells, warranting the use of EMab-51 to detect EGFR in pathological analyses of EGFR-expressing cancers.

High throughput, real-time detection of Naegleria lovaniensis in natural river water using LED-illuminated Fountain Flow Cytometry.

PubMed

Johnson, P E; Deromedi, A J; Lebaron, P; Catala, P; Havens, C; Pougnard, C

2007-09-01

To test Fountain Flow Cytometry (FFC) for the rapid and sensitive detection of Naegleria lovaniensis amoebae (an analogue for Naegleria fowleri) in natural river waters. Samples were incubated with one of two fluorescent labels to facilitate detection: ChemChrome V6, a viability indicator, and an R-phycoerytherin (RPE) immunolabel to detect N. lovaniensis specifically. The resulting aqueous sample was passed as a stream in front of a light-emitting diode, which excited the fluorescent labels. The fluorescence was detected with a digital camera as the sample flowed toward the imager. Detections of N. lovaniensis were made in inoculated samples of natural water from eight rivers in France and the United States. FFC enumeration yielded results that are consistent with other counting methods: solid-phase cytometry, flow cytometry, and hemocytometry, down to concentrations of 0.06 amoebae ml(-1), using a flow rate of 15 ml min(-1). This study supports the efficacy of using FFC for the detection of viable protozoa in natural waters and indicates that use of RPE illuminated at 530 nm and detected at 585 nm provides a satisfactory means of attenuating background. Because of the severe global public health issues with drinking water and sanitation, there is an urgent need to develop a technique for the real-time detection of viable pathogens in environmental samples at low concentrations. FFC addresses this need.

Discovering cell types in flow cytometry data with random matrix theory

NASA Astrophysics Data System (ADS)

Shen, Yang; Nussenblatt, Robert; Losert, Wolfgang

Flow cytometry is a widely used experimental technique in immunology research. During the experiments, peripheral blood mononuclear cells (PBMC) from a single patient, labeled with multiple fluorescent stains that bind to different proteins, are illuminated by a laser. The intensity of each stain on a single cell is recorded and reflects the amount of protein expressed by that cell. The data analysis focuses on identifying specific cell types related to a disease. Different cell types can be identified by the type and amount of protein they express. To date, this has most often been done manually by labelling a protein as expressed or not while ignoring the amount of expression. Using a cross correlation matrix of stain intensities, which contains both information on the proteins expressed and their amount, has been largely ignored by researchers as it suffers from measurement noise. Here we present an algorithm to identify cell types in flow cytometry data which uses random matrix theory (RMT) to reduce noise in a cross correlation matrix. We demonstrate our method using a published flow cytometry data set. Compared with previous analysis techniques, we were able to rediscover relevant cell types in an automatic way. Department of Physics, University of Maryland, College Park, MD 20742.

Detecting specific cytotoxic T lymphocytes against SARS-coronavirus with DimerX HLA-A2:Ig fusion protein.

PubMed

Wang, Yue-Dan; Chen, Wei Feng

2004-11-01

To assess specific cytotoxic T lymphocytes (CTLs) against Severe acute respiratory syndrome (SARS)-coronavirus, a modified DimerX flow cytometry assay was performed with peripheral blood mononuclear cell (PBMC) from HLA-A2+ SARS-recovered donors at different time points post disease. CD8+DimerX-S1203+ CTLs were detected in the PBMC from these donors up to 3 months after recovery. The percentages of CD8+DimerX-S1203+ cells paralleled the numbers of interferon-gamma-positive spots in an ELISPOT assay using the same antigenic peptide. In conclusion, DimerX-based flow cytometry staining may prove to be a real-time method to screen for CTL directed at epitopes from a newly identified virus.

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.

Topically Delivered Adipose Derived Stem Cells Show an Activated-Fibroblast Phenotype and Enhance Granulation Tissue Formation in Skin Wounds

DTIC Science & Technology

2013-01-31

have similar surface markers . We found that topically delivered ASCs are engrafted and proliferate in the wounds. We showed that transplanted ASCs...Material Command (W81XWH-10-2-0054). Flow cytometry was supported by the Northwestern University Flow Cytometry Facility and a Cancer Center Support...blasticidin. GFP expressing cells were further selected by flow cytometry using the Northwestern University Flow Cytometry Facility. Treatment of MSCs

Cytobank: providing an analytics platform for community cytometry data analysis and collaboration.

PubMed

Chen, Tiffany J; Kotecha, Nikesh

2014-01-01

Cytometry is used extensively in clinical and laboratory settings to diagnose and track cell subsets in blood and tissue. High-throughput, single-cell approaches leveraging cytometry are developed and applied in the computational and systems biology communities by researchers, who seek to improve the diagnosis of human diseases, map the structures of cell signaling networks, and identify new cell types. Data analysis and management present a bottleneck in the flow of knowledge from bench to clinic. Multi-parameter flow and mass cytometry enable identification of signaling profiles of patient cell samples. Currently, this process is manual, requiring hours of work to summarize multi-dimensional data and translate these data for input into other analysis programs. In addition, the increase in the number and size of collaborative cytometry studies as well as the computational complexity of analytical tools require the ability to assemble sufficient and appropriately configured computing capacity on demand. There is a critical need for platforms that can be used by both clinical and basic researchers who routinely rely on cytometry. Recent advances provide a unique opportunity to facilitate collaboration and analysis and management of cytometry data. Specifically, advances in cloud computing and virtualization are enabling efficient use of large computing resources for analysis and backup. An example is Cytobank, a platform that allows researchers to annotate, analyze, and share results along with the underlying single-cell data.

Principles and applications of flow cytometry and cell sorting in companion animal medicine.

PubMed

Wilkerson, Melinda J

2012-01-01

Flow cytometry measures multiple characteristic of single cells using light scatter properties and fluorescence properties of fluorescent probes with specificity to cellular constituents. The use of flow cytometry in the veterinary clinical laboratory has become more routine in veterinary diagnostic laboratories and institutions (http://www.vet.k-state.edu/depts/dmp/service/immunology/index.htm), and reference laboratories. The most common applications in small animal medicine includes quantitation of erythrocytes and leukocytes in automated hematology instruments, detection of antibodies to erythrocytes and platelets in cases of immune-mediated diseases, immunophenotyping of leukocytes and lymphocytes in immunodeficiency syndromes, or leukemias and lymphomas. DNA content analysis to identify aneuploidy or replicating cells in tumor preparations has not gained routine acceptance because of the variability of prognostic results. Other applications including cell sorting and multiplexing using microspheres are potential assays of the future once they become validated and the instrumentation footprint becomes more and more compact, less expensive, and easier to use.

Rapid Flow Cytometry-Based Test for the Diagnosis of Lipopolysaccharide Responsive Beige-Like Anchor (LRBA) Deficiency.

PubMed

Gámez-Díaz, Laura; Sigmund, Elena C; Reiser, Veronika; Vach, Werner; Jung, Sophie; Grimbacher, Bodo

2018-01-01

The diagnosis of lipopolysaccharide-responsive beige-like-anchor-protein (LRBA) deficiency currently relies on gene sequencing approaches that do not support a timely diagnosis and clinical management. We developed a rapid and sensitive test for clinical implementation based on the detection of LRBA protein by flow cytometry in peripheral blood cells after stimulation. LRBA protein was assessed in a prospective cohort of 54 healthy donors and 57 patients suspected of LRBA deficiency. Receiver operating characteristics analysis suggested an LRBA:MFI ratio cutoff point of 2.6 to identify LRBA-deficient patients by FACS with 94% sensitivity and 80% specificity and to discriminate them from patients with a similar clinical picture but other disease-causing mutations. This easy flow cytometry-based assay allows a fast screening of patients with suspicion of LRBA deficiency reducing therefore the number of patients requiring LRBA sequencing and accelerating the treatment implementation. Detection of biallelic mutations in LRBA is however required for a definitive diagnosis.

Detection of antibody to Purpureocillium lilacinum by immunofluorescent assay and flow cytometry in serum of infected C57BL/6 mice.

PubMed

de Sequeira, Danielly C M; Peixoto, Mariana L P; De Luca, Paula M; Oliveira-Ferreira, Joseli; Antas, Paulo R Z; Borba, Cintia M

2013-10-31

Purpureocillium lilacinum is an emerging pathogenic fungus that can cause different clinical manifestations ranging from cutaneous and sub-cutaneous infections to severe oculomycosis. In this study, using both conventional indirect immunofluorescence and non-conventional flow cytometry approaches, IgG antibodies were readily detected in both C57BL/6 immunocompetent and immunosuppressed mice after i.v. infection with P. lilacinum. The humoral immune response was specific, since virtually no antibodies were detected in the serum of control mice. Flow cytometry assays also showed both quantitative and qualitative differences in total IgG and its isotypes in sera of immunocompetent and immunosupressed infected mice. Although a good starting point, it is clear that the effectiveness of serological assays for P. lilacinum hyalohyphomycosis identification in clinical studies still requires further standardization. Upon further validation in humans, these techniques have the potential to be suitable to detect P. lilacinum infection in patients, thereby avoiding current laborious and time-consuming culture techniques. © 2013.

In vitro flow cytometry-based screening platform for cellulase engineering

PubMed Central

Körfer, Georgette; Pitzler, Christian; Vojcic, Ljubica; Martinez, Ronny; Schwaneberg, Ulrich

2016-01-01

Ultrahigh throughput screening (uHTS) plays an essential role in directed evolution for tailoring biocatalysts for industrial applications. Flow cytometry-based uHTS provides an efficient coverage of the generated protein sequence space by analysis of up to 107 events per hour. Cell-free enzyme production overcomes the challenge of diversity loss during the transformation of mutant libraries into expression hosts, enables directed evolution of toxic enzymes, and holds the promise to efficiently design enzymes of human or animal origin. The developed uHTS cell-free compartmentalization platform (InVitroFlow) is the first report in which a flow cytometry-based screened system has been combined with compartmentalized cell-free expression for directed cellulase enzyme evolution. InVitroFlow was validated by screening of a random cellulase mutant library employing a novel screening system (based on the substrate fluorescein-di-β-D-cellobioside), and yielded significantly improved cellulase variants (e.g. CelA2-H288F-M1 (N273D/H288F/N468S) with 13.3-fold increased specific activity (220.60 U/mg) compared to CelA2 wildtype: 16.57 U/mg). PMID:27184298

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.

Annexin-V/quantum dot probes for multimodal apoptosis monitoring in living cells: improving bioanalysis using electrochemistry

NASA Astrophysics Data System (ADS)

Montón, Helena; Parolo, Claudio; Aranda-Ramos, Antonio; Merkoçi, Arben; Nogués, Carme

2015-02-01

There is a great demand to develop novel techniques that allow useful and complete monitoring of apoptosis, which is a key factor of several diseases and a target for drug development. Here, we present the use of a novel dual electrochemical/optical label for the detection and study of apoptosis. We combined the specificity of Annexin-V for phosphatidylserine, a phospholipid expressed in the outer membrane of apoptotic cells, with the optical and electrochemical properties of quantum dots to create a more efficient label. Using this conjugate we addressed three important issues: (i) we made the labeling of apoptotic cells faster (30 min) and easier; (ii) we fully characterized the samples by common cell biological techniques (confocal laser scanning microscopy, scanning electron microscopy and flow cytometry); and (iii) we developed a fast, cheap and quantitative electrochemical detection method for apoptotic cells with results in full agreement with those obtained by flow cytometry.There is a great demand to develop novel techniques that allow useful and complete monitoring of apoptosis, which is a key factor of several diseases and a target for drug development. Here, we present the use of a novel dual electrochemical/optical label for the detection and study of apoptosis. We combined the specificity of Annexin-V for phosphatidylserine, a phospholipid expressed in the outer membrane of apoptotic cells, with the optical and electrochemical properties of quantum dots to create a more efficient label. Using this conjugate we addressed three important issues: (i) we made the labeling of apoptotic cells faster (30 min) and easier; (ii) we fully characterized the samples by common cell biological techniques (confocal laser scanning microscopy, scanning electron microscopy and flow cytometry); and (iii) we developed a fast, cheap and quantitative electrochemical detection method for apoptotic cells with results in full agreement with those obtained by flow cytometry. Electronic supplementary information (ESI) available: Optical microscopy images of apoptotic induced cell cultures at different times and negative control of flow cytometry. See DOI: 10.1039/c4nr07191c

Optimizing transformations for automated, high throughput analysis of flow cytometry data

PubMed Central

2010-01-01

Background In a high throughput setting, effective flow cytometry data analysis depends heavily on proper data preprocessing. While usual preprocessing steps of quality assessment, outlier removal, normalization, and gating have received considerable scrutiny from the community, the influence of data transformation on the output of high throughput analysis has been largely overlooked. Flow cytometry measurements can vary over several orders of magnitude, cell populations can have variances that depend on their mean fluorescence intensities, and may exhibit heavily-skewed distributions. Consequently, the choice of data transformation can influence the output of automated gating. An appropriate data transformation aids in data visualization and gating of cell populations across the range of data. Experience shows that the choice of transformation is data specific. Our goal here is to compare the performance of different transformations applied to flow cytometry data in the context of automated gating in a high throughput, fully automated setting. We examine the most common transformations used in flow cytometry, including the generalized hyperbolic arcsine, biexponential, linlog, and generalized Box-Cox, all within the BioConductor flowCore framework that is widely used in high throughput, automated flow cytometry data analysis. All of these transformations have adjustable parameters whose effects upon the data are non-intuitive for most users. By making some modelling assumptions about the transformed data, we develop maximum likelihood criteria to optimize parameter choice for these different transformations. Results We compare the performance of parameter-optimized and default-parameter (in flowCore) data transformations on real and simulated data by measuring the variation in the locations of cell populations across samples, discovered via automated gating in both the scatter and fluorescence channels. We find that parameter-optimized transformations improve visualization, reduce variability in the location of discovered cell populations across samples, and decrease the misclassification (mis-gating) of individual events when compared to default-parameter counterparts. Conclusions Our results indicate that the preferred transformation for fluorescence channels is a parameter- optimized biexponential or generalized Box-Cox, in accordance with current best practices. Interestingly, for populations in the scatter channels, we find that the optimized hyperbolic arcsine may be a better choice in a high-throughput setting than current standard practice of no transformation. However, generally speaking, the choice of transformation remains data-dependent. We have implemented our algorithm in the BioConductor package, flowTrans, which is publicly available. PMID:21050468

Optimizing transformations for automated, high throughput analysis of flow cytometry data.

PubMed

Finak, Greg; Perez, Juan-Manuel; Weng, Andrew; Gottardo, Raphael

2010-11-04

In a high throughput setting, effective flow cytometry data analysis depends heavily on proper data preprocessing. While usual preprocessing steps of quality assessment, outlier removal, normalization, and gating have received considerable scrutiny from the community, the influence of data transformation on the output of high throughput analysis has been largely overlooked. Flow cytometry measurements can vary over several orders of magnitude, cell populations can have variances that depend on their mean fluorescence intensities, and may exhibit heavily-skewed distributions. Consequently, the choice of data transformation can influence the output of automated gating. An appropriate data transformation aids in data visualization and gating of cell populations across the range of data. Experience shows that the choice of transformation is data specific. Our goal here is to compare the performance of different transformations applied to flow cytometry data in the context of automated gating in a high throughput, fully automated setting. We examine the most common transformations used in flow cytometry, including the generalized hyperbolic arcsine, biexponential, linlog, and generalized Box-Cox, all within the BioConductor flowCore framework that is widely used in high throughput, automated flow cytometry data analysis. All of these transformations have adjustable parameters whose effects upon the data are non-intuitive for most users. By making some modelling assumptions about the transformed data, we develop maximum likelihood criteria to optimize parameter choice for these different transformations. We compare the performance of parameter-optimized and default-parameter (in flowCore) data transformations on real and simulated data by measuring the variation in the locations of cell populations across samples, discovered via automated gating in both the scatter and fluorescence channels. We find that parameter-optimized transformations improve visualization, reduce variability in the location of discovered cell populations across samples, and decrease the misclassification (mis-gating) of individual events when compared to default-parameter counterparts. Our results indicate that the preferred transformation for fluorescence channels is a parameter- optimized biexponential or generalized Box-Cox, in accordance with current best practices. Interestingly, for populations in the scatter channels, we find that the optimized hyperbolic arcsine may be a better choice in a high-throughput setting than current standard practice of no transformation. However, generally speaking, the choice of transformation remains data-dependent. We have implemented our algorithm in the BioConductor package, flowTrans, which is publicly available.

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.

Advances in small animal mesentery models for in vivo flow cytometry, dynamic microscopy, and drug screening

PubMed Central

Galanzha, Ekaterina I; Tuchin, Valery V; Zharov, Vladimir P

2007-01-01

Using animal mesentery with intravital optical microscopy is a well-established experimental model for studying blood and lymph microcirculation in vivo. Recent advances in cell biology and optical techniques provide the basis for extending this model for new applications, which should generate significantly improved experimental data. This review summarizes the achievements in this specific area, including in vivo label-free blood and lymph photothermal flow cytometry, super-sensitive fluorescence image cytometry, light scattering and speckle flow cytometry, microvessel dynamic microscopy, infrared (IR) angiography, and high-speed imaging of individual cells in fast flow. The capabilities of these techniques, using the rat mesentery model, were demonstrated in various studies; e.g., real-time quantitative detection of circulating and migrating individual blood and cancer cells, studies on vascular dynamics with a focus on lymphatics under normal conditions and under different interventions (e.g. lasers, drugs, nicotine), assessment of lymphatic disturbances from experimental lymphedema, monitoring cell traffic between blood and lymph systems, and high-speed imaging of cell transient deformability in flow. In particular, the obtained results demonstrated that individual cell transportation in living organisms depends on cell type (e.g., normal blood or leukemic cells), the cell’s functional state (e.g., live, apoptotic, or necrotic), and the functional status of the organism. Possible future applications, including in vivo early diagnosis and prevention of disease, monitoring immune response and apoptosis, chemo- and radio-sensitivity tests, and drug screening, are also discussed. PMID:17226898

In Vivo Flow Cytometry: A Horizon of Opportunities

PubMed Central

Tuchin, Valery V.; Tárnok, Attila; Zharov, Vladimir P.

2012-01-01

Flow cytometry has been a fundamental tool of biological discovery for many years. Invasive extraction of cells from a living organism, however, may lead to changes in cell properties and prevents studying cells in their native environment. These problems can be overcome by use of in vivo flow cytometry which provides detection and imaging of circulating normal and abnormal cells directlyin blood or lymph flow. The goal of this mini-review is to provide a brief history, features and challenges of this new generation of flow cytometry methods and instruments. Spectrum of possibilities of in vivo flow cytometry in biological science (e.g., cell metabolism, immune function, or apoptosis) and medical fields (e.g., cancer, infection, cardiovascular disorder) including integrated photoacoustic-photothermal theranostics of circulating abnormal cells are discussed with focus on recent advances of this new platform. PMID:21915991

Monoclonal Antibody L1Mab-13 Detected Human PD-L1 in Lung Cancers.

PubMed

Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Yanaka, Miyuki; Chang, Yao-Wen; Suzuki, Hiroyoshi; Kaneko, Mika K; Kato, Yukinari

2018-04-01

Programmed cell death ligand-1 (PD-L1) is a type I transmembrane glycoprotein expressed on antigen-presenting cells. It is also expressed in several tumor cells such as melanoma and lung cancer cells. A strong correlation has been reported between human PD-L1 (hPD-L1) expression in tumor cells and negative prognosis in cancer patients. Here, a novel anti-hPD-L1 monoclonal antibody (mAb) L 1 Mab-13 (IgG 1 , kappa) was produced using a cell-based immunization and screening (CBIS) method. We investigated hPD-L1 expression in lung cancer using flow cytometry, Western blot, and immunohistochemical analyses. L 1 Mab-13 specifically reacted hPD-L1 of hPD-L1-overexpressed Chinese hamster ovary (CHO)-K1 cells and endogenous hPD-L1 of KMST-6 (human fibroblast) in flow cytometry and Western blot. Furthermore, L 1 Mab-13 reacted with lung cancer cell lines (EBC-1, Lu65, and Lu99) in flow cytometry and stained lung cancer tissues in a membrane-staining pattern in immunohistochemical analysis. These results indicate that a novel anti-hPD-L1 mAb, L 1 Mab-13, is very useful for detecting hPD-L1 of lung cancers in flow cytometry, Western blot, and immunohistochemical analyses.

Isomorphic red blood cells using automated urine flow cytometry is a reliable method in diagnosis of bladder cancer.

PubMed

Muto, Satoru; Sugiura, Syo-Ichiro; Nakajima, Akiko; Horiuchi, Akira; Inoue, Masahiro; Saito, Keisuke; Isotani, Shuji; Yamaguchi, Raizo; Ide, Hisamitsu; Horie, Shigeo

2014-10-01

We aimed to identify patients with a chief complaint of hematuria who could safely avoid unnecessary radiation and instrumentation in the diagnosis of bladder cancer (BC), using automated urine flow cytometry to detect isomorphic red blood cells (RBCs) in urine. We acquired urine samples from 134 patients over the age of 35 years with a chief complaint of hematuria and a positive urine occult blood test or microhematuria. The data were analyzed using the UF-1000i (®) (Sysmex Co., Ltd., Kobe, Japan) automated urine flow cytometer to determine RBC morphology, which was classified as isomorphic or dysmorphic. The patients were divided into two groups (BC versus non-BC) for statistical analysis. Multivariate logistic regression analysis was used to determine the predictive value of flow cytometry versus urine cytology, the bladder tumor antigen test, occult blood in urine test, and microhematuria test. BC was confirmed in 26 of 134 patients (19.4 %). The area under the curve for RBC count using the automated urine flow cytometer was 0.94, representing the highest reference value obtained in this study. Isomorphic RBCs were detected in all patients in the BC group. On multivariate logistic regression analysis, only isomorphic RBC morphology was significantly predictive for BC (p < 0.001). Analytical parameters such as sensitivity, specificity, positive predictive value, and negative predictive value of isomorphic RBCs in urine were 100.0, 91.7, 74.3, and 100.0 %, respectively. Detection of urinary isomorphic RBCs using automated urine flow cytometry is a reliable method in the diagnosis of BC with hematuria.

Separating the signal from the noise: Expanding flow cytometry into the sub-micron range.

EPA Science Inventory

Cytometry Part A Special Section: Separating the signal from the noise: Expanding flow cytometry into the sub-micron range. The current Cytometry Part A Special Section presents three studies that utilize cytometers to study sub-micron particles. The three studies involve the 1...

Red blood cell microparticles and blood group antigens: an analysis by flow cytometry

PubMed Central

Canellini, Giorgia; Rubin, Olivier; Delobel, Julien; Crettaz, David; Lion, Niels; Tissot, Jean-Daniel

2012-01-01

Background The storage of blood induces the formation of erythrocytes-derived microparticles. Their pathogenic role in blood transfusion is not known so far, especially the risk to trigger alloantibody production in the recipient. This work aims to study the expression of clinically significant blood group antigens on the surface of red blood cells microparticles. Material and methods Red blood cells contained in erythrocyte concentrates were stained with specific antibodies directed against blood group antigens and routinely used in immunohematology practice. After inducing erythrocytes vesiculation with calcium ionophore, the presence of blood group antigens was analysed by flow cytometry. Results The expression of several blood group antigens from the RH, KEL, JK, FY, MNS, LE and LU systems was detected on erythrocyte microparticles. The presence of M (MNS1), N (MNS2) and s (MNS4) antigens could not be demonstrated by flow cytometry, despite that glycophorin A and B were identified on microparticles using anti-CD235a and anti-MNS3. Discussion We conclude that blood group antigens are localized on erythrocytes-derived microparticles and probably keep their immunogenicity because of their capacity to bind specific antibody. Selective segregation process during vesiculation or their ability to elicit an immune response in vivo has to be tested by further studies. PMID:22890266

An imaging flow cytometry method to assess ricin trafficking in A549 human lung epithelial cells.

PubMed

Jenner, Dominic; Chong, Damien; Walker, Nicola; Green, A Christopher

2018-02-01

The endocytosis and trafficking of ricin in mammalian cells is an important area of research for those producing ricin anti-toxins and other ricin therapeutics. Ricin trafficking is usually observed by fluorescence microscopy techniques. This gives good resolution and leads to a detailed understanding of the internal movement of ricin within cells. However, microscopy techniques are often hampered by complex analysis and quantification techniques, and the inability to look at ricin trafficking in large populations of cells. In these studies we have directly labelled ricin and assessed if its trafficking can be observed using Imaging Flow Cytometry (IFC) both to the cytoplasmic region of cells and specifically to the Golgi apparatus. Using IDEAS® data analysis software the specific fluorescence location of the ricin within the cells was analysed. Then, using cytoplasmic masking techniques to quantify the number of cells with endocytosed cytoplasmic ricin or cells with Golgi-associated ricin, kinetic endocytosis curves were generated. Here we present, to the authors' knowledge, the first example of using imaging flow cytometry for evaluating the subcellular transport of protein cargo, using the trafficking of ricin toxin in lung cells as a model. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

Development of an Anti-HER2 Monoclonal Antibody H2Mab-139 Against Colon Cancer.

PubMed

Kaneko, Mika K; Yamada, Shinji; Itai, Shunsuke; Kato, Yukinari

2018-02-01

Human epidermal growth factor receptor 2 (HER2) expression has been reported in several cancers, such as breast, gastric, lung, pancreatic, and colorectal cancers. HER2 is overexpressed in those cancers and is associated with poor clinical outcomes. Trastuzumab, a humanized anti-HER2 antibody, provides significant survival benefits for patients with HER2-overexpressing breast cancers and gastric cancers. In this study, we developed a novel anti-HER2 monoclonal antibody (mAb), H 2 Mab-139 (IgG 1 , kappa) and investigated it against colon cancers using flow cytometry, western blot, and immunohistochemical analyses. Flow cytometry analysis revealed that H 2 Mab-139 reacted with colon cancer cell lines, such as Caco-2, HCT-116, HCT-15, HT-29, LS 174T, COLO 201, COLO 205, HCT-8, SW1116, and DLD-1. Although H 2 Mab-139 strongly reacted with LN229/HER2 cells on the western blot, we did not observe a specific signal for HER2 in colon cancer cell lines. Immunohistochemical analyses revealed sensitive and specific reactions of H 2 Mab-139 against colon cancers, indicating that H 2 Mab-139 is useful in detecting HER2 overexpression in colon cancers using flow cytometry and immunohistochemical analyses.

FuGEFlow: data model and markup language for flow cytometry.

PubMed

Qian, Yu; Tchuvatkina, Olga; Spidlen, Josef; Wilkinson, Peter; Gasparetto, Maura; Jones, Andrew R; Manion, Frank J; Scheuermann, Richard H; Sekaly, Rafick-Pierre; Brinkman, Ryan R

2009-06-16

Flow cytometry technology is widely used in both health care and research. The rapid expansion of flow cytometry applications has outpaced the development of data storage and analysis tools. Collaborative efforts being taken to eliminate this gap include building common vocabularies and ontologies, designing generic data models, and defining data exchange formats. The Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard was recently adopted by the International Society for Advancement of Cytometry. This standard guides researchers on the information that should be included in peer reviewed publications, but it is insufficient for data exchange and integration between computational systems. The Functional Genomics Experiment (FuGE) formalizes common aspects of comprehensive and high throughput experiments across different biological technologies. We have extended FuGE object model to accommodate flow cytometry data and metadata. We used the MagicDraw modelling tool to design a UML model (Flow-OM) according to the FuGE extension guidelines and the AndroMDA toolkit to transform the model to a markup language (Flow-ML). We mapped each MIFlowCyt term to either an existing FuGE class or to a new FuGEFlow class. The development environment was validated by comparing the official FuGE XSD to the schema we generated from the FuGE object model using our configuration. After the Flow-OM model was completed, the final version of the Flow-ML was generated and validated against an example MIFlowCyt compliant experiment description. The extension of FuGE for flow cytometry has resulted in a generic FuGE-compliant data model (FuGEFlow), which accommodates and links together all information required by MIFlowCyt. The FuGEFlow model can be used to build software and databases using FuGE software toolkits to facilitate automated exchange and manipulation of potentially large flow cytometry experimental data sets. Additional project documentation, including reusable design patterns and a guide for setting up a development environment, was contributed back to the FuGE project. We have shown that an extension of FuGE can be used to transform minimum information requirements in natural language to markup language in XML. Extending FuGE required significant effort, but in our experiences the benefits outweighed the costs. The FuGEFlow is expected to play a central role in describing flow cytometry experiments and ultimately facilitating data exchange including public flow cytometry repositories currently under development.

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

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

Flow Cytometry and Solid Organ Transplantation: A Perfect Match

PubMed Central

Maguire, Orla; Tario, Joseph D.; Shanahan, Thomas C.; Wallace, Paul K.; Minderman, Hans

2015-01-01

In the field of transplantation, flow cytometry serves a well-established role in pre-transplant crossmatching and monitoring immune reconstitution following hematopoietic stem cell transplantation. The capabilities of flow cytometers have continuously expanded and this combined with more detailed knowledge of the constituents of the immune system, their function and interaction and newly developed reagents to study these parameters have led to additional utility of flow cytometry-based analyses, particularly in the post-transplant setting. This review discusses the impact of flow cytometry on managing alloantigen reactions, monitoring opportunistic infections and graft rejection and gauging immunosuppression in the context of solid organ transplantation. PMID:25296232

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.

Pumpless Microflow Cytometry Enabled by Viscosity Modulation and Immunobead Labeling.

PubMed

Kim, Byeongyeon; Oh, Sein; Shin, Suyeon; Yim, Sang-Gu; Yang, Seung Yun; Hahn, Young Ki; Choi, Sungyoung

2018-06-19

Major challenges of miniaturizing flow cytometry include obviating the need for bulky, expensive, and complex pump-based fluidic and laser-based optical systems while retaining the ability to detect target cells based on their unique surface receptors. We addressed these critical challenges by (i) using a viscous liquid additive to control flow rate passively, without external pumping equipment, and (ii) adopting an immunobead assay that can be quantified with a portable fluorescence cell counter based on a blue light-emitting diode. Such novel features enable pumpless microflow cytometry (pFC) analysis by simply dropping a sample solution onto the inlet reservoir of a disposable cell-counting chamber. With our pFC platform, we achieved reliable cell counting over a dynamic range of 9-298 cells/μL. We demonstrated the practical utility of the platform by identifying a type of cancer cell based on CD326, the epithelial cell adhesion molecule. This portable microflow cytometry platform can be applied generally to a range of cell types using immunobeads labeled with specific antibodies, thus making it valuable for cell-based and point-of-care diagnostics.

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.

A benchmark for evaluation of algorithms for identification of cellular correlates of clinical outcomes.

PubMed

Aghaeepour, Nima; Chattopadhyay, Pratip; Chikina, Maria; Dhaene, Tom; Van Gassen, Sofie; Kursa, Miron; Lambrecht, Bart N; Malek, Mehrnoush; McLachlan, G J; Qian, Yu; Qiu, Peng; Saeys, Yvan; Stanton, Rick; Tong, Dong; Vens, Celine; Walkowiak, Sławomir; Wang, Kui; Finak, Greg; Gottardo, Raphael; Mosmann, Tim; Nolan, Garry P; Scheuermann, Richard H; Brinkman, Ryan R

2016-01-01

The Flow Cytometry: Critical Assessment of Population Identification Methods (FlowCAP) challenges were established to compare the performance of computational methods for identifying cell populations in multidimensional flow cytometry data. Here we report the results of FlowCAP-IV where algorithms from seven different research groups predicted the time to progression to AIDS among a cohort of 384 HIV+ subjects, using antigen-stimulated peripheral blood mononuclear cell (PBMC) samples analyzed with a 14-color staining panel. Two approaches (FlowReMi.1 and flowDensity-flowType-RchyOptimyx) provided statistically significant predictive value in the blinded test set. Manual validation of submitted results indicated that unbiased analysis of single cell phenotypes could reveal unexpected cell types that correlated with outcomes of interest in high dimensional flow cytometry datasets. © 2015 International Society for Advancement of Cytometry.

FISH-Flow, a protocol for the concurrent detection of mRNA and protein in single cells using fluorescence in situ hybridization and flow cytometry

PubMed Central

Arrigucci, Riccardo; Bushkin, Yuri; Radford, Felix; Lakehal, Karim; Vir, Pooja; Pine, Richard; Martin, December; Sugarman, Jeffrey; Zhao, Yanlin; Yap, George S; Lardizabal, Alfred A; Tyagi, Sanjay; Gennaro, Maria Laura

2017-01-01

We describe a flow-cytometry-based protocol for intracellular mRNA measurements in nonadherent mammalian cells using fluorescence in situ hybridization (FISH) probes. The method, which we call FISH-Flow, allows for high-throughput multiparametric measurements of gene expression, a task that was not feasible with earlier, microscopy-based approaches. The FISH-Flow protocol involves cell fixation, permeabilization and hybridization with a set of fluorescently labeled oligonucleotide probes. In this protocol, surface and intracellular protein markers can also be stained with fluorescently labeled antibodies for simultaneous protein and mRNA measurement. Moreover, a semiautomated, single-tube version of the protocol can be performed with a commercially available cell-wash device that reduces cell loss, operator time and interoperator variability. It takes ~30 h to perform this protocol. An example of FISH-Flow measurements of cytokine mRNA induction by ex vivo stimulation of primed T cells with specific antigens is described. PMID:28518171

Tips and tricks for flow cytometry-based analysis and counting of microparticles.

PubMed

Poncelet, Philippe; Robert, Stéphane; Bailly, Nicolas; Garnache-Ottou, Francine; Bouriche, Tarik; Devalet, Bérangère; Segatchian, Jerard H; Saas, Philippe; Mullier, François

2015-10-01

Submicron-sized extra-cellular vesicles generated by budding from the external cell membranes, microparticles (MPs) are important actors in transfusion as well as in other medical specialties. After briefly positioning their role in the characterization of labile blood products, this technically oriented chapter aims to review practical points that need to be considered when trying to use flow cytometry for the analysis, characterization and absolute counting of MP subsets. Subjects of active discussions relative to instrumentation will include the choice of the trigger parameter, possible standardization approaches requiring instrument quality-control, origin and control of non-specific background and of coincidence artifacts, choice of the type of electronic signals, optimal sheath fluid and sample speed. Questions related to reagents will cover target antigens and receptors, multi-color reagents, negative controls, enumeration of MPs and limiting artifacts due to unexpected (micro-) coagulation of plasma samples. Newly detected problems are generating innovative solutions and flow cytometry will continue to remain the technology of choice for the analysis of MPs, in the domain of transfusion as well as in many diverse specialties. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

The development and validation of different decision-making tools to predict urine culture growth out of urine flow cytometry parameter.

PubMed

Müller, Martin; Seidenberg, Ruth; Schuh, Sabine K; Exadaktylos, Aristomenis K; Schechter, Clyde B; Leichtle, Alexander B; Hautz, Wolf E

2018-01-01

Patients presenting with suspected urinary tract infection are common in every day emergency practice. Urine flow cytometry has replaced microscopic urine evaluation in many emergency departments, but interpretation of the results remains challenging. The aim of this study was to develop and validate tools that predict urine culture growth out of urine flow cytometry parameter. This retrospective study included all adult patients that presented in a large emergency department between January and July 2017 with a suspected urinary tract infection and had a urine flow cytometry as well as a urine culture obtained. The objective was to identify urine flow cytometry parameters that reliably predict urine culture growth and mixed flora growth. The data set was split into a training (70%) and a validation set (30%) and different decision-making approaches were developed and validated. Relevant urine culture growth (respectively mixed flora growth) was found in 40.2% (7.2% respectively) of the 613 patients included. The number of leukocytes and bacteria in flow cytometry were highly associated with urine culture growth, but mixed flora growth could not be sufficiently predicted from the urine flow cytometry parameters. A decision tree, predictive value figures, a nomogram, and a cut-off table to predict urine culture growth from bacteria and leukocyte count were developed, validated and compared. Urine flow cytometry parameters are insufficient to predict mixed flora growth. However, the prediction of urine culture growth based on bacteria and leukocyte count is highly accurate and the developed tools should be used as part of the decision-making process of ordering a urine culture or starting an antibiotic therapy if a urogenital infection is suspected.

The development and validation of different decision-making tools to predict urine culture growth out of urine flow cytometry parameter

PubMed Central

Seidenberg, Ruth; Schuh, Sabine K.; Exadaktylos, Aristomenis K.; Schechter, Clyde B.; Leichtle, Alexander B.; Hautz, Wolf E.

2018-01-01

Objective Patients presenting with suspected urinary tract infection are common in every day emergency practice. Urine flow cytometry has replaced microscopic urine evaluation in many emergency departments, but interpretation of the results remains challenging. The aim of this study was to develop and validate tools that predict urine culture growth out of urine flow cytometry parameter. Methods This retrospective study included all adult patients that presented in a large emergency department between January and July 2017 with a suspected urinary tract infection and had a urine flow cytometry as well as a urine culture obtained. The objective was to identify urine flow cytometry parameters that reliably predict urine culture growth and mixed flora growth. The data set was split into a training (70%) and a validation set (30%) and different decision-making approaches were developed and validated. Results Relevant urine culture growth (respectively mixed flora growth) was found in 40.2% (7.2% respectively) of the 613 patients included. The number of leukocytes and bacteria in flow cytometry were highly associated with urine culture growth, but mixed flora growth could not be sufficiently predicted from the urine flow cytometry parameters. A decision tree, predictive value figures, a nomogram, and a cut-off table to predict urine culture growth from bacteria and leukocyte count were developed, validated and compared. Conclusions Urine flow cytometry parameters are insufficient to predict mixed flora growth. However, the prediction of urine culture growth based on bacteria and leukocyte count is highly accurate and the developed tools should be used as part of the decision-making process of ordering a urine culture or starting an antibiotic therapy if a urogenital infection is suspected. PMID:29474463

Imaging flow cytometry for phytoplankton analysis.

PubMed

Dashkova, Veronika; Malashenkov, Dmitry; Poulton, Nicole; Vorobjev, Ivan; Barteneva, Natasha S

2017-01-01

This review highlights the concepts and instrumentation of imaging flow cytometry technology and in particular its use for phytoplankton analysis. Imaging flow cytometry, a hybrid technology combining speed and statistical capabilities of flow cytometry with imaging features of microscopy, is rapidly advancing as a cell imaging platform that overcomes many of the limitations of current techniques and contributed significantly to the advancement of phytoplankton analysis in recent years. This review presents the various instrumentation relevant to the field and currently used for assessment of complex phytoplankton communities' composition and abundance, size structure determination, biovolume estimation, detection of harmful algal bloom species, evaluation of viability and metabolic activity and other applications. Also we present our data on viability and metabolic assessment of Aphanizomenon sp. cyanobacteria using Imagestream X Mark II imaging cytometer. Herein, we highlight the immense potential of imaging flow cytometry for microalgal research, but also discuss limitations and future developments. Copyright © 2016 Elsevier Inc. All rights reserved.

FuGEFlow: data model and markup language for flow cytometry

PubMed Central

Qian, Yu; Tchuvatkina, Olga; Spidlen, Josef; Wilkinson, Peter; Gasparetto, Maura; Jones, Andrew R; Manion, Frank J; Scheuermann, Richard H; Sekaly, Rafick-Pierre; Brinkman, Ryan R

2009-01-01

Background Flow cytometry technology is widely used in both health care and research. The rapid expansion of flow cytometry applications has outpaced the development of data storage and analysis tools. Collaborative efforts being taken to eliminate this gap include building common vocabularies and ontologies, designing generic data models, and defining data exchange formats. The Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard was recently adopted by the International Society for Advancement of Cytometry. This standard guides researchers on the information that should be included in peer reviewed publications, but it is insufficient for data exchange and integration between computational systems. The Functional Genomics Experiment (FuGE) formalizes common aspects of comprehensive and high throughput experiments across different biological technologies. We have extended FuGE object model to accommodate flow cytometry data and metadata. Methods We used the MagicDraw modelling tool to design a UML model (Flow-OM) according to the FuGE extension guidelines and the AndroMDA toolkit to transform the model to a markup language (Flow-ML). We mapped each MIFlowCyt term to either an existing FuGE class or to a new FuGEFlow class. The development environment was validated by comparing the official FuGE XSD to the schema we generated from the FuGE object model using our configuration. After the Flow-OM model was completed, the final version of the Flow-ML was generated and validated against an example MIFlowCyt compliant experiment description. Results The extension of FuGE for flow cytometry has resulted in a generic FuGE-compliant data model (FuGEFlow), which accommodates and links together all information required by MIFlowCyt. The FuGEFlow model can be used to build software and databases using FuGE software toolkits to facilitate automated exchange and manipulation of potentially large flow cytometry experimental data sets. Additional project documentation, including reusable design patterns and a guide for setting up a development environment, was contributed back to the FuGE project. Conclusion We have shown that an extension of FuGE can be used to transform minimum information requirements in natural language to markup language in XML. Extending FuGE required significant effort, but in our experiences the benefits outweighed the costs. The FuGEFlow is expected to play a central role in describing flow cytometry experiments and ultimately facilitating data exchange including public flow cytometry repositories currently under development. PMID:19531228

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.

Flow cytometry analysis of inflammatory cells isolated from the sciatic nerve and DRG after chronic constriction injury in mice.

PubMed

Liu, Liping; Yin, Yan; Li, Fei; Malhotra, Charvi; Cheng, Jianguo

2017-06-01

Cellular responses to nerve injury play a central role in the pathogenesis of neuropathic pain. However, the analysis of site specific cellular responses to nerve injury and neuropathic pain is limited to immunohistochemistry staining with numerous limitations. We proposed to apply flow cytometry to overcome some of the limitations and developed two protocols for isolation of cells from small specimens of the sciatic nerve and dorsal root ganglion (DRG) in mice. RESULTS AND COMPARASION WITH EXISTING: methods We found that both the non-enzymatic and enzymatic approaches were highly effective in harvesting a sufficient number of cells for flow cytometry analysis in normal and pathological conditions. The total number of cells in the injury site of the sciatic and its DRGs increased significantly 14days after chronic constriction injury (CCI) of the sciatic nerve, compared to sham surgery control or the contralateral control. The enzymatic approach yielded a significantly higher total number of cells and CD45 negative cells, suggesting that this approach allows for harvest of more resident cells, compared to the non-enzymatic method. The percentage of CD45 + /CD11b + cells was significantly increased in the sciatic nerve but not in the DRG. These results were consistent with both protocols. We thus offer two simple and effective protocols that allow for application of flow cytometry to the investigation of cellular and molecular mechanisms of neuropathic pain. Copyright © 2017 Elsevier B.V. All rights reserved.

Label-free high-throughput imaging flow cytometry

NASA Astrophysics Data System (ADS)

Mahjoubfar, A.; Chen, C.; Niazi, K. R.; Rabizadeh, S.; Jalali, B.

2014-03-01

Flow cytometry is an optical method for studying cells based on their individual physical and chemical characteristics. It is widely used in clinical diagnosis, medical research, and biotechnology for analysis of blood cells and other cells in suspension. Conventional flow cytometers aim a laser beam at a stream of cells and measure the elastic scattering of light at forward and side angles. They also perform single-point measurements of fluorescent emissions from labeled cells. However, many reagents used in cell labeling reduce cellular viability or change the behavior of the target cells through the activation of undesired cellular processes or inhibition of normal cellular activity. Therefore, labeled cells are not completely representative of their unaltered form nor are they fully reliable for downstream studies. To remove the requirement of cell labeling in flow cytometry, while still meeting the classification sensitivity and specificity goals, measurement of additional biophysical parameters is essential. Here, we introduce an interferometric imaging flow cytometer based on the world's fastest continuous-time camera. Our system simultaneously measures cellular size, scattering, and protein concentration as supplementary biophysical parameters for label-free cell classification. It exploits the wide bandwidth of ultrafast laser pulses to perform blur-free quantitative phase and intensity imaging at flow speeds as high as 10 meters per second and achieves nanometer-scale optical path length resolution for precise measurements of cellular protein concentration.

Multinode acoustic focusing for parallel flow cytometry

PubMed Central

Piyasena, Menake E.; Suthanthiraraj, Pearlson P. Austin; Applegate, Robert W.; Goumas, Andrew M.; Woods, Travis A.; López, Gabriel P.; Graves, Steven W.

2012-01-01

Flow cytometry can simultaneously measure and analyze multiple properties of single cells or particles with high sensitivity and precision. Yet, conventional flow cytometers have fundamental limitations with regards to analyzing particles larger than about 70 microns, analyzing at flow rates greater than a few hundred microliters per minute, and providing analysis rates greater than 50,000 per second. To overcome these limits, we have developed multi-node acoustic focusing flow cells that can position particles (as small as a red blood cell and as large as 107 microns in diameter) into as many as 37 parallel flow streams. We demonstrate the potential of such flow cells for the development of high throughput, parallel flow cytometers by precision focusing of flow cytometry alignment microspheres, red blood cells, and the analysis of CD4+ cellular immunophenotyping assay. This approach will have significant impact towards the creation of high throughput flow cytometers for rare cell detection applications (e.g. circulating tumor cells), applications requiring large particle analysis, and high volume flow cytometry. PMID:22239072

Peripheral T-cell lymphomas of follicular helper T-cell type frequently display an aberrant CD3(-/dim)CD4(+) population by flow cytometry: an important clue to the diagnosis of a Hodgkin lymphoma mimic.

PubMed

Alikhan, Mir; Song, Joo Y; Sohani, Aliyah R; Moroch, Julien; Plonquet, Anne; Duffield, Amy S; Borowitz, Michael J; Jiang, Liuyan; Bueso-Ramos, Carlos; Inamdar, Kedar; Menon, Madhu P; Gurbuxani, Sandeep; Chan, Ernest; Smith, Sonali M; Nicolae, Alina; Jaffe, Elaine S; Gaulard, Philippe; Venkataraman, Girish

2016-10-01

Nodal follicular helper T-cell-derived lymphoproliferations (specifically the less common peripheral T-cell lymphomas of follicular type) exhibit a spectrum of histologic features that may mimic reactive hyperplasia or Hodgkin lymphoma. Even though angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma of follicular type share a common biologic origin from follicular helper T-cells and their morphology has been well characterized, flow cytometry of peripheral T-cell lymphomas of follicular type has not been widely discussed as a tool for identifying this reactive hyperplasia/Hodgkin lymphoma mimic. We identified 10 peripheral T-cell lymphomas of follicular type with available flow cytometry data from five different institutions, including two cases with peripheral blood evaluation. For comparison, we examined flow cytometry data for 8 classical Hodgkin lymphomas (including 1 lymphocyte-rich classical Hodgkin lymphoma), 15 nodular lymphocyte predominant Hodgkin lymphomas, 15 angioimmunoblastic T-cell lymphomas, and 26 reactive nodes. Lymph node histology and flow cytometry data were reviewed, specifically for the presence of a CD3(-/dim)CD4(+) aberrant T-cell population (described in angioimmunoblastic T-cell lymphomas), besides other T-cell aberrancies. Nine of 10 (90%) peripheral T-cell lymphomas of follicular type showed a CD3(-/dim)CD4(+) T-cell population constituting 29.3% (range 7.9-62%) of all lymphocytes. Five of 10 (50%) had nodular lymphocyte predominant Hodgkin lymphoma or lymphocyte-rich classical Hodgkin lymphoma-like morphology with scattered Hodgkin-like cells that expressed CD20, CD30, CD15, and MUM1. Three cases had a nodular growth pattern and three others exhibited a perifollicular growth pattern without Hodgkin-like cells. Epstein-Barr virus was positive in 1 of 10 cases (10%). PCR analysis showed clonal T-cell receptor gamma gene rearrangement in all 10 peripheral T-cell lymphomas of follicular type. By flow cytometry, 11 of 15 (73.3%) angioimmunoblastic T-cell lymphomas showed the CD3(-/dim)CD4(+) population (mean: 19.5%, range: 3-71.8%). Using a threshold of 3% for CD3(-/dim)CD4(+) T cells, all 15 nodular lymphocyte predominant Hodgkin lymphoma controls and 8 classical Hodgkin lymphomas were negative (Mann-Whitney P=0.01, F-PTCL vs Hodgkin lymphomas), as were 25 of 26 reactive lymph nodes. The high frequency of CD3(-/dim)CD4(+) aberrant T cells is similar in angioimmunoblastic T-cell lymphomas and peripheral T-cell lymphomas of follicular type, and is a useful feature in distinguishing peripheral T-cell lymphomas of follicular type from morphologic mimics such as reactive hyperplasia or Hodgkin lymphoma.

A new "Logicle" display method avoids deceptive effects of logarithmic scaling for low signals and compensated data.

PubMed

Parks, David R; Roederer, Mario; Moore, Wayne A

2006-06-01

In immunofluorescence measurements and most other flow cytometry applications, fluorescence signals of interest can range down to essentially zero. After fluorescence compensation, some cell populations will have low means and include events with negative data values. Logarithmic presentation has been very useful in providing informative displays of wide-ranging flow cytometry data, but it fails to adequately display cell populations with low means and high variances and, in particular, offers no way to include negative data values. This has led to a great deal of difficulty in interpreting and understanding flow cytometry data, has often resulted in incorrect delineation of cell populations, and has led many people to question the correctness of compensation computations that were, in fact, correct. We identified a set of criteria for creating data visualization methods that accommodate the scaling difficulties presented by flow cytometry data. On the basis of these, we developed a new data visualization method that provides important advantages over linear or logarithmic scaling for display of flow cytometry data, a scaling we refer to as "Logicle" scaling. Logicle functions represent a particular generalization of the hyperbolic sine function with one more adjustable parameter than linear or logarithmic functions. Finally, we developed methods for objectively and automatically selecting an appropriate value for this parameter. The Logicle display method provides more complete, appropriate, and readily interpretable representations of data that includes populations with low-to-zero means, including distributions resulting from fluorescence compensation procedures, than can be produced using either logarithmic or linear displays. The method includes a specific algorithm for evaluating actual data distributions and deriving parameters of the Logicle scaling function appropriate for optimal display of that data. It is critical to note that Logicle visualization does not change the data values or the descriptive statistics computed from them. Copyright 2006 International Society for Analytical Cytology.

Accuracy of Automated Flow Cytometry-Based Leukocyte Counts To Rule Out Urinary Tract Infection in Febrile Children: a Prospective Cross-Sectional Study

PubMed Central

Duong, Hong Phuoc; Wissing, Karl Martin; Tram, Nathalie; Mascart, Georges; Lepage, Philippe

2016-01-01

Automated flow cytometry of urine remains an incompletely validated method to rule out urinary tract infection (UTI) in children. This cross-sectional analytical study was performed to compare the predictive values of flow cytometry and a dipstick test as initial diagnostic tests for UTI in febrile children and prospectively included 1,106 children (1,247 episodes). Urine culture was used as the gold standard test for diagnosing UTI. The performance of screening tests to diagnose UTI were established using receiver operating characteristic (ROC) analysis. Among these 1,247 febrile episodes, 221 UTIs were diagnosed (17.7% [95% confidence interval {CI}, 15.6 to 19.8%]). The area under the ROC curve for flow cytometry white blood cell (WBC) counts (0.99 [95% CI, 0.98 to 0.99]) was significantly superior to that for red blood cell (0.74 [95% CI, 0.70 to 0.78]) and bacterial counts (0.89 [95% CI, 0.87 to 0.92]) (P < 0.001). Urinary WBC counts also had a significantly higher area under the ROC curve than that of the leukocyte esterase (LE) dipstick (0.92 [95% CI, 0.90 to 0.94]), nitrite dipstick (0.83 [95% CI, 0.80 to 0.87]), or the combination of positive LE and/or nitrite dipstick (0.91 [95% CI, 0.89 to 0.93]) test (P < 0.001). The presence of ≥35 WBC/μl of urine was the best cutoff point, yielding both a high sensitivity (99.5% [95% CI, 99 to 100%]) and an acceptable specificity (80.6% [95% CI, 78 to 83%]). Using this cutoff point would have reduced the number of samples sent to the laboratory for culture by 67%. In conclusion, the determination of urinary WBC counts by flow cytometry provides optimal performance as an initial diagnostic test for UTI in febrile children. PMID:27682127

Biomarkers of Selenium Chemoprevention of Prostate Cancer

DTIC Science & Technology

2005-01-01

than Se-Met in inhibiting Flow Kit from BD Pharmigen (San Diego, CA). Stained cells were then quantified by flow cytometry , and the data were analyzed...decrease in Quantitation of Apoptosis by Flow Cytometry . PC-3 cells were plated at cell number accumulation by MSA was related to cell cycle arrest, we... flow exposed to either 5 or 10Mm MSA for 48 or 72 h. Adherent cells harvested by mild cytometry of ethanol-permeabilized cells stained with Pl. Synchro

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

Small lasers in flow cytometry.

PubMed

Telford, William G

2004-01-01

Laser technology has made tremendous advances in recent years, particularly in the area of diode and diode-pumped solid state sources. Flow cytometry has been a direct beneficiary of these advances, as these small, low-maintenance, inexpensive lasers with reasonable power outputs are integrated into flow cytometers. In this chapter we review the contribution and potential of solid-state lasers to flow cytometry, and show several examples of these novel sources integrated into production flow cytometers. Technical details and critical parameters for successful application of these lasers for biomedical analysis are reviewed.

Development of a Fluorescent Based Immunosensor for the Serodiagnosis of Canine Leishmaniasis Combining Immunomagnetic Separation and Flow Cytometry

PubMed Central

Sousa, Susana; Cardoso, Luís; Reed, Steven G.; Reis, Alexandre B.; Martins-Filho, Olindo A.; Silvestre, Ricardo; Cordeiro da Silva, Anabela

2013-01-01

Background An accurate diagnosis is essential for the control of infectious diseases. In the search for effective and efficient tests, biosensors have increasingly been exploited for the development of new and highly sensitive diagnostic methods. Here, we describe a new fluorescent based immunosensor comprising magnetic polymer microspheres coated with recombinant antigens to improve the detection of specific antibodies generated during an infectious disease. As a challenging model, we used canine leishmaniasis due to the unsatisfactory sensitivity associated with the detection of infection in asymptomatic animals where the levels of pathogen-specific antibodies are scarce. Methodology Ni-NTA magnetic microspheres with 1,7 µm and 8,07 µm were coated with the Leishmania recombinant proteins LicTXNPx and rK39, respectively. A mixture of equal proportions of both recombinant protein-coated microspheres was used to recognize and specifically bind anti-rK39 and anti-LicTNXPx antibodies present in serum samples of infected dogs. The microspheres were recovered by magnetic separation and the percentage of fluorescent positive microspheres was quantified by flow cytometry. Principal Findings A clinical evaluation carried out with 129 dog serum samples using the antigen combination demonstrated a sensitivity of 98,8% with a specificity of 94,4%. rK39 antigen alone demonstrated a higher sensitivity for symptomatic dogs (96,9%), while LicTXNPx antigen showed a higher sensitivity for asymptomatic (94,4%). Conclusions Overall, our results demonstrated the potential of a magnetic microsphere associated flow cytometry methodology as a viable tool for highly sensitive laboratorial serodiagnosis of both clinical and subclinical forms of canine leishmaniasis. PMID:23991232

Flow cytometry as an improved method for the titration of Chlamydiaceae and other intracellular bacteria.

PubMed

Käser, T; Pasternak, J A; Hamonic, G; Rieder, M; Lai, K; Delgado-Ortega, M; Gerdts, V; Meurens, F

2016-05-01

Chlamydiaceae is a family of intracellular bacteria causing a range of diverse pathological outcomes. The most devastating human diseases are ocular infections with C. trachomatis leading to blindness and genital infections causing pelvic inflammatory disease with long-term sequelae including infertility and chronic pelvic pain. In order to enable the comparison of experiments between laboratories investigating host-chlamydia interactions, the infectious titer has to be determined. Titer determination of chlamydia is most commonly performed via microscopy of host cells infected with a serial dilution of chlamydia. However, other methods including fluorescent ELISpot (Fluorospot) and DNA Chip Scanning Technology have also been proposed to enumerate chlamydia-infected cells. For viruses, flow cytometry has been suggested as a superior alternative to standard titration methods. In this study we compared the use of flow cytometry with microscopy and Fluorospot for the titration of C. suis as a representative of other intracellular bacteria. Titer determination via Fluorospot was unreliable, while titration via microscopy led to a linear read-out range of 16 - 64 dilutions and moderate reproducibility with acceptable standard deviations within and between investigators. In contrast, flow cytometry had a vast linear read-out range of 1,024 dilutions and the lowest standard deviations given a basic training in these methods. In addition, flow cytometry was faster and material costs were lower compared to microscopy. Flow cytometry offers a fast, cheap, precise, and reproducible alternative for the titration of intracellular bacteria like C. suis. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

Cytometry standards continuum

NASA Astrophysics Data System (ADS)

Leif, Robert C.; Spidlen, Josef; Brinkman, Ryan R.

2008-02-01

Introduction: The International Society for Analytical Cytology, ISAC, is developing a new combined flow and image Analytical Cytometry Standard (ACS). This standard needs to serve both the research and clinical communities. The clinical medicine and clinical research communities have a need to exchange information with hospital and other clinical information systems. Methods: 1) Prototype the standard by creating CytometryML and a RAW format for binary data. 2) Join the ISAC Data Standards Task Force. 3) Create essential project documentation. 4) Cooperate with other groups by assisting in the preparation of the DICOM Supplement 122: Specimen Module and Pathology Service-Object Pair Classes. Results: CytometryML has been created and serves as a prototype and source of experience for the following: the Analytical Cytometry Standard (ACS) 1.0, the ACS container, Minimum Information about a Flow Cytometry Experiment (MIFlowCyt), and Requirements for a Data File Standard Format to Describe Flow Cytometry and Related Analytical Cytology Data. These requirements provide a means to judge the appropriateness of design elements and to develop tests for the final ACS. The requirements include providing the information required for understanding and reproducing a cytometry experiment or clinical measurement, and for a single standard for both flow and digital microscopic cytometry. Schemas proposed by other members of the ISAC Data Standards Task Force (e.g, Gating-ML) have been independently validated and have been integrated with CytometryML. The use of netCDF as an element of the ACS container has been proposed by others and a suggested method of its use is proposed.

Fundamentals of flow cytometry.

PubMed

Jaroszeski, M J; Radcliff, G

1999-02-01

Flow cytometers are instruments that are used primarily to measure the physical and biochemical characteristics of biological particles. This technology is used to perform measurements on whole cells as well as prepared cellular constituents, such as nuclei and organelles. Flow cytometers are investigative tools for a broad range of scientific disciplines because they make measurements on thousands of individual cells/particles in a matter of seconds. This is a unique advantage relative to other detection instruments that provide bulk particle measurements. Flow cytometry is a complex and highly technical field; therefore, a basic understanding of the technology is essential for all users. The purpose of this article is to provide fundamental information about the instrumentation used for flow cytometry as well as the methods used to analyze and interpret data. This information will provide a foundation to use flow cytometry effectively as a research tool.

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

Diagnosis of Plasma Cell Dyscrasias and Monitoring of Minimal Residual Disease by Multiparametric Flow Cytometry

PubMed Central

Soh, Kah Teong; Tario, Joseph D.; Wallace, Paul K.

2018-01-01

Synopsis Plasma cell dyscrasia (PCD) is a heterogeneous disease which has seen a tremendous change in outcomes due to improved therapies. Over the last few decades, multiparametric flow cytometry has played an important role in the detection and monitoring of PCDs. Flow cytometry is a high sensitivity assay for early detection of minimal residual disease (MRD) that correlates well with progression-free survival and overall survival. Before flow cytometry can be effectively implemented in the clinical setting sample preparation, panel configuration, analysis, and gating strategies must be optimized to ensure accurate results. Current consensus methods and reporting guidelines for MRD testing are discussed. PMID:29128071

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.

Monitoring survival and function of transfused platelets in Bernard-Soulier syndrome by flow cytometry and a cone and plate(let) analyzer (Impact-R).

PubMed

Panzer, Simon; Eichelberger, Beate; Koren, Daniela; Kaufmann, Karin; Male, Christoph

2007-01-01

Bernard-Soulier syndrome (BSS) patients may repeatedly require transfusion of platelets (PLTs). The hemostatic competence of transfused PLTs requires monitoring. Flow cytometry and a cone and plate(let) analyzer (Impact-R, DiaMed) were used to monitor survival and function of transfused PLTs in a 7-year-old girl with BSS undergoing surgery. Flow cytometry was applied to differentiate autologous PLTs from transfused PLTs by staining for CD42b. The Impact, which measures PLT adhesion and aggregation in response to high shear stress, was used to evaluate PLT function. Transfused PLTs were detectable by flow cytometry for 1 week after transfusion. While the patient's PLTs did not respond to high shear stress before transfusion, a normal response was documented by the Impact on the day after transfusion and 1 week thereafter. Transfused PLTs were detectable by flow cytometry, and their functional activity was demonstrated by the Impact.

A Method for the Interpretation of Flow Cytometry Data Using Genetic Algorithms.

PubMed

Angeletti, Cesar

2018-01-01

Flow cytometry analysis is the method of choice for the differential diagnosis of hematologic disorders. It is typically performed by a trained hematopathologist through visual examination of bidimensional plots, making the analysis time-consuming and sometimes too subjective. Here, a pilot study applying genetic algorithms to flow cytometry data from normal and acute myeloid leukemia subjects is described. Initially, Flow Cytometry Standard files from 316 normal and 43 acute myeloid leukemia subjects were transformed into multidimensional FITS image metafiles. Training was performed through introduction of FITS metafiles from 4 normal and 4 acute myeloid leukemia in the artificial intelligence system. Two mathematical algorithms termed 018330 and 025886 were generated. When tested against a cohort of 312 normal and 39 acute myeloid leukemia subjects, both algorithms combined showed high discriminatory power with a receiver operating characteristic (ROC) curve of 0.912. The present results suggest that machine learning systems hold a great promise in the interpretation of hematological flow cytometry data.

Characterization of neutrophils and macrophages from ex vivo cultured murine bone marrow for morphologic maturation and functional responses by imaging flow cytometry

PubMed Central

Pelletier, Margery G. H.; Szymczak, Klaudia; Barbeau, Anna M.; Prata, Gianna N.; O’Fallon, Kevin S.; Gaines, Peter

2016-01-01

Neutrophils and macrophages differentiate from common myeloid progenitors in the bone marrow, where they undergo nuclear morphologic changes during maturation. During this process, both cell types acquire critical innate immune functions that include phagocytosis of pathogens, and for neutrophils the release of nuclear material called nuclear extracellular traps (NETs). Primary cells used to study these functions are typically purified from mature mouse tissues, but bone marrow-derived ex vivo cultures provide more abundant numbers of progenitors and functionally mature cells. Routine analyses of these cells use conventional microscopy and flow cytometry, which present limitations; microscopy is laborious and subjective, whereas flow cytometry lacks spatial resolution. Here we describe methods to generate enriched populations of neutrophils or macrophages from cryopreserved mouse bone marrow cultured ex vivo, and to use imaging flow cytometry that combines the resolution of microscopy with flow cytometry to analyze cells for morphologic features, phagocytosis, and NETosis. PMID:27663441

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

Improved panels for clinical immune phenotyping: Utilization of the violet laser.

PubMed

Ryherd, Mark; Plassmeyer, Matthew; Alexander, Connor; Eugenio, Ines; Kleschenko, Yuliya; Badger, Ariel; Gupta, Raavi; Alpan, Oral; Sønder, Søren Ulrik

2017-05-10

Clinical diagnostic laboratories are subject to numerous regulations imposed by government agencies. Laboratory developed tests for flow cytometry panels are essentially restricted to the use of analyte-specific reagents (ASR) antibodies. With the advances in clinical flow cytometry systems, there is a trend toward the utilization of blue/red/violet laser flow systems and 8 to 10-color panels. Currently, the selection of commercially available ASR antibodies for the violet laser is very limited. The market is dominated by Brilliant Violet 421 (BV421) manufactured by BD Biosciences and Pacific Blue (PB) manufactured by Beckman Coulter. In this study, we compare BV421 and PB conjugated ASR antibodies. Whole blood was stained and acquired on a Gallios flow cytometer system. For single color staining, the stain index (SI) was calculated. For the two panels, the compensation matrix was calculated and the performance of the antibody cocktails analyzed in FCS Express. The results show that five out of six tested BV421 conjugated antibodies have significantly higher SI than their PB counterparts. Furthermore, BV421 antibodies require less compensation for spillover than PB. Finally, BV421 conjugated antibodies give better separation between negative and positive populations in the context of an 8 and 10 color panel without affecting the intensity of the other dyes. Overall, using BV421 conjugated antibodies results in better separation between populations compared to PB conjugated antibodies without negatively affecting other fluorochromes in our panels. We conclude that the BV421 conjugated ASR antibodies are currently the better available option for clinical flow panels. © 2017 International Clinical Cytometry Society. © 2017 International Clinical Cytometry Society.

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.

Development of a Flow Cytometry-Based Method for Rapid Detection of Escherichia coli and Shigella Spp. Using an Oligonucleotide Probe

PubMed Central

Xue, Yong; Wilkes, Jon G.; Moskal, Ted J.; Williams, Anna J.; Cooper, Willie M.; Nayak, Rajesh; Rafii, Fatemeh; Buzatu, Dan A.

2016-01-01

Standard methods to detect Escherichia coli contamination in food use the polymerase chain reaction (PCR) and agar culture plates. These methods require multiple incubation steps and take a long time to results. An improved rapid flow-cytometry based detection method was developed, using a fluorescence-labeled oligonucleotide probe specifically binding a16S rRNA sequence. The method positively detected 51 E. coli isolates as well as 4 Shigella species. All 27 non-E. coli strains tested gave negative results. Comparison of the new genetic assay with a total plate count (TPC) assay and agar plate counting indicated similar sensitivity, agreement between cytometry cell and colony counts. This method can detect a small number of E.coli cells in the presence of large numbers of other bacteria. This method can be used for rapid, economical, and stable detection of E. coli and Shigella contamination in the food industry and other contexts. PMID:26913737

Development of a Flow Cytometry-Based Method for Rapid Detection of Escherichia coli and Shigella Spp. Using an Oligonucleotide Probe.

PubMed

Xue, Yong; Wilkes, Jon G; Moskal, Ted J; Williams, Anna J; Cooper, Willie M; Nayak, Rajesh; Rafii, Fatemeh; Buzatu, Dan A

2016-01-01

Standard methods to detect Escherichia coli contamination in food use the polymerase chain reaction (PCR) and agar culture plates. These methods require multiple incubation steps and take a long time to results. An improved rapid flow-cytometry based detection method was developed, using a fluorescence-labeled oligonucleotide probe specifically binding a16S rRNA sequence. The method positively detected 51 E. coli isolates as well as 4 Shigella species. All 27 non-E. coli strains tested gave negative results. Comparison of the new genetic assay with a total plate count (TPC) assay and agar plate counting indicated similar sensitivity, agreement between cytometry cell and colony counts. This method can detect a small number of E.coli cells in the presence of large numbers of other bacteria. This method can be used for rapid, economical, and stable detection of E. coli and Shigella contamination in the food industry and other contexts.

DNA polymorphism identity determination using flow cytometry

DOEpatents

Nolan, John P.; White, P. Scott; Cai, Hong

2001-01-01

DNA polymorphism identity determination using flow cytometry. Primers designed to be immobilized on microspheres are allowed to anneal to the DNA strand under investigation, and are extended by either DNA polymerase using fluorescent dideoxynucleotides or ligated by DNA ligase to fluorescent reporter oligonucleotides. The fluorescence of either the dideoxynucleotide or the reporter oligonucleotide attached to the immobilized primer is measured by flow cytometry, thereby identifying the nucleotide polymorphism on the DNA strand.

Early Detection of NSCLC Using Stromal Markers in Peripheral Blood

DTIC Science & Technology

2016-09-01

circulating myeloid cells, flow cytometry, RNA -sequencing, expression profiling. 3. ACCOMPLISHMENTS:  What were the major goals of the project...Subtask 2: Flow cytometry sorting of circulating myeloid cells. Subtask 3: RNA -Sequencing Subtask 4: RNA -seq data analysis Subtask 5: Feasible RT-PCR...accomplished the patient recruitment, flow cytometry sorting of circulating myeloid cells, RNA -sequencing of the samples. During the RNA - seq data analysis, we

CD200 is a useful diagnostic marker for identifying atypical chronic lymphocytic leukemia by flow cytometry.

PubMed

Ting, Y S; Smith, S A B C; Brown, D A; Dodds, A J; Fay, K C; Ma, D D F; Milliken, S; Moore, J J; Sewell, W A

2018-05-27

Immunophenotyping by flow cytometry is routinely employed in distinguishing between chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). Inclusion of CD200 has been reported to contribute to more reliable differentiation between CLL and MCL. We investigated the value of CD200 in assessment of atypical CLL cases. CD200 expression on mature B cell neoplasms was studied by eight-color flow cytometry in combination with a conventional panel of flow cytometry markers. The study included 70 control samples, 63 samples with CLL or atypical CLL phenotype, 6 MCL samples, and 40 samples of other mature B cell neoplasms. All CLL samples were positive for CD200, whereas MCL samples were dim or negative for CD200. Of the CLL samples, 7 were atypical by conventional flow cytometry, with Matutes scores ≤3. These cases were tested for evidence of a t(11;14) translocation, characteristic of MCL, and all were negative, consistent with their classification as atypical CLL. All these atypical CLL samples were strongly positive for CD200. CD200 proved to be a useful marker for differentiation between CLL and MCL by flow cytometry. In particular, CD200 was useful in distinguishing CLL samples with atypical immunophenotypes from MCL. © 2018 John Wiley & Sons Ltd.

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.

High-throughput autofluorescence flow cytometry of breast cancer metabolism (Conference Presentation)

NASA Astrophysics Data System (ADS)

Shah, Amy T.; Cannon, Taylor M.; Higginbotham, Jim N.; Skala, Melissa C.

2016-02-01

Tumor heterogeneity poses challenges for devising optimal treatment regimens for cancer patients. In particular, subpopulations of cells can escape treatment and cause relapse. There is a need for methods to characterize tumor heterogeneity of treatment response. Cell metabolism is altered in cancer (Warburg effect), and cells use the autofluorescent cofactor NADH in numerous metabolic reactions. Previous studies have shown that microscopy measurements of NADH autofluorescence are sensitive to treatment response in breast cancer, and these techniques typically assess hundreds of cells per group. An alternative approach is flow cytometry, which measures fluorescence on a single-cell level and is attractive for characterizing tumor heterogeneity because it achieves high-throughput analysis and cell sorting in millions of cells per group. Current applications for flow cytometry rely on staining with fluorophores. This study characterizes flow cytometry measurements of NADH autofluorescence in breast cancer cells. Preliminary results indicate flow cytometry of NADH is sensitive to cyanide perturbation, which inhibits oxidative phosphorylation, in nonmalignant MCF10A cells. Additionally, flow cytometry is sensitive to higher NADH intensity for HER2-positive SKBr3 cells compared with triple-negative MDA-MB-231 cells. These results agree with previous microscopy studies. Finally, a mixture of SKBr3 and MDA-MB-231 cells were sorted into each cell type using NADH intensity. Sorted cells were cultured, and microscopy validation showed the expected morphology for each cell type. Ultimately, flow cytometry could be applied to characterize tumor heterogeneity based on treatment response and sort cell subpopulations based on metabolic profile. These achievements could enable individualized treatment strategies and improved patient outcomes.

Ultrasensitive automated RNA in situ hybridization for kappa and lambda light chain mRNA detects B-cell clonality in tissue biopsies with performance comparable or superior to flow cytometry.

PubMed

Guo, Ling; Wang, Zhen; Anderson, Courtney M; Doolittle, Emerald; Kernag, Siobhan; Cotta, Claudiu V; Ondrejka, Sarah L; Ma, Xiao-Jun; Cook, James R

2018-03-01

The assessment of B-cell clonality is a critical component of the evaluation of suspected lymphoproliferative disorders, but analysis from formalin-fixed, paraffin-embedded tissues can be challenging if fresh tissue is not available for flow cytometry. Immunohistochemical and conventional bright field in situ hybridization stains for kappa and lambda are effective for evaluation of plasma cells but are often insufficiently sensitive to detect the much lower abundance of light chains present in B-cells. We describe an ultrasensitive RNA in situ hybridization assay that has been adapted for use on an automated immunohistochemistry platform and compare results with flow cytometry in 203 consecutive tissues and 104 consecutive bone marrows. Overall, in 203 tissue biopsies, RNA in situ hybridization identified light chain-restricted B-cells in 85 (42%) vs 58 (29%) by flow cytometry. Within 83 B-cell non-Hodgkin lymphomas, RNA in situ hybridization identified restricted B-cells in 74 (89%) vs 56 (67%) by flow cytometry. B-cell clonality could be evaluated in only 23/104 (22%) bone marrow cases owing to poor RNA preservation, but evaluable cases showed 91% concordance with flow cytometry. RNA in situ hybridization allowed for recognition of biclonal/composite lymphomas not identified by flow cytometry and highlighted unexpected findings, such as coexpression of kappa and lambda RNA in 2 cases and the presence of lambda light chain RNA in a T lymphoblastic lymphoma. Automated RNA in situ hybridization showed excellent interobserver reproducibility for manual evaluation (average K=0.92), and an automated image analysis system showed high concordance (97%) with manual evaluation. Automated RNA in situ hybridization staining, which can be adopted on commonly utilized immunohistochemistry instruments, allows for the interpretation of clonality in the context of the morphological features in formalin-fixed, paraffin-embedded tissues with a clinical sensitivity similar or superior to flow cytometry.

Ultrasensitive Automated RNA in situ Hybridization for Kappa and Lambda Light Chain mRNA Detects B-cell Clonality in Tissue Biopsies with Performance Comparable or Superior to Flow Cytometry

PubMed Central

Guo, Ling; Wang, Zhen; Anderson, Courtney M.; Doolittle, Emerald; Kernag, Siobhan; Cotta, Claudiu V.; Ondrejka, Sarah L.; Ma, Xiao-Jun; Cook, James R.

2017-01-01

The assessment of B-cell clonality is a critical component of the evaluation of suspected lymphoproliferative disorders, but analysis from formalin fixed paraffin embedded tissues can be challenging if fresh tissue is not available for flow cytometry. Immunohistochemical and conventional bright field in situ hybridization stains for kappa and lambda are effective for evaluation of plasma cells, but are often insufficiently sensitive to detect the much lower abundance of light chains present in B cells. We describe an ultrasensitive RNA in situ hybridization assay which has been adapted for use on an automated immunohistochemistry platform and compare results with flow cytometry in 203 consecutive tissues and 104 consecutive bone marrows. Overall, in 203 tissue biopsies, RNA in situ hybridization identified light chain restricted B-cells in 85 (42%) vs. 58 (29%) by flow cytometry. Within 83 B-cell non-Hodgkin lymphomas, RNA in situ hybridization identified a restricted B-cells in 74 (89%) vs. 56 (67%) by flow cytometry. B-cell clonality could be evaluated in only 23/104 (22%) bone marrow cases due to poor RNA preservation, but evaluable cases showed 91% concordance with flow cytometry. RNA in situ hybridization allowed for recognition of biclonal/composite lymphomas not identified by flow cytometry, and highlighted unexpected findings, such as coexpression of kappa and lambda RNA in 2 cases and the presence of lambda light chain RNA in a T lymphoblastic lymphoma. Automated RNA in situ hybridization showed excellent interobserver reproducibility for manual evaluation (average K=0.92), and an automated image analysis system showed high concordance (97%) with manual evaluation. Automated RNA in situ hybridization staining, which can be adopted on commonly utilized immunohistochemistry instruments, allows for the interpretation of clonality in the context of the morphologic features in formalin fixed, paraffin embedded tissues with a clinical sensitivity similar or superior to flow cytometry. PMID:29052600

Two-Photon Flow Cytometry

NASA Technical Reports Server (NTRS)

Zhog, Cheng Frank; Ye, Jing Yong; Norris, Theodore B.; Myc, Andrzej; Cao, Zhengyl; Bielinska, Anna; Thomas, Thommey; Baker, James R., Jr.

2004-01-01

Flow cytometry is a powerful technique for obtaining quantitative information from fluorescence in cells. Quantitation is achieved by assuring a high degree of uniformity in the optical excitation and detection, generally by using a highly controlled flow such as is obtained via hydrodynamic focusing. In this work, we demonstrate a two-beam, two- channel detection and two-photon excitation flow cytometry (T(sup 3)FC) system that enables multi-dye analysis to be performed very simply, with greatly relaxed requirements on the fluid flow. Two-photon excitation using a femtosecond near-infrared (NIR) laser has the advantages that it enables simultaneous excitation of multiple dyes and achieves very high signal-to-noise ratio through simplified filtering and fluorescence background reduction. By matching the excitation volume to the size of a cell, single-cell detection is ensured. Labeling of cells by targeted nanoparticles with multiple fluorophores enables normalization of the fluorescence signal and thus ratiometric measurements under nonuniform excitation. Quantitative size measurements can also be done even under conditions of nonuniform flow via a two-beam layout. This innovative detection scheme not only considerably simplifies the fluid flow system and the excitation and collection optics, it opens the way to quantitative cytometry in simple and compact microfluidics systems, or in vivo. Real-time detection of fluorescent microbeads in the vasculature of mouse ear demonstrates the ability to do flow cytometry in vivo. The conditions required to perform quantitative in vivo cytometry on labeled cells will be presented.

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.

Identification of contact and respiratory sensitizers using flow cytometry

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

Goutet, Michele; Pepin, Elsa; Langonne, Isabelle

2005-06-15

Identification of the chemicals responsible for respiratory and contact allergies in the industrial area is an important occupational safety issue. This study was conducted in mice to determine whether flow cytometry is an appropriate method to analyze and differentiate the specific immune responses to the respiratory sensitizer trimellitic anhydride (TMA) and to the contact sensitizer dinitrochlorobenzene (DNCB) used at concentrations with comparable immunogenic potential. Mice were exposed twice on the flanks (days 0, 5) to 10% TMA or 1% DNCB and challenged three times on the ears (days 10, 11, 12) with 2.5% TMA or 0.25% DNCB. Flow cytometry analysesmore » were conducted on draining lymph node cells harvested on days 13 and 18. Comparing TMA and DNCB immune responses on day 13, we found obvious differences that persisted for most of them on day 18. An increased proportion of IgE+ cells correlated to total serum IgE level and an enhancement of MHC II molecule expression were observed in the lymph node B lymphocytes from TMA-treated mice. The percentage of IL-4-producing CD4+ lymphocytes and the IL-4 receptor expression were clearly higher following TMA exposure. In contrast, higher proportions of IL-2-producing cells were detected in CD4+ and CD8+ cells from DNCB-treated mice. Both chemicals induced a significant increase in the percentage of IFN-{gamma}-producing cells among CD8+ lymphocytes but to a greater proportion following TMA treatment. In conclusion, this study encourages the use of flow cytometry to discriminate between contact and respiratory sensitizers by identifying divergent expression of immune response parameters.« less

Polystyrene microspheres enable 10‐color compensation for immunophenotyping of primary human leukocytes

PubMed Central

Carr, Karen D.; Norman, John C.; Huye, Leslie; Hegde, Meenakshi

2015-01-01

Abstract Compensation is a critical process for the unbiased analysis of flow cytometry data. Numerous compensation strategies exist, including the use of bead‐based products. The purpose of this study was to determine whether beads, specifically polystyrene microspheres (PSMS) compare to the use of primary leukocytes for single color based compensation when conducting polychromatic flow cytometry. To do so, we stained individual tubes of both PSMS and leukocytes with panel specific antibodies conjugated to fluorochromes corresponding to fluorescent channels FL1‐FL10. We compared the matrix generated by PSMS to that generated using peripheral blood mononuclear cells (PBMC). Ideal for compensation is a sample with both a discrete negative population and a bright positive population. We demonstrate that PSMS display autofluorescence properties similar to PBMC. When comparing PSMS to PBMC for compensation PSMS yielded more evenly distributed and discrete negative and positive populations to use for compensation. We analyzed three donors' PBMC stained with our 10‐color T cell subpopulation panel using compensation generated by PSMS vs.PBMC and detected no significant differences in the population distribution. Panel specific antibodies bound to PSMS represent an invaluable valid tool to generate suitable compensation matrices especially when sample material is limited and/or the sample requires analysis of dynamically modulated or rare events. © 2015 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. PMID:26202733

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.

One-dimensional acoustic standing waves in rectangular channels for flow cytometry.

PubMed

Austin Suthanthiraraj, Pearlson P; Piyasena, Menake E; Woods, Travis A; Naivar, Mark A; Lόpez, Gabriel P; Graves, Steven W

2012-07-01

Flow cytometry has become a powerful analytical tool for applications ranging from blood diagnostics to high throughput screening of molecular assemblies on microsphere arrays. However, instrument size, expense, throughput, and consumable use limit its use in resource poor areas of the world, as a component in environmental monitoring, and for detection of very rare cell populations. For these reasons, new technologies to improve the size and cost-to-performance ratio of flow cytometry are required. One such technology is the use of acoustic standing waves that efficiently concentrate cells and particles to the center of flow channels for analysis. The simplest form of this method uses one-dimensional acoustic standing waves to focus particles in rectangular channels. We have developed one-dimensional acoustic focusing flow channels that can be fabricated in simple capillary devices or easily microfabricated using photolithography and deep reactive ion etching. Image and video analysis demonstrates that these channels precisely focus single flowing streams of particles and cells for traditional flow cytometry analysis. Additionally, use of standing waves with increasing harmonics and in parallel microfabricated channels is shown to effectively create many parallel focused streams. Furthermore, we present the fabrication of an inexpensive optical platform for flow cytometry in rectangular channels and use of the system to provide precise analysis. The simplicity and low-cost of the acoustic focusing devices developed here promise to be effective for flow cytometers that have reduced size, cost, and consumable use. Finally, the straightforward path to parallel flow streams using one-dimensional multinode acoustic focusing, indicates that simple acoustic focusing in rectangular channels may also have a prominent role in high-throughput flow cytometry. Copyright © 2012 Elsevier Inc. All rights reserved.

Genomic Instability at Premalignant and Early Stages of Breast Cancer Development

DTIC Science & Technology

1999-08-01

by routine DNA flow cytometry vation. ERBB2 expression was detected with a to determine DNA index (DI). commercially available antibody (Oncogene Sci...supplements the information gained from ic microsatellite primers. We observed that the ploidy analysis by DNA flow cytometry alone. In DNA so obtained...preserved the proportionality of many cases where flow cytometry could not be per- the different alleles as found in the original sample. formed because the

Inhibition of Breast Cancer-Induced Angiogenesis by a Diverged Homeobox Gene

DTIC Science & Technology

2006-05-01

and then harvested for flow cytometry using appropriate antibodies. Ad.Gax blocked the expression of VCAM-1, E-selectin, and ICAM-1. DOD Idea Award...solution. Bands were visualized by chemiluminescence using the ECL-Plus reagent (Amersham, Piscataway, NJ). Flow Cytometry Cells were harvested after...33), all of whose down-regulation we have confirmed using real time quantitative RT-PCR, Western blot, and flow cytometry (Fig. 5). Moreover, Gax

Mesenchymal Stem Cells for Vascular Target Discovery in Breast Cancer-Associated Angiogenesis

DTIC Science & Technology

2005-09-01

demonstrating this marker as demonstrated by flow cytometry . These GFP+ MSCs were subsequently analyzed for expression of commonly reported markers of...phenotypically and genotypically analyzed by flow cytometry and gene chip analysis, respectively. We have also shown that MSCs can then be stimulated to...positive MSCs retrieved by collagenase digestion of the Matrigel plug and sorted by flow cytometry . Sorting of these retrieved cells based on co-expression

Probing Tumor Microenvironment With In Vivo Phage Display

DTIC Science & Technology

2014-10-01

C). (C) Dot plots showing mCherry expression on the X axis and fibroblast activation protein ( FAP ) or rabbit isotype control staining in the Y...by flow cytometry-based cell sorting using an antibody against fibroblast activation protein ( FAP ). During the optimization steps, flow cytometry...expression of αvβ3 and αvβ5 integrins, neuropilin-1 (NRP-1), and fibroblast activation protein ( FAP ) in hb6011 CAFs was analyzed by flow cytometry

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.

Flow Cytometry Technician | Center for Cancer Research

Cancer.gov

PROGRAM DESCRIPTION The Basic Science Program (BSP) pursues independent, multidisciplinary research in basic and applied molecular biology, immunology, retrovirology, cancer biology, and human genetics. Research efforts and support are an integral part of the Center for Cancer Research (CCR) at the Frederick National Laboratory for Cancer Research (FNLCR). KEY ROLES/RESPONSIBILITIES The Flow Cytometry Core (Flow Core) of the Cancer and Inflammation Program (CIP) is a service core which supports the research efforts of the CCR by providing expertise in the field of flow cytometry (using analyzers and sorters) with the goal of gaining a more thorough understanding of the biology of cancer and cancer cells. The Flow Core provides service to 12-15 CIP laboratories and more than 22 non-CIP laboratories. Flow core staff provide technical advice on the experimental design of applications, which include immunological phenotyping, cell function assays, and cell cycle analysis. Work is performed per customer requirements, and no independent research is involved. The Flow Cytometry Technician will be responsible for: Monitor performance of and maintain high dimensional flow cytometer analyzers and cell sorters Operate high dimensional flow cytometer analyzers and cell sorters Monitoring lab supply levels and order lab supplies, perform various record keeping responsibilities Assist in the training of scientific end users on the use of flow cytometry in their research, as well as how to operate and troubleshoot the bench-top analyzer instruments Experience with sterile technique and tissue culture

Near infrared lasers in flow cytometry.

PubMed

Telford, William G

2015-07-01

Technology development in flow cytometry has closely tracked laser technology, the light source that flow cytometers almost exclusively use to excite fluorescent probes. The original flow cytometers from the 1970s and 1980s used large water-cooled lasers to produce only one or two laser lines at a time. Modern cytometers can take advantage of the revolution in solid state laser technology to use almost any laser wavelength ranging from the ultraviolet to the near infrared. Commercial cytometers can now be equipped with many small solid state lasers, providing almost any wavelength needed for cellular analysis. Flow cytometers are now equipped to analyze 20 or more fluorescent probes simultaneously, requiring multiple laser wavelengths. Instrument developers are now trying to increase this number by designing fluorescent probes that can be excited by laser wavelength at the "edges" of the visible light range, in the near ultraviolet and near-infrared region. A variety of fluorescent probes have been developed that excite with violet and long wavelength ultraviolet light; however, the near-infrared range (660-800 nm) has yet seen only exploitation in flow cytometry. Fortunately, near-infrared laser diodes and other solid state laser technologies appropriate for flow cytometry have been in existence for some time, and can be readily incorporated into flow cytometers to accelerate fluorescent probe development. The near infrared region represents one of the last "frontiers" to maximize the number of fluorescent probes that can be analyzed by flow cytometry. In addition, near infrared fluorescent probes used in biomedical tracking and imaging could also be employed for flow cytometry with the correct laser wavelengths. This review describes the available technology, including lasers, fluorescent probes and detector technology optimal for near infrared signal detection. Published by Elsevier Inc.

Detection of EpCAM-positive microparticles in pleural fluid: A new approach to mini-invasively identify patients with malignant pleural effusions

PubMed Central

Roca, Elisa; Lacroix, Romaric; Judicone, Coralie; Laroumagne, Sophie; Robert, Stéphane; Cointe, Sylvie; Muller, Alexandre; Kaspi, Elise; Roll, Patrice; Brisson, Alain R.; Tantucci, Claudio

2016-01-01

Pleural biomarkers allowing to mini-invasively discriminate benign from malignant pleural effusions are needed. Among potential candidates, microparticles (MPs) are extracellular vesicles that vectorize antigen derived from the parent cell. We hypothesized that tumor-derived MPs could be present in the pleural liquid and help to identify patients with malignant pleural effusions. Using highly sensitive flow cytometry and cryo-electron microscopy, we showed that large amounts of MPs from hematopoïetic and vascular origin could be detectable in pleural fluids. Their level did not differ between benign (n = 14) and malignant (n = 71) pleural effusions. Analysis of selected tumoral associated antigens (podoplanin, mucin 1 and EpCAM, epithelial-cell-adhesion-molecule) evidenced for the first time the presence of tumor-derived MPs expressing EpCAM in malignant pleural fluids only (Specificity = 93%, Sensitivity = 49% and 45% for flow cytometry and ELISA, respectively). The detection of EpCAM-positive-MPs (EpCAM + MPs) by flow cytometry showed a better specificity and sensitivity than ELISA to distinguish between pleural carcinoma and the others malignant pleural effusions (MPE; Sp: 96% vs 89%; Se: 79% vs 66%). Combining EpCAM+ MPs and cytology improved the diagnosis of MPE compared to cytology alone. This study establishes the basis for using EpCAM+ MPs as a promising new biomarker that could be added to the armamentarium to mini-invasively identify patients with malignant pleural effusions. PMID:26689993

Detection of EpCAM-positive microparticles in pleural fluid: A new approach to mini-invasively identify patients with malignant pleural effusions.

PubMed

Roca, Elisa; Lacroix, Romaric; Judicone, Coralie; Laroumagne, Sophie; Robert, Stéphane; Cointe, Sylvie; Muller, Alexandre; Kaspi, Elise; Roll, Patrice; Brisson, Alain R; Tantucci, Claudio; Astoul, Philippe; Dignat-George, Françoise

2016-01-19

Pleural biomarkers allowing to mini-invasively discriminate benign from malignant pleural effusions are needed. Among potential candidates, microparticles (MPs) are extracellular vesicles that vectorize antigen derived from the parent cell. We hypothesized that tumor-derived MPs could be present in the pleural liquid and help to identify patients with malignant pleural effusions. Using highly sensitive flow cytometry and cryo-electron microscopy, we showed that large amounts of MPs from hematopoïetic and vascular origin could be detectable in pleural fluids. Their level did not differ between benign (n = 14) and malignant (n = 71) pleural effusions. Analysis of selected tumoral associated antigens (podoplanin, mucin 1 and EpCAM, epithelial-cell-adhesion-molecule) evidenced for the first time the presence of tumor-derived MPs expressing EpCAM in malignant pleural fluids only (Specificity = 93%, Sensitivity = 49% and 45% for flow cytometry and ELISA, respectively). The detection of EpCAM-positive-MPs (EpCAM + MPs) by flow cytometry showed a better specificity and sensitivity than ELISA to distinguish between pleural carcinoma and the others malignant pleural effusions (MPE; Sp: 96% vs 89%; Se: 79% vs 66%). Combining EpCAM+ MPs and cytology improved the diagnosis of MPE compared to cytology alone. This study establishes the basis for using EpCAM+ MPs as a promising new biomarker that could be added to the armamentarium to mini-invasively identify patients with malignant pleural effusions.

Validation of Flow Cytometry and Magnetic Bead-Based Methods to Enrich CNS Single Cell Suspensions for Quiescent Microglia.

PubMed

Volden, T A; Reyelts, C D; Hoke, T A; Arikkath, J; Bonasera, S J

2015-12-01

Microglia are resident mononuclear phagocytes within the CNS parenchyma that intimately interact with neurons and astrocytes to remodel synapses and extracellular matrix. We briefly review studies elucidating the molecular pathways that underlie microglial surveillance, activation, chemotaxis, and phagocytosis; we additionally place these studies in a clinical context. We describe and validate an inexpensive and simple approach to obtain enriched single cell suspensions of quiescent parenchymal and perivascular microglia from the mouse cerebellum and hypothalamus. Following preparation of regional CNS single cell suspensions, we remove myelin debris, and then perform two serial enrichment steps for cells expressing surface CD11b. Myelin depletion and CD11b enrichment are both accomplished using antigen-specific magnetic beads in an automated cell separation system. Flow cytometry of the resultant suspensions shows a significant enrichment for CD11b(+)/CD45(+) cells (perivascular microglia) and CD11b(+)/CD45(-) cells (parenchymal microglia) compared to starting suspensions. Of note, cells from these enriched suspensions minimally express Aif1 (aka Iba1), suggesting that the enrichment process does not evoke significant microglial activation. However, these cells readily respond to a functional challenge (LPS) with significant changes in the expression of molecules specifically associated with microglia. We conclude that methods employing a combination of magnetic-bead based sorting and flow cytometry produce suspensions highly enriched for microglia that are appropriate for a variety of molecular and cellular assays.

Blockade of Tumor Cell TGF-Betas: A Strategy to Reverse Antiestrogen Resistance in Human Breast Cancer

DTIC Science & Technology

2002-01-01

the TM- FKHRL1 construct exhibited exclusive nuclear localization Cell Cycle Analysis by Flow Cytometry of the HA-tagged mutant under any experimental...distribution as measured by flow cytometry (Figure 8A). ALS AND METHODS. Consistent with its antiapoptotic effect, these results, addi- tion of TGFI3... flow cytometry . Under these conditions more than 95% of selected cells expressed GFP at the time of experiments. Immunoblot Analysis. Cells were

Slow-Adhering Stem Cells Derived from Injured Skeletal Muscle Have Improved Regenerative Capacity

DTIC Science & Technology

2011-08-01

images. Flow Cytometry Assay of Stem Cell Markers SASCs (1 105) isolated from noninjured or injured muscle were collected and washed twice with...muscle. Results of flow cytometry further verified Sca-1 and CD34 expression in isolated SASCs, and a greater percentage of cells were positive for Sca-1...from both injured and control noninjured muscle were analyzed using flow cytometry for the immunofluorescent signal of Sca-1 and CD34. Results

Augmenting Trastuzumab Therapy against Breast Cancer through Selective Activation of NK Cells

DTIC Science & Technology

2014-12-01

purity as defined by CD3-CD56+ flow cytometry ) and activation (>50% expression of CD137). Breast cancer cell lines including MCF7 (A and E...purity as defined by CD3-CD56+ flow cytometry ) and activation (>50% expression of CD137). Chromium-labeled breast cancer cell lines including MCF7 (A...and Whiteside, T.L. 2007. A novel multiparametric flow cytometry -based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons

Hypoxia and Prx1 in Malignant Progression of Prostate Cancer

DTIC Science & Technology

2006-09-01

Species (ROS) Formation The rate of ROS formation was determined by flow cytometry analysis using the probe 20,70-dichlorofluorescin diacetate (DCFH-DA...DA were subjected to 4-h hypoxia treatment. After the indicated time, fluorescent cells were analyzed by flow cytometry . Western Blot Analysis Equal...species (ROS) generation was measured by flow cytometry at 0.5, 1, 2, 3, 6, 12, or 24 h after hypoxia treatment. The rate of ROS generation increased

Significances of red cell bound immunoglobulin G as detected by flow cytometry in patients with Coombs-negative immune hemolysis.

PubMed

Thedsawad, A; Taka, O; Wanachiwanawin, W

2016-04-01

This study was to investigate the use of flow cytometry for detection and quantitation of red blood cells (RBC) bound IgG in immune hemolysis of patients with autoimmune hemolytic anaemia (AIHA) and systematic lupus erythematosus (SLE). Two to ten percent of patients with warm-autoimmune hemolytic anaemia (WAIHA) exhibit a negative direct Coombs test. Flow cytometry has been applied to detect RBC bound IgG with high accuracy, reproducibility and sensitivity. In this study 45 and 75 patients with AIHA and SLE, respectively were evaluated for RBC bound IgG by direct Coombs test and flow cytometry. Seventy-one percent (32/45) and 31% (23/75) of patients with AIHA and SLE respectively, had laboratory evidence of hemolysis. A positive flow cytometry, as defined by mean fluorescent intensity (MFI) values >0·21 and IgG molecules >28, was found in 4 of 32 (12·5%) and 4 of 23 (17·4%) patients with AIHA and SLE who had hemolysis with a negative direct Coombs test. There were very strong and strong correlations between the strength of direct Coombs test with MFI values and IgG molecules in patients with AIHA and SLE, respectively. Flow cytometry can be applied in the diagnosis of Coombs-negative hemolytic anaemia in patients with AIHA and SLE. © 2016 British Blood Transfusion Society.

Immunophenotyping of acute leukaemias by flow cytometry: a review.

PubMed

Pamnani, R

2009-12-01

To provide an overview of the utility of flow cytometry for phenotyping of acute leukaemias and selection-of monoclonal antibodies. The literature review was obtained through internet, journals and chapters in the relevant books. Relevant articles and chapters on immunophenotyping of acute leukaemias were selected from respected international journals and books in the field of haematology and were reviewed. Complete articles relevant to the topic were selected and reviewed and the necessary information extracted for this review. Flow cytometry has been used extensively in recent years to characterise haemopoeitic malignancies and done routinely in the developed world. This technique has greatly improved the diagnosis and classification of haemopoeitic malignancies and has been recommended by World Health Organisation classification (WHO) of tumours of haemopoeitic and lymphoid tissue. Application of flow cytometry for the diagnosis of leukaemias has been recently introduced in Kenya and is currently being undertaken in research using limited but appropriate panels of monoclonal antibodies. It is hoped that findings of this research will inform the use of flow cytometry as an ancillary diagnostic technique in our resource-constrained set up.

A classification tree approach for improving the utilization of flow cytometry testing of blood specimens for B-cell non-Hodgkin lymphoproliferative disorders.

PubMed

Healey, Ryan; Naugler, Christopher; de Koning, Lawrence; Patel, Jay L

2015-01-01

We sought to improve the diagnostic efficiency of flow cytometry investigation on blood by developing data-driven ordering guidelines. Our goal was to improve flow cytometry utilization by decreasing negative testing, therefore reducing healthcare costs. We investigated several laboratory tests performed alongside flow cytometry to identify biomarkers useful in excluding non-leukemic bloods. Test results and patient demographic features were subjected to receiver-operator characteristic (ROC) curve, logistic regression and classification tree analyses to find significant predictors and develop decision rules. Our data show that, in the absence of a compelling clinical indication, flow cytometry testing is largely non-informative on bloods from patients less than 50 years of age having an absolute lymphocyte count (ALC) below 5.0 × 10(9)/L. For patients over age 50 having an ALC below this value, a ferritin value above 450 μg/L is counter-indicative of B-cell clonality. Using these guidelines, 26% of cases were correctly predicted as negative with greater than 97% accuracy.

DNA Detection by Flow Cytometry using PNA-Modified Metal-Organic Framework Particles.

PubMed

Mejia-Ariza, Raquel; Rosselli, Jessica; Breukers, Christian; Manicardi, Alex; Terstappen, Leon W M M; Corradini, Roberto; Huskens, Jurriaan

2017-03-23

A DNA-sensing platform is developed by exploiting the easy surface functionalization of metal-organic framework (MOF) particles and their highly parallelized fluorescence detection by flow cytometry. Two strategies were employed to functionalize the surface of MIL-88A, using either covalent or non-covalent interactions, resulting in alkyne-modified and biotin-modified MIL-88A, respectively. Covalent surface coupling of an azide-dye and the alkyne-MIL-88A was achieved by means of a click reaction. Non-covalent streptavidin-biotin interactions were employed to link biotin-PNA to biotin-MIL-88A particles mediated by streptavidin. Characterization by confocal imaging and flow cytometry demonstrated that DNA can be bound selectively to the MOF surface. Flow cytometry provided quantitative data of the interaction with DNA. Making use of the large numbers of particles that can be simultaneously processed by flow cytometry, this MOF platform was able to discriminate between fully complementary, single-base mismatched, and randomized DNA targets. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Design and first results of CytoBuoy: a wireless flow cytometer for in situ analysis of marine and fresh waters.

PubMed

Dubelaar, G B; Gerritzen, P L; Beeker, A E; Jonker, R R; Tangen, K

1999-12-01

The high costs of microscopical determination and counting of phytoplankton often limit sampling frequencies below an acceptable level for the monitoring of dynamic ecosystems. Although having a limited discrimination power, flow cytometry allows the analysis of large numbers of samples to a level that is sufficient for many basic monitoring jobs. For this purpose, flow cytometers should not be restricted to research laboratories. We report here on the development of an in situ flow cytometer for autonomous operation inside a small moored buoy or on other platforms. Operational specifications served a wide range of applications in the aquatic field. Specific conditions had to be met with respect to the operation platform and autonomy. A small, battery-operated flow cytometer resulted, requiring no external sheath fluid supply. Because it was designed to operate in a buoy, we call it CytoBuoy. Sampling, analysis, and radio transmission of the data proceed automatically at user-defined intervals. A powerful feature is the acquisition and radio transmission of full detector pulse shapes of each particle. This provides valuable morphological information for particles larger than the 5-microm laser focus. CytoBuoy allows on-line in situ particle analysis, estimation of phytoplankton biomass, and discrimination between different phytoplankton groups. This will increase the applicability of flow cytometry in the field of environmental monitoring. Copyright 1999 Wiley-Liss, Inc.

Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art

PubMed Central

Hossain, Md. Sharoare; Johannisson, Anders; Wallgren, Margareta; Nagy, Szabolcs; Siqueira, Amanda Pimenta; Rodriguez-Martinez, Heriberto

2011-01-01

Flow cytometry is now a recognized methodology within animal spermatology, and has moved from being a research tool to become routine in the assessment of animal semen destined to breeding. The availability of ‘bench-top' flow cytometers and of newer and versatile markers for cell structure and function had allowed the instrumentation to measure more sperm parameters, from viability to reactiveness when exposed to exogenous stimuli, and to increase our capabilities to sort spermatozoa for potential fertilizing capacity, or chromosomal sex. The present review summarizes the state of the art regarding flow cytometry applied to animal andrology, albeit keeping an open comparative intent. It critically evaluates the present and future capabilities of flow cytometry for the diagnostics of potential fertility and for the development of current reproductive technologies such as sperm freezing, sperm selection and sperm sorting. The flow cytometry methods will probably further revolutionize our understanding of the sperm physiology and their functionality, and will undoubtedly extend its application in isolating many uncharacterized features of spermatozoa. However, continuous follow-up of the methods is a necessity owing to technical developments and the complexity of mapping spermatozoa. PMID:21478895

CytometryML: a markup language for analytical cytology

NASA Astrophysics Data System (ADS)

Leif, Robert C.; Leif, Stephanie H.; Leif, Suzanne B.

2003-06-01

Cytometry Markup Language, CytometryML, is a proposed new analytical cytology data standard. CytometryML is a set of XML schemas for encoding both flow cytometry and digital microscopy text based data types. CytometryML schemas reference both DICOM (Digital Imaging and Communications in Medicine) codes and FCS keywords. These schemas provide representations for the keywords in FCS 3.0 and will soon include DICOM microscopic image data. Flow Cytometry Standard (FCS) list-mode has been mapped to the DICOM Waveform Information Object. A preliminary version of a list mode binary data type, which does not presently exist in DICOM, has been designed. This binary type is required to enhance the storage and transmission of flow cytometry and digital microscopy data. Index files based on Waveform indices will be used to rapidly locate the cells present in individual subsets. DICOM has the advantage of employing standard file types, TIF and JPEG, for Digital Microscopy. Using an XML schema based representation means that standard commercial software packages such as Excel and MathCad can be used to analyze, display, and store analytical cytometry data. Furthermore, by providing one standard for both DICOM data and analytical cytology data, it eliminates the need to create and maintain special purpose interfaces for analytical cytology data thereby integrating the data into the larger DICOM and other clinical communities. A draft version of CytometryML is available at www.newportinstruments.com.

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

Elastomeric negative acoustic contrast particles for affinity capture assays.

PubMed

Cushing, Kevin W; Piyasena, Menake E; Carroll, Nick J; Maestas, Gian C; López, Beth Ann; Edwards, Bruce S; Graves, Steven W; López, Gabriel P

2013-02-19

This report describes the development of elastomeric capture microparticles (ECμPs) and their use with acoustophoretic separation to perform microparticle assays via flow cytometry.We have developed simple methods to form ECμPs by cross-linking droplets of common commercially available silicone precursors in suspension followed by surface functionalization with biomolecular recognition reagents. The ECμPs are compressible particles that exhibit negative acoustic contrast in ultrasound when suspended in aqueous media, blood serum, or diluted blood. In this study, these particles have been functionalized with antibodies to bind prostate specific antigen and immunoglobulin (IgG). Specific separation of the ECμPs from blood cells is achieved by flowing them through a microfluidic acoustophoretic device that uses an ultrasonic standing wave to align the blood cells, which exhibit positive acoustic contrast, at a node in the acoustic pressure distribution while aligning the negative acoustic contrast ECμPs at the antinodes. Laminar flow of the separated particles to downstream collection ports allows for collection of the separated negative contrast (ECμPs) and positive contrast particles (cells). Separated ECμPs were analyzed via flow cytometry to demonstrate nanomolar detection for prostate specific antigen in aqueous buffer and picomolar detection for IgG in plasma and diluted blood samples. This approach has potential applications in the development of rapid assays that detect the presence of low concentrations of biomarkers in a number of biological sample types.

Elastomeric Negative Acoustic Contrast Particles for Affinity Capture Assays

PubMed Central

Cushing, Kevin W.; Piyasena, Menake E.; Carroll, Nick J.; Maestas, Gian C.; López, Beth Ann; Edwards, Bruce S.; Graves, Steven W.; López, Gabriel P.

2013-01-01

This report describes the development of elastomeric capture microparticles (ECμPs) and their use with acoustophoretic separation to perform microparticle assays via flow cytometry. We have developed simple methods to form ECμPsby crosslinking droplets of common commercially available silicone precursors in suspension followed by surface functionalization with biomolecular recognition reagents. The ECμPs are compressible particles that exhibit negative acoustic contrast in ultrasound when suspended in aqueous media, blood serum or diluted blood. In this study, these particles have been functionalized with antibodies to bind prostate specific antigen and immunoglobulin (IgG). Specific separation of the ECμPs from blood cells is achieved by flowing them through a microfluidic acoustophoretic device that uses an ultrasonic standing wave to align the blood cells, which exhibit positive acoustic contrast, at a node in the acoustic pressure distribution while aligning the negative acoustic contrast ECμPs at the antinodes. Laminar flow of the separated particles to downstream collection ports allows for collection of the separated negative contrast (ECμPs) and positive contrast particles (cells). Separated ECμPs were analyzed via flow cytometry to demonstrate nanomolar detection for prostate specific antigen in aqueous buffer and picomolar detection for IgG in plasma and diluted blood samples. This approach has potential applications in the development of rapid assays that detect the presence of low concentrations of biomarkers in a number of biological sample types. PMID:23331264

The role of flow cytometry in companion animal diagnostic medicine.

PubMed

Tarrant, Jacqueline M

2005-11-01

Flow cytometry is a powerful tool for characterising the composition of complex cell populations. The accuracy and precision of this technology for describing and enumerating cells exceeds traditional methods. The number of diagnostic veterinary laboratories with access to a dedicated machine is increasing, and there is the potential to offer a clinical flow cytometry service. The improved availability of monoclonal antibodies (mAb) to cell markers expressed by the leukocytes of companion animals, permits the implementation of comprehensive mAb panels suitable for diagnosis of lympho- and myeloproliferative disease. Reticulated erythrocyte and platelet quantification, antiglobulin assays for immune-mediated cytopenias, lymphocyte subset analysis, and immunophenotyping of lymphoma and leukemia, have been validated for companion animal samples on the flow cytometer. It is now timely to consider the role of flow cytometry in diagnostic practice, and the requirement for quality assurance and standardization of testing procedures.

FlowSOM: Using self-organizing maps for visualization and interpretation of cytometry data.

PubMed

Van Gassen, Sofie; Callebaut, Britt; Van Helden, Mary J; Lambrecht, Bart N; Demeester, Piet; Dhaene, Tom; Saeys, Yvan

2015-07-01

The number of markers measured in both flow and mass cytometry keeps increasing steadily. Although this provides a wealth of information, it becomes infeasible to analyze these datasets manually. When using 2D scatter plots, the number of possible plots increases exponentially with the number of markers and therefore, relevant information that is present in the data might be missed. In this article, we introduce a new visualization technique, called FlowSOM, which analyzes Flow or mass cytometry data using a Self-Organizing Map. Using a two-level clustering and star charts, our algorithm helps to obtain a clear overview of how all markers are behaving on all cells, and to detect subsets that might be missed otherwise. R code is available at https://github.com/SofieVG/FlowSOM and will be made available at Bioconductor. © 2015 International Society for Advancement of Cytometry.

Web-based analysis and publication of flow cytometry experiments.

PubMed

Kotecha, Nikesh; Krutzik, Peter O; Irish, Jonathan M

2010-07-01

Cytobank is a Web-based application for storage, analysis, and sharing of flow cytometry experiments. Researchers use a Web browser to log in and use a wide range of tools developed for basic and advanced flow cytometry. In addition to providing access to standard cytometry tools from any computer, Cytobank creates a platform and community for developing new analysis and publication tools. Figure layouts created on Cytobank are designed to allow transparent access to the underlying experiment annotation and data processing steps. Since all flow cytometry files and analysis data are stored on a central server, experiments and figures can be viewed or edited by anyone with the proper permission, from any computer with Internet access. Once a primary researcher has performed the initial analysis of the data, collaborators can engage in experiment analysis and make their own figure layouts using the gated, compensated experiment files. Cytobank is available to the scientific community at http://www.cytobank.org. (c) 2010 by John Wiley & Sons, Inc.

Web-Based Analysis and Publication of Flow Cytometry Experiments

PubMed Central

Kotecha, Nikesh; Krutzik, Peter O.; Irish, Jonathan M.

2014-01-01

Cytobank is a web-based application for storage, analysis, and sharing of flow cytometry experiments. Researchers use a web browser to log in and use a wide range of tools developed for basic and advanced flow cytometry. In addition to providing access to standard cytometry tools from any computer, Cytobank creates a platform and community for developing new analysis and publication tools. Figure layouts created on Cytobank are designed to allow transparent access to the underlying experiment annotation and data processing steps. Since all flow cytometry files and analysis data are stored on a central server, experiments and figures can be viewed or edited by anyone with the proper permissions from any computer with Internet access. Once a primary researcher has performed the initial analysis of the data, collaborators can engage in experiment analysis and make their own figure layouts using the gated, compensated experiment files. Cytobank is available to the scientific community at www.cytobank.org PMID:20578106

Confocal Microscopy and Flow Cytometry System Performance: Assessment of QA Parameters that affect data Quanitification

EPA Science Inventory

Flow and image cytometers can provide useful quantitative fluorescence data. We have devised QA tests to be used on both a flow cytometer and a confocal microscope to assure that the data is accurate, reproducible and precise. Flow Cytometry: We have provided two simple perform...

Generation and Characterization of Siglec-F-Specific Monoclonal Antibodies.

PubMed

Shahmohammadi-Farid, Sima; Ghods, Roya; Jeddi-Tehrani, Mahmood; Bayat, Ali-Ahmad; Mojtabavi, Nazanin; Razavi, Alireza; Zarnani, Amir-Hassan

2017-12-01

Siglec-F (SF) is a surface glycoprotein expressed by mouse eosinophils and induces caspase- and mitochondria-dependent apoptosis after engagement with its cognate ligand or specific antibodies. This targeting eosinophils by monoclonal antibodies may help diverse diseases associated with increased frequency of eosinophils including allergy and asthma. In this paper, production of murine and rat monoclonal antibodies (mAbs) against Siglec-F has been addressed. Balb/c mice were immunized with siglec-F1 (SF1) and siglec-F2 (SF2) synthetic peptides conjugated to a carrier protein. Rats were immunized with Chinese hamster ovary CHO cells overexpressing Siglec-F (CHO-SF) or with Siglec-F-human immunoglobulin FC fusion protein (CHO-SF-Ig). Hybridomas were produced by standard protocol and screened for their reactivity by enzyme-linked immunosorbent assay (ELISA), western blotting (WB), and flow cytometry. In parallel, polyclonal antibodies were generated in New Zealand White rabbits immunized with SF1 and SF2 peptides. Three mouse and three rat mAbs were generated against synthetic peptides and SF-Ig, respectively. All mouse monoclonal and rabbit polyclonal antibodies reacted well with immunizing molecules in ELISA and detected specific band of Siglec-F in WB. However, they failed to detect native molecule in flow cytometry analysis. Quite the contrary, rat mAbs did not reacted with the denatured protein in WB, instead exhibited significant reactivity with CHO-SF cells in flow cytometry. Based on the heavily glycosylated nature of Siglec-F, it seems that generation of anti-SF antibodies able to detect native protein needs a properly folded molecule for immunization. Monoclonal antibodies reported here are invaluable tools for studying linear and conformation epitopes of SF and tracing mouse eosinophils.

Biocompatible water soluble quantum dots as new biophotonic tools for hematologic cells: applications for flow cell cytometry

NASA Astrophysics Data System (ADS)

Lira, Rafael B.; de Sales Neto, Antonio T.; Carvalho, Kilmara K. H. G.; Leite, Elisa S.; Brasil, Aluizio G., Jr.; Azevedo, Denise P. L.; Cabral Filho, Paulo E.; Cavalcanti, Mariana B.; Amaral, Ademir J.; Farias, Patricía M. A.; Santos, Beate S.; Fontes, Adriana

2010-02-01

Quantum dots (QDs) are a promising class of fluorescent probes that can be conjugated to a variety of specific cell antibodies. For this reason, simple, cheap and reproducible routes of QDśs syntheses are the main goal of many researches in this field. The main objective of this work was to demonstrate the ability of QDs as biolabels for flow cell cytometry analysis. We have synthesized biocompatible water soluble CdS/Cd(OH)2 and CdTe/CdS QDs and applied them as fluorescent labels of hematologic cells. CdTe/CdS QDs was prepared using using a simple aqueous route with mercaptoacetic acid and mercaptopropionic acid as stabilizing agents. The resulting CdTe/CdS QDs can target biological membrane proteins and can also be internalized by cells. We applied the CdTe/CdS QDs as biolabels of human lymphocytes and compared the results obtained for lymphocytes treated and non-treated with permeabilizing agents for cell membranes. Permeabilized cells present higher fluorescence pattern than non permeabilized ones. We associated antibody A to the CdS/Cd(OH)2 QDs to label type A red blood cell (RBC). In this case, the O erythrocytes were used as the negative control. The results demonstrate that QDs were successfully functionalized with antibody A. There was a specific binding of QDs-antibody A to RBC membrane antigen only for A RBCs. We have also monitored QDs-hematologic cell interaction by using fluorescence microscopy. Our method shows that QDs can be conjugated to a variety of specific cell antibodies and can become a potential, highly efficient and low cost diagnostic tool for flow cell cytometry, very compatible with the lasers and filters used in this kind of equipments.

Therapy of Prostate Cancer Using a Human Antibody Targeting the Type 1 Insulin-Like Growth Factor Receptor (IGF-IR)

DTIC Science & Technology

2009-09-01

euthanized, tumors harvested and portions processed for IHC, Western blot, flow cytometry , culture, and RNA analysis. If not enough tissue is available...temperature for 60 minutes. Samples were analyzed by flow cytometry using a BD FACScan. Data were analyzed with CellQuestPRO software. Evaluation of BrdUrd...were approved by the University of Washington Institutional Animal Care and Use Committee (IACUC). Flow cytometry . To measure tumor IGF-IR expression

Micro and Nano-mediated 3D Cardiac Tissue Engineering

DTIC Science & Technology

2009-10-01

in both flow cytometry and Western Blot applications. The CD34 antigen is important in stem cell research due to its widespread use in identifying...for further characterization. We generated a pCD34 expressing CHO cell line (CHO-CD34) and analyzed pCD34 expression by flow cytometry (Figure 1A... flow cytometry using the 3G7 antibody and co-stained with an anti-CD31 antibody (AbD Serotec; FITC conjugated). CD31 (PECAM) is a pan endothelial

Dose-Dependent Thresholds of 10-ns Electric Pulse Induced Plasma Membrane Disruption and Cytotoxicity in Multiple Cell Lines

DTIC Science & Technology

2011-01-01

normalized to parallel controls. Flow Cytometry and Confocal Microscopy Upon exposure to 10-ns EP, aliquots of the cellular suspension were added to a tube...Survival data was processed and plotted using GrapherH software (Golden Software, Golden, Colorado). Flow cytometry results were processed in C6 software...Accuri Cytometers, Inc., Ann Arbor, MI) and FCSExpress software (DeNovo Software, Los Angeles, CA). Final analysis and presentation of flow cytometry

Sensitivity of Breast Cancer Stem Cells to TRA-8 Anti-DR5 Monoclonal Antibody

DTIC Science & Technology

2012-02-01

cytotoxicity and reduction in BrCSC marker expression. A. 2LMP cells were sorted using flow cytometry for CD44+/CD24-/ALDHhigh. Cells were pre...cells were sorted using flow cytometry for ALDH? cells and allowed to form primary tumorspheres for 3 days. After tumorspheres were mechanically...n =5 ) Day Fig. 5 Effect of ex vivo treatment of BrCSC enriched cells on tumorgenicity in NOD/SCID mice. 2LMP cells were sorted using flow cytometry

Articular Cartilage Repair Through Muscle Cell-Based Tissue Engineering

DTIC Science & Technology

2010-03-01

results suggest that sFlt-1 has more of an enhancing effect in vivo. With cell markers and flow cytometry , investiga- tors at our laboratory have...were analyzed by flow cytometry . They were immunostained by desmin, vimentin and MyoD and their chondrogenic potential was evaluated under the...M1, M2, and M3) and 3 F-MDSC populations (F1, F2, and F3) were characterized by flow cytometry for CD34 and Sca1 expression. MDSCs were labeled with

Development of small and inexpensive digital data acquisition systems using a microcontroller-based approach†

PubMed Central

Naivar, Mark A.; Wilder, Mark E.; Habbersett, Robert C.; Woods, Travis A.; Sebba, David S.; Nolan, John P.; Graves, Steven W.

2014-01-01

Fully digital data acquisition systems for use in flow cytometry provide excellent flexibility and precision. Here, we demonstrate the development of a low cost, small, and low power digital flow cytometry data acquisition system using a single microcontroller chip with an integrated analog to digital converter (ADC). Our demonstration system uses a commercially available evaluation board making the system simple to integrate into a flow cytometer. We have evaluated this system using calibration microspheres analyzed on commercial, slow-flow, and CCD based flow cytometers. In our evaluations, our demonstration data system clearly resolves all eight peaks of a Rainbow microsphere set on both a slow-flow flow cytometer and a retrofitted BD FACScalibur, which indicates it has the sensitivity and resolution required for most flow cytometry applications. It is also capable of millisecond time resolution, full waveform collection, and selective triggering of data collection from a CCD camera. The capability of our demonstration system suggests that the use of microcontrollers for flow cytometry digital data-acquisition will be increasingly valuable for extending the life of older cytometers and provides a compelling data-system design approach for low-cost, portable flow cytometers. PMID:19852060

Development of small and inexpensive digital data acquisition systems using a microcontroller-based approach.

PubMed

Naivar, Mark A; Wilder, Mark E; Habbersett, Robert C; Woods, Travis A; Sebba, David S; Nolan, John P; Graves, Steven W

2009-12-01

Fully digital data acquisition systems for use in flow cytometry provide excellent flexibility and precision. Here, we demonstrate the development of a low cost, small, and low power digital flow cytometry data acquisition system using a single microcontroller chip with an integrated analog to digital converter (ADC). Our demonstration system uses a commercially available evaluation board making the system simple to integrate into a flow cytometer. We have evaluated this system using calibration microspheres analyzed on commercial, slow-flow, and CCD-based flow cytometers. In our evaluations, our demonstration data system clearly resolves all eight peaks of a Rainbow microsphere set on both a slow-flow flow cytometer and a retrofitted BD FACScalibur, which indicates it has the sensitivity and resolution required for most flow cytometry applications. It is also capable of millisecond time resolution, full waveform collection, and selective triggering of data collection from a CCD camera. The capability of our demonstration system suggests that the use of microcontrollers for flow cytometry digital data-acquisition will be increasingly valuable for extending the life of older cytometers and provides a compelling data-system design approach for low-cost, portable flow cytometers.

Flow Cytometry Scientist | Center for Cancer Research

Cancer.gov

PROGRAM DESCRIPTION The Basic Science Program (BSP) pursues independent, multidisciplinary research in basic and applied molecular biology, immunology, retrovirology, cancer biology, and human genetics. Research efforts and support are an integral part of the Center for Cancer Research (CCR) at the Frederick National Laboratory for Cancer Research (FNLCR). KEY ROLES/RESPONSIBILITIES The Flow Cytometry Core (Flow Core) in the Cancer and Inflammation Program (CIP) is a service core which supports the research efforts of the CCR by providing expertise in the field of flow cytometry (using analyzers and sorters) with the goal of gaining a more thorough understanding of the biology of the immune system, cancer, and inflammation processes. The Flow Core provides service to 12-15 CIP laboratories and more than 22 non-CIP laboratories. Flow core staff provide technical advice on the experimental design of applications, which include immunological phenotyping, cell function assays, and cell cycle analysis. Work is performed per customer requirements, and no independent research is involved. The Flow Cytometry Scientist will be responsible for: Daily management of the Flow Cytometry Core, to include the supervision and guidance of technical staff members Monitor performance of and maintain high dimensional flow cytometer analyzers and cell sorters Operate high dimensional flow cytometer analyzers and cell sorters Provide scientific expertise to the user community and facilitate the development of cutting edge technologies Interact with Flow Core users and customers, and provide technical and scientific advice, and guidance regarding their experiments, including possible collaborations Train staff and scientific end users on the use of flow cytometry in their research, as well as teach them how to operate and troubleshoot the bench-top analyzer instruments Prepare and deliver lectures, as well as one-on-one training sessions, with customers/users Ensure that protocols are up-to-date, and appropriately adhered to Experience with sterile technique and tissue culture

Spaceflight Flow Cytometry: Design Challenges and Applications

NASA Technical Reports Server (NTRS)

Pappas, Dimitri; Kao, Shih-Hsin; Jeevarajan, Antony S.

2004-01-01

Future space exploration missions will require analytical technology capable of providing both autonomous medical care to the crew and investigative capabilities to researchers. While several promising candidate technologies exist for further development, flow cytometry is an attractive technology as it offers both crew health and a wide array of biochemistry and immunology assays. While flow cytometry has been widely used for cellular analysis in both clinical and research settings, the requirements for proper operation in spaceflight impose constraints on any instrument designs. The challenges of designing a spaceflight-ready flow cytometer are discussed, as well as some preliminary results using a prototype system.

Where Do All the Phytoplankton Go? Challenges in Keeping Track of Viable Cells in Phytoplankton Communities Using Flow Cytometry and Cell Staining

NASA Astrophysics Data System (ADS)

Simmons, L. J.; Fobbe, D. J.; Berges, J. A.

2016-02-01

Understanding the dynamics of phytoplankton communities has traditionally focused on differences in growth and related processes among taxa. It is now appreciated that differences in mortality could be equally important in contributing to these dynamics. Studying mortality in communities is difficult, especially on relevant time scales, which could be as short as hours to days. Flow cytometry can potentially provide solutions, because it can allow discrimination of different taxa, and when combined with staining, distinguish live and dead cells. We applied flow cytometry and staining to phytoplankton communities in a model system: a small, well-studied, urban pond in southeastern Wisconsin. Using flow cytometry, it was possible to resolve up to six dominant taxa (most <37 µm) and track them through an annual cycle. However, the axes traditionally used, forward scatter (FSC, related to cell size) and red fluorescence (FL3, related to chlorophyll a content) offered poor discrimination. Addition of orange fluorescence (FL2, traditionally related to phycobilipigments) and side scatter (SSC, related to cell surface characteristics) improved separation of taxa, but reproducibility (i.e. the specific position of the taxa on axes) was also more sensitive to environmental variation in the case of the fluorescence parameters. Dead cells could be distinguished by green fluorescence (FL1, using SYTOX Green©), but the stain also affected other fluorescence channels, requiring compensation. Correlations of numbers of dead cells with environmental factors (e.g. temperature, nutrient concentrations, irradiance) were generally poor, suggesting the greater importance of biotic versus abiotic variables in community mortality dynamics. Ongoing work is focusing on the effects of viral pathogens, grazing and allelopathic interactions using experimental manipulations and individual-based modeling.

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.

High resolution light-sheet based high-throughput imaging cytometry system enables visualization of intra-cellular organelles

NASA Astrophysics Data System (ADS)

Regmi, Raju; Mohan, Kavya; Mondal, Partha Pratim

2014-09-01

Visualization of intracellular organelles is achieved using a newly developed high throughput imaging cytometry system. This system interrogates the microfluidic channel using a sheet of light rather than the existing point-based scanning techniques. The advantages of the developed system are many, including, single-shot scanning of specimens flowing through the microfluidic channel at flow rate ranging from micro- to nano- lit./min. Moreover, this opens-up in-vivo imaging of sub-cellular structures and simultaneous cell counting in an imaging cytometry system. We recorded a maximum count of 2400 cells/min at a flow-rate of 700 nl/min, and simultaneous visualization of fluorescently-labeled mitochondrial network in HeLa cells during flow. The developed imaging cytometry system may find immediate application in biotechnology, fluorescence microscopy and nano-medicine.

Role of CD81 and CD58 in minimal residual disease detection in pediatric B lymphoblastic leukemia.

PubMed

Tsitsikov, E; Harris, M H; Silverman, L B; Sallan, S E; Weinberg, O K

2018-06-01

Minimal residual disease (MRD) in B lymphoblastic leukemia has been demonstrated to be a powerful predictor of clinical outcome in numerous studies in both children and adults. In this study, we evaluated 86 pediatric patients with both diagnostic and remission flow cytometry studies and compared expression of CD81, CD58, CD19, CD34, CD20, and CD38 in the detection of MRD. We evaluated 86 patients with B lymphoblastic leukemia who had both diagnostic studies and remission studies for the presence of MRD using multicolor flow cytometry. We established our detection limit for identifying abnormal lymphoblasts using serial dilutions. We also compared flow cytometry findings with molecular MRD detection in a subset of patients. We found that we can resolve differences between hematogones and lymphoblasts in 85 of 86 cases using a combination of CD45, CD19, CD34, CD10, CD20, CD38, CD58, and CD81. Our detection limit using flow cytometry is 0.002% for detecting a population of abnormal B lymphoblasts. Comparison with MRD assessment by molecular methods showed a high concordance rate with flow cytometry findings. Our study highlights importance of using multiple markers to detect MRD in B lymphoblastic leukemia. Our findings indicate that including both CD58 and CD81 markers in addition to CD19, CD34, CD20, CD38, and CD10 are helpful in MRD detection by flow cytometry. © 2018 John Wiley & Sons Ltd.

Techniques to improve the direct ex vivo detection of low frequency antigen-specific CD8+ T cells with peptide-major histocompatibility complex class I tetramers

PubMed Central

Chattopadhyay, Pratip K.; Melenhorst, J. Joseph; Ladell, Kristin; Gostick, Emma; Scheinberg, Philip; Barrett, A. John; Wooldridge, Linda; Roederer, Mario; Sewell, Andrew K.; Price, David A.

2008-01-01

The ability to quantify and characterize antigen-specific CD8+ T cells irrespective of functional readouts using fluorochrome-conjugated tetrameric peptide-MHC class I (pMHCI) complexes in conjunction with flow cytometry has transformed our understanding of cellular immune responses over the past decade. In the case of prevalent CD8+ T cell populations that engage cognate pMHCI tetramers with high avidities, direct ex vivo identification and subsequent data interpretation is relatively straightforward. However, the accurate identification of low frequency antigen-specific CD8+ T cell populations can be complicated, especially in situations where TCR-mediated tetramer binding occurs at low avidities. Here, we highlight a few simple techniques that can be employed to improve the visual resolution, and hence the accurate quantification, of tetramer-binding CD8+ T cell populations by flow cytometry. These methodological modifications enhance signal intensity, especially in the case of specific CD8+ T cell populations that bind cognate antigen with low avidity, minimize background noise and enable improved discrimination of true pMHCI tetramer binding events from nonspecific uptake. PMID:18836993

An Active, Collaborative Approach to Learning Skills in Flow Cytometry

ERIC Educational Resources Information Center

Fuller, Kathryn; Linden, Matthew D.; Lee-Pullen, Tracey; Fragall, Clayton; Erber, Wendy N.; Röhrig, Kimberley J.

2016-01-01

Advances in science education research have the potential to improve the way students learn to perform scientific interpretations and understand science concepts. We developed active, collaborative activities to teach skills in manipulating flow cytometry data using FlowJo software. Undergraduate students were given compensated clinical flow…

CytometryML, an XML format based on DICOM and FCS for analytical cytology data.

PubMed

Leif, Robert C; Leif, Suzanne B; Leif, Stephanie H

2003-07-01

Flow Cytometry Standard (FCS) was initially created to standardize the software researchers use to analyze, transmit, and store data produced by flow cytometers and sorters. Because of the clinical utility of flow cytometry, it is necessary to have a standard consistent with the requirements of medical regulatory agencies. We extended the existing mapping of FCS to the Digital Imaging and Communications in Medicine (DICOM) standard to include list-mode data produced by flow cytometry, laser scanning cytometry, and microscopic image cytometry. FCS list-mode was mapped to the DICOM Waveform Information Object. We created a collection of Extensible Markup Language (XML) schemas to express the DICOM analytical cytologic text-based data types except for large binary objects. We also developed a cytometry markup language, CytometryML, in an open environment subject to continuous peer review. The feasibility of expressing the data contained in FCS, including list-mode in DICOM, was demonstrated; and a preliminary mapping for list-mode data in the form of XML schemas and documents was completed. DICOM permitted the creation of indices that can be used to rapidly locate in a list-mode file the cells that are members of a subset. DICOM and its coding schemes for other medical standards can be represented by XML schemas, which can be combined with other relevant XML applications, such as Mathematical Markup Language (MathML). The use of XML format based on DICOM for analytical cytology met most of the previously specified requirements and appears capable of meeting the others; therefore, the present FCS should be retired and replaced by an open, XML-based, standard CytometryML. Copyright 2003 Wiley-Liss, Inc.

Antimicrobial Activity of Rhoeo discolor Phenolic Rich Extracts Determined by Flow Cytometry.

PubMed

García-Varela, Rebeca; García-García, Rebeca M; Barba-Dávila, Bertha A; Fajardo-Ramírez, Oscar R; Serna-Saldívar, Sergio O; Cardineau, Guy A

2015-10-14

Traditional medicine has led to the discovery of important active substances used in several health-related areas. Phytochemicals in Rhoeo discolor extracts have proven to have important antimicrobial activity. In the present study, our group determined the antimicrobial effects of extracts of Rhoeo discolor, a plant commonly used in Mexico for both medicinal and ornamental purposes. We evaluated the in vitro activity of phenolic rich extracts against specifically chosen microorganisms of human health importance by measuring their susceptibility via agar-disc diffusion assay and flow cytometry: Gram-positive Listeria innocua and Streptococcus mutans, Gram-negative Escherichia coli and Pseudomonas aeruginosa, and lastly a fungal pathogen Candida albicans. Ten different extracts were tested in eight different doses on all the microorganisms. Analytical data revealed a high content of phenolic compounds. Both agar-disc diffusion assay and flow cytometry results demonstrated that Pseudomonas aeruginosa was the least affected by extract exposure. However, low doses of these extracts (predominantly polar), in a range from 1 to 4 μg/mL, did produce a statistically significant bacteriostatic and bactericidal effect on the rest of the microorganisms. These results suggest the addition of certain natural extracts from Rhoeo discolor could act as antibacterial and antimycotic drugs or additives for foods and cosmetics.

Generation and characterization of monoclonal antibodies against Giardia muris trophozoites.

PubMed

Heyworth, M F; Ho, K E; Pappo, J

1989-11-01

Mouse monoclonal antibodies (mAb) were produced against Giardia muris trophozoite surface antigens. To generate B-cell hybridomas, P3/NS1/1-Ag4-1 myeloma cells were fused with splenic lymphocytes from BALB/c mice that had been immunized parenterally with G. muris trophozoites. Hybridoma culture supernatants were screened for mAb by flow cytometry of G. muris trophozoites incubated with culture supernatant followed by fluorescein-conjugated anti-mouse IgG and IgM. Flow cytometry showed three types of trophozoite staining by mAb: (i) bright staining of greater than 90% of trophozoites, with aggregation of the organisms; (ii) bright staining of approximately 90% of trophozoites, with little or no aggregation; (iii) dull staining of approximately 20% of trophozoites, without aggregation. Western blotting of mAb on G. muris trophozoite antigens separated by polyacrylamide gel electrophoresis showed that a mAb exhibiting the third of these flow cytometry staining patterns recognized trophozoite antigens of MW approximately 31,000 and 35,000. Immunoprecipitation studies indicated that the same mAb specifically precipitated two 125I-labelled trophozoite surface antigens of MW approximately 30,000. Monoclonal antibodies generated in this study may facilitate the purification and biochemical characterization of trophozoite antigens that are targets for protective intestinal antibody in G. muris-infected mice.

Quantification of Circulating Clonal Plasma Cells via Multiparametric Flow Cytometry Identifies Patients with Smoldering Multiple Myeloma at High Risk of Progression

PubMed Central

Gonsalves, Wilson I.; Rajkumar, S. Vincent; Dispenzieri, Angela; Dingli, David; Timm, Michael M.; Morice, William G.; Lacy, Martha Q.; Buadi, Francis K.; Go, Ronald S.; Leung, Nelson; Kapoor, Prashant; Hayman, Suzanne R.; Lust, John A.; Russell, Stephen J.; Zeldenrust, Steven R.; Hwa, Lisa; Kourelis, Taxiarchis V.; Kyle, Robert A.; Gertz, Morie A.; Kumar, Shaji K.

2017-01-01

The presence of high numbers of circulating clonal plasma cells (cPCs) in patients with smoldering multiple myeloma (SMM), detected by a slide-based immunofluorescence assay, has been associated with a shorter time to progression (TTP) to multiple myeloma (MM). The significance of quantifying cPCs via multiparameter flow cytometry, a much more readily available diagnostic modality, in patients with SMM has not been evaluated. This study evaluated 100 patients with a known or new diagnosis of SMM who were seen at the Mayo Clinic, Rochester from January 2008 until December 2013. Patients with ≥ 150 cPCs (N = 9) were considered to have high number of cPCs based on the 97% specificity and 78% PPV of progression to MM within 2 years of cPC assessment. The median TTP of patients with ≥ 150 cPCs was 9 months compared to not reached for patients with < 150 cPCs (P < 0.001). Thus, quantification of cPCs via multiparametric flow cytometry identifies patients with SMM at very high risk of progression to MM within 2 years and warrants confirmation in larger studies. In the future, this may allow reclassification of such patients as having MM requiring therapy prior to them enduring end-organ damage. PMID:27457702

Establishment of H2Mab-119, an Anti-Human Epidermal Growth Factor Receptor 2 Monoclonal Antibody, Against Pancreatic Cancer.

PubMed

Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Chang, Yao-Wen; Harada, Hiroyuki; Suzuki, Hiroyoshi; Kaneko, Mika K; Kato, Yukinari

2017-12-01

Human epidermal growth factor receptor 2 (HER2) is overexpressed in breast cancer and is associated with poor clinical outcomes. In addition, HER2 expression has been reported in other cancers, such as gastric, colorectal, lung, and pancreatic cancers. An anti-HER2 humanized antibody, trastuzumab, leads to significant survival benefits in patients with HER2-overexpressing breast cancers and gastric cancers. Herein, we established a novel anti-HER2 monoclonal antibody (mAb), H 2 Mab-119 (IgG 1 , kappa), and characterized its efficacy against pancreatic cancers using flow cytometry, Western blot, and immunohistochemical analyses. H 2 Mab-119 reacted with pancreatic cancer cell lines, such as KLM-1, Capan-2, and MIA PaCa-2, but did not react with PANC-1 in flow cytometry analysis. Western blot analysis also revealed a moderate signal for KLM-1 and a weak signal for MIA PaCa-2, although H 2 Mab-119 reacted strongly with LN229/HER2 cells. Finally, immunohistochemical analyses with H 2 Mab-119 revealed sensitive and specific reactions against breast and colon cancers but did not react with pancreatic cancers, indicating that H 2 Mab-119 is useful for detecting HER2 overexpression in pancreatic cancers using flow cytometry and Western blot analyses.

Flow cytometry in the post fluorescence era.

PubMed

Nolan, Garry P

2011-12-01

While flow cytometry once enabled researchers to examine 10--15 cell surface parameters, new mass flow cytometry technology enables interrogation of up to 45 parameters on a single cell. This new technology has increased understanding of cell expression and how cells differentiate during hematopoiesis. Using this information, knowledge of leukemia cell biology has also increased. Other new technologies, such as SPADE analysis and single cell network profiling (SCNP), are enabling researchers to put different cancers into more biologically similar categories and have the potential to enable more personalized medicine. Copyright © 2011. Published by Elsevier Ltd.

Novel Therapeutic and Prophylactic Modalities to Protect U.S. Armed Forces Against Major Biological Threat Agents

DTIC Science & Technology

2004-10-01

using flow cytometry after staining with CBA kit produced by BD-Pharmingen. The CBA kit can simultaneously test 5 inflammatory cytokines that include...or TLR4 transfected cells using flow cytometry . CHOK1 and CHOR1.1 cells were plated out in a 24-well dish and transfected 24 h later with either TLR2...with PE-labeled anti-hTLR2 antibody or PE-isotype control antibody (eBiosciences, CA), and cells were analyzed by flow cytometry . 39 The expression

Rapid susceptibility testing of fungi by flow cytometry using vital staining.

PubMed Central

Wenisch, C; Linnau, K F; Parschalk, B; Zedtwitz-Liebenstein, K; Georgopoulos, A

1997-01-01

A 1-h assay for antifungal susceptibility testing measuring the impairment of fungal metabolic activity was developed. Yeast viability was analyzed by flow cytometry with a novel fluorescent probe, FUN-1, which emits a red fluorescence when the yeast is metabolically active. For nine Candida albicans strains tested, this method yielded results comparable to those obtained by the standard M27 procedure for amphotericin B, flucytosine, fluconazole, and ketoconazole. Whether the flow cytometry antifungal susceptibility test results correlate with the in vivo activities of the drugs remains to determined. PMID:8968873

Examination pf Potential Anti-Tumor Activity of N-Thiolated B-Lactam Antibiotics in Nude Mice Bearing Human Breast Tumors

DTIC Science & Technology

2005-08-01

temperature in the dark, and then analyzed by flow cytometry within 3 hr of staining. 2.7. Caspase-3/-7 activity assay To measure cell-free caspase-3/-7...were treated with 50 mM of lactam 12 for the indicated hours. (A) Measurement of sub-G1 DNA content by flow cytometry analysis. The percentage of sub...Daniel for critical reading of the manuscript. We also appreciate the assistance of the Flow Cytometry Core at H. Lee Moffitt Cancer Center

The use of flow cytometry to monitor chitin synthesis in regenerating protoplasts of Candida albicans.

PubMed

Hector, R F; Braun, P C; Hart, J T; Kamarck, M E

1990-01-01

Flow cytometry was used to monitor chitin synthesis in regenerating protoplasts of the yeast Candida albicans. Comparisons of cells stained with Calcofluor White, a fluorochrome with known affinity for chitin, and cells incubated in the presence of N-[3H]-acetylglucosamine, the precursor substrate for chitin, showed a linear relationship between fluorescence and incorporation of label over time. Changes in both the fluorescence and light scatter of regenerating protoplasts treated with inhibitors of fungal chitin synthase were also quantitated by flow cytometry.

Measurement of basophil-activating capacity of grass pollen allergens, allergoids and hypoallergenic recombinant derivatives by flow cytometry using anti-CD203c.

PubMed

Kahlert, H; Cromwell, O; Fiebig, H

2003-09-01

The assessment of the basophil-activating potential is an important aspect in the development of improved preparations for specific immunotherapy. The aim of the study was to evaluate the suitability of CD203c expression as a measure of basophil activation to compare allergoids with original allergen extracts, and recombinant hypoallergenic allergen derivatives with recombinant wild-type and natural allergens. Heparinized whole blood samples from grass pollen allergic subjects were stimulated with grass pollen allergens and allergen derivatives followed by labelling of the basophils with PE-conjugated anti-CD203c. After lysis of the erythrocytes and fixation, the basophils were detected by flow cytometry. In some experiments, histamine release was determined simultaneously. Grass pollen allergoids revealed a 10-10 000-fold reduction of basophil-activating capacity measured by CD203c expression. The deletion mutant DM4 of rPhl p 5b showed stronger hypoallergenic characteristics in a range of 50-10 000-fold reduction, whereas a combination mutant of rPhl p 5b and Phl p 6 revealed less hypoallergenic features. Histamine release experiments led to a similar outcome as CD203c measurement. The measurement of CD203c expression on basophils by flow cytometry provides a rapid and sensitive method for the estimation of the allergic or hypoallergenic features of allergen preparations. The results demonstrated the hypoallergenicity of grass pollen allergoids and of the rPhl p 5b variant DM4, which may be a candidate in future preparations for specific immunotherapy.

In vivo plant flow cytometry: A first proof-of-concept

PubMed Central

Nedosekin, Dmitry A.; Khodakovskaya, Mariya V.; Biris, Alexandru S.; Wang, Daoyuan; Xu, Yang; Villagarcia, Hector; Galanzha, Ekaterina I.; Zharov, Vladimir P.

2011-01-01

In vivo flow cytometry has facilitated advances in the ultrasensitive detection of tumor cells, bacteria, nanoparticles, dyes, and other normal and abnormal objects directly in blood and lymph circulatory systems. Here, we propose in vivo plant flow cytometry for the real-time noninvasive study of nanomaterial transport in xylem and phloem plant vascular systems. As a proof of this concept, we demonstrate in vivo real-time photoacoustic monitoring of quantum dot-carbon nanotube conjugate uptake and uptake by roots and spreading through stem to leaves in a tomato plant. In addition, in vivo scanning cytometry using multimodal photoacoustic, photothermal, and fluorescent detection schematics provided multiplex detection and identification of nanoparticles accumulated in plant leaves in the presence of intensive absorption, scattering, and autofluorescent backgrounds. The use of a portable fiber-based photoacoustic flow cytometer for studies of plant vasculature was demonstrated. These integrated cytometry modalities using both endogenous and exogenous contrast agents have a potential to open new avenues of in vivo study of the nutrients, products of photosynthesis and metabolism, nanoparticles, infectious agents, and other objects transported through plant vasculature. PMID:21905208

Magnetic fingerprints of rolling cells for quantitative flow cytometry in whole blood

NASA Astrophysics Data System (ADS)

Reisbeck, Mathias; Helou, Michael Johannes; Richter, Lukas; Kappes, Barbara; Friedrich, Oliver; Hayden, Oliver

2016-09-01

Over the past 50 years, flow cytometry has had a profound impact on preclinical and clinical applications requiring single cell function information for counting, sub-typing and quantification of epitope expression. At the same time, the workflow complexity and high costs of such optical systems still limit flow cytometry applications to specialized laboratories. Here, we present a quantitative magnetic flow cytometer that incorporates in situ magnetophoretic cell focusing for highly accurate and reproducible rolling of the cellular targets over giant magnetoresistance sensing elements. Time-of-flight analysis is used to unveil quantitative single cell information contained in its magnetic fingerprint. Furthermore, we used erythrocytes as a biological model to validate our methodology with respect to precise analysis of the hydrodynamic cell diameter, quantification of binding capacity of immunomagnetic labels, and discrimination of cell morphology. The extracted time-of-flight information should enable point-of-care quantitative flow cytometry in whole blood for clinical applications, such as immunology and primary hemostasis.

Isolation of purified oocyst walls and sporocysts from Toxoplasma gondii.

PubMed

Everson, William V; Ware, Michael W; Dubey, J P; Lindquist, H D Alan

2002-01-01

Toxoplasma gondii oocysts are environmentally resistant and can infect virtually all warm-blooded hosts, including humans and livestock. Little is known about the biochemical basis for this resistance of oocysts, and mechanism for excystation of T. gondii sporozoites. The objective of the present study was to evaluate different methods (mechanical fragmentation, gradients, flow cytometry) to separate and purify T. gondii oocyst walls and sporocysts. Oocyst walls were successfully separated and purified using iodixanol gradients. Sporocysts were successfully separated and purified using iodixanol and Percoll gradients. Purification was also achieved by flow cytometry. Flow cytometry with fluorescence-activated cell sorting (FACS) yielded analytical quantities of oocyst walls and intact sporocysts. Flow cytometry with FACS also proved useful for quantitation of purity obtained following iodixanol gradient fractionation. Methods reported in this paper will be useful for analytical purposes, such as proteomic analysis of components unique to this life cycle stage, development of detection methods, or excystation studies.

Methodology and application of flow cytometry for investigation of human malaria parasites.

PubMed

Grimberg, Brian T

2011-03-31

Historically, examinations of the inhibition of malaria parasite growth/invasion, whether using drugs or antibodies, have relied on the use of microscopy or radioactive hypoxanthine uptake. These are considered gold standards for measuring the effectiveness of antimalarial treatments, however, these methods have well known shortcomings. With the advent of flow cytometry coupled with the use of fluorescent DNA stains allowed for increased speed, reproducibility, and qualitative estimates of the effectiveness of antibodies and drugs to limit malaria parasite growth which addresses the challenges of traditional techniques. Because materials and machines available to research facilities are so varied, different methods have been developed to investigate malaria parasites by flow cytometry. This review is intended to serve as a reference guide for advanced users and importantly, as a primer for new users, to support expanded use and improvements to malaria flow cytometry, particularly in endemic countries. Copyright © 2011 Elsevier B.V. All rights reserved.

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.

Flow cytometry analysis of cell cycle and specific cell synchronization with butyrate

USDA-ARS?s Scientific Manuscript database

Synchronized cells have been invaluable in many kinds of cell cycle and cell proliferation studies. Butyrate induces cell cycle arrest and apoptosis in MDBK cells. The possibility of using butyrate-blocked cells to obtain synchronized cells was explored and the properties of butyrate-induced cell ...

Novel Prostate Cancer Pathway Modeling using Boolean Implication

DTIC Science & Technology

2012-09-01

cause of cancer deaths in men. Diagnosis and pathogenesis of this disease is poorly understood. Prostate specific antigen (PSA) test is still the... specific database, I combined 14 different datasets (global prostate cancer database, total n=891) that are in Affymetrix U133A (n=456), U133A 2.0...immunohistochemistry and flow cytometry are limited by the availability of antigen - specific monoclonal antibodies and by the small number of parallel

[Identification of Vibrio cholerae O1 by flow cytometry].

PubMed

Alvarado-Alemán, F J; González-Bonilla, C; Wong-Arambula, C; Gutiérrez-Cogco, L; Sepúlveda-Amor, J; Kumate-Rodríguez, J

1994-01-01

A total of 72 peptonated water samples suspected of carrying Vibrio cholerae were assessed by laser flow cytometry (LFC) and compared with positive culture. We used a direct fluorescence technique using polyclonal (PolAb) and monoclonal antibodies (MoAb) conjugated to fluorescein. The PolAb were able to detect 33 positive samples. A clear difference among the 20 positive samples was found with only three V. cholerae O1 false negatives when MoAb were used whereas all 13 V. cholerae Non O1 samples were detected. The correlation index comparing control autofluorescence with peptonated water samples show a R = 0.69, versus 0.96 with pure V. cholerae O1 strains. Our data suggest that the LFC technique is able to recognize V. cholerae O1 from a mixture of microorganisms with high sensitivity and specificity in a few hours.

DICOM-compatible format for analytical cytology data

NASA Astrophysics Data System (ADS)

Leif, Robert C.; Leif, Suzanne B.

1998-04-01

The addition of a list mode data type to the Digital Imaging and Communications in Medicine standard, DICOM will enhance the storage and transmission of digital microscopy data and extend DICOM to include flow cytometry data. This would permit the present International Society for analytical Cytology Flow Cytometry Standard to be retired. DICOM includes: image graphics objects, specifications for describing: studies, reports, the acquisition of the data, work list management, and the individuals involved (physician, patient, etc.) The glossary of terms (objects) suitable for use with DICOM has been extended to include the collaborative effort of Logical Observation Identifier Names and Codes (LOINC) and Systematized Nomenclature of Human and Veterinary Medicine (SNOMED) to create a consistent, unambiguous clinical reference terminology. It also appears that DICOM will be a significant part of the Common Object Request Broker Architecture, CORBA.

Alternatives to current flow cytometry data analysis for clinical and research studies.

PubMed

Gondhalekar, Carmen; Rajwa, Bartek; Patsekin, Valery; Ragheb, Kathy; Sturgis, Jennifer; Robinson, J Paul

2018-02-01

Flow cytometry has well-established methods for data analysis based on traditional data collection techniques. These techniques typically involved manual insertion of tube samples into an instrument that, historically, could only measure 1-3 colors. The field has since evolved to incorporate new technologies for faster and highly automated sample preparation and data collection. For example, the use of microwell plates on benchtop instruments is now a standard on virtually every new instrument, and so users can easily accumulate multiple data sets quickly. Further, because the user must carefully define the layout of the plate, this information is already defined when considering the analytical process, expanding the opportunities for automated analysis. Advances in multi-parametric data collection, as demonstrated by the development of hyperspectral flow-cytometry, 20-40 color polychromatic flow cytometry, and mass cytometry (CyTOF), are game-changing. As data and assay complexity increase, so too does the complexity of data analysis. Complex data analysis is already a challenge to traditional flow cytometry software. New methods for reviewing large and complex data sets can provide rapid insight into processes difficult to define without more advanced analytical tools. In settings such as clinical labs where rapid and accurate data analysis is a priority, rapid, efficient and intuitive software is needed. This paper outlines opportunities for analysis of complex data sets using examples of multiplexed bead-based assays, drug screens and cell cycle analysis - any of which could become integrated into the clinical environment. Copyright © 2017. Published by Elsevier Inc.

Flow Cytometry Sorting to Separate Viable Giant Viruses from Amoeba Co-culture Supernatants

PubMed Central

Khalil, Jacques Y. B.; Langlois, Thierry; Andreani, Julien; Sorraing, Jean-Marc; Raoult, Didier; Camoin, Laurence; La Scola, Bernard

2017-01-01

Flow cytometry has contributed to virology but has faced many drawbacks concerning detection limits, due to the small size of viral particles. Nonetheless, giant viruses changed many concepts in the world of viruses, as a result of their size and hence opened up the possibility of using flow cytometry to study them. Recently, we developed a high throughput isolation of viruses using flow cytometry and protozoa co-culture. Consequently, isolating a viral mixture in the same sample became more common. Nevertheless, when one virus multiplies faster than others in the mixture, it is impossible to obtain a pure culture of the minority population. Here, we describe a robust sorting system, which can separate viable giant virus mixtures from supernatants. We tested three flow cytometry sorters by sorting artificial mixtures. Purity control was assessed by electron microscopy and molecular biology. As proof of concept, we applied the sorting system to a co-culture supernatant taken from a sample containing a viral mixture that we couldn't separate using end point dilution. In addition to isolating the quick-growing Mimivirus, we sorted and re-cultured a new, slow-growing virus, which we named “Cedratvirus.” The sorting assay presented in this paper is a powerful and versatile tool for separating viral populations from amoeba co-cultures and adding value to the new field of flow virometry. PMID:28111619

Flow Cytometry Sorting to Separate Viable Giant Viruses from Amoeba Co-culture Supernatants.

PubMed

Khalil, Jacques Y B; Langlois, Thierry; Andreani, Julien; Sorraing, Jean-Marc; Raoult, Didier; Camoin, Laurence; La Scola, Bernard

2016-01-01

Flow cytometry has contributed to virology but has faced many drawbacks concerning detection limits, due to the small size of viral particles. Nonetheless, giant viruses changed many concepts in the world of viruses, as a result of their size and hence opened up the possibility of using flow cytometry to study them. Recently, we developed a high throughput isolation of viruses using flow cytometry and protozoa co-culture. Consequently, isolating a viral mixture in the same sample became more common. Nevertheless, when one virus multiplies faster than others in the mixture, it is impossible to obtain a pure culture of the minority population. Here, we describe a robust sorting system, which can separate viable giant virus mixtures from supernatants. We tested three flow cytometry sorters by sorting artificial mixtures. Purity control was assessed by electron microscopy and molecular biology. As proof of concept, we applied the sorting system to a co-culture supernatant taken from a sample containing a viral mixture that we couldn't separate using end point dilution. In addition to isolating the quick-growing Mimivirus , we sorted and re-cultured a new, slow-growing virus, which we named "Cedratvirus." The sorting assay presented in this paper is a powerful and versatile tool for separating viral populations from amoeba co-cultures and adding value to the new field of flow virometry.

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.

Label-free counting of circulating cells by in vivo photoacoustic flow cytometry

NASA Astrophysics Data System (ADS)

Zhou, Quanyu; Yang, Ping; Wang, Qiyan; Pang, Kai; Zhou, Hui; He, Hao; Wei, Xunbin

2018-02-01

Melanoma, developing from melanocytes, is the most serious type of skin cancer. Circulating melanoma cells, the prognosis marker for metastasis, are present in the circulation at the early stage. Thus, quantitative detection of rare circulating melanoma cells is essential for monitoring tumor metastasis and prognosis evaluation. Compared with in vitro assays, in vivo flow cytometry is able to identify circulating tumor cells without drawing blood. Here, we built in vivo photoacoustic flow cytometry based on the high absorption coefficient of melanoma cells, which is applied to labelfree counting of circulating melanoma cells in tumor-bearing mice.

Critical Role of CD8 T Cells in Mediating Sex-Based Differences in a Murine Model of Lupus

DTIC Science & Technology

2009-08-21

into  female  transfers  (fF)  mice  that  was  reduced  in  CD8  depleted  fF  mice.  Flow   cytometry   analysis  showed increased numbers of splenic...splenocytes were first analyzed by flow cytometry for CD4 and CD8 T cells and F1 mice received either: a) unfractionated splenocytes (CD8 intactF1...using magnetic beads purchased from Invitrogen (Carlsbad, CA) according to the manufacturer’s instructions. Flow cytometry analysis before cell

Value of HIV patients with regular follow-up as in-house internal controls of flow cytometry measurement of lymphocyte subsets.

PubMed

de Carvalho Bittencourt, Marcelo; Kohler, Chantal; Henard, Sandrine; Rabaud, Christian; Béné, Marie C; Faure, Gilbert C

2013-01-01

Quality assessment in flow cytometry cannot obey the same rules as those applicable to the measurement of chemical analytes. However, regular follow-up of known patients may provide a robust in-house control of cell subsets evaluation. Sequential blood samples assessed for 32 HIV patients over several years and showing good stability were retrospectively assessed to establish coefficient of variations of the percentages of CD3+, CD4+, CD8+ cells, and CD4+ absolute counts (ACs). Mean relative standard variations for the whole cohort were of 0.04, 0.14, 0.08, and 0.18 for CD3%, CD4%, CD8%, and CD4 ACs, respectively. In-house follow-up of regularly checked compliant patients is a good alternative to traditional and costly repeatability and reproducibility studies for the validation of routine flow cytometry. © 2013 International Clinical Cytometry Society. Copyright © 2013 International Clinical Cytometry Society.

Value of HIV patients with regular follow-up as in-house internal controls of flow cytometry measurement of lymphocyte subsets.

PubMed

de Carvalho Bittencourt, Marcelo; Kohler, Chantal; Henard, Sandrine; Rabaud, Christian; Béné, Marie C; Faure, Gilbert C

2013-07-08

Background. Quality assessment in flow cytometry cannot obey the same rules as those applicable to the measurement of chemical analytes. However, regular follow-up of known patients may provide a robust in-house control of cell subsets evaluation. Methods. Sequential blood samples assessed for 32 HIV patients over several years and showing good stability were retrospectively assessed to establish coefficient of variations of the percentages of CD3+, CD4+, CD8+ cells and CD4+ absolute counts. Results. Mean relative standard variations for the whole cohort were of 0.04, 0.14, 0.08 and 0.18 for CD3%, CD4% CD8% and CD4 absolute counts respectively. Discussion. In-house follow up of regularly checked compliant patients is a good alternative to traditional and costly repeatability and reproducibility studies for the validation of routine flow cytometry. © 2013 Clinical Cytometry Society. Copyright © 2013 Clinical Cytometry Society.

Organizing the Cellular and Molecular Heterogeneity in High-Grade Serous Ovarian Cancer by Mass Cytometry

DTIC Science & Technology

2014-10-01

Bendall SC, Sung P, Nolan GP, Arvin AM. Single-cell mass cytometry analysis of human tonsil T cell remodeling by varicella zoster virus. Cell Rep...Perspectives on Flow Cytometry 2013, September 20, 2013, Mass Cytometry and Cell Cycle, Mexico City, Mexico (by Web Conference) Nolan: Nuclear

Flow cytometry microscopy and hyperspectral imaging of microcystis, cyanobacteria and algae

EPA Science Inventory

The detection of algae and cyanobacteria is an important step in assessing water quality. Studies were initiated using microscopy, flow cytometry and hyperspectral imaging with two fresh water species that could be grown in the laboratory: Microcystis Aeruginosa (cyanobacteria),...

Highly Specific Binding on Antifouling Zwitterionic Polymer-Coated Microbeads as Measured by Flow Cytometry.

PubMed

van Andel, Esther; de Bus, Ian; Tijhaar, Edwin J; Smulders, Maarten M J; Savelkoul, Huub F J; Zuilhof, Han

2017-11-08

Micron- and nano-sized particles are extensively used in various biomedical applications. However, their performance is often drastically hampered by the nonspecific adsorption of biomolecules, a process called biofouling, which can cause false-positive and false-negative outcomes in diagnostic tests. Although antifouling coatings have been extensively studied on flat surfaces, their use on micro- and nanoparticles remains largely unexplored, despite the widespread experimental (specifically, clinical) uncertainties that arise because of biofouling. Here, we describe the preparation of magnetic micron-sized beads coated with zwitterionic sulfobetaine polymer brushes that display strong antifouling characteristics. These coated beads can then be equipped with recognition elements of choice, to enable the specific binding of target molecules. First, we present a proof of principle with biotin-functionalized beads that are able to specifically bind fluorescently labeled streptavidin from a complex mixture of serum proteins. Moreover, we show the versatility of the method by demonstrating that it is also possible to functionalize the beads with mannose moieties to specifically bind the carbohydrate-binding protein concanavalin A. Flow cytometry was used to show that thus-modified beads only bind specifically targeted proteins, with minimal/near-zero nonspecific protein adsorption from other proteins that are present. These antifouling zwitterionic polymer-coated beads, therefore, provide a significant advancement for the many bead-based diagnostic and other biosensing applications that require stringent antifouling conditions.

Highly Specific Binding on Antifouling Zwitterionic Polymer-Coated Microbeads as Measured by Flow Cytometry

PubMed Central

2017-01-01

Micron- and nano-sized particles are extensively used in various biomedical applications. However, their performance is often drastically hampered by the nonspecific adsorption of biomolecules, a process called biofouling, which can cause false-positive and false-negative outcomes in diagnostic tests. Although antifouling coatings have been extensively studied on flat surfaces, their use on micro- and nanoparticles remains largely unexplored, despite the widespread experimental (specifically, clinical) uncertainties that arise because of biofouling. Here, we describe the preparation of magnetic micron-sized beads coated with zwitterionic sulfobetaine polymer brushes that display strong antifouling characteristics. These coated beads can then be equipped with recognition elements of choice, to enable the specific binding of target molecules. First, we present a proof of principle with biotin-functionalized beads that are able to specifically bind fluorescently labeled streptavidin from a complex mixture of serum proteins. Moreover, we show the versatility of the method by demonstrating that it is also possible to functionalize the beads with mannose moieties to specifically bind the carbohydrate-binding protein concanavalin A. Flow cytometry was used to show that thus-modified beads only bind specifically targeted proteins, with minimal/near-zero nonspecific protein adsorption from other proteins that are present. These antifouling zwitterionic polymer-coated beads, therefore, provide a significant advancement for the many bead-based diagnostic and other biosensing applications that require stringent antifouling conditions. PMID:29064669

Mass cytometry: a highly multiplexed single-cell technology for advancing drug development.

PubMed

Atkuri, Kondala R; Stevens, Jeffrey C; Neubert, Hendrik

2015-02-01

Advanced single-cell analysis technologies (e.g., mass cytometry) that help in multiplexing cellular measurements in limited-volume primary samples are critical in bridging discovery efforts to successful drug approval. Mass cytometry is the state-of-the-art technology in multiparametric single-cell analysis. Mass cytometers (also known as cytometry by time-of-flight or CyTOF) combine the cellular analysis principles of traditional fluorescence-based flow cytometry with the selectivity and quantitative power of inductively coupled plasma-mass spectrometry. Standard flow cytometry is limited in the number of parameters that can be measured owing to the overlap in signal when detecting fluorescently labeled antibodies. Mass cytometry uses antibodies tagged to stable isotopes of rare earth metals, which requires minimal signal compensation between the different metal tags. This unique feature enables researchers to seamlessly multiplex up to 40 independent measurements on single cells. In this overview we first present an overview of mass cytometry and compare it with traditional flow cytometry. We then discuss the emerging and potential applications of CyTOF technology in the pharmaceutical industry, including quantitative and qualitative deep profiling of immune cells and their applications in assessing drug immunogenicity, extensive mapping of signaling networks in single cells, cell surface receptor quantification and multiplexed internalization kinetics, multiplexing sample analysis by barcoding, and establishing cell ontologies on the basis of phenotype and/or function. We end with a discussion of the anticipated impact of this technology on drug development lifecycle with special emphasis on the utility of mass cytometry in deciphering a drug's pharmacokinetics and pharmacodynamics relationship. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Cytometry metadata in XML

NASA Astrophysics Data System (ADS)

Leif, Robert C.; Leif, Stephanie H.

2016-04-01

Introduction: The International Society for Advancement of Cytometry (ISAC) has created a standard for the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt 1.0). CytometryML will serve as a common metadata standard for flow and image cytometry (digital microscopy). Methods: The MIFlowCyt data-types were created, as is the rest of CytometryML, in the XML Schema Definition Language (XSD1.1). The datatypes are primarily based on the Flow Cytometry and the Digital Imaging and Communication (DICOM) standards. A small section of the code was formatted with standard HTML formatting elements (p, h1, h2, etc.). Results:1) The part of MIFlowCyt that describes the Experimental Overview including the specimen and substantial parts of several other major elements has been implemented as CytometryML XML schemas (www.cytometryml.org). 2) The feasibility of using MIFlowCyt to provide the combination of an overview, table of contents, and/or an index of a scientific paper or a report has been demonstrated. Previously, a sample electronic publication, EPUB, was created that could contain both MIFlowCyt metadata as well as the binary data. Conclusions: The use of CytometryML technology together with XHTML5 and CSS permits the metadata to be directly formatted and together with the binary data to be stored in an EPUB container. This will facilitate: formatting, data- mining, presentation, data verification, and inclusion in structured research, clinical, and regulatory documents, as well as demonstrate a publication's adherence to the MIFlowCyt standard, promote interoperability and should also result in the textual and numeric data being published using web technology without any change in composition.

Differences in leukocyte differentiation molecule abundances on domestic sheep (Ovis aries) and bighorn sheep (Ovis canadensis) neutrophils identified by flow cytometry

USDA-ARS?s Scientific Manuscript database

Abundance was assessed by utilizing a panel of cross-reactive monoclonal antibodies (mAbs) tested in this study. Characterization of multichannel autofluorescence of eosinophils permitted cell-type specific gating of granulocytes for quantification of LDMs on neutrophils and eosinophils by indirect,...

Flow Cytometry, Microscopy and Hyperspectral Imaging of microcystis, Cyanobacteria, and Algae- SETAC

EPA Science Inventory

The detection of cyanobacteria algae, and picoplankton, in water is an important step in assessing water quality. Studies were initiated using fluorescence microscopy, flow cytometry and hyperspectral imaging with two fresh water species that were cultured in the laboratory:Micr...

Flow cytometry enables identification of sporophytic eliciting stress treatments in gametic cells.

PubMed

Ribalta, F M; Croser, J S; Ochatt, S J

2012-01-01

Flow cytometry was used to quantify the effect of individual and combined stress treatments on elicitation of androgenesis by analyzing the relative nuclear DNA content of in vitro cultured microspores of Pisum sativum L. Differences in relative nuclear DNA content of microspores within anthers after stress treatments were clearly evident from the flow cytometry profiles, and permitted us to predict whether a combination of stresses were elicitors or enhancers of androgenesis. This is the first report to assess the effect of various stress treatments in a plant species based on relative nuclear DNA content and to use this information to categorize them as 'elicitors' or 'enhancers'. Flow cytometry represents a simple, quick and reliable way to analyze and discriminate the effect of various stress treatments on elicitation of androgenesis. These results form a solid basis for further efforts designed to enhance responses and to extend double haploid technology to other legumes. Copyright © 2011 Elsevier GmbH. All rights reserved.

Integration of lyoplate based flow cytometry and computational analysis for standardized immunological biomarker discovery.

PubMed

Villanova, Federica; Di Meglio, Paola; Inokuma, Margaret; Aghaeepour, Nima; Perucha, Esperanza; Mollon, Jennifer; Nomura, Laurel; Hernandez-Fuentes, Maria; Cope, Andrew; Prevost, A Toby; Heck, Susanne; Maino, Vernon; Lord, Graham; Brinkman, Ryan R; Nestle, Frank O

2013-01-01

Discovery of novel immune biomarkers for monitoring of disease prognosis and response to therapy in immune-mediated inflammatory diseases is an important unmet clinical need. Here, we establish a novel framework for immunological biomarker discovery, comparing a conventional (liquid) flow cytometry platform (CFP) and a unique lyoplate-based flow cytometry platform (LFP) in combination with advanced computational data analysis. We demonstrate that LFP had higher sensitivity compared to CFP, with increased detection of cytokines (IFN-γ and IL-10) and activation markers (Foxp3 and CD25). Fluorescent intensity of cells stained with lyophilized antibodies was increased compared to cells stained with liquid antibodies. LFP, using a plate loader, allowed medium-throughput processing of samples with comparable intra- and inter-assay variability between platforms. Automated computational analysis identified novel immunophenotypes that were not detected with manual analysis. Our results establish a new flow cytometry platform for standardized and rapid immunological biomarker discovery with wide application to immune-mediated diseases.

Integration of Lyoplate Based Flow Cytometry and Computational Analysis for Standardized Immunological Biomarker Discovery

PubMed Central

Villanova, Federica; Di Meglio, Paola; Inokuma, Margaret; Aghaeepour, Nima; Perucha, Esperanza; Mollon, Jennifer; Nomura, Laurel; Hernandez-Fuentes, Maria; Cope, Andrew; Prevost, A. Toby; Heck, Susanne; Maino, Vernon; Lord, Graham; Brinkman, Ryan R.; Nestle, Frank O.

2013-01-01

Discovery of novel immune biomarkers for monitoring of disease prognosis and response to therapy in immune-mediated inflammatory diseases is an important unmet clinical need. Here, we establish a novel framework for immunological biomarker discovery, comparing a conventional (liquid) flow cytometry platform (CFP) and a unique lyoplate-based flow cytometry platform (LFP) in combination with advanced computational data analysis. We demonstrate that LFP had higher sensitivity compared to CFP, with increased detection of cytokines (IFN-γ and IL-10) and activation markers (Foxp3 and CD25). Fluorescent intensity of cells stained with lyophilized antibodies was increased compared to cells stained with liquid antibodies. LFP, using a plate loader, allowed medium-throughput processing of samples with comparable intra- and inter-assay variability between platforms. Automated computational analysis identified novel immunophenotypes that were not detected with manual analysis. Our results establish a new flow cytometry platform for standardized and rapid immunological biomarker discovery with wide application to immune-mediated diseases. PMID:23843942

Vitrectomy for the diagnosis and management of uveitis of unknown cause.

PubMed

Margolis, Ron; Brasil, Oswaldo F M; Lowder, Careen Y; Singh, Rishi P; Kaiser, Peter K; Smith, Scott D; Perez, Victor L; Sonnie, Christine; Sears, Jonathan E

2007-10-01

To determine the diagnostic yield of tests commonly used for vitreous fluid analysis in eyes with suspected intraocular infection or malignancy. Noncomparative interventional case series. Forty-four consecutive patients (45 eyes) treated from 1998 through 2006 with posterior segment inflammation who underwent pars plana vitrectomy for diagnostic purposes. Vitreous specimens obtained via pars plana vitrectomy were analyzed by microbiologic culture, cytologic analysis, and flow cytometry. Diagnostic yield and sensitivity of each test performed on vitreous specimens and visual outcomes of eyes that underwent diagnostic vitrectomy (DVx). Preoperative diagnoses were infection in 15 eyes and malignancy in 30 eyes. Overall, vitreous analysis identified a specific cause in 9 (20%) of 45 eyes. The overall sensitivity of DVx was 63.6%. The sensitivities of individual tests were: culture, 50%; cytologic analysis, 66.7%; and flow cytometry, 83.3%. The yields of diagnostic tests were: culture, 5.7%; cytologic analysis, 14.3%; and flow cytometry, 20.6%. Final diagnoses were infection in 6 eyes, malignancy in 9 eyes, and idiopathic in 30 eyes. Mean visual acuity improved significantly in the first 6 months after DVx. Visual acuity improved in 60% of eyes, with 37.8% of eyes improving by 3 lines or more. Analysis of vitreous fluid by widely available tests is useful in identifying intraocular infection or malignancy. Most patients experienced a substantial improvement in vision.

Generation and characterization of monoclonal antibodies against Giardia muris trophozoites.

PubMed Central

Heyworth, M F; Ho, K E; Pappo, J

1989-01-01

Mouse monoclonal antibodies (mAb) were produced against Giardia muris trophozoite surface antigens. To generate B-cell hybridomas, P3/NS1/1-Ag4-1 myeloma cells were fused with splenic lymphocytes from BALB/c mice that had been immunized parenterally with G. muris trophozoites. Hybridoma culture supernatants were screened for mAb by flow cytometry of G. muris trophozoites incubated with culture supernatant followed by fluorescein-conjugated anti-mouse IgG and IgM. Flow cytometry showed three types of trophozoite staining by mAb: (i) bright staining of greater than 90% of trophozoites, with aggregation of the organisms; (ii) bright staining of approximately 90% of trophozoites, with little or no aggregation; (iii) dull staining of approximately 20% of trophozoites, without aggregation. Western blotting of mAb on G. muris trophozoite antigens separated by polyacrylamide gel electrophoresis showed that a mAb exhibiting the third of these flow cytometry staining patterns recognized trophozoite antigens of MW approximately 31,000 and 35,000. Immunoprecipitation studies indicated that the same mAb specifically precipitated two 125I-labelled trophozoite surface antigens of MW approximately 30,000. Monoclonal antibodies generated in this study may facilitate the purification and biochemical characterization of trophozoite antigens that are targets for protective intestinal antibody in G. muris-infected mice. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:2592009

Effect of Medium pH on Rhodosporidium toruloides NCYC 921 Carotenoid and Lipid Production Evaluated by Flow Cytometry.

PubMed

Dias, Carla; Silva, Corália; Freitas, Claudia; Reis, Alberto; da Silva, Teresa Lopes

2016-07-01

The effect of the culture medium pH (3.5-6.0) on the carotenoid and lipid (as fatty acids) production by the yeast Rhodosporidium toruloides NCYC 921 was studied. Flow cytometry was used to evaluate the yeast's physiological response to different culture medium pH values. The yeast biomass concentration and lipid content were maxima at pH 4.0 (5.90 g/L and 21.85 % w/w, respectively), while the maximum carotenoid content (63.37 μg/g) was obtained at pH 5.0. At the exponential phase, the yeast cell size and internal complexity were similar, at different medium pH. At the stationary phase, the yeast cell size and internal complexity decreased as the medium pH increased. At the exponential phase, the proportion of cells with polarized membranes was always high (>80 %) but at the stationary phase, the proportion of yeast cells with depolarized membranes was dominant (>65 %) and increased with the medium pH increase. The results here reported may contribute for yeast bioprocesses optimization. For the first time, multiparameter flow cytometry was used to evaluate the impact of medium pH changes on the yeast cell physiological status, specifically on the yeast membrane potential, membrane integrity, cell size and internal complexity.

HSV-1 infection of human corneal epithelial cells: receptor-mediated entry and trends of re-infection.

PubMed

Shah, Arpeet; Farooq, Asim V; Tiwari, Vaibhav; Kim, Min-Jung; Shukla, Deepak

2010-11-20

The human cornea is a primary target for herpes simplex virus-1 (HSV-1) infection. The goals of the study were to determine the cellular modalities of HSV-1 entry into human corneal epithelial (HCE) cells. Specific features of the study included identifying major entry receptors, assessing pH dependency, and determining trends of re-infection. A recombinant HSV-1 virus expressing beta-galactosidase was used to ascertain HSV-1 entry into HCE cells. Viral replication within cells was confirmed using a time point plaque assay. Lysosomotropic agents were used to test for pH dependency of entry. Flow cytometry and immunocytochemistry were used to determine expression of three cellular receptors--nectin-1, herpesvirus entry mediator (HVEM), and paired immunoglobulin-like 2 receptor alpha (PILR-a). The necessity of these receptors for viral entry was tested using antibody-blocking. Finally, trends of re-infection were investigated using viral entry assay and flow cytometry post-primary infection. Cultured HCE cells showed high susceptibility to HSV-1 entry and replication. Entry was demonstrated to be pH dependent as blocking vesicular acidification decreased entry. Entry receptors expressed on the cell membrane include nectin-1, HVEM, and PILR-α. Receptor-specific antibodies blocked entry receptors, reduced viral entry and indicated nectin-1 as the primary receptor used for entry. Cells re-infected with HSV-1 showed a decrease in entry, which was correlated to decreased levels of nectin-1 as demonstrated by flow cytometry. HSV-1 is capable of developing an infection in HCE cells using a pH dependent entry process that involves primarily nectin-1 but also the HVEM and PILR-α receptors. Re-infected cells show decreased levels of entry, correlated with a decreased level of nectin-1 receptor expression.

Establishment of EMab-134, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody for Detecting Squamous Cell Carcinoma Cells of the Oral Cavity.

PubMed

Itai, Shunsuke; Yamada, Shinji; Kaneko, Mika K; Chang, Yao-Wen; Harada, Hiroyuki; Kato, Yukinari

2017-12-01

Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, activates downstream signaling cascades in many tumors. In this study, we established novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. We immunized mice with a combination of the extracellular domain of EGFR and EGFR-overexpressing LN229 glioblastoma cells (LN229/EGFR) and performed the first screening using enzyme-linked immunosorbent assay. Next, we selected mAbs using flow cytometry. Among 156 established clones, two mAbs, EMab-51 (IgG 1 , kappa) and EMab-134 (IgG 1 , kappa), reacted with EGFR in Western blot analysis; EMab-134 showed a much higher sensitivity compared with EMab-51. We compared the binding affinities of EMab-51 and EMab-134 using flow cytometry; the calculated K D values for EMab-51 and EMab-134 against SAS cells/HSC-2 cells were 9.2 × 10 -9 M/9.9 × 10 -9 M and 2.6 × 10 -9 M/8.3 × 10 -9 M, respectively, indicating that EMab-134 has a higher affinity to EGFR-expressing cells. Immunohistochemical analysis of EMab-51 and EMab-134 showed sensitive and specific reactions against oral cancer cells; EMab-134 demonstrated a much higher sensitivity (36/38 cases; 94.7%) to oral squamous cell carcinomas compared with EMab-51 (6/38 cases; 15.8%). This novel anti-EGFR mAb, EMab-134, could be advantageous for detecting EGFR in the pathological analysis of EGFR-expressing cancers.

Quantum-dot-based quantitative identification of pathogens in complex mixture

NASA Astrophysics Data System (ADS)

Lim, Sun Hee; Bestwater, Felix; Buchy, Philippe; Mardy, Sek; Yu, Alexey Dan Chin

2010-02-01

In the present study we describe sandwich design hybridization probes consisting of magnetic particles (MP) and quantum dots (QD) with target DNA, and their application in the detection of avian influenza virus (H5N1) sequences. Hybridization of 25-, 40-, and 100-mer target DNA with both probes was analyzed and quantified by flow cytometry and fluorescence microscopy on the scale of single particles. The following steps were used in the assay: (i) target selection by MP probes and (ii) target detection by QD probes. Hybridization efficiency between MP conjugated probes and target DNA hybrids was controlled by a fluorescent dye specific for nucleic acids. Fluorescence was detected by flow cytometry to distinguish differences in oligo sequences as short as 25-mer capturing in target DNA and by gel-electrophoresis in the case of QD probes. This report shows that effective manipulation and control of micro- and nanoparticles in hybridization assays is possible.

Detection of ASC Speck Formation by Flow Cytometry and Chemical Cross-linking.

PubMed

Hoss, Florian; Rolfes, Verena; Davanso, Mariana R; Braga, Tarcio T; Franklin, Bernardo S

2018-01-01

Assembly of a relatively large protein aggregate or "speck" formed by the adaptor protein ASC is a common downstream step in the activation of most inflammasomes. This unique feature of ASC allows its visualization by several imaging techniques and constitutes a reliable and feasible readout for inflammasome activation in cells and tissues. We have previously described step-by-step protocols to generate immortalized cell lines stably expressing ASC fused to a fluorescent protein for measuring inflammasome activation by confocal microscopy, and immunofluorescence of endogenous ASC in primary cells. Here, we present two more methods to detect ASC speck formation: (1) Assessment of ASC speck formation by flow cytometry; and (2) Chemical cross-linking of ASC followed by immunoblotting. These methods allow for the discrimination of inflammasome-activated versus non-activated cells, the identification of lineage-specific inflammasome activation in complex cell mixtures, and sorting of inflammasome-activated cells for further analysis.

High-Throughput Particle Uptake Analysis by Imaging Flow Cytometry

PubMed Central

Smirnov, Asya; Solga, Michael D.; Lannigan, Joanne; Criss, Alison K.

2017-01-01

Quantifying the efficiency of particle uptake by host cells is important in fields including infectious diseases, autoimmunity, cancer, developmental biology, and drug delivery. Here we present a protocol for high-throughput analysis of particle uptake using imaging flow cytometry, using the bacterium Neisseria gonorrhoeae attached and internalized to neutrophils as an example. Cells are exposed to fluorescently labeled bacteria, fixed, and stained with a bacteria-specific antibody of a different fluorophore. Thus in the absence of a permeabilizing agent, extracellular bacteria are double-labeled with two fluorophores while intracellular bacteria remain single-labeled. A spot count algorithm is used to determine the number of single- and double-labeled bacteria in individual cells, to calculate the percent of cells associated with bacteria, percent of cells with internalized bacteria, and percent of cell-associated bacteria that are internalized. These analyses quantify bacterial association and internalization across thousands of cells and can be applied to diverse experimental systems. PMID:28369762

Immune Response to Mycobacterial Infection: Lessons from Flow Cytometry

PubMed Central

Rovina, Nikoletta; Panagiotou, Marios; Koulouris, Nikolaos G.

2013-01-01

Detecting and treating active and latent tuberculosis are pivotal elements for effective infection control; yet, due to their significant inherent limitations, the diagnostic means for these two stages of tuberculosis (TB) to date remain suboptimal. This paper reviews the current diagnostic tools for mycobacterial infection and focuses on the application of flow cytometry as a promising method for rapid and reliable diagnosis of mycobacterial infection as well as discrimination between active and latent TB: it summarizes diagnostic biomarkers distinguishing the two states of infection and also features of the distinct immune response against Mycobacterium tuberculosis (Mtb) at certain stages of infection as revealed by flow cytometry to date. PMID:24376464

Immune response to mycobacterial infection: lessons from flow cytometry.

PubMed

Rovina, Nikoletta; Panagiotou, Marios; Pontikis, Konstantinos; Kyriakopoulou, Magdalini; Koulouris, Nikolaos G; Koutsoukou, Antonia

2013-01-01

Detecting and treating active and latent tuberculosis are pivotal elements for effective infection control; yet, due to their significant inherent limitations, the diagnostic means for these two stages of tuberculosis (TB) to date remain suboptimal. This paper reviews the current diagnostic tools for mycobacterial infection and focuses on the application of flow cytometry as a promising method for rapid and reliable diagnosis of mycobacterial infection as well as discrimination between active and latent TB: it summarizes diagnostic biomarkers distinguishing the two states of infection and also features of the distinct immune response against Mycobacterium tuberculosis (Mtb) at certain stages of infection as revealed by flow cytometry to date.

Flow cytometry as a rapid test for detection of penicillin resistance directly in bacterial cells in Enterococcus faecalis and Staphylococcus aureus.

PubMed

Jarzembowski, T; Wiśniewska, K; Józwik, A; Bryl, E; Witkowski, J

2008-08-01

We studied the usefulness of flow cytometry for detection of penicillin resistance in E. faecalis and S. aureus by direct binding of commercially available fluorescent penicillin, Bocillin FL, to cells obtained from culture. There were significantly lower percentages of fluorescent cells and median and mean fluorescence values per particle in penicillin-resistant than in penicillin-sensitive strains of both species observed. The method allows rapid detection of penicillin resistance in S. aureus and E. faecalis. The results encourage further investigations on the detection of antibiotic resistance in bacteria using flow cytometry.

Gating mass cytometry data by deep learning.

PubMed

Li, Huamin; Shaham, Uri; Stanton, Kelly P; Yao, Yi; Montgomery, Ruth R; Kluger, Yuval

2017-11-01

Mass cytometry or CyTOF is an emerging technology for high-dimensional multiparameter single cell analysis that overcomes many limitations of fluorescence-based flow cytometry. New methods for analyzing CyTOF data attempt to improve automation, scalability, performance and interpretation of data generated in large studies. Assigning individual cells into discrete groups of cell types (gating) involves time-consuming sequential manual steps, untenable for larger studies. We introduce DeepCyTOF, a standardization approach for gating, based on deep learning techniques. DeepCyTOF requires labeled cells from only a single sample. It is based on domain adaptation principles and is a generalization of previous work that allows us to calibrate between a target distribution and a source distribution in an unsupervised manner. We show that DeepCyTOF is highly concordant (98%) with cell classification obtained by individual manual gating of each sample when applied to a collection of 16 biological replicates of primary immune blood cells, even when measured across several instruments. Further, DeepCyTOF achieves very high accuracy on the semi-automated gating challenge of the FlowCAP-I competition as well as two CyTOF datasets generated from primary immune blood cells: (i) 14 subjects with a history of infection with West Nile virus (WNV), (ii) 34 healthy subjects of different ages. We conclude that deep learning in general, and DeepCyTOF specifically, offers a powerful computational approach for semi-automated gating of CyTOF and flow cytometry data. Our codes and data are publicly available at https://github.com/KlugerLab/deepcytof.git. yuval.kluger@yale.edu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

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.

Characterizing the glycocalyx of poultry spermatozoa: I. Identification and distribution of carbohydrate residues using flow cytometry and epifluorescence microscopy.

PubMed

Peláez, Jesús; Long, Julie A

2007-01-01

The aim of the present work was to use a battery of lectins to 1) delineate the carbohydrate content of sperm glycocalyx in the turkey and chicken using flow cytometry analysis, and 2) evaluate the distribution of existing sugars over the sperm plasma membrane surface with epifluorescent microscopy. Carbohydrate groups (corresponding lectins) that were investigated included galactose (GS-I, Jacalin, RCA-I, PNA), glucose and/or mannose (Con A, PSA, GNA), N-acetyl-glucosamine (GS-II, s-WGA, STA), N-acetyl-galactosamine (SBA, WFA), fucose (Lotus, UEA-I), sialic acid (LFA, LPA), and N-acetyl-lactosamine (ECA). Spermatozoa were assessed before and after treatment with neuraminidase to remove sialic acid. Mean fluorescence intensity (MnFI) was used as indicator of lectin binding for flow cytometry analysis. Nontreated spermatozoa from both species showed high MnFI when incubated with RCA-I, Con A, LFA, and LPA, as did chicken spermatozoa incubated with s-WGA. Neuraminidase treatment increased the MnFI for most lectins except LFA and LPA, as expected. Differences in MnFI between species included higher values for s-WGA and ECA in chicken spermatozoa and for WFA in turkey spermatozoa. Microscopy revealed segregation of some sugar residues into membrane-specific domains; however, the 2 staining techniques (cell suspension vs fixed preparation) differed in identifying lectin binding patterns, with fixed preparations yielding a high degree of nonspecific binding. We conclude that 1) the glycocalyx of turkey and chicken spermatozoa contains a diversity of carbohydrate groups, 2) these residues are extensively masked by sialic acid, 3) the glycocalyx composition is species-specific, and 4) some glycoconjugates appear to be segregated into membrane-specific domains. Characterization of the poultry sperm glycocalyx is the first step in identifying the physiological impact of semen storage on sperm function.

Application of flow cytometry with a fluorescent dye to measurement of intracellular nitric oxide in plant cells.

PubMed

Kępczyński, Jan; Cembrowska-Lech, Danuta

2018-04-27

A simple and rapid method involving flow cytometry and NO-specific probe (DAF-FM DA) proved useful for detection and determination of intracellular NO production in Medicago truncatula suspension cells and leaves as well as in cells of Avena fatua, Amaranthus retroflexus embryos and leaves. The measurement of nitric oxide (NO) in plant material is important for examining the regulatory roles of endogenous NO in various physiological processes. The possibility of detecting and determining intracellular NO production by flow cytometry (FCM) with 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM DA), an NO-specific probe in Medicago truncatula cells in suspension and leaves as well as in cells of embryos and leaves of Avena fatua L. or Amaranthus retroflexus L. was explored. To detect and measure NO production by cell suspension or embryos and leaves, the recommended DAF-FM DA concentration is 5 or 10 µM, respectively, applied for 30 min. Exogenous NO increased the intensity of the fluorescent signal in embryos and leaves of both plants, while carboxy-PTIO (cPTIO), an NO scavenger, decreased it. Thus, these results demonstrate that NO can be detected and an increase and a decrease of its intracellular level can be estimated. Wounding was observed to increase the fluorescence signal, indicating an increase in the intracellular NO level. In addition, the levels of exogenous and endogenous ascorbic acid were demonstrated to have no effect on the NO-related fluorescence signal, indicating the signal's specificity only in relation with NO. The applicability of the proposed method for detection and determination of NO was confirmed (1) by in situ NO imaging in cell suspensions and (2) by determining the NO concentration in embryos and leaves using the Griess reagent. In view of the data obtained, FCM is recommended as a rapid and simple method with which to detect and determine intracellular NO production in plant cells.

Use of flow cytometry to monitor cell damage and predict fermentation activity of dried yeasts.

PubMed

Attfield, P V; Kletsas, S; Veal, D A; van Rooijen, R; Bell, P J

2000-08-01

Viable dried yeast is used as an inoculum for many fermentations in the baking and wine industries. The fermentative activity of yeast in bread dough or grape must is a critical parameter of process efficiency. Here, it is shown that fluorescent stains and flow cytometry can be used in concert to predict the abilities of populations of dried bakers' and wine yeasts to ferment after rehydration. Fluorescent dyes that stain cells only if they have damaged membrane potential (oxonol) or have increased membrane permeability (propidium iodide) were used to analyse, by flow cytometry, populations of rehydrated yeasts. A strong relationship (r2 = 0.99) was found between the percentages of populations staining with the oxonol and the degree of cell membrane damage as measured by the more traditional method of leakage of intracellular compounds. There were also were good negative relationships (r2 > or = 0.83) between fermentation by rehydrated bakers' or wine dry yeasts and percentage of populations staining with either oxonol or propidium iodide. Fluorescent staining with flow cytometry confirmed that factors such as vigour of dried yeast mixing in water, soaking before stirring, rehydration in water or fermentation medium and temperature of rehydration have profound effects on subsequent yeast vitality. These experiments indicate the potential of flow cytometry as a rapid means of predicting the fermentation performance of dried bakers' and wine yeasts.

Novel Methods of Determining Urinary Calculi Composition: Petrographic Thin Sectioning of Calculi and Nanoscale Flow Cytometry Urinalysis

PubMed Central

Gavin, Carson T; Ali, Sohrab N; Tailly, Thomas; Olvera-Posada, Daniel; Alenezi, Husain; Power, Nicholas E; Hou, Jinqiang; St. Amant, Andre H; Luyt, Leonard G; Wood, Stephen; Wu, Charles; Razvi, Hassan; Leong, Hon S

2016-01-01

Accurate determination of urinary stone composition has significant bearing on understanding pathophysiology, choosing treatment modalities and preventing recurrence. A need exists for improved methods to determine stone composition. Urine of 31 patients with known renal calculi was examined with nanoscale flow cytometry and the calculi collected during surgery subsequently underwent petrographic thin sectioning with polarized and fluorescent microscopy. Fluorescently labeled bisphosphonate probes (Alendronate-fluorescein/Alendronate-Cy5) were developed for nanoscale flow cytometry to enumerate nanocrystals that bound the fluorescent probes. Petrographic sections of stones were also imaged by fluorescent and polarized light microscopy with composition analysis correlated to alendronate +ve nanocrystal counts in corresponding urine samples. Urine samples from patients with Ca2+ and Mg2+ based calculi exhibited the highest alendronate +ve nanocrystal counts, ranging from 100–1000 nm in diameter. This novel urine based assay was in agreement with composition determined by petrographic thin sections with Alendronate probes. In some cases, high alendronate +ve nanocrystal counts indicated a Ca2+ or Mg2+ composition, as confirmed by petrographic analysis, overturning initial spectrophotometric diagnosis of stone composition. The combination of nanoscale flow cytometry and petrographic thin sections offer an alternative means for determining stone composition. Nanoscale flow cytometry of alendronate +ve nanocrystals alone may provide a high-throughput means of evaluating stone burden. PMID:26771074

Quantitative analysis of cellular glutathione by flow cytometry utilizing monochlorobimane: some applications to radiation and drug resistance in vitro and in vivo.

PubMed

Rice, G C; Bump, E A; Shrieve, D C; Lee, W; Kovacs, M

1986-12-01

An assay using a bimane derivative has been developed to detect free glutathione (GSH) in individual viable cells by flow cytometry. Monochlorobimane [syn-(ClCH2CH3)-1,5-diazabicycla[3.30]acta-3,6-diene-2,8-dio ne], itself nonfluorescent, reacts with GSH to form a highly fluorescent derivative. High pressure liquid chromatography analysis showed that, using specific staining conditions, the only low molecular weight fluorescent derivative formed in Chinese hamster ovary cells was that formed with GSH. Very little reaction with protein sulfhydryls was observed. Rates of GSH depletion in Chinese hamster ovary cells exposed to diethylmaleate were essentially the same, whether measured by relative fluorescence intensity, by flow cytometry or by enzymatic assay on cellular extracts. This method was shown to be useful for measurement of GSH resynthesis, uptake, and depletion by prolonged hypoxia and misonidazole treatment. Since measurements are made on individual cells, cell-to-cell variation and populational heterogeneity in GSH content are revealed by flow cytometry. Although under most conditions in vitro GSH content is relatively homogeneous, under certain circumstances, such as release from hypoxia, heterogeneity in populational GSH levels was observed. The significance of this heterogeneity is discussed in regard to the induction of gene amplification and drug resistance by transient hypoxia. Numerous subclones of Chinese hamster ovary cells selected by growth in Adriamycin or methotrexate-containing medium express elevated levels of GSH per cell. The method was extended to quantitate the GSH content of cells excised from EMT-6/SF mouse tumors that had been treated in vivo with L-buthionine-S-R-sulfoximine, an inhibitor of GSH synthesis. The bivariate analysis (forward angle light scatter versus monochlorobimane fluorescence) of cells derived from these tumors gave excellent resolution of normal and tumor cells and demonstrated extensive heterogeneity in the tumor cell population with respect to GSH content per cell.

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy (ATOM).

PubMed

Tang, Anson H L; Lai, Queenie T K; Chung, Bob M F; Lee, Kelvin C M; Mok, Aaron T Y; Yip, G K; Shum, Anderson H C; Wong, Kenneth K Y; Tsia, Kevin K

2017-06-28

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the main trend in the advanced development of flow cytometry. Notably, adding high-resolution imaging capabilities allows for the complex morphological analysis of cellular/sub-cellular structures. This is not possible with standard flow cytometers. However, it is valuable for advancing our knowledge of cellular functions and can benefit life science research, clinical diagnostics, and environmental monitoring. Incorporating imaging capabilities into flow cytometry compromises the assay throughput, primarily due to the limitations on speed and sensitivity in the camera technologies. To overcome this speed or throughput challenge facing imaging flow cytometry while preserving the image quality, asymmetric-detection time-stretch optical microscopy (ATOM) has been demonstrated to enable high-contrast, single-cell imaging with sub-cellular resolution, at an imaging throughput as high as 100,000 cells/s. Based on the imaging concept of conventional time-stretch imaging, which relies on all-optical image encoding and retrieval through the use of ultrafast broadband laser pulses, ATOM further advances imaging performance by enhancing the image contrast of unlabeled/unstained cells. This is achieved by accessing the phase-gradient information of the cells, which is spectrally encoded into single-shot broadband pulses. Hence, ATOM is particularly advantageous in high-throughput measurements of single-cell morphology and texture - information indicative of cell types, states, and even functions. Ultimately, this could become a powerful imaging flow cytometry platform for the biophysical phenotyping of cells, complementing the current state-of-the-art biochemical-marker-based cellular assay. This work describes a protocol to establish the key modules of an ATOM system (from optical frontend to data processing and visualization backend), as well as the workflow of imaging flow cytometry based on ATOM, using human cells and micro-algae as the examples.

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

Occurrence of specific humoral non-responsiveness to swine antigens following administration of GalT-KO bone marrow to baboons

PubMed Central

Griesemer, Adam; Liang, Fan; Hirakata, Atsushi; Hirsh, Erica; Lo, Diana; Okumi, Masayoshi; Sykes, Megan; Yamada, Kazuhiko; Huang, Christene A.; Sachs, David H.

2010-01-01

Background Hematopoietic chimerism induces transplantation tolerance across allogeneic and xenogeneic barriers, but has been difficult to achieve in the pig-to-primate model. We have now utilized swine with knockout of the gene coding for α-1,3-galactosyltransferase (GalT-KO pigs) as bone marrow donors in an attempt to achieve chimerism and tolerance by avoiding the effects of natural antibodies to Gal determinants on pig hematopoietic cells. Methods Baboons (n = 4; Baboons 1 to 4 = B156, B158, B167, and B175, respectively) were splenectomized and conditioned with TBI (150 cGy), thymic irradiation (700 cGy), T cell depletion with rabbit anti-thymocyte globulin (rATG) and rat anti-primate CD2 (LoCD2b), and received FK506 and supportive therapy for 28 days. All animals received GalT-KO bone marrow (1 to 2 × 109 cells/kg) in two fractions on days 0 and 2, and were thereafter monitored for the presence of pig cells by flow cytometry, for porcine progenitor cells by PCR of BM colony-forming units, and for cellular reactivity to pig cells by mixed lymphocyte reaction (MLR). In vitro antibody formation to LoCD2b and rATG was tested by ELISA; antibody reactivity to GalT-KO pig cells was tested by flow cytometry and cytotoxicity assays. Additionally, Baboons 3 and 4 received orthotopic kidney transplants on days 17 and 2, respectively, to test the potential impact of the protocol on renal transplantation. Results None of the animals showed detectable pig cells by flow cytometry for more than 12 h post-BM infusion. However, porcine progenitor cell engraftment, as evidenced by pig-derived colony forming units in the BM, as well as peripheral microchimerism in the thymus, lymph node, and peripheral blood was detected by PCR in baboons 1 and 2 for at least 28 days post-transplant. ELISA results confirmed humoral immunocompetence at time of transplantation as antibody titers to rat (LoCD2b) and rabbit (ATG) increased within 2 weeks. However, no induced antibodies to GalT-KO pig cells or increased donor specific cytotoxicity was detectable by flow cytometry. In contrast, baboons 3 and 4 developed serum antibodies to pig cells as well as to rat and rabbit immunoglobulin by day 14. Retrospective analysis revealed that although all four baboons possessed low levels of antibody-mediated cytotoxicity to GalT-KO cells prior to transplantation, the two baboons (3 and 4) that became sensitized to pig cells (and rejected pig kidneys) had relatively high pre-transplantation titers of anti–non-Gal IgG detectable by flow cytometry, whereas baboons 1 and 2 had undetectable titers. Conclusions Engraftment and specific non-responsiveness to pig cells has been achieved in two of four baboons following GalT-KO pig-to-baboon BMT. Engraftment correlated with absence of preformed anti–non-Gal IgG serum antibodies. These results are encouraging with regard to the possibility of achieving transplantation tolerance across this xenogeneic barrier. PMID:20723202

Detection of activated basophils using flow cytometry for diagnosis in atopic patients.

PubMed

Cozon, G; Ferrándiz, J; Peyramond, D; Brunet, J

1999-01-01

human basophils release mediators of allergy after cross-linking of IgE receptors by allergens. Specific activation of basophils is detectable through flow cytometry (FCM) using an anti-CD63 fluorescein-conjugated monoclonal antibody. this study evaluate the detection of activated basophils by FCM in routine diagnosis of atopic diseases as regard to skin prick tests and specific immunoglobulin E antibodies. whole blood from twenty patients suspected of atopy was preincubated with interleukin-3 (IL-3), then incubated with specific allergens. After staining using anti-CD63 antibodies, activated basophils were detected through FCM. IL-3-preincubation increases the spontaneous expression of CD63 even at low concentrations (0.1 ng/ml) on the basophils of 2 patients out of 20. The sensitivity and specificity of FCM were respectively 0.56 +/- 0.17 (m +/- SD) and 1.0 +/- 0.0 for the detection of dust mite-activated basophils without IL-3 preincubation, and 0.73 +/- 0.13 and 1.0 +/- 0.0 for the detention of grass pollen-activated basophils. IL-3-preincubation increased the sensitivity in a dose-dependent manner but decreased the specificity fo FCM for detecting dust mite hypersensitivity. this method allow for rapid and easy detection of activated basophils from whole blood, and could be of interest for detecting allergies to non-conventional allergens such as pharmaceutical drugs.

Noninvasive imaging of multiple myeloma using near infrared fluorescent molecular probe

NASA Astrophysics Data System (ADS)

Hathi, Deep; Zhou, Haiying; Bollerman-Nowlis, Alex; Shokeen, Monica; Akers, Walter J.

2016-03-01

Multiple myeloma is a plasma cell malignancy characterized by monoclonal gammopathy and osteolytic bone lesions. Multiple myeloma is most commonly diagnosed in late disease stages, presenting with pathologic fracture. Early diagnosis and monitoring of disease status may improve quality of life and long-term survival for multiple myeloma patients from what is now a devastating and fatal disease. We have developed a near-infrared targeted fluorescent molecular probe with high affinity to the α4β1 integrin receptor (VLA-4)overexpressed by a majority of multiple myeloma cells as a non-radioactive analog to PET/CT tracer currently being developed for human diagnostics. A near-infrared dye that emits about 700 nm was conjugated to a high affinity peptidomimmetic. Binding affinity and specificity for multiple myeloma cells was investigated in vitro by tissue staining and flow cytometry. After demonstration of sensitivity and specificity, preclinical optical imaging studies were performed to evaluate tumor specificity in murine subcutaneous and metastatic multiple myeloma models. The VLA-4-targeted molecular probe showed high affinity for subcutaneous MM tumor xenografts. Importantly, tumor cells specific accumulation in the bone marrow of metastatic multiple myeloma correlated with GFP signal from transfected cells. Ex vivo flow cytometry of tumor tissue and bone marrow further corroborated in vivo imaging data, demonstrating the specificity of the novel agent and potential for quantitative imaging of multiple myeloma burden in these models.

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

Teaching the Microbial Growth Curve Concept Using Microalgal Cultures and Flow Cytometry

ERIC Educational Resources Information Center

Forget, Nathalie; Belzile, Claude; Rioux, Pierre; Nozais, Christian

2010-01-01

The microbial growth curve is widely studied within microbiology classes and bacteria are usually the microbial model used. Here, we describe a novel laboratory protocol involving flow cytometry to assess the growth dynamics of the unicellular microalgae "Isochrysis galbana." The algal model represents an appropriate alternative to…

Targeting the Nociceptin/Orphanin FQ Receptor for Scleroderma Therapy

DTIC Science & Technology

2015-12-01

bottom well; the number of migrating cells is quantified by flow cytometry. In the aortic ring assay, freshly isolated thoracic aorta rings will...quantified by flow cytometry. In the aortic ring assay, freshly isolated thoracic aorta rings will be harvested and mounted in a small-vessel myograph. KO

Flow cytometry of sputum: assessing inflammation and immune response elements in the bronchial airways**

EPA Science Inventory

Rationale: 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. ...

Flow cytometry as a tool for analyzing changes in Plasmodium falciparum cell cycle following treatment with indol compounds.

PubMed

Schuck, Desirée Cigaran; Ribeiro, Ramira Yuri; Nery, Arthur A; Ulrich, Henning; Garcia, Célia R S

2011-11-01

Melatonin and its derivatives modulate the Plasmodium falciparum and Plasmodium chabaudi cell cycle. Flow cytometry was employed together with the nucleic acid dye YOYO-1 allowing precise discrimination between mono- and multinucleated forms of P. falciparum-infected red blood cell. The use of YOYO-1 permitted excellent discrimination between uninfected and infected red blood cells as well as between early and late parasite stages. Fluorescence intensities of schizont-stage parasites were about 10-fold greater than those of ring-trophozoite form parasites. Melatonin and related indolic compounds including serotonin, N-acetyl-serotonin and tryptamine induced an increase in the percentage of multinucleated forms compared to non-treated control cultures. YOYO-1 staining of infected erythrocyte and subsequent flow cytometry analysis provides a powerful tool in malaria research for screening of bioactive compounds. Copyright © 2011 International Society for Advancement of Cytometry.

Semi-quantitative estimation of cellular SiO2 nanoparticles using flow cytometry combined with X-ray fluorescence measurements.

PubMed

Choi, Seo Yeon; Yang, Nuri; Jeon, Soo Kyung; Yoon, Tae Hyun

2014-09-01

In this study, we have demonstrated feasibility of a semi-quantitative approach for the estimation of cellular SiO2 nanoparticles (NPs), which is based on the flow cytometry measurements of their normalized side scattering intensity. In order to improve our understanding on the quantitative aspects of cell-nanoparticle interactions, flow cytometry, transmission electron microscopy, and X-ray fluorescence experiments were carefully performed for the HeLa cells exposed to SiO2 NPs with different core diameters, hydrodynamic sizes, and surface charges. Based on the observed relationships among the experimental data, a semi-quantitative cellular SiO2 NPs estimation method from their normalized side scattering and core diameters was proposed, which can be applied for the determination of cellular SiO2 NPs within their size-dependent linear ranges. © 2014 International Society for Advancement of Cytometry.

Time-Domain Microfluidic Fluorescence Lifetime Flow Cytometry for High-Throughput Förster Resonance Energy Transfer Screening

PubMed Central

Nedbal, Jakub; Visitkul, Viput; Ortiz-Zapater, Elena; Weitsman, Gregory; Chana, Prabhjoat; Matthews, Daniel R; Ng, Tony; Ameer-Beg, Simon M

2015-01-01

Sensing ion or ligand concentrations, physico-chemical conditions, and molecular dimerization or conformation change is possible by assays involving fluorescent lifetime imaging. The inherent low throughput of imaging impedes rigorous statistical data analysis on large cell numbers. We address this limitation by developing a fluorescence lifetime-measuring flow cytometer for fast fluorescence lifetime quantification in living or fixed cell populations. The instrument combines a time-correlated single photon counting epifluorescent microscope with microfluidics cell-handling system. The associated computer software performs burst integrated fluorescence lifetime analysis to assign fluorescence lifetime, intensity, and burst duration to each passing cell. The maximum safe throughput of the instrument reaches 3,000 particles per minute. Living cells expressing spectroscopic rulers of varying peptide lengths were distinguishable by Förster resonant energy transfer measured by donor fluorescence lifetime. An epidermal growth factor (EGF)-stimulation assay demonstrated the technique's capacity to selectively quantify EGF receptor phosphorylation in cells, which was impossible by measuring sensitized emission on a standard flow cytometer. Dual-color fluorescence lifetime detection and cell-specific chemical environment sensing were exemplified using di-4-ANEPPDHQ, a lipophilic environmentally sensitive dye that exhibits changes in its fluorescence lifetime as a function of membrane lipid order. To our knowledge, this instrument opens new applications in flow cytometry which were unavailable due to technological limitations of previously reported fluorescent lifetime flow cytometers. The presented technique is sensitive to lifetimes of most popular fluorophores in the 0.5–5 ns range including fluorescent proteins and is capable of detecting multi-exponential fluorescence lifetime decays. This instrument vastly enhances the throughput of experiments involving fluorescence lifetime measurements, thereby providing statistically significant quantitative data for analysis of large cell populations. © 2014 International Society for Advancement of Cytometry PMID:25523156

Biochemical characterization of prostate-specific membrane antigen from canine prostate carcinoma cells.

PubMed

Wu, Lisa Y; Johnson, Jacqueline M; Simmons, Jessica K; Mendes, Desiree E; Geruntho, Jonathan J; Liu, Tiancheng; Dirksen, Wessel P; Rosol, Thomas J; Davis, William C; Berkman, Clifford E

2014-05-01

Prostate-specific membrane antigen (PSMA) remains an important target for diagnostic and therapeutic application for human prostate cancer. Model cell lines have been recently developed to study canine prostate cancer but their PSMA expression and enzymatic activity have not been elucidated. The present study was focused on determining PSMA expression in these model canine cell lines and the use of fluorescent small-molecule enzyme inhibitors to detect canine PSMA expression by flow cytometry. Western blot and RT-PCR were used to determine the transcriptional and translational expression of PSMA on the canine cell lines Leo and Ace-1. An endpoint HPLC-based assay was used to monitor the enzymatic activity of canine PSMA and the potency of enzyme inhibitors. Flow cytometry was used to detect the PSMA expressed on Leo and Ace-1 cells using a fluorescently tagged PSMA enzyme inhibitor. Canine PSMA expression on the Leo cell line was confirmed by Western blot and RT-PCR, the enzyme activity, and flow cytometry. Kinetic parameters Km and Vmax of PSMA enzymatic activity for the synthetic substrate (PABGγG) were determined to be 393 nM and 220 pmol min(-1)  mg protein(-1) , respectively. The inhibitor core 1 and fluorescent inhibitor 2 were found to be potent reversible inhibitors (IC50  = 13.2 and 1.6 nM, respectively) of PSMA expressed on the Leo cell line. Fluorescent labeling of Leo cells demonstrated that the fluorescent PSMA inhibitor 2 can be used for the detection of PSMA-positive canine prostate tumor cells. Expression of PSMA on Ace-1 was low and not detectable by flow cytometry. The results described herein have demonstrated that PSMA is expressed on canine prostate tumor cells and exhibits similar enzymatic characteristics as human PSMA. The findings show that the small molecule enzyme inhibitors currently being studied for use in diagnosis and therapy of human prostate cancer can also be extended to include canine prostate cancer. Importantly, the findings demonstrate that the potential of the inhibitors for use in diagnosis and therapy can be evaluated in an immunocompetent animal model that naturally develops prostate cancer before use in humans. © 2014 Wiley Periodicals, Inc.

FlowCal: A user-friendly, open source software tool for automatically converting flow cytometry data from arbitrary to calibrated units

PubMed Central

Castillo-Hair, Sebastian M.; Sexton, John T.; Landry, Brian P.; Olson, Evan J.; Igoshin, Oleg A.; Tabor, Jeffrey J.

2017-01-01

Flow cytometry is widely used to measure gene expression and other molecular biological processes with single cell resolution via fluorescent probes. Flow cytometers output data in arbitrary units (a.u.) that vary with the probe, instrument, and settings. Arbitrary units can be converted to the calibrated unit molecules of equivalent fluorophore (MEF) using commercially available calibration particles. However, there is no convenient, non-proprietary tool available to perform this calibration. Consequently, most researchers report data in a.u., limiting interpretation. Here, we report a software tool named FlowCal to overcome current limitations. FlowCal can be run using an intuitive Microsoft Excel interface, or customizable Python scripts. The software accepts Flow Cytometry Standard (FCS) files as inputs and is compatible with different calibration particles, fluorescent probes, and cell types. Additionally, FlowCal automatically gates data, calculates common statistics, and produces publication quality plots. We validate FlowCal by calibrating a.u. measurements of E. coli expressing superfolder GFP (sfGFP) collected at 10 different detector sensitivity (gain) settings to a single MEF value. Additionally, we reduce day-to-day variability in replicate E. coli sfGFP expression measurements due to instrument drift by 33%, and calibrate S. cerevisiae mVenus expression data to MEF units. Finally, we demonstrate a simple method for using FlowCal to calibrate fluorescence units across different cytometers. FlowCal should ease the quantitative analysis of flow cytometry data within and across laboratories and facilitate the adoption of standard fluorescence units in synthetic biology and beyond. PMID:27110723

International Society for the Advancement of Cytometry Cell Sorter Biosafety Standards

PubMed Central

Holmes, Kevin L.; Fontes, Benjamin; Hogarth, Philip; Konz, Richard; Monard, Simon; Pletcher, Charles H.; Wadley, Robert B.; Schmid, Ingrid; Perfetto, Stephen P.

2014-01-01

Flow cytometric cell sorting of biological specimens has become prevalent in basic and clinical research laboratories. These specimens may contain known or unknown infectious agents, necessitating precautions to protect instrument operators and the environment from biohazards arising from the use of sorters. To this end the International Society of Analytical Cytology (ISAC) was proactive in establishing biosafety guidelines in 1997 (Schmid et al., Cytometry 1997;28:99–117) and subsequently published revised biosafety standards for cell sorting of unfixed samples in 2007 (Schmid et al., Cytometry Part A J Int Soc Anal Cytol 2007;71A:414–437). Since their publication, these documents have become recognized worldwide as the standard of practice and safety precautions for laboratories performing cell sorting experiments. However, the field of cytometry has progressed since 2007, and the document requires an update. The new Standards provides guidance: (1) for laboratory design for cell sorter laboratories; (2) for the creation of laboratory or instrument specific Standard Operating Procedures (SOP); and (3) on procedures for the safe operation of cell sorters, including personal protective equipment (PPE) and validation of aerosol containment. PMID:24634405

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.

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

Characterization and use of new monoclonal antibodies to CD11c,CD14, and CD163 to analyze the phenotypic complexity of ruminantmonocyte subsets

USDA-ARS?s Scientific Manuscript database

The sequencing of the bovine genome and development of mass spectrometry, in conjunction with flow cytometry (FC), have afforded an opportunity to complete the characterization of the specificity of monoclonal antibodies (mAbs), only partially characterized during previous international workshops fo...

Structure of the Global Nanoscience and Nanotechnology Research Literature

DTIC Science & Technology

2006-01-01

Transistors, Nature, 424 (6949): 654-657, 2003. Joannopoulos, JD, Meade, RD, Winn, JN, Photonic Crystals: Molding the Flow of Light, Princeton...1.27 Force Microscopy 40 0.10 0.00 Electron Spectroscopy 40 0.10 0.00 Rutherford backscattering spectrometry 38 0.10 0.00 flow cytometry 36 0.09...Backscattering Spectroscopy/Spectrometry • Flow Cytometry • Spectrophotometry (UV-Visible) • Deep Level Transient Spectroscopy • Inductively

Altered immunophenotypic features of peripheral blood platelets in myelodysplastic syndromes

PubMed Central

Sandes, Alex F.; Yamamoto, Mihoko; Matarraz, Sergio; Chauffaille, Maria de Lourdes L.F.; Quijano, Sandra; López, Antonio; Oguro, Tsutomu; Kimura, Eliza Y. S.; Orfao, Alberto

2012-01-01

Background Multiparameter flow cytometric analysis of bone marrow and peripheral blood cells has proven to be of help in the diagnostic workup of myelodysplastic syndromes. However, the usefulness of flow cytometry for the detection of megakaryocytic and platelet dysplasia has not yet been investigated. The aim of this pilot study was to evaluate by flow cytometry the diagnostic and prognostic value of platelet dysplasia in myelodysplastic syndromes. Design and Methods We investigated the pattern of expression of distinct surface glycoproteins on peripheral blood platelets from a series of 44 myelodysplastic syndrome patients, 20 healthy subjects and 19 patients with platelet alterations associated to disease conditions other than myelodysplastic syndromes. Quantitative expression of CD31, CD34, CD36, CD41a, CD41b, CD42a, CD42b and CD61 glycoproteins together with the PAC-1, CD62-P, fibrinogen and CD63 platelet activation-associated markers and platelet light scatter properties were systematically evaluated. Results Overall, flow cytometry identified multiple immunophenotypic abnormalities on platelets of myelodysplastic syndrome patients, including altered light scatter characteristics, over-and under expression of specific platelet glycoproteins and asynchronous expression of CD34; decreased expression of CD36 (n=5), CD42a (n=1) and CD61 (n=2), together with reactivity for CD34 (n=1) were only observed among myelodysplastic syndrome cases, while other alterations were also found in other platelet disorders. Based on the overall platelet alterations detected for each patient, an immunophenotypic score was built which identified a subgroup of myelodysplastic syndrome patients with a high rate of moderate to severe alterations (score>1.5; n=16) who more frequently showed thrombocytopenia, megakaryocytic dysplasia and high-risk disease, together with a shorter overall survival. Conclusions Our results show the presence of altered phenotypes by flow cytometry on platelets from around half of the myelodysplastic syndrome patients studied. If confirmed in larger series of patients, these findings may help refine the diagnostic and prognostic assessment of this group of disorders. PMID:22271903

Mobile flow cytometer for mHealth.

PubMed

Balsam, Joshua; Bruck, Hugh Alan; Rasooly, Avraham

2015-01-01

Flow cytometry is used for cell counting and analysis in numerous clinical and environmental applications. However flow cytometry is not used in mHealth mainly because current flow cytometers are large, expensive, power-intensive devices designed to operate in a laboratory. Their design results in a lack of portability and makes them unsuitable for mHealth applications. Another limitation of current technology is the low volumetric throughput rates that are not suitable for rapid detection of rare cells.To address these limitations, we describe here a novel, low-cost, mobile flow cytometer based on wide-field imaging with a webcam for large volume and high throughput fluorescence detection of rare cells as a simulation for circulating tumor cells (CTCs) detection. The mobile flow cytometer uses a commercially available webcam capable of 187 frames per second video capture at a resolution of 320 × 240 pixels. For fluorescence detection, a 1 W 450 nm blue laser is used for excitation of Syto-9 fluorescently stained cells detected at 535 nm. A wide-field flow cell was developed for large volume analysis that allows for the linear velocity of target cells to be lower than in conventional hydrodynamic focusing flow cells typically used in cytometry. The mobile flow cytometer was found to be capable of detecting low concentrations at flow rates of 500 μL/min, suitable for rare cell detection in large volumes. The simplicity and low cost of this device suggests that it may have a potential clinical use for mHealth flow cytometry for resource-poor settings associated with global health.

Annual Progress Report FY-91. Volume 1 and 2.

DTIC Science & Technology

1992-03-12

Pulmonary Med Tech 11 0644 GS Berger, TA Allergy Microbiologist 12 0403 GS Billups, L Flow Cytom Chemist 12 1320 GS Vacant Pulmonary Kyle Metabolic Unit...Reactions 11 (11/89) 3349 Salata, Kalman PhD. Mitogen-Inducible T Suppressor Cell 12 Assay by Flow Cytometry (12/89) 3350 Salata, Kalman PhD. Flow ...3/90) 3354 Salata, Kalman PhD. Two Way Mixed Lymphocyte Culture: 17 Analysis by Two Color Flow Cytometry (4/90) 3355 Salata, Kalman PhD. Effect of

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.

78 FR 5186 - Clinical Flow Cytometry in Hematologic Malignancies; Public Workshop; Request for Comments

Federal Register 2010, 2011, 2012, 2013, 2014

2013-01-24

... in the diagnosis of leukemia and lymphoma and more recently in the detection of minimal residual... chronic lymphocytic leukemia (CLL); (3) Third-party flow cytometry data analysis software; and (4... held February 27, 2013 (77 FR 76051, December 26, 2012). An FDA workshop for acute lymphocytic leukemia...

Applications of flow cytometry to toxicological mycotoxin effects in cultured mammalian cells: a review.

PubMed

Juan-García, Ana; Manyes, Lara; Ruiz, María-José; Font, Guillermina

2013-06-01

This review gives an overview of flow cytometry applications to toxicological studies of several physiological target sites of mycotoxins on different mammalian cell lines. Mycotoxins are secondary metabolites of fungi that may be present in food, feed, air and water. The increasing presence of mycotoxins in crops, their wide distribution in the food chain, and their potential for toxicity demonstrate the need for further knowledge. Flow cytometry has become a valuable tool in mycotoxin studies in recent years for the rapid analysis of single cells in a mixture. In toxicology, the power of these methods lies in the possibility of determining a wide range of cell parameters, providing valuable information to elucidate cell growth and viability, metabolic activity, mitochondrial membrane potential and membrane integrity mechanisms. There are studies using flow cytometry technique on Alternaria, Aspergillus, Fusarium and Penicillium mycotoxins including information about cell type, assay conditions and functional parameters. Most of the studies collected in the literature are on deoxynivalenol and zearalenone mycotoxins. Cell cycle analysis and apoptosis are the processes more widely investigated. Copyright © 2013 Elsevier Ltd. All rights reserved.

[Fiat Lux. May be no more true in cytometry! Go to mass and spectrum but still stay classic].

PubMed

Idziorek, Thierry; Cazareth, Julie; Blanc, Catherine; Jouy, Nathalie; Bourdely, Pierre; Corneau, Aurélien

2018-05-01

The last decade has been an era of accelerated technological progress for flow cytometry. New technologies have been developed such as mass cytometry in which standard fluorochromes have been replaced by lanthanide-based non-radioactive metals and by spectral cytometry that measures the complete fluorescence spectrum. In this review, we schematically describe conventional, mass and spectral cytometry and present the plus and minus of each technology. © 2018 médecine/sciences – Inserm.

Functional expression of Ca²⁺ dependent mammalian transmembrane gap junction protein Cx43 in slime mold Dictyostelium discoideum.

PubMed

Kaufmann, Stefan; Weiss, Ingrid M; Eckstein, Volker; Tanaka, Motomu

2012-03-09

In this paper, we expressed murine gap junction protein Cx43 in Dictyostelium discoideum by introducing the specific vector pDXA. In the first step, the successful expression of Cx43 and Cx43-eGFP was verified by (a) Western blot (anti-Cx43, anti-GFP), (b) fluorescence microscopy (eGFP-Cx43 co-expression, Cx43 immunostaining), and (c) flow cytometry analysis (eGFP-Cx43 co-expression). Although the fluorescence signals from cells expressing Cx43-eGFP detected by fluorescence microscopy seem relatively low, analysis by flow cytometry demonstrated that more than 60% of cells expressed Cx43-eGFP. In order to evaluate the function of expressed Cx43 in D. discoideum, we examined the hemi-channel function of Cx43. In this series of experiments, the passive uptake of carboxyfluorescein was monitored using flow cytometric analysis. A significant number of the transfected cells showed a prominent dye uptake in the absence of Ca(2+). The dye uptake by transfected cells in the presence of Ca(2+) was even lower than the non-specific dye uptake by non-transformed Ax3 orf+ cells, confirming that Cx43 expressed in D. discoideum retains its Ca(2+)-dependent, specific gating function. The expression of gap junction proteins expressed in slime molds opens a possibility to the biological significance of intercellular communications in development and maintenance of multicellular organisms. Copyright © 2012 Elsevier Inc. All rights reserved.

flowVS: channel-specific variance stabilization in flow cytometry.

PubMed

Azad, Ariful; Rajwa, Bartek; Pothen, Alex

2016-07-28

Comparing phenotypes of heterogeneous cell populations from multiple biological conditions is at the heart of scientific discovery based on flow cytometry (FC). When the biological signal is measured by the average expression of a biomarker, standard statistical methods require that variance be approximately stabilized in populations to be compared. Since the mean and variance of a cell population are often correlated in fluorescence-based FC measurements, a preprocessing step is needed to stabilize the within-population variances. We present a variance-stabilization algorithm, called flowVS, that removes the mean-variance correlations from cell populations identified in each fluorescence channel. flowVS transforms each channel from all samples of a data set by the inverse hyperbolic sine (asinh) transformation. For each channel, the parameters of the transformation are optimally selected by Bartlett's likelihood-ratio test so that the populations attain homogeneous variances. The optimum parameters are then used to transform the corresponding channels in every sample. flowVS is therefore an explicit variance-stabilization method that stabilizes within-population variances in each channel by evaluating the homoskedasticity of clusters with a likelihood-ratio test. With two publicly available datasets, we show that flowVS removes the mean-variance dependence from raw FC data and makes the within-population variance relatively homogeneous. We demonstrate that alternative transformation techniques such as flowTrans, flowScape, logicle, and FCSTrans might not stabilize variance. Besides flow cytometry, flowVS can also be applied to stabilize variance in microarray data. With a publicly available data set we demonstrate that flowVS performs as well as the VSN software, a state-of-the-art approach developed for microarrays. The homogeneity of variance in cell populations across FC samples is desirable when extracting features uniformly and comparing cell populations with different levels of marker expressions. The newly developed flowVS algorithm solves the variance-stabilization problem in FC and microarrays by optimally transforming data with the help of Bartlett's likelihood-ratio test. On two publicly available FC datasets, flowVS stabilizes within-population variances more evenly than the available transformation and normalization techniques. flowVS-based variance stabilization can help in performing comparison and alignment of phenotypically identical cell populations across different samples. flowVS and the datasets used in this paper are publicly available in Bioconductor.

Rapid assessment of viable but non-culturable Bacillus coagulans MTCC 5856 in commercial formulations using Flow cytometry.

PubMed

Majeed, Muhammed; Majeed, Shaheen; Nagabhushanam, Kalyanam; Punnapuzha, Ardra; Philip, Sheena; Mundkur, Lakshmi

2018-01-01

Accurate enumeration of bacterial count in probiotic formulation is imperative to ensure that the product adheres to regulatory standards and citation in consumer product label. Standard methods like plate count, can enumerate only replicating bacterial population under selected culture conditions. Viable but non culturable bacteria (VBNC) retain characteristics of living cells and can regain cultivability by a process known as resuscitation. This is a protective mechanism adapted by bacteria to evade stressful environmental conditions. B. coagulans MTCC 5856(LactoSpore®) is a probiotic endospore which can survive for decades in hostile environments without dividing. In the present study, we explored the use of flow cytometry to enumerate the viable count of B. coagulans MTCC 5856 under acidic and alkaline conditions, high temperature and in commercial formulations like compressed tablets and capsules. Flow cytometry (FCM) was comparable to plate count method when the spores were counted at physiological conditions. We show that VBNC state is induced in B. coagulans MTCC 5856by high temperature and acidic pH. The cells get resuscitated under physiological conditions and FCM was sensitive to detect the VBNC spores. Flow cytometry showed excellent ability to assess the viable spore count in commercial probiotic formulations of B. coagulans MTCC 5856. The results establish Flow cytometry as a reliable method to count viable bacteria in commercial probiotic preparations. Sporulation as well as existence as VBNC could contribute to the extreme stability of B. coagulans MTCC 5856.

Rapid assessment of viable but non-culturable Bacillus coagulans MTCC 5856 in commercial formulations using Flow cytometry

PubMed Central

Majeed, Muhammed; Majeed, Shaheen; Nagabhushanam, Kalyanam; Punnapuzha, Ardra; Philip, Sheena

2018-01-01

Accurate enumeration of bacterial count in probiotic formulation is imperative to ensure that the product adheres to regulatory standards and citation in consumer product label. Standard methods like plate count, can enumerate only replicating bacterial population under selected culture conditions. Viable but non culturable bacteria (VBNC) retain characteristics of living cells and can regain cultivability by a process known as resuscitation. This is a protective mechanism adapted by bacteria to evade stressful environmental conditions. B. coagulans MTCC 5856(LactoSpore®) is a probiotic endospore which can survive for decades in hostile environments without dividing. In the present study, we explored the use of flow cytometry to enumerate the viable count of B. coagulans MTCC 5856 under acidic and alkaline conditions, high temperature and in commercial formulations like compressed tablets and capsules. Flow cytometry (FCM) was comparable to plate count method when the spores were counted at physiological conditions. We show that VBNC state is induced in B. coagulans MTCC 5856by high temperature and acidic pH. The cells get resuscitated under physiological conditions and FCM was sensitive to detect the VBNC spores. Flow cytometry showed excellent ability to assess the viable spore count in commercial probiotic formulations of B. coagulans MTCC 5856. The results establish Flow cytometry as a reliable method to count viable bacteria in commercial probiotic preparations. Sporulation as well as existence as VBNC could contribute to the extreme stability of B. coagulans MTCC 5856. PMID:29474436

Highly multiplexed simultaneous detection of RNAs and proteins in single cells.

PubMed

Frei, Andreas P; Bava, Felice-Alessio; Zunder, Eli R; Hsieh, Elena W Y; Chen, Shih-Yu; Nolan, Garry P; Gherardini, Pier Federico

2016-03-01

To enable the detection of expression signatures specific to individual cells, we developed PLAYR (proximity ligation assay for RNA), a method for highly multiplexed transcript quantification by flow and mass cytometry that is compatible with standard antibody staining. When used with mass cytometry, PLAYR allowed for the simultaneous quantification of more than 40 different mRNAs and proteins. In primary cells, we quantified multiple transcripts, with the identity and functional state of each analyzed cell defined on the basis of the expression of a separate set of transcripts or proteins. By expanding high-throughput deep phenotyping of cells beyond protein epitopes to include RNA expression, PLAYR opens a new avenue for the characterization of cellular metabolism.

Does the high nucleic acid content of individual bacterial cells allow us to discriminate between active cells and inactive cells in aquatic systems?

PubMed

Lebaron, P; Servais, P; Agogué, H; Courties, C; Joux, F

2001-04-01

The nucleic acid contents of individual bacterial cells as determined with three different nucleic acid-specific fluorescent dyes (SYBR I, SYBR II, and SYTO 13) and flow cytometry were compared for different seawater samples. Similar fluorescence patterns were observed, and bacteria with high apparent nucleic acid contents (HNA) could be discriminated from bacteria with low nucleic acid contents (LNA). The best discrimination between HNA and LNA cells was found when cells were stained with SYBR II. Bacteria in different water samples collected from seven freshwater, brackish water, and seawater ecosystems were prelabeled with tritiated leucine and then stained with SYBR II. After labeling and staining, HNA, LNA, and total cells were sorted by flow cytometry, and the specific activity of each cellular category was determined from leucine incorporation rates. The HNA cells were responsible for most of the total bacterial production, and the specific activities of cells in the HNA population varied between samples by a factor of seven. We suggest that nucleic acid content alone can be a better indicator of the fraction of growing cells than total counts and that this approach should be combined with other fluorescent physiological probes to improve detection of the most active cells in aquatic systems.

Does the High Nucleic Acid Content of Individual Bacterial Cells Allow Us To Discriminate between Active Cells and Inactive Cells in Aquatic Systems?

PubMed Central

Lebaron, Philippe; Servais, Pierre; Agogué, Helene; Courties, Claude; Joux, Fabien

2001-01-01

The nucleic acid contents of individual bacterial cells as determined with three different nucleic acid-specific fluorescent dyes (SYBR I, SYBR II, and SYTO 13) and flow cytometry were compared for different seawater samples. Similar fluorescence patterns were observed, and bacteria with high apparent nucleic acid contents (HNA) could be discriminated from bacteria with low nucleic acid contents (LNA). The best discrimination between HNA and LNA cells was found when cells were stained with SYBR II. Bacteria in different water samples collected from seven freshwater, brackish water, and seawater ecosystems were prelabeled with tritiated leucine and then stained with SYBR II. After labeling and staining, HNA, LNA, and total cells were sorted by flow cytometry, and the specific activity of each cellular category was determined from leucine incorporation rates. The HNA cells were responsible for most of the total bacterial production, and the specific activities of cells in the HNA population varied between samples by a factor of seven. We suggest that nucleic acid content alone can be a better indicator of the fraction of growing cells than total counts and that this approach should be combined with other fluorescent physiological probes to improve detection of the most active cells in aquatic systems. PMID:11282632

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

Vasdekis, Andreas E.; Stephanopoulos, Gregory

The sampling and manipulation of cells down to the individual has been of substantial interest since the very beginning of Life Sciences. Herein, our objective is to highlight the most recent developments in single cell manipulation, as well as pioneering ones. First, flow-through methods will be discussed, namely methods in which the single cells flow continuously in an ordered manner during their analysis. This section will be followed by confinement techniques that enable cell isolation and confinement in one, two- or three-dimensions. Flow cytometry and droplet microfluidics are the two most common methods of flow-through analysis. While both are high-throughputmore » techniques, their difference lays in the fact that the droplet encapsulated cells experience a restricted and personal microenvironment, while in flow cytometry cells experience similar nutrient and stimuli initial concentrations. These methods are rather well established; however, they recently enabled immense strides in single cell phenotypic analysis, namely the identification and analysis of metabolically distinct individuals from an isogenic population using both droplet microfluidics and flow cytometry.« less

Impact of Two Measures of Micrometastatic Disease on Clinical Outcomes in Patients with Newly Diagnosed Ewing Sarcoma: A Report from the Children’s Oncology Group

PubMed Central

Vo, Kieuhoa T.; Edwards, Jeremy V.; Epling, C. Lorrie; Sinclair, Elizabeth; Hawkins, Douglas S.; Grier, Holcombe E.; Janeway, Katherine A.; Barnette, Phillip; McIlvaine, Elizabeth; Krailo, Mark D.; Barkauskas, Donald A.; Matthay, Katherine K.; Womer, Richard B.; Gorlick, Richard G.; Lessnick, Stephen L.; Mackall, Crystal L.; DuBois, Steven G.

2016-01-01

Purpose Flow cytometry and RT-PCR can detect occult Ewing sarcoma (ES) cells in the blood and bone marrow (BM). These techniques were used to evaluate the prognostic significance of micrometastatic disease in ES. Experimental Design Newly diagnosed patients with ES were enrolled on two prospective multi-center studies. In the flow cytometry cohort, patients were defined as “positive” for BM micrometastatic disease if their CD99+/CD45− values were above the upper limit in 22 control patients. In the PCR cohort, RT-PCR on blood or BM samples classified the patients as “positive” or “negative” for EWSR1/FLI1 translocations. The association between micrometastatic disease burden with clinical features and outcome was assessed. Co-expression of IGF-1R on detected tumor cells was performed in a subset of flow cytometry samples. Results The median total BM CD99+CD45− percent was 0.0012% (range 0–1.10%) in the flow cytometry cohort, with 14/109 (12.8%) of ES patients defined as “positive.” In the PCR cohort, 19.6% (44/225) patients were “positive” for any EWSR1/FLI1 translocation in blood or BM. There were no differences in baseline clinical features or event-free or overall survival between patients classified as “positive” vs. “negative” by either method. CD99+CD45− cells had significantly higher IGF-1R expression compared to CD45+ hematopoietic cells (mean geometric mean fluorescence intensity 982.7 vs. 190.9; p<0.001). Conclusion The detection of micrometastatic disease at initial diagnosis by flow cytometry or RT-PCR is not associated with outcome in newly diagnosed patients with ES. Flow cytometry provides a tool to characterize occult micrometastatic tumor cells for proteins of interest. PMID:26861456

SCENERY: a web application for (causal) network reconstruction from cytometry data

PubMed Central

Papoutsoglou, Georgios; Athineou, Giorgos; Lagani, Vincenzo; Xanthopoulos, Iordanis; Schmidt, Angelika; Éliás, Szabolcs; Tegnér, Jesper

2017-01-01

Abstract Flow and mass cytometry technologies can probe proteins as biological markers in thousands of individual cells simultaneously, providing unprecedented opportunities for reconstructing networks of protein interactions through machine learning algorithms. The network reconstruction (NR) problem has been well-studied by the machine learning community. However, the potentials of available methods remain largely unknown to the cytometry community, mainly due to their intrinsic complexity and the lack of comprehensive, powerful and easy-to-use NR software implementations specific for cytometry data. To bridge this gap, we present Single CEll NEtwork Reconstruction sYstem (SCENERY), a web server featuring several standard and advanced cytometry data analysis methods coupled with NR algorithms in a user-friendly, on-line environment. In SCENERY, users may upload their data and set their own study design. The server offers several data analysis options categorized into three classes of methods: data (pre)processing, statistical analysis and NR. The server also provides interactive visualization and download of results as ready-to-publish images or multimedia reports. Its core is modular and based on the widely-used and robust R platform allowing power users to extend its functionalities by submitting their own NR methods. SCENERY is available at scenery.csd.uoc.gr or http://mensxmachina.org/en/software/. PMID:28525568

Semi-automatized segmentation method using image-based flow cytometry to study sperm physiology: the case of capacitation-induced tyrosine phosphorylation.

PubMed

Matamoros-Volante, Arturo; Moreno-Irusta, Ayelen; Torres-Rodriguez, Paulina; Giojalas, Laura; Gervasi, María G; Visconti, Pablo E; Treviño, Claudia L

2018-02-01

Is image-based flow cytometry a useful tool to study intracellular events in human sperm such as protein tyrosine phosphorylation or signaling processes? Image-based flow cytometry is a powerful tool to study intracellular events in a relevant number of sperm cells, which enables a robust statistical analysis providing spatial resolution in terms of the specific subcellular localization of the labeling. Sperm capacitation is required for fertilization. During this process, spermatozoa undergo numerous physiological changes, via activation of different signaling pathways, which are not completely understood. Classical approaches for studying sperm physiology include conventional microscopy, flow cytometry and Western blotting. These techniques present disadvantages for obtaining detailed subcellular information of signaling pathways in a relevant number of cells. This work describes a new semi-automatized analysis using image-based flow cytometry which enables the study, at the subcellular and population levels, of different sperm parameters associated with signaling. The increase in protein tyrosine phosphorylation during capacitation is presented as an example. Sperm cells were isolated from seminal plasma by the swim-up technique. We evaluated the intensity and distribution of protein tyrosine phosphorylation in sperm incubated in non-capacitation and capacitation-supporting media for 1 and 18 h under different experimental conditions. We used an antibody against FER kinase and pharmacological inhibitors in an attempt to identify the kinases involved in protein tyrosine phosphorylation during human sperm capacitation. Semen samples from normospermic donors were obtained by masturbation after 2-3 days of sexual abstinence. We used the innovative technique image-based flow cytometry and image analysis tools to segment individual images of spermatozoa. We evaluated and quantified the regions of sperm where protein tyrosine phosphorylation takes place at the subcellular level in a large number of cells. We also used immunocytochemistry and Western blot analysis. Independent experiments were performed with semen samples from seven different donors. Using image analysis tools, we developed a completely novel semi-automatic strategy useful for segmenting thousands of individual cell images obtained using image-based flow cytometry. Contrary to immunofluorescence which relies on the analysis of a limited sperm population and also on the observer, image-based flow cytometry allows for unbiased quantification and simultaneous localization of post-translational changes in an extended sperm population. Interestingly, important data can be independently analyzed by looking to the frame of interest. As an example, we evaluated the capacitation-associated increase in tyrosine phosphorylation in sperm incubated in non-capacitation and capacitation-supporting media for 1 and 18 h. As previously reported, protein tyrosine phosphorylation increases in a time-depending manner, but our method revealed that this increase occurs differentially among distinct sperm segments. FER kinase is reported to be the enzyme responsible for the increase in protein tyrosine phosphorylation in mouse sperm. Our Western blot analysis revealed for the first time the presence of this enzyme in human sperm. Using our segmentation strategy, we aimed to quantify the effect of pharmacological inhibition of FER kinase and found a marked reduction of protein tyrosine phosphorylation only in the flagellum, which corresponded to the physical localization of FER in human sperm. Our method provides an alternative strategy to study signaling markers associated with capacitation, such as protein tyrosine phosphorylation, in a fast and quantitative manner. None. This is an in vitro study performed under controlled conditions. Chemical inhibitors are not completely specific for the intended target; the possibility of side effects cannot be discarded. Our results demonstrate that the use of image-based flow cytometry is a very powerful tool to study sperm physiology. A large number of cells can be easily analyzed and information at the subcellular level can be obtained. As the segmentation process works with bright-field images, it can be extended to study expression of other proteins of interest using different antibodies or it can be used in living sperm to study intracellular parameters that can be followed using fluorescent dyes sensitive to the parameter of interest (e.g. pH, Ca2+). Therefore, this a versatile method that can be exploited to study several aspects of sperm physiology. This work was supported DGAPA (IN203116 to C. Treviño), Fronteras-CONACyT No. 71 and Eunice Kennedy Shriver National Institute of Child Health and Human Development NIH (RO1 HD38082) to P.E. Visconti and by a Lalor Foundation fellowship to M.G. Gervasi. A. Matamoros is a student of the Maestría en Ciencias Bioquímicas-UNAM program supported by CONACyT (416400) and DGAPA-UNAM. A. Moreno obtained a scholarship from Red MacroUniversidades and L. Giojalas obtained a schloarhip from CONICET and Universidad Nacional de Cordoba. The authors declare there are not conflicts of interest. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email:journals.permissions@oup.com

Clinical Investigation Program. Annual Progress Report. Volume 1

DTIC Science & Technology

1994-01-20

Suport Labs Resch Chemist 13 0644 GS Salata, KF Allergy Microbiologist 12 0403 CS Billups, L Flow Cytom Microbiologist 12 0403 GS Dobek, AS Inf Disease 5...continued to increase laboratory research support to principal investigators throughout the medical center. The DCI Flow Cytometry Laboratory provided...Kalman PhD. Mitogen-Inducible T Suppressor Cell 12 Assay by Flow Cytometry (12/89) * Reference is to page number(s) in Volume II. 30 PROTOCOL NUMBER

Cardiomyocyte differentiation of rat bone marrow multipotent progenitor cells is associated with downregulation of Oct-4 expression.

PubMed

Lu, Tiewei; Pelacho, Beatriz; Hao, Hong; Luo, Min; Zhu, Jing; Verfaillie, Catherine M; Tian, Jie; Liu, Zhenguo

2010-10-01

This study was to determine if bone marrow multipotent adult progenitor cells (MAPCs) underwent cardiac specification and Oct-4 expression during their cardiomyocyte differentiation in vitro. MAPCs were isolated from rat bone marrow, treated with 5-azacytidine (5-aza, 1μM) for 24h, and cultured in a serum-free medium for cardiac differentiation for up to 35 days. The cells started to express early cardiac-specific genes Nkx2.5 and GATA-4 with a significant increase in their mRNA level within 24h after 5-aza treatment. Western blotting analysis and immunofluorescence staining revealed that the cardiac-specific proteins connexin-43 and troponin I were expressed in the cells 7 days after 5-aza treatment. Flow cytometry analysis demonstrated that over 37% of the cells were positive for troponin I by 35 days of differentiation, although the cells did not display spontaneous contraction. On the other hand, the undifferentiated MAPCs expressed a significant level of the stem-cell-specific marker Oct-4 that was dramatically decreased in the cells shortly after the initiation of cardiomyocyte differentiation as evaluated using real-time (RT)-polymerase chain reaction, Western blotting, immunofluorescence staining, and flow cytometry. These data indicated that MAPCs were able to effectively differentiate into cardiomyocyte-like cells after 5-aza induction in association with downregulation of Oct-4 expression.

Development and validation of a bioassay to evaluate binding of adalimumab to cell membrane-anchored TNFα using flow cytometry detection.

PubMed

Camacho-Sandoval, Rosa; Sosa-Grande, Eréndira N; González-González, Edith; Tenorio-Calvo, Alejandra; López-Morales, Carlos A; Velasco-Velázquez, Marco; Pavón-Romero, Lenin; Pérez-Tapia, Sonia Mayra; Medina-Rivero, Emilio

2018-06-05

Physicochemical and structural properties of proteins used as active pharmaceutical ingredients of biopharmaceuticals are determinant to carry out their biological activity. In this regard, the assays intended to evaluate functionality of biopharmaceuticals provide confirmatory evidence that they contain the appropriate physicochemical properties and structural conformation. The validation of the methodologies used for the assessment of critical quality attributes of biopharmaceuticals is a key requirement for manufacturing under GMP environments. Herein we present the development and validation of a flow cytometry-based methodology for the evaluation of adalimumab's affinity towards membrane-bound TNFα (mTNFα) on recombinant CHO cells. This in vitro methodology measures the interaction between an in-solution antibody and its target molecule onto the cell surface through a fluorescent signal. The characteristics evaluated during the validation exercise showed that this methodology is suitable for its intended purpose. The assay demonstrated to be accurate (r 2  = 0.92, slope = 1.20), precise (%CV ≤ 18.31) and specific (curve fitting, r 2  = 0.986-0.997) to evaluate binding of adalimumab to mTNFα. The results obtained here provide evidence that detection by flow cytometry is a viable alternative for bioassays used in the pharmaceutical industry. In addition, this methodology could be standardized for the evaluation of other biomolecules acting through the same mechanism of action. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

RchyOptimyx: Cellular Hierarchy Optimization for Flow Cytometry

PubMed Central

Aghaeepour, Nima; Jalali, Adrin; O’Neill, Kieran; Chattopadhyay, Pratip K.; Roederer, Mario; Hoos, Holger H.; Brinkman, Ryan R.

2013-01-01

Analysis of high-dimensional flow cytometry datasets can reveal novel cell populations with poorly understood biology. Following discovery, characterization of these populations in terms of the critical markers involved is an important step, as this can help to both better understand the biology of these populations and aid in designing simpler marker panels to identify them on simpler instruments and with fewer reagents (i.e., in resource poor or highly regulated clinical settings). However, current tools to design panels based on the biological characteristics of the target cell populations work exclusively based on technical parameters (e.g., instrument configurations, spectral overlap, and reagent availability). To address this shortcoming, we developed RchyOptimyx (cellular hieraRCHY OPTIMization), a computational tool that constructs cellular hierarchies by combining automated gating with dynamic programming and graph theory to provide the best gating strategies to identify a target population to a desired level of purity or correlation with a clinical outcome, using the simplest possible marker panels. RchyOptimyx can assess and graphically present the trade-offs between marker choice and population specificity in high-dimensional flow or mass cytometry datasets. We present three proof-of-concept use cases for RchyOptimyx that involve 1) designing a panel of surface markers for identification of rare populations that are primarily characterized using their intracellular signature; 2) simplifying the gating strategy for identification of a target cell population; 3) identification of a non-redundant marker set to identify a target cell population. PMID:23044634

Peripheral blood biomarkers of solid tumor angiogenesis in dogs: A polychromatic flow cytometry pilot study

PubMed Central

Bentley, R. Timothy; Mund, Julie A.; Pollok, Karen E.; Childress, Michael O.; Case, Jamie

2012-01-01

A subset of peripheral blood hematopoietic stem and progenitor cells of bone marrow origin is elevated in humans with solid cancers before treatment and declines with therapy. This biomarker of angiogenesis is not specific to tumor type and has great potential in the objective assessment of treatment response in clinical trials. This pilot study was designed to develop a biomarker of neoangiogenesis in dogs for the diagnosis of cancer, the measurement of treatment response, and the provision of objective data in clinical trials. Polychromatic flow cytometry was used to quantify two subsets of circulating hematopoietic stem and progenitor cells in dogs with spontaneous solid tumors before (n = 8) and after (n = 3) treatment, and normal controls (n = 6). Pro-angiogenic peripheral blood cells of bone marrow origin were detected in all eight cases and the six normal controls; however, there was no statistically significant difference between the two groups. Interestingly, an apparent decline in pro-angiogenic cells was observed after treatment. Bone marrow derived hematopoietic cells appear to contribute to tumor angiogenesis in dogs, as has been previously reported in humans. While the methodology for pro-angiogenic cell quantification in a small number of dogs in the current study did not result in a significant difference from normal controls, an optimized canine polychromatic flow cytometry protocol holds great promise in the development of a canine cancer model and for the objective measurements of treatment response in clinical trials. PMID:23063489

Waveguide detection of right-angle-scattered light in flow cytometry

DOEpatents

Mariella, Jr., Raymond P.

2000-01-01

A transparent flow cell is used as an index-guided optical waveguide. A detector for the flow cell but not the liquid stream detects the Right-Angle-Scattered (RAS) Light exiting from one end of the flow cell. The detector(s) could view the trapped RAS light from the flow cell either directly or through intermediate optical light guides. If the light exits one end of the flow cell, then the other end of the flow cell can be given a high-reflectivity coating to approximately double the amount of light collected. This system is more robust in its alignment than the traditional flow cytometry systems which use imaging optics, such as microscope objectives.

Immunological Tools: Engaging Students in the Use and Analysis of Flow Cytometry and Enzyme-linked Immunosorbent Assay (ELISA)

ERIC Educational Resources Information Center

Ott, Laura E.; Carson, Susan

2014-01-01

Flow cytometry and enzyme-linked immunosorbent assay (ELISA) are commonly used techniques associated with clinical and research applications within the immunology and medical fields. The use of these techniques is becoming increasingly valuable in many life science and engineering disciplines as well. Herein, we report the development and…

Differences in leukocyte differentiation molecule abundances on domestic sheep (Ovis aries) and bighorn sheep (Ovis canadensis) neutrophils identified by flow cytometry

USDA-ARS?s Scientific Manuscript database

Although both domestic sheep (DS) and bighorn sheep (BHS) are affected by similar respiratory bacterial pathogens, experimental and field data indicate BHS are more susceptible to pneumonia. Cross-reactive monoclonal antibodies (mAbs) for use in flow cytometry (FC) are valuable reagents for interspe...

Utilization of flow cytometry to identify chimeral sectors in leaf tissue of Lolium multiflorum x L. arundinaceum hybrids

USDA-ARS?s Scientific Manuscript database

We have identified a method whereby Lolium multiflorum (Lm) or L. arundinaceum (Fa) genomes are preferentially eliminated through a mitotic loss behavior in interspecific Lm x Fa F1 hybrids, generating either dihaploid Lm lines or Fa lines. Flow cytometry, a method for rapidly characterizing optical...

Flow cytometry quality requirements for monitoring of minimal disease in plasma cell myeloma.

PubMed

Oldaker, Teri A; Wallace, Paul K; Barnett, David

2016-01-01

Current therapeutic approaches for plasma cell myeloma (PCM) attain an overall survival of more than 6 years for the majority of newly diagnosed patients. However, PFS and OS are the only accepted FDA clinical endpoints for demonstrating drug efficacy before they can be become frontline therapeutic options. There is, however, recognition that the increasing gap between drug development and approval for mainstream therapeutic use needs to be shortened. As such regulatory bodies such as the FDA are now considering whether biomarker response evaluation, as in measurement of minimal residual disease (MRD) as assessed by flow cytometry (FC), can provide an early, robust prediction of survival and therefore improve the drug approval process. Recently, FC MRD using a standardized eight-color antibody methodology has been shown to have a minimum sensitivity of 0.01% and an upper sensitivity of 0.001%. To ensure that all laboratories using this approach achieve the same levels of sensitivity it is crucially important to have standardized quality management procedures in place. This manuscript accompanies those published in this special issue and describes the minimum that is required for validating and quality monitoring of this highly specific test to ensure any laboratory, irrespective of location, will achieve the expected quality standards required. © 2015 International Clinical Cytometry Society.

Monitoring Extracellular Vesicle Cargo Active Uptake by Imaging Flow Cytometry.

PubMed

Ofir-Birin, Yifat; Abou Karam, Paula; Rudik, Ariel; Giladi, Tal; Porat, Ziv; Regev-Rudzki, Neta

2018-01-01

Extracellular vesicles are essential for long distance cell-cell communication. They function as carriers of different compounds, including proteins, lipids and nucleic acids. Pathogens, like malaria parasites ( Plasmodium falciparum, Pf ), excel in employing vesicle release to mediate cell communication in diverse processes, particularly in manipulating the host response. Establishing research tools to study the interface between pathogen-derived vesicles and their host recipient cells will greatly benefit the scientific community. Here, we present an imaging flow cytometry (IFC) method for monitoring the uptake of malaria-derived vesicles by host immune cells. By staining different cargo components, we were able to directly track the cargo's internalization over time and measure the kinetics of its delivery. Impressively, we demonstrate that this method can be used to specifically monitor the translocation of a specific protein within the cellular milieu upon internalization of parasitic cargo; namely, we were able to visually observe how uptaken parasitic Pf -DNA cargo leads to translocation of transcription factor IRF3 from the cytosol to the nucleus within the recipient immune cell. Our findings demonstrate that our method can be used to study cellular dynamics upon vesicle uptake in different host-pathogen and pathogen-pathogen systems.

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.

Flow cytometry sorting of nuclei enables the first global characterization of Paramecium germline DNA and transposable elements.

PubMed

Guérin, Frédéric; Arnaiz, Olivier; Boggetto, Nicole; Denby Wilkes, Cyril; Meyer, Eric; Sperling, Linda; Duharcourt, Sandra

2017-04-26

DNA elimination is developmentally programmed in a wide variety of eukaryotes, including unicellular ciliates, and leads to the generation of distinct germline and somatic genomes. The ciliate Paramecium tetraurelia harbors two types of nuclei with different functions and genome structures. The transcriptionally inactive micronucleus contains the complete germline genome, while the somatic macronucleus contains a reduced genome streamlined for gene expression. During development of the somatic macronucleus, the germline genome undergoes massive and reproducible DNA elimination events. Availability of both the somatic and germline genomes is essential to examine the genome changes that occur during programmed DNA elimination and ultimately decipher the mechanisms underlying the specific removal of germline-limited sequences. We developed a novel experimental approach that uses flow cell imaging and flow cytometry to sort subpopulations of nuclei to high purity. We sorted vegetative micronuclei and macronuclei during development of P. tetraurelia. We validated the method by flow cell imaging and by high throughput DNA sequencing. Our work establishes the proof of principle that developing somatic macronuclei can be sorted from a complex biological sample to high purity based on their size, shape and DNA content. This method enabled us to sequence, for the first time, the germline DNA from pure micronuclei and to identify novel transposable elements. Sequencing the germline DNA confirms that the Pgm domesticated transposase is required for the excision of all ~45,000 Internal Eliminated Sequences. Comparison of the germline DNA and unrearranged DNA obtained from PGM-silenced cells reveals that the latter does not provide a faithful representation of the germline genome. We developed a flow cytometry-based method to purify P. tetraurelia nuclei to high purity and provided quality control with flow cell imaging and high throughput DNA sequencing. We identified 61 germline transposable elements including the first Paramecium retrotransposons. This approach paves the way to sequence the germline genomes of P. aurelia sibling species for future comparative genomic studies.

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

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

PubMed Central

Zhu, Hongying; Ozcan, Aydogan

2013-01-01

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. ~ 10 μm over a very large field-of-view of ~ 81 mm2. This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water. PMID:23603893

Wide-field fluorescent microscopy and fluorescent imaging flow cytometry on a cell-phone.

PubMed

Zhu, Hongying; Ozcan, Aydogan

2013-04-11

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. - 10 μm over a very large field-of-view of - 81 mm(2). This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water.

Trace detection of specific viable bacteria using tetracysteine-tagged bacteriophages.

PubMed

Wu, Lina; Luan, Tian; Yang, Xiaoting; Wang, Shuo; Zheng, Yan; Huang, Tianxun; Zhu, Shaobin; Yan, Xiaomei

2014-01-07

Advanced methods are urgently needed to determine the identity and viability of trace amounts of pathogenic bacteria in a short time. Existing approaches either fall short in the accurate assessment of microbial viability or lack specificity in bacterial identification. Bacteriophages (or phages for short) are viruses that exclusively infect bacterial host cells with high specificity. As phages infect and replicate only in living bacterial hosts, here we exploit the strategy of using tetracysteine (TC)-tagged phage in combination with biarsenical dye to the discriminative detection of viable target bacteria from dead target cells and other viable but nontarget bacterial cells. Using recombinant M13KE-TC phage and Escherichia coli ER2738 as a model system, distinct differentiation between individual viable target cells from dead target cells was demonstrated by flow cytometry and fluorescence microscopy. As few as 1% viable E. coli ER2738 can be accurately quantified in a mix with dead E. coli ER2738 by flow cytometry. With fluorescence microscopic measurement, specific detection of as rare as 1 cfu/mL original viable target bacteria was achieved in the presence of a large excess of dead target cells and other viable but nontarget bacterial cells in 40 mL artificially contaminated drinking water sample in less than 3 h. This TC-phage-FlAsH approach is sensitive, specific, rapid, and simple, and thus shows great potential in water safety monitoring, health surveillance, and clinical diagnosis of which trace detection and identification of viable bacterial pathogens is highly demanded.

Non-linear optical flow cytometry using a scanned, Bessel beam light-sheet.

PubMed

Collier, Bradley B; Awasthi, Samir; Lieu, Deborah K; Chan, James W

2015-05-29

Modern flow cytometry instruments have become vital tools for high-throughput analysis of single cells. However, as issues with the cellular labeling techniques often used in flow cytometry have become more of a concern, the development of label-free modalities for cellular analysis is increasingly desired. Non-linear optical phenomena (NLO) are of growing interest for label-free analysis because of the ability to measure the intrinsic optical response of biomolecules found in cells. We demonstrate that a light-sheet consisting of a scanned Bessel beam is an optimal excitation geometry for efficiently generating NLO signals in a microfluidic environment. The balance of photon density and cross-sectional area provided by the light-sheet allowed significantly larger two-photon fluorescence intensities to be measured in a model polystyrene microparticle system compared to measurements made using other excitation focal geometries, including a relaxed Gaussian excitation beam often used in conventional flow cytometers.

Stability validation of paraformaldehyde-fixed samples for the assessment of the platelet PECAM-1, P-selectin, and PAR-1 thrombin receptor by flow cytometry.

PubMed

Atar, Oliver D; Eisert, Christian; Pokov, Ilya; Serebruany, Victor L

2010-07-01

Sample fixation for storage and/or transportation represents an unsolved challenge for multicenter clinical trials assessing serial changes in platelet activity, or monitoring various antiplatelet regimens. Whole blood flow cytometry represents a major advance in defining platelet function, although special training and expensive equipment is required. We sought to determine how fixation with 2% paraformaldehyde (PFA), and storage of blood samples over 1 week affects the flow cytometry readings for both intact and thrombin-activating four major surface platelet receptors. Whole blood platelet expression of PECAM-1, P-selectin, PAR-1 inactive receptor (SPAN-12), and cleaved (WEDE-15) epitope was assessed immediately after blood draw, after staining with 2% PFA, and at day 1, 3, 5, and 7. The study was performed in 6 volunteers with multiple risk factors for vascular disease, not receiving any antiplatelet agents. Staining with PFA resulted in a slight decrease of fluorescence intensity, especially for PECAM-1, while antigen expression at day 1, 3 and 5 remains consistent, and highly reproducible. At day 7 there was a small but inconsistent trend towards diminished fluorescence intensity. The platelet data were consistent while validated with the isotype-matched irrelevant antibody. These data suggest that there is a 5 day window to perform final flow cytometry readings of whole blood PFA-fixed inactivated platelet samples. In contrast, thrombin activation cause gradual loss of flow cytometry signal, and cannot be recommended for long-term storage. This is critical logistic information for conducting multicenter platelet substudies within the framework of major clinical trials.

Progress on an implementation of MIFlowCyt in XML

NASA Astrophysics Data System (ADS)

Leif, Robert C.; Leif, Stephanie H.

2015-03-01

Introduction: The International Society for Advancement of Cytometry (ISAC) Data Standards Task Force (DSTF) has created a standard for the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt 1.0). The CytometryML schemas, are based in part upon the Flow Cytometry Standard and Digital Imaging and Communication (DICOM) standards. CytometryML has and will be extended and adapted to include MIFlowCyt, as well as to serve as a common standard for flow and image cytometry (digital microscopy). Methods: The MIFlowCyt data-types were created, as is the rest of CytometryML, in the XML Schema Definition Language (XSD1.1). Individual major elements of the MIFlowCyt schema were translated into XML and filled with reasonable data. A small section of the code was formatted with HTML formatting elements. Results: The differences in the amount of detail to be recorded for 1) users of standard techniques including data analysts and 2) others, such as method and device creators, laboratory and other managers, engineers, and regulatory specialists required that separate data-types be created to describe the instrument configuration and components. A very substantial part of the MIFlowCyt element that describes the Experimental Overview part of the MIFlowCyt and substantial parts of several other major elements have been developed. Conclusions: The future use of structured XML tags and web technology should facilitate searching of experimental information, its presentation, and inclusion in structured research, clinical, and regulatory documents, as well as demonstrate in publications adherence to the MIFlowCyt standard. The use of CytometryML together with XML technology should also result in the textual and numeric data being published using web technology without any change in composition. Preliminary testing indicates that CytometryML XML pages can be directly formatted with the combination of HTML and CSS.

[Isolation and identification of human periodontal ligament stem cells in vitro].

PubMed

Shen, Tao; Chang, Hui-jun; Jian, Cong-xiang; Yang, Yan-chun; Zhou, Ji-xiang

2011-02-01

To isolate and identify human periodontal ligament stem cells (PDLSC) by improved methods and assess the characteristics of PDLSC ex vivo. The periodontal ligament cells were obtained from the healthy impacted third molars and teeth extracted for orthodontic purposes and used to isolate PDLSC by limiting dilution assay. PDLSC were cultured and expanded in alpha-MEM supplemented with 10% FBS. Colony-forming assay, immunohistochemistry, flow cytometry, osteogenic and adipogenic induction were used to identify PDLSC. The obtained cells had high colony-forming efficiency and were positive staining for vimentin and negative for pancytokeratin. Flow cytometry revealed that the isolated cells were positive for STRO-1 and CD146 antibodies and most were in the G0/G1 phase of cell cycle. Under specific conditions, they could differentiate to the osteoblast and adipocyte lineages in vitro. Limiting dilution assay is an effective method to isolate PDLSC and the single-cell-derived colonies demonstrate the properties of stem cells in vitro.

The characterization of exosome from blood plasma of patients with colorectal cancer

NASA Astrophysics Data System (ADS)

Yunusova, N. V.; Tamkovich, S. N.; Stakheeva, M. N.; Afanas'ev, S. G.; Frolova, A. Y.; Kondakova, I. V.

2016-08-01

Exosomes are extracellular membrane structures involved in many physiological and pathological processes including cancerogenesis and metastasis. The clarification of the criteria for exosome isolating and identifying is the purpose of this study. Exosome samples from the plasma of patients with colorectal cancer and healthy donors were examined using transmission electron microscopy and flow cytometry in accordance with the minimum requirements of "International Society for Extracellular Vesicles". The choice of the method for isolation of exosomes from the blood plasma by ultrafiltration and ultracentrifugation allowed obtaining highly purified samples of exosomes, in which all the structural components were clearly seen. The results obtained with flow cytometry suggest that exosomes of blood plasma from patients with colorectal cancer can be produced by epithelial cells. Moreover, cells produce different types of exosomes, which correspond to different mechanisms in sorting macromolecules in the membrane of multivesicular bodies. Determination of significant differences in the expression of specific exosomal proteins from colorectal cancer patients compared to healthy donors suggests a high diagnostic potential significance of circulating exosomes.

Human immune cell targeting of protein nanoparticles - caveospheres

NASA Astrophysics Data System (ADS)

Glass, Joshua J.; Yuen, Daniel; Rae, James; Johnston, Angus P. R.; Parton, Robert G.; Kent, Stephen J.; de Rose, Robert

2016-04-01

Nanotechnology has the power to transform vaccine and drug delivery through protection of payloads from both metabolism and off-target effects, while facilitating specific delivery of cargo to immune cells. However, evaluation of immune cell nanoparticle targeting is conventionally restricted to monocultured cell line models. We generated human caveolin-1 nanoparticles, termed caveospheres, which were efficiently functionalized with monoclonal antibodies. Using this platform, we investigated CD4+ T cell and CD20+ B cell targeting within physiological mixtures of primary human blood immune cells using flow cytometry, imaging flow cytometry and confocal microscopy. Antibody-functionalization enhanced caveosphere binding to targeted immune cells (6.6 to 43.9-fold) within mixed populations and in the presence of protein-containing fluids. Moreover, targeting caveospheres to CCR5 enabled caveosphere internalization by non-phagocytic CD4+ T cells--an important therapeutic target for HIV treatment. This efficient and flexible system of immune cell-targeted caveosphere nanoparticles holds promise for the development of advanced immunotherapeutics and vaccines.

Multivariate data analysis methods for the interpretation of microbial flow cytometric data.

PubMed

Davey, Hazel M; Davey, Christopher L

2011-01-01

Flow cytometry is an important technique in cell biology and immunology and has been applied by many groups to the analysis of microorganisms. This has been made possible by developments in hardware that is now sensitive enough to be used routinely for analysis of microbes. However, in contrast to advances in the technology that underpin flow cytometry, there has not been concomitant progress in the software tools required to analyse, display and disseminate the data and manual analysis, of individual samples remains a limiting aspect of the technology. We present two new data sets that illustrate common applications of flow cytometry in microbiology and demonstrate the application of manual data analysis, automated visualisation (including the first description of a new piece of software we are developing to facilitate this), genetic programming, principal components analysis and artificial neural nets to these data. The data analysis methods described here are equally applicable to flow cytometric applications with other cell types.

High sensitivity detection and sorting of infectious human immunodeficiency virus (HIV-1) particles by flow virometry

PubMed Central

Bonar, Micha M.; Tilton, John C.

2017-01-01

Detection of viruses by flow cytometry is complicated by their small size. Here, we characterized the ability of a standard (FACSAria II) and a sub-micron flow cytometer (A50 Micro) to resolve HIV-1 viruses. The A50 was superior at resolving small particles but did not reliably distinguish HIV-1, extracellular vesicles, and laser noise by light scatter properties alone. However, single fluorescent HIV-1 particles could readily be detected by both cytometers. Fluorescent particles were sorted and retained infectivity, permitting further exploration of the functional consequences of HIV-1 heterogeneity. Finally, flow cytometry had a limit of detection of 80 viruses/ml, nearly equal to PCR assays. These studies demonstrate the power of flow cytometry to detect and sort viral particles and provide a critical toolkit to validate methods to label wild-type HIV-1; quantitatively assess integrity and aggregation of viruses and virus-based therapeutics; and efficiently screen drugs inhibiting viral assembly and release. PMID:28235684

High sensitivity detection and sorting of infectious human immunodeficiency virus (HIV-1) particles by flow virometry.

PubMed

Bonar, Michał M; Tilton, John C

2017-05-01

Detection of viruses by flow cytometry is complicated by their small size. Here, we characterized the ability of a standard (FACSAria II) and a sub-micron flow cytometer (A50 Micro) to resolve HIV-1 viruses. The A50 was superior at resolving small particles but did not reliably distinguish HIV-1, extracellular vesicles, and laser noise by light scatter properties alone. However, single fluorescent HIV-1 particles could readily be detected by both cytometers. Fluorescent particles were sorted and retained infectivity, permitting further exploration of the functional consequences of HIV-1 heterogeneity. Finally, flow cytometry had a limit of detection of 80 viruses/ml, nearly equal to PCR assays. These studies demonstrate the power of flow cytometry to detect and sort viral particles and provide a critical toolkit to validate methods to label wild-type HIV-1; quantitatively assess integrity and aggregation of viruses and virus-based therapeutics; and efficiently screen drugs inhibiting viral assembly and release. Copyright © 2017 Elsevier Inc. All rights reserved.

Usefullness of IGH/TCR PCR studies in lymphoproliferative disorders with inconclusive clonality by flow cytometry.

PubMed

Ribera, Jordi; Zamora, Lurdes; Juncà, Jordi; Rodríguez, Inés; Marcé, Silvia; Cabezón, Marta; Millá, Fuensanta

2013-07-25

In up to 5-15% of studies of lymphoproliferative disorders (LPD) flow cytometry (FCM) or immunomorphologic methods cannot discriminate malignant from reactive processes. The aim of this work was to determine the usefulness of PCR for solving these diagnostic uncertainties. We analyzed IGH and TCRγ genes by PCR in 106 samples with inconclusive FCM results. A clonal result was registered in 36/106 studies, with a LPD being confirmed in 27 (75%) of these cases. Specifically, 9/9 IGH clonal and 16/25 TCRγ clonal results were finally diagnosed with LPD. Additionally, 2 clonal TCRγ samples with suspicion of undefined LPD were finally diagnosed with T LPD. Although polyclonal results were obtained in 47 of the cases studied (38 IGH and 9 TCRγ), hematologic neoplasms were diagnosed in 4/38 IGH polyclonal and in 1/9 TCRγ polyclonal studies. There were also 14 PCR polyclonal results (4 IGH, 10 TCRγ), albeit non-conclusive. Of these, 2/4 were eventually diagnosed with B-cell lymphoma and 3/10 with T-cell LPD. In 8 IGH samples the results of PCR techniques were non-informative but in 3/8 cases a B lymphoma was finally confirmed. We concluded that PCR is a useful technique to identify LPD when FCM is inconclusive. A PCR clonal B result is indicative of malignancy but IGH polyclonal and non-conclusive results do not exclude lymphoid neoplasms. Interpretation of T-cell clonality should be based on all the available clinical and analytical data. © 2013 Clinical Cytometry Society. Copyright © 2013 Clinical Cytometry Society.

Use of flow cytometry, fluorescence microscopy, and PCR-based techniques to assess intraspecific and interspecific matings of Armillaria species

Treesearch

Mee-Sook Kim; Ned B. Klopfenstein; Geral I. McDonald; Kathiravetpillai Arumuganathan

2001-01-01

For assessments of intraspecific mating using flow cytometry and fluorescence microscopy, two compatible basidiospore-derived isolates were selected from each of four parental basidiomata of North American Biological Species (NABS) X. The nuclear status in NABS X varied with basidiospore-derived isolates. Nuclei within basidiospore-derived isolates existed as haploids...

Candidiasis and the impact of flow cytometry on antifungal drug discovery.

PubMed

Ku, Tsun Sheng N; Bernardo, Stella; Walraven, Carla J; Lee, Samuel A

2017-11-01

Invasive candidiasis continues to be associated with significant morbidity and mortality as well as substantial health care costs nationally and globally. One of the contributing factors is the development of resistance to antifungal agents that are already in clinical use. Moreover, there are known treatment limitations with all of the available antifungal agents. Since traditional techniques in novel drug discovery are time consuming, high-throughput screening using flow cytometry presents as a potential tool to identify new antifungal agents that would be useful in the management of these patients. Areas covered: In this review, the authors discuss the use of automated high-throughput screening assays based upon flow cytometry to identify potential antifungals from a library comprised of a large number of bioactive compounds. They also review studies that employed the use of this research methodology that has identified compounds with antifungal activity. Expert opinion: High-throughput screening using flow cytometry has substantially decreased the processing time necessary for screening thousands of compounds, and has helped enhance our understanding of fungal pathogenesis. Indeed, the authors see this technology as a powerful tool to help scientists identify new antifungal agents that can be added to the clinician's arsenal in their fight against invasive candidiasis.

Assessment of cell concentration and viability of isolated hepatocytes using flow cytometry.

PubMed

Wigg, Alan J; Phillips, John W; Wheatland, Loretta; Berry, Michael N

2003-06-01

The assessment of cell concentration and viability of freshly isolated hepatocyte preparations has been traditionally performed using manual counting with a Neubauer counting chamber and staining for trypan blue exclusion. Despite the simple and rapid nature of this assessment, concerns about the accuracy of these methods exist. Simple flow cytometry techniques which determine cell concentration and viability are available yet surprisingly have not been extensively used or validated with isolated hepatocyte preparations. We therefore investigated the use of flow cytometry using TRUCOUNT Tubes and propidium iodide staining to measure cell concentration and viability of isolated rat hepatocytes in suspension. Analysis using TRUCOUNT Tubes provided more accurate and reproducible measurement of cell concentration than manual cell counting. Hepatocyte viability, assessed using propidium iodide, correlated more closely than did trypan blue exclusion with all indicators of hepatocyte integrity and function measured (lactate dehydrogenase leakage, cytochrome p450 content, cellular ATP concentration, ammonia and lactate removal, urea and albumin synthesis). We conclude that flow cytometry techniques can be used to measure cell concentration and viability of isolated hepatocyte preparations. The techniques are simple, rapid, and more accurate than manual cell counting and trypan blue staining and the results are not affected by protein-containing media.

[Flow cytometry in datecting lymph node micrometastasis in colorectal cancer].

PubMed

Sun, Q; Ding, Y; Zhang, J

2001-01-25

To study the methodology and significance of flow cytometry in detecting lymph node micrometastasis of colorectal cancer. One hundred sixty-two cellular suspensions were prepared with lymph nodes which were resected radically on 25 patients with colorectal cancer and in which no cancer cells were found by HE staining. Different concentrations of cultured Lovo colorectal cancer cells were added into the celular suspension prepared from lymph node tissue of persons without colorectal cancer in order to prepare a control model. Dual staining with CK/FTTC and PI was made to the sedimetns from those 2 kinds of suspension. Flow cytometry was used to detect cancer cells. An ideal correlation was obtained between the detection value and the theoretical value of cancer cells in the specimen suspensions and control models (r = 0.097 6) with a sensitivity rate of 10/10(5). Cancer cells were detected from 7 out of the 25 patients and 30 of the 162 cellular suspensions. The detection rate was correlated with the size and infiltrating depth of the cancer. Flow cytometry is a reliable, rapid, and quantitative method for detecting lymph node micrometastasis in colorectal cancer.

Characterization of subpopulations and immune-related parameters of hemocytes in the green-lipped mussel Perna viridis.

PubMed

Wang, Youji; Hu, Menghong; Chiang, M W L; Shin, P K S; Cheung, S G

2012-03-01

The green-lipped mussel Perna viridis is distributed widely in the estuarine and coastal areas of the Indo-Pacific region and extensively cultured as an inexpensive protein source. Morphology and immunological activities of hemocytes of P. viridis were investigated using flow cytometry and light and electron microscopy. Three major types of hemocytes were identified in the hemolymph, including dense-granulocyte, semi-granulocyte (small and large size) and hyalinocyte. Other hemocytes, which occurred in low numbers, included granulocytes with different electron-dense/lucent granules and hemoblast-like cells. Based on flow cytometry, two subpopulations were identified. Granulocytes were larger cells, and the more abundant, containing numerous granules in the cytoplasm, and hyalinocytes were the smaller and less abundant with the fewest granules. Flow cytometry revealed that the granulocytes were more active in cell phagocytosis, contained the higher lysosomal content, and showed higher esterase activity and reactive oxygen species (ROS) generation compared with hyalinocytes. Immune functions assessed by the flow cytometry indicated that the granulocytes were the main hemocytes involved in the cellular defence in P. viridis. Copyright © 2011. Published by Elsevier Ltd.

Verification and characterization of chromosome duplication in haploid maize.

PubMed

de Oliveira Couto, E G; Resende Von Pinho, E V; Von Pinho, R G; Veiga, A D; de Carvalho, M R; de Oliveira Bustamante, F; Nascimento, M S

2015-06-26

Doubled haploid technology has been used by various private companies. However, information regarding chromosome duplication methodologies, particularly those concerning techniques used to identify duplication in cells, is limited. Thus, we analyzed and characterized artificially doubled haploids using microsatellites molecular markers, pollen viability, and flow cytometry techniques. Evaluated material was obtained using two different chromosome duplication protocols in maize seeds considered haploids, resulting from the cross between the haploid inducer line KEMS and 4 hybrids (GNS 3225, GNS 3032, GNS 3264, and DKB 393). Fourteen days after duplication, plant samples were collected and assessed by flow cytometry. Further, the plants were transplanted to a field, and samples were collected for DNA analyses using microsatellite markers. The tassels were collected during anthesis for pollen viability analyses. Haploid, diploid, and mixoploid individuals were detected using flow cytometry, demonstrating that this technique was efficient for identifying doubled haploids. The microsatellites markers were also efficient for confirming the ploidies preselected by flow cytometry and for identifying homozygous individuals. Pollen viability showed a significant difference between the evaluated ploidies when the Alexander and propionic-carmin stains were used. The viability rates between the plodies analyzed show potential for fertilization.

Toward development of a comprehensive external quality assurance program for polyfunctional intracellular cytokine staining assays

PubMed Central

Staats, Janet S.; Enzor, Jennifer H.; Sanchez, Ana M.; Rountree, Wes; Chan, Cliburn; Jaimes, Maria; Chan, Ray Chun-Fai; Gaur, Amitabh; Denny, Thomas N.; Weinhold, Kent J.

2014-01-01

The External Quality Assurance Program Oversight Laboratory (EQAPOL) Flow Cytometry Program assesses the proficiency of NIH/NIAID/DAIDS-supported and potentially other interested research laboratories in performing Intracellular Cytokine Staining (ICS) assays. The goal of the EQAPOL Flow Cytometry External Quality Assurance Program (EQAP) is to provide proficiency testing and remediation for participating sites. The program is not punitive; rather, EQAPOL aims to help sites identify areas for improvement. EQAPOL utilizes a highly standardized ICS assay to minimize variability and readily identify those sites experiencing technical difficulties with their assays. Here, we report the results of External Proficiency 3 (EP3) where participating sites performed a 7-color ICS assay. On average, sites perform well in the Flow Cytometry EQAP (median score is “Good”). The most common technical issues identified by the program involve protocol adherence and data analysis; these areas have been the focus of site remediation. The EQAPOL Flow Cytometry team is now in the process of expanding the program to 8-color ICS assays. Evaluating polyfunctional ICS responses would align the program with assays currently being performed in support of HIV immune monitoring assays. PMID:24968072

Flow cytometry and conventional enumeration of microorganisms in ships' ballast water and marine samples.

PubMed

Joachimsthal, Eva L; Ivanov, Volodymyr; Tay, Joo-Hwa; Tay, Stephen T-L

2003-03-01

Conventional methods for bacteriological testing of water quality take long periods of time to complete. This makes them inappropriate for a shipping industry that is attempting to comply with the International Maritime Organization's anticipated regulations for ballast water discharge. Flow cytometry for the analysis of marine and ship's ballast water is a comparatively fast and accurate method. Compared to a 5% standard error for flow cytometry analysis the standard methods of culturing and epifluorescence analysis have errors of 2-58% and 10-30%, respectively. Also, unlike culturing methods, flow cytometry is capable of detecting both non-viable and viable but non-culturable microorganisms which can still pose health risks. The great variability in both cell concentrations and microbial content for the samples tested is an indication of the difficulties facing microbial monitoring programmes. The concentration of microorganisms in the ballast tank was generally lower than in local seawater. The proportion of aerobic, microaerophilic, and facultative anaerobic microorganisms present appeared to be influenced by conditions in the ballast tank. The gradual creation of anaerobic conditions in a ballast tank could lead to the accumulation of facultative anaerobic microorganisms, which might represent a potential source of pathogenic species.

International Society for the Advancement of Cytometry cell sorter biosafety standards.

PubMed

Holmes, Kevin L; Fontes, Benjamin; Hogarth, Philip; Konz, Richard; Monard, Simon; Pletcher, Charles H; Wadley, Robert B; Schmid, Ingrid; Perfetto, Stephen P

2014-05-01

Flow cytometric cell sorting of biological specimens has become prevalent in basic and clinical research laboratories. These specimens may contain known or unknown infectious agents, necessitating precautions to protect instrument operators and the environment from biohazards arising from the use of sorters. To this end the International Society of Analytical Cytology (ISAC) was proactive in establishing biosafety guidelines in 1997 (Schmid et al., Cytometry 1997;28:99-117) and subsequently published revised biosafety standards for cell sorting of unfixed samples in 2007 (Schmid et al., Cytometry Part A J Int Soc Anal Cytol 2007;71A:414-437). Since their publication, these documents have become recognized worldwide as the standard of practice and safety precautions for laboratories performing cell sorting experiments. However, the field of cytometry has progressed since 2007, and the document requires an update. The new Standards provides guidance: (1) for laboratory design for cell sorter laboratories; (2) for the creation of laboratory or instrument specific Standard Operating Procedures (SOP); and (3) on procedures for the safe operation of cell sorters, including personal protective equipment (PPE) and validation of aerosol containment. Published © 2014 Wiley Periodicals Inc.

Review of methods to probe single cell metabolism and bioenergetics

DOE PAGES

Vasdekis, Andreas E.; Stephanopoulos, Gregory

2014-10-31

The sampling and manipulation of cells down to the individual has been of substantial interest since the very beginning of Life Sciences. Herein, our objective is to highlight the most recent developments in single cell manipulation, as well as pioneering ones. First, flow-through methods will be discussed, namely methods in which the single cells flow continuously in an ordered manner during their analysis. This section will be followed by confinement techniques that enable cell isolation and confinement in one, two- or three-dimensions. Flow cytometry and droplet microfluidics are the two most common methods of flow-through analysis. While both are high-throughputmore » techniques, their difference lays in the fact that the droplet encapsulated cells experience a restricted and personal microenvironment, while in flow cytometry cells experience similar nutrient and stimuli initial concentrations. These methods are rather well established; however, they recently enabled immense strides in single cell phenotypic analysis, namely the identification and analysis of metabolically distinct individuals from an isogenic population using both droplet microfluidics and flow cytometry.« less

Flow Cytometry Data Preparation Guidelines for Improved Automated Phenotypic Analysis.

PubMed

Jimenez-Carretero, Daniel; Ligos, José M; Martínez-López, María; Sancho, David; Montoya, María C

2018-05-15

Advances in flow cytometry (FCM) increasingly demand adoption of computational analysis tools to tackle the ever-growing data dimensionality. In this study, we tested different data input modes to evaluate how cytometry acquisition configuration and data compensation procedures affect the performance of unsupervised phenotyping tools. An analysis workflow was set up and tested for the detection of changes in reference bead subsets and in a rare subpopulation of murine lymph node CD103 + dendritic cells acquired by conventional or spectral cytometry. Raw spectral data or pseudospectral data acquired with the full set of available detectors by conventional cytometry consistently outperformed datasets acquired and compensated according to FCM standards. Our results thus challenge the paradigm of one-fluorochrome/one-parameter acquisition in FCM for unsupervised cluster-based analysis. Instead, we propose to configure instrument acquisition to use all available fluorescence detectors and to avoid integration and compensation procedures, thereby using raw spectral or pseudospectral data for improved automated phenotypic analysis. Copyright © 2018 by The American Association of Immunologists, Inc.

Optimization of flow cytometric detection and cell sorting of transgenic Plasmodium parasites using interchangeable optical filters

PubMed Central

2012-01-01

Background Malaria remains a major cause of morbidity and mortality worldwide. Flow cytometry-based assays that take advantage of fluorescent protein (FP)-expressing malaria parasites have proven to be valuable tools for quantification and sorting of specific subpopulations of parasite-infected red blood cells. However, identification of rare subpopulations of parasites using green fluorescent protein (GFP) labelling is complicated by autofluorescence (AF) of red blood cells and low signal from transgenic parasites. It has been suggested that cell sorting yield could be improved by using filters that precisely match the emission spectrum of GFP. Methods Detection of transgenic Plasmodium falciparum parasites expressing either tdTomato or GFP was performed using a flow cytometer with interchangeable optical filters. Parasitaemia was evaluated using different optical filters and, after optimization of optics, the GFP-expressing parasites were sorted and analysed by microscopy after cytospin preparation and by imaging cytometry. Results A new approach to evaluate filter performance in flow cytometry using two-dimensional dot blot was developed. By selecting optical filters with narrow bandpass (BP) and maximum position of filter emission close to GFP maximum emission in the FL1 channel (510/20, 512/20 and 517/20; dichroics 502LP and 466LP), AF was markedly decreased and signal-background improve dramatically. Sorting of GFP-expressing parasite populations in infected red blood cells at 90 or 95% purity with these filters resulted in 50-150% increased yield when compared to the standard filter set-up. The purity of the sorted population was confirmed using imaging cytometry and microscopy of cytospin preparations of sorted red blood cells infected with transgenic malaria parasites. Discussion Filter optimization is particularly important for applications where the FP signal and percentage of positive events are relatively low, such as analysis of parasite-infected samples with in the intention of gene-expression profiling and analysis. The approach outlined here results in substantially improved yield of GFP-expressing parasites, and requires decreased sorting time in comparison to standard methods. It is anticipated that this protocol will be useful for a wide range of applications involving rare events. PMID:22950515

Minimal Residual Disease Evaluation in Childhood Acute Lymphoblastic Leukemia: An Economic Analysis

PubMed Central

Gajic-Veljanoski, O.; Pham, B.; Pechlivanoglou, P.; Krahn, M.; Higgins, Caroline; Bielecki, Joanna

2016-01-01

Background Minimal residual disease (MRD) testing by higher performance techniques such as flow cytometry and polymerase chain reaction (PCR) can be used to detect the proportion of remaining leukemic cells in bone marrow or peripheral blood during and after the first phases of chemotherapy in children with acute lymphoblastic leukemia (ALL). The results of MRD testing are used to reclassify these patients and guide changes in treatment according to their future risk of relapse. We conducted a systematic review of the economic literature, cost-effectiveness analysis, and budget-impact analysis to ascertain the cost-effectiveness and economic impact of MRD testing by flow cytometry for management of childhood precursor B-cell ALL in Ontario. Methods A systematic literature search (1998–2014) identified studies that examined the incremental cost-effectiveness of MRD testing by either flow cytometry or PCR. We developed a lifetime state-transition (Markov) microsimulation model to quantify the cost-effectiveness of MRD testing followed by risk-directed therapy to no MRD testing and to estimate its marginal effect on health outcomes and on costs. Model input parameters were based on the literature, expert opinion, and data from the Pediatric Oncology Group of Ontario Networked Information System. Using predictions from our Markov model, we estimated the 1-year cost burden of MRD testing versus no testing and forecasted its economic impact over 3 and 5 years. Results In a base-case cost-effectiveness analysis, compared with no testing, MRD testing by flow cytometry at the end of induction and consolidation was associated with an increased discounted survival of 0.0958 quality-adjusted life-years (QALYs) and increased discounted costs of $4,180, yielding an incremental cost-effectiveness ratio (ICER) of $43,613/QALY gained. After accounting for parameter uncertainty, incremental cost-effectiveness of MRD testing was associated with an ICER of $50,249/QALY gained. In the budget-impact analysis, the 1-year cost expenditure for MRD testing by flow cytometry in newly diagnosed patients with precursor B-cell ALL was estimated at $340,760. We forecasted that the province would have to pay approximately $1.3 million over 3 years and $2.4 million over 5 years for MRD testing by flow cytometry in this population. Conclusions Compared with no testing, MRD testing by flow cytometry in newly diagnosed patients with precursor B-cell ALL represents good value for money at commonly used willingness-to-pay thresholds of $50,000/QALY and $100,000/QALY. PMID:27099644

Minimal Residual Disease Evaluation in Childhood Acute Lymphoblastic Leukemia: An Economic Analysis.

PubMed

2016-01-01

Minimal residual disease (MRD) testing by higher performance techniques such as flow cytometry and polymerase chain reaction (PCR) can be used to detect the proportion of remaining leukemic cells in bone marrow or peripheral blood during and after the first phases of chemotherapy in children with acute lymphoblastic leukemia (ALL). The results of MRD testing are used to reclassify these patients and guide changes in treatment according to their future risk of relapse. We conducted a systematic review of the economic literature, cost-effectiveness analysis, and budget-impact analysis to ascertain the cost-effectiveness and economic impact of MRD testing by flow cytometry for management of childhood precursor B-cell ALL in Ontario. A systematic literature search (1998-2014) identified studies that examined the incremental cost-effectiveness of MRD testing by either flow cytometry or PCR. We developed a lifetime state-transition (Markov) microsimulation model to quantify the cost-effectiveness of MRD testing followed by risk-directed therapy to no MRD testing and to estimate its marginal effect on health outcomes and on costs. Model input parameters were based on the literature, expert opinion, and data from the Pediatric Oncology Group of Ontario Networked Information System. Using predictions from our Markov model, we estimated the 1-year cost burden of MRD testing versus no testing and forecasted its economic impact over 3 and 5 years. In a base-case cost-effectiveness analysis, compared with no testing, MRD testing by flow cytometry at the end of induction and consolidation was associated with an increased discounted survival of 0.0958 quality-adjusted life-years (QALYs) and increased discounted costs of $4,180, yielding an incremental cost-effectiveness ratio (ICER) of $43,613/QALY gained. After accounting for parameter uncertainty, incremental cost-effectiveness of MRD testing was associated with an ICER of $50,249/QALY gained. In the budget-impact analysis, the 1-year cost expenditure for MRD testing by flow cytometry in newly diagnosed patients with precursor B-cell ALL was estimated at $340,760. We forecasted that the province would have to pay approximately $1.3 million over 3 years and $2.4 million over 5 years for MRD testing by flow cytometry in this population. Compared with no testing, MRD testing by flow cytometry in newly diagnosed patients with precursor B-cell ALL represents good value for money at commonly used willingness-to-pay thresholds of $50,000/QALY and $100,000/QALY.

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.

Isolating LacZ-expressing cells from mouse inner ear tissues using flow cytometry.

PubMed

Jan, Taha A; Chai, Renjie; Sayyid, Zahra N; Cheng, Alan G

2011-12-23

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae. The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown. To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Analysis of cellular autofluorescence in touch samples by flow cytometry: implications for front end separation of trace mixture evidence.

PubMed

Katherine Philpott, M; Stanciu, Cristina E; Kwon, Ye Jin; Bustamante, Eduardo E; Greenspoon, Susan A; Ehrhardt, Christopher J

2017-07-01

The goal of this study was to survey optical and biochemical variation in cell populations deposited onto a surface through touch or contact and identify specific features that may be used to distinguish and then sort cell populations from separate contributors in a trace biological mixture. Although we were not able to detect meaningful biochemical variation in touch samples deposited by different contributors through preliminary antibody surveys, we did observe distinct differences in red autofluorescence emissions (650-670 nm), with as much as a tenfold difference in mean fluorescence intensities observed between certain pairs of donors. Results indicate that the level of red autofluorescence in touch samples can be influenced by a donor's contact with specific material prior to handling the substrate from which cells were collected. In particular, we observed increased red autofluorescence in cells deposited subsequent to handling laboratory gloves, plant material, and certain types of marker ink, which could be easily visualized microscopically or using flow cytometry, and persisted after hand washing. To test whether these observed optical differences could potentially be used as the basis for a cell separation workflow, a controlled two-person touch mixture was separated into two fractions via fluorescence-activated cell sorting (FACS) using gating criteria based on intensity of 650-670 nm emissions and then subjected to DNA analysis. Genetic analysis of the sorted fractions provided partial DNA profiles that were consistent with separation of individual contributors from the mixture suggesting that variation in autofluorescence signatures, even if driven by extrinsic factors, may nonetheless be a useful means of isolating contributors to some touch mixtures. Graphical Abstract Conceptual workflow diagram. Trace biological mixtures containing cells from multiple individuals are analyzed by flow cytometry. Cells are then physically separated into two populations based on intensity of red autofluorescence using Fluorescence Activated Cell Sorting. Each isolated cell fraction is subjected to DNA analysis resulting in a DNA profile for each contributor.

SCENERY: a web application for (causal) network reconstruction from cytometry data.

PubMed

Papoutsoglou, Georgios; Athineou, Giorgos; Lagani, Vincenzo; Xanthopoulos, Iordanis; Schmidt, Angelika; Éliás, Szabolcs; Tegnér, Jesper; Tsamardinos, Ioannis

2017-07-03

Flow and mass cytometry technologies can probe proteins as biological markers in thousands of individual cells simultaneously, providing unprecedented opportunities for reconstructing networks of protein interactions through machine learning algorithms. The network reconstruction (NR) problem has been well-studied by the machine learning community. However, the potentials of available methods remain largely unknown to the cytometry community, mainly due to their intrinsic complexity and the lack of comprehensive, powerful and easy-to-use NR software implementations specific for cytometry data. To bridge this gap, we present Single CEll NEtwork Reconstruction sYstem (SCENERY), a web server featuring several standard and advanced cytometry data analysis methods coupled with NR algorithms in a user-friendly, on-line environment. In SCENERY, users may upload their data and set their own study design. The server offers several data analysis options categorized into three classes of methods: data (pre)processing, statistical analysis and NR. The server also provides interactive visualization and download of results as ready-to-publish images or multimedia reports. Its core is modular and based on the widely-used and robust R platform allowing power users to extend its functionalities by submitting their own NR methods. SCENERY is available at scenery.csd.uoc.gr or http://mensxmachina.org/en/software/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Synergistic growth inhibition in HL-60 cells by the combination of acyclic retinoid and vitamin K2.

PubMed

Kitagawa, Junichi; Hara, Takeshi; Tsurumi, Hisashi; Ninomiya, Soranobu; Ogawa, Kengo; Adachi, Seiji; Kanemura, Nobuhiro; Kasahara, Senji; Shimizu, Masahito; Moriwaki, Hisataka

2011-05-01

The aim of this study was to assess the effects of acyclic retinoid (ACR) and vitamin K(2) (VK(2)) in HL-60 cells. We used HL-60 cells, and the Trypan Blue dye exclusion method was used for cell proliferation assays. For detection of apoptosis, the Annexin V-binding capacity of treated cells was examined by flow cytometry. To evaluate the cell cycle, we used a FITC BrdU Flow KIT and flow cytometry. Total extracted and equivalent amounts of protein were examined by Western blotting using specific antibodies. ACR and VK(2) dose dependently inhibited the proliferation of HL-60 cells. These two agents in combination synergistically inhibited cell growth and induced apoptosis. VK(2) inhibited activation of the Ras/MAPK signaling pathway, and ACR plus VK(2) cooperatively inhibited phosphorylation of RXRα and the growth of HL-60 cells. Moreover, ACR and VK(2) induced increases in G0/G1 phase HL-60 cells, alone and synergistically in combination. The synergistic effects of ACR and VK(2) on HL-60 cells may provide a novel strategy for treating leukemia.

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.

An introduction to mass cytometry: fundamentals and applications.

PubMed

Tanner, Scott D; Baranov, Vladimir I; Ornatsky, Olga I; Bandura, Dmitry R; George, Thaddeus C

2013-05-01

Mass cytometry addresses the analytical challenges of polychromatic flow cytometry by using metal atoms as tags rather than fluorophores and atomic mass spectrometry as the detector rather than photon optics. The many available enriched stable isotopes of the transition elements can provide up to 100 distinguishable reporting tags, which can be measured simultaneously because of the essential independence of detection provided by the mass spectrometer. We discuss the adaptation of traditional inductively coupled plasma mass spectrometry to cytometry applications. We focus on the generation of cytometry-compatible data and on approaches to unsupervised multivariate clustering analysis. Finally, we provide a high-level review of some recent benchmark reports that highlight the potential for massively multi-parameter mass cytometry.

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

Rapid detection of total and viable Legionella pneumophila in tap water by immunomagnetic separation, double fluorescent staining and flow cytometry.

PubMed

Keserue, Hans-Anton; Baumgartner, Andreas; Felleisen, Richard; Egli, Thomas

2012-11-01

We developed a rapid detection method for Legionella pneumophila (Lp) by filtration, immunomagnetic separation, double fluorescent staining, and flow cytometry (IMS-FCM method). The method requires 120 min and can discriminate 'viable' and 'membrane-damaged' cells. The recovery is over 85% of spiked Lp SG 1 cells in 1 l of tap water and detection limits are around 50 and 15 cells per litre for total and viable Lp, respectively. The method was compared using water samples from house installations in a blind study with three environmental laboratories performing the ISO 11731 plating method. In 53% of the water samples from different taps and showers significantly higher concentrations of Lp were detected by flow cytometry. No correlation to the plate culture method was found. Since also 'viable but not culturable' (VNBC) cells are detected by our method, this result was expected. The IMS-FCM method is limited by the specificity of the used antibodies; in the presented case they target Lp serogroups 1-12. This and the fact that no Lp-containing amoebae are detected may explain why in 21% of all samples higher counts were observed using the plate culture method. Though the IMS-FCM method is not yet fit to completely displace the established plating method (ISO 11731) for routine Lp monitoring, it has major advantages to plating and can quickly provide important insights into the ecology of this pathogen in water distribution systems. © 2012 The Authors. Microbial Biotechnology © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

A novel mAb against a human CD34 peptide reacts with the native protein on CD34+ cells.

PubMed

Shams, Mahmood; Jeddi-Tehrani, Mahmood; Notash Haghighat, Farzaneh; Bayat, Ali Ahmad; Mahmoudian, Jafar; Rezvani, Mohammad Reza

2013-12-01

‎Human CD34 is a transmembrane glycoprotein which is expressed in human hematopoietic stem ‎cells (HSCs) and the small-‎vessel endothelial cells of a variety of tissues. CD34 plays a critical role as a ‎marker for diagnosis ‎and classification of leukemia. Anti CD34 antibodies are used for isolation and ‎purification ‎of HSCs from bone marrow, peripheral blood and cord blood. To characterize a newly produced monoclonal antibody against a human CD34 peptide. Anti CD34 monoclonal antibody (Clone 2C10-D3) was purified from mouse ascitic fluid and hybridoma cell culture supernatants by affinity chromatography and its immune reactivity was examined by ELISA. The purified antibody was further characterized using Western blot and flow cytometry on TF1 (Human Erythroblast) cell line. ‎ELISA experiment revealed that the antibody recognized CD34 peptide. Western ‎blot analysis on TF1 ‎cell lysate confirmed the reactivity of the antibody with a 42 KDa protein. Blocking the antibody with a saturating concentration of specific CD34 peptide resulted in loss of its activity with TF1 lysate in Western blot. The 2C10-D3 antibody reacted with TF1 cells in flow cytometry in a similar manner to a commercial anti CD34 monoclonal antibody.‎ ‎Our data suggest that the anti CD34 monoclonal antibody (Clone 2C10-D3) is an appropriate antibody to study the CD34+ cells by flow cytometry and Western blot.

Phagocytosis and Respiratory Burst Activity in Lumpsucker (Cyclopterus lumpus L.) Leucocytes Analysed by Flow Cytometry

PubMed Central

Haugland, Gyri T.; Jakobsen, Ragnhild Aakre; Vestvik, Nils; Ulven, Kristian; Stokka, Lene; Wergeland, Heidrun I.

2012-01-01

In the present study, we have isolated leucocytes from peripheral blood, head kidney and spleen from lumpsucker (Cyclopterus lumpus L.), and performed functional studies like phagocytosis and respiratory burst, as well as morphological and cytochemical analyses. Different leucocytes were identified, such as lymphocytes, monocytes/macrophages and polymorphonuclear cells with bean shaped or bilobed nuclei. In addition, cells with similar morphology as described for dendritic cells in trout were abundant among the isolated leucocytes. Flow cytometry was successfully used for measuring phagocytosis and respiratory burst activity. The phagocytic capacity and ability were very high, and cells with different morphology in all three leucocyte preparations phagocytised beads rapidly. Due to lack of available cell markers, the identity of the phagocytic cells could not be determined. The potent non-specific phagocytosis was in accordance with a high number of cells positive for myeloperoxidase, an enzyme involved in oxygen-dependent killing mechanism present in phagocytic cells. Further, high respiratory burst activity was present in the leucocytes samples, verifying a potent oxygen- dependent degradation. At present, the specific antibody immune response could not be measured, as immunoglobulin or B-cells have not yet been isolated. Therefore, analyses of the specific immune response in this fish species await further clarification. The present study presents the first analyses of lumpsucker immunity and also the first within the order Scopaeniformes. PMID:23112870

Characterisation of monoclonal antibodies specific for hamster leukocyte differentiation molecules.

PubMed

Rees, Jennifer; Haig, David; Mack, Victoria; Davis, William C

2017-01-01

Flow cytometry was used to identify mAbs that recognize conserved epitopes on hamster leukocyte differentiation molecules (hLDM) and also to characterize mAbs developed against hLDM. Initial screening of mAbs developed against LDMs in other species yielded mAbs specific for the major histocompatibility (MHC) II molecule, CD4 and CD18. Screening of sets of mAbs developed against hLDM yielded 22 new mAbs, including additional mAbs to MHC II molecules and mAbs that recognize LDMs expressed on all leukocytes, granulocytes, all lymphocytes, all T cells, a subset of T cells, or on all B cells. Based on comparison of the pattern of expression of LDMs expressed on all hamster leukocytes with the patterns of expression of known LDMs in other species, as detected by flow cytometry (FC), four mAbs are predicted to recognize CD11a, CD44, and CD45. Cross comparison of mAbs specific for a subset of hamster T cells with a cross reactive mAb known to recognize CD4 in mice and one recognising CD8 revealed they recognize CD4. The characterization of these mAbs expands opportunities to use hamsters as an additional model species to investigate the mechanisms of immunopathogenesis of infectious diseases. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

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.

Comparison of flow cytometry, fluorescence microscopy and spectrofluorometry for analysis of gene electrotransfer efficiency.

PubMed

Marjanovič, Igor; Kandušer, Maša; Miklavčič, Damijan; Keber, Mateja Manček; Pavlin, Mojca

2014-12-01

In this study, we compared three different methods used for quantification of gene electrotransfer efficiency: fluorescence microscopy, flow cytometry and spectrofluorometry. We used CHO and B16 cells in a suspension and plasmid coding for GFP. The aim of this study was to compare and analyse the results obtained by fluorescence microscopy, flow cytometry and spectrofluorometry and in addition to analyse the applicability of spectrofluorometry for quantifying gene electrotransfer on cells in a suspension. Our results show that all the three methods detected similar critical electric field strength, around 0.55 kV/cm for both cell lines. Moreover, results obtained on CHO cells showed that the total fluorescence intensity and percentage of transfection exhibit similar increase in response to increase electric field strength for all the three methods. For B16 cells, there was a good correlation at low electric field strengths, but at high field strengths, flow cytometer results deviated from results obtained by fluorescence microscope and spectrofluorometer. Our study showed that all the three methods detected similar critical electric field strengths and high correlations of results were obtained except for B16 cells at high electric field strengths. The results also demonstrated that flow cytometry measures higher values of percentage transfection compared to microscopy. Furthermore, we have demonstrated that spectrofluorometry can be used as a simple and consistent method to determine gene electrotransfer efficiency on cells in a suspension.

Imaging Flow Cytometry Analysis to Identify Differences of Survival Motor Neuron Protein Expression in Patients With Spinal Muscular Atrophy.

PubMed

Arakawa, Reiko; Arakawa, Masayuki; Kaneko, Kaori; Otsuki, Noriko; Aoki, Ryoko; Saito, Kayoko

2016-08-01

Spinal muscular atrophy is a neurodegenerative disorder caused by the deficient expression of survival motor neuron protein in motor neurons. A major goal of disease-modifying therapy is to increase survival motor neuron expression. Changes in survival motor neuron protein expression can be monitored via peripheral blood cells in patients; therefore we tested the sensitivity and utility of imaging flow cytometry for this purpose. After the immortalization of peripheral blood lymphocytes from a human healthy control subject and two patients with spinal muscular atrophy type 1 with two and three copies of SMN2 gene, respectively, we used imaging flow cytometry analysis to identify significant differences in survival motor neuron expression. A bright detail intensity analysis was used to investigate differences in the cellular localization of survival motor neuron protein. Survival motor neuron expression was significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject. Moreover, survival motor neuron expression correlated with the clinical severity of spinal muscular atrophy according to SMN2 copy number. The cellular accumulation of survival motor neuron protein was also significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject. The benefits of imaging flow cytometry for peripheral blood analysis include its capacities for analyzing heterogeneous cell populations; visualizing cell morphology; and evaluating the accumulation, localization, and expression of a target protein. Imaging flow cytometry analysis should be implemented in future studies to optimize its application as a tool for spinal muscular atrophy clinical trials. Copyright © 2016 Elsevier Inc. All rights reserved.

Single cell analysis using surface enhanced Raman scattering (SERS) tags

PubMed Central

Nolan, John P.; Duggan, Erika; Liu, Er; Condello, Danilo; Dave, Isha; Stoner, Samuel A.

2013-01-01

Fluorescence is a mainstay of bioanalytical methods, offering sensitive and quantitative reporting, often in multiplexed or multiparameter assays. Perhaps the best example of the latter is flow cytometry, where instruments equipped with multiple lasers and detectors allow measurement of 15 or more different fluorophores simultaneously, but increases beyond this number are limited by the relatively broad emission spectra. Surface enhanced Raman scattering (SERS) from metal nanoparticles can produce signal intensities that rival fluorescence, but with narrower spectral features that allow a greater degree of multiplexing. We are developing nanoparticle SERS tags as well as Raman flow cytometers for multiparameter single cell analysis of suspension or adherent cells. SERS tags are based on plasmonically active nanoparticles (gold nanorods) whose plasmon resonance can be tuned to give optimal SERS signals at a desired excitation wavelength. Raman resonant compounds are adsorbed on the nanoparticles to confer a unique spectral fingerprint on each SERS tag, which are then encapsulated in a polymer coating for conjugation to antibodies or other targeting molecules. Raman flow cytometry employs a high resolution spectral flow cytometer capable of measuring the complete SERS spectra, as well as conventional flow cytometry measurements, from thousands of individual cells per minute. Automated spectral unmixing algorithms extract the contributions of each SERS tag from each cell to generate high content, multiparameter single cell population data. SERS-based cytometry is a powerful complement to conventional fluorescence-based cytometry. The narrow spectral features of the SERS signal enables more distinct probes to be measured in a smaller region of the optical spectrum with a single laser and detector, allowing for higher levels of multiplexing and multiparameter analysis. PMID:22498143

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

B-ALL minimal residual disease flow cytometry: an application of a novel method for optimization of a single-tube model.

PubMed

Shaver, Aaron C; Greig, Bruce W; Mosse, Claudio A; Seegmiller, Adam C

2015-05-01

Optimizing a clinical flow cytometry panel can be a subjective process dependent on experience. We develop a quantitative method to make this process more rigorous and apply it to B lymphoblastic leukemia/lymphoma (B-ALL) minimal residual disease (MRD) testing. We retrospectively analyzed our existing three-tube, seven-color B-ALL MRD panel and used our novel method to develop an optimized one-tube, eight-color panel, which was tested prospectively. The optimized one-tube, eight-color panel resulted in greater efficiency of time and resources with no loss in diagnostic power. Constructing a flow cytometry panel using a rigorous, objective, quantitative method permits optimization and avoids problems of interdependence and redundancy in a large, multiantigen panel. Copyright© by the American Society for Clinical Pathology.

A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes using flow cytometry.

PubMed

Alves, L P S; Almeida, A T; Cruz, L M; Pedrosa, F O; de Souza, E M; Chubatsu, L S; Müller-Santos, M; Valdameri, G

2017-01-16

The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC) is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB) production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i) cell permeabilization, ii) Nile red staining, and iii) analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2=0.99) compared to a standard method based on methanolysis and GC. This method was successfully applied to the quantification of PHB in epiphytic bacteria isolated from rice roots.

Flow cytometry as a method for the evaluation of raw material, product and process in the dairy industry.

PubMed

Ruszczyńska, A; Szteyn, J; Wiszniewska-Laszczych, A

2007-01-01

Producing dairy products which are safe for consumers requires the constant monitoring of the microbiological quality of raw material, the production process itself and the end product. Traditional methods, still a "gold standard", require a specialized laboratory working on recognized and validated methods. Obtaining results is time- and labor-consuming and do not allow rapid evaluation. Hence, there is a need for a rapid, precise method enabling the real-time monitoring of microbiological quality, and flow cytometry serves this function well. It is based on labeling cells suspended in a solution with fluorescent dyes and pumping them into a measurement zone where they are exposed to a precisely focused laser beam. This paper is aimed at presenting the possibilities of applying flow cytometry in the dairy industry.

Line-Focused Optical Excitation of Parallel Acoustic Focused Sample Streams for High Volumetric and Analytical Rate Flow Cytometry.

PubMed

Kalb, Daniel M; Fencl, Frank A; Woods, Travis A; Swanson, August; Maestas, Gian C; Juárez, Jaime J; Edwards, Bruce S; Shreve, Andrew P; Graves, Steven W

2017-09-19

Flow cytometry provides highly sensitive multiparameter analysis of cells and particles but has been largely limited to the use of a single focused sample stream. This limits the analytical rate to ∼50K particles/s and the volumetric rate to ∼250 μL/min. Despite the analytical prowess of flow cytometry, there are applications where these rates are insufficient, such as rare cell analysis in high cellular backgrounds (e.g., circulating tumor cells and fetal cells in maternal blood), detection of cells/particles in large dilute samples (e.g., water quality, urine analysis), or high-throughput screening applications. Here we report a highly parallel acoustic flow cytometer that uses an acoustic standing wave to focus particles into 16 parallel analysis points across a 2.3 mm wide optical flow cell. A line-focused laser and wide-field collection optics are used to excite and collect the fluorescence emission of these parallel streams onto a high-speed camera for analysis. With this instrument format and fluorescent microsphere standards, we obtain analysis rates of 100K/s and flow rates of 10 mL/min, while maintaining optical performance comparable to that of a commercial flow cytometer. The results with our initial prototype instrument demonstrate that the integration of key parallelizable components, including the line-focused laser, particle focusing using multinode acoustic standing waves, and a spatially arrayed detector, can increase analytical and volumetric throughputs by orders of magnitude in a compact, simple, and cost-effective platform. Such instruments will be of great value to applications in need of high-throughput yet sensitive flow cytometry analysis.

Addressing the malaria drug resistance challenge using flow cytometry to discover new antimalarials.

PubMed

Grimberg, Brian T; Jaworska, Maria M; Hough, Lindsay B; Zimmerman, Peter A; Phillips, James G

2009-09-15

A new flow cytometry method that uses an optimized DNA and RNA staining strategy to monitor the growth and development of the Plasmodium falciparum strain W2mef has been used in a pilot study and has identified Bay 43-9006 1, SU 11274 2, and TMC 125 5 as compounds that exhibit potent (<1 microM) overall and ring stage in vitro antimalarial activity.

Stochastic Individual-Based Modeling of Bacterial Growth and Division Using Flow Cytometry.

PubMed

García, Míriam R; Vázquez, José A; Teixeira, Isabel G; Alonso, Antonio A

2017-01-01

A realistic description of the variability in bacterial growth and division is critical to produce reliable predictions of safety risks along the food chain. Individual-based modeling of bacteria provides the theoretical framework to deal with this variability, but it requires information about the individual behavior of bacteria inside populations. In this work, we overcome this problem by estimating the individual behavior of bacteria from population statistics obtained with flow cytometry. For this objective, a stochastic individual-based modeling framework is defined based on standard assumptions during division and exponential growth. The unknown single-cell parameters required for running the individual-based modeling simulations, such as cell size growth rate, are estimated from the flow cytometry data. Instead of using directly the individual-based model, we make use of a modified Fokker-Plank equation. This only equation simulates the population statistics in function of the unknown single-cell parameters. We test the validity of the approach by modeling the growth and division of Pediococcus acidilactici within the exponential phase. Estimations reveal the statistics of cell growth and division using only data from flow cytometry at a given time. From the relationship between the mother and daughter volumes, we also predict that P. acidilactici divide into two successive parallel planes.

In vivo, label-free, and noninvasive detection of melanoma metastasis by photoacoustic flow cytometry

NASA Astrophysics Data System (ADS)

Liu, Rongrong; Wang, Cheng; Hu, Cheng; Wang, Xueding; Wei, Xunbin

2014-02-01

Melanoma, a malignant tumor of melanocytes, is the most serious type of skin cancer in the world. It accounts for about 80% of deaths of all skin cancer. For cancer detection, circulating tumor cells (CTCs) serve as a marker for metastasis development, cancer recurrence, and therapeutic efficacy. Melanoma tumor cells have high content of melanin, which has high light absorption and can serve as endogenous biomarker for CTC detection without labeling. Here, we have developed an in vivo photoacoustic flow cytometry (PAFC) to monitor the metastatic process of melanoma cancer by counting CTCs of melanoma tumor bearing mice in vivo. To test in vivo PAFC's capability of detecting melanoma cancer, we have constructed a melanoma tumor model by subcutaneous inoculation of highly metastatic murine melanoma cancer cells, B16F10. In order to effectively distinguish the targeting PA signals from background noise, we have used the algorithm of Wavelet denoising method to reduce the background noise. The in vivo flow cytometry (IVFC) has shown a great potential for detecting circulating tumor cells quantitatively in the blood stream. Compared with fluorescence-based in vivo flow cytometry (IVFC), PAFC technique can be used for in vivo, label-free, and noninvasive detection of circulating tumor cells (CTCs).

Advantages of flow cytometry immunophenotyping for the diagnosis of central nervous system non-Hodgkin's lymphoma in AIDS patients.

PubMed

Subirá, D; Górgolas, M; Castañón, S; Serrano, C; Román, A; Rivas, F; Tomás, J F

2005-01-01

Neurological disorders are common in HIV-infected patients. Central nervous system (CNS) lymphoma should always be considered because it is an important cause of morbidity and mortality. To investigate the clinical utility of flow cytometry immunophenotyping (FCI) in diagnosing or discarding leptomeningeal involvement in HIV-infected patients and to compare its sensitivity with that of conventional cytological methods. Fifty-six cerebrospinal fluid (CSF) samples from 29 HIV-infected patients were independently evaluated by flow cytometry and cytology. The description of an aberrant immunophenotype was the criterion used to define the malignant nature of any CSF cell population. FCI and cytology gave concordant results for 48 of the 56 CSF samples studied: 37 were negative for malignancy and 11 had evidence of CNS lymphoma. Discordant results were obtained for eight CSF samples, and the accuracy of the FCI findings could be demonstrated for four CSF samples described as positive for malignancy according to the FCI criteria. A high level of agreement was found between the results obtained using the two methods, but FCI gave at least 25% higher sensitivity than conventional cytomorphological methods for the detection of malignant cells. This advantage suggests that, in case of negative flow cytometry results, disorders other than non-Hodgkin's lymphoma should be strongly considered.

Drug testing and flow cytometry analysis on a large number of uniform sized tumor spheroids using a microfluidic device

NASA Astrophysics Data System (ADS)

Patra, Bishnubrata; Peng, Chien-Chung; Liao, Wei-Hao; Lee, Chau-Hwang; Tung, Yi-Chung

2016-02-01

Three-dimensional (3D) tumor spheroid possesses great potential as an in vitro model to improve predictive capacity for pre-clinical drug testing. In this paper, we combine advantages of flow cytometry and microfluidics to perform drug testing and analysis on a large number (5000) of uniform sized tumor spheroids. The spheroids are formed, cultured, and treated with drugs inside a microfluidic device. The spheroids can then be harvested from the device without tedious operation. Due to the ample cell numbers, the spheroids can be dissociated into single cells for flow cytometry analysis. Flow cytometry provides statistical information in single cell resolution that makes it feasible to better investigate drug functions on the cells in more in vivo-like 3D formation. In the experiments, human hepatocellular carcinoma cells (HepG2) are exploited to form tumor spheroids within the microfluidic device, and three anti-cancer drugs: Cisplatin, Resveratrol, and Tirapazamine (TPZ), and their combinations are tested on the tumor spheroids with two different sizes. The experimental results suggest the cell culture format (2D monolayer vs. 3D spheroid) and spheroid size play critical roles in drug responses, and also demonstrate the advantages of bridging the two techniques in pharmaceutical drug screening applications.

Immunological tools: engaging students in the use and analysis of flow cytometry and enzyme-linked immunosorbent assay (ELISA).

PubMed

Ott, Laura E; Carson, Susan

2014-01-01

Flow cytometry and enzyme-linked immunosorbent assay (ELISA) are commonly used techniques associated with clinical and research applications within the immunology and medical fields. The use of these techniques is becoming increasingly valuable in many life science and engineering disciplines as well. Herein, we report the development and evaluation of a novel half-semester course that focused on introducing undergraduate and graduate students to advance conceptual and technical skills associated with flow cytometry and ELISA, with emphasis on applications, experimental design, and data analysis. This course was offered in the North Carolina State University Biotechnology Program over three semesters and consisted of weekly lectures and laboratories. Students performed and/or analyzed flow cytometry and ELISA in three separate laboratory exercises: (1) identification of transgenic zebrafish hematopoietic cells, (2) analysis of transfection efficiency, and (3) analysis of cytokine production upon lipopolysaccharide stimulation. Student learning outcomes were achieved as demonstrated by multiple means of assessment, including three laboratory reports, a data analysis laboratory practicum, and a cumulative final exam. Further, anonymous student self-assessment revealed increased student confidence in the knowledge and skill sets defined in the learning outcomes. Copyright © 2014 The International Union of Biochemistry and Molecular Biology.

Canonical Wnt Signaling as a Specific Marker for Normal and Tumorigenic Mammary Stem Cells

DTIC Science & Technology

2012-02-01

These cells were identified by  flow   cytometry  to detect cells that were positive for CD24 and CD49f.   2. We have established that activation of Wnt...09-1-0072 TITLE: Canonical Wnt Signaling as a Specific marker for Normal and Tumorigenic Mammary Stem Cells PRINCIPAL...activation of canonical Wnt signaling may be a very specific marker for mammary stem cells and be a target for transformation that results in the

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): A Method for Bacterial Small RNA Detection

DTIC Science & Technology

2012-01-10

flow cytometry, locked nucleic acid, sRNA, Vibrio , Date Published: 1/10/2012 This is an open-access article distributed under the terms of the Creative...solubilization process to maintain a 10 mL volume. Aliquot the 60% dextran sulfate solution and store at -20 °C until use. 1. Harvest 1x108 cells of...bioluminescent Vibrio campbellii or your bacteria of interest and transfer them into a 1.5 mL microcentrifuge tube. This quantity of cells provides

Measuring sickle cell morphology in flow using spectrally encoded flow cytometry (Conference Presentation)

NASA Astrophysics Data System (ADS)

Kviatkovsky, Inna; Zeidan, Adel; Yeheskely-Hayon, Daniella; Dann, Eldad J.; Yelin, Dvir

2017-02-01

During a sickle cell crisis in sickle cell anemia patients, deoxygenated red blood cells may change their mechanical properties and block small blood vessels, causing pain, local tissue damage and even organ failure. Measuring these cellular structural and morphological changes is important for understanding the factors contributing to vessel blockage and developing an effective treatment. In this work, we use spectrally encoded flow cytometry for confocal, high-resolution imaging of flowing blood cells from sickle cell anemia patients. A wide variety of cell morphologies were observed by analyzing the interference patterns resulting from reflections from the front and back faces of the cells' membrane. Using numerical simulation for calculating the two-dimensional reflection pattern from the cells, we propose an analytical expression for the three-dimensional shape of a characteristic sickle cell and compare it to a previous from the literature. In vitro spectrally encoded flow cytometry offers new means for analyzing the morphology of sickle cells in stress-free environment, and could provide an effective tool for studying the unique physiological properties of these cells.

Multiplexed and Microparticle-based Analyses: Quantitative Tools for the Large-Scale Analysis of Biological Systems

PubMed Central

Nolan, John P.; Mandy, Francis

2008-01-01

While the term flow cytometry refers to the measurement of cells, the approach of making sensitive multiparameter optical measurements in a flowing sample stream is a very general analytical approach. The past few years have seen an explosion in the application of flow cytometry technology for molecular analysis and measurements using micro-particles as solid supports. While microsphere-based molecular analyses using flow cytometry date back three decades, the need for highly parallel quantitative molecular measurements that has arisen from various genomic and proteomic advances has driven the development in particle encoding technology to enable highly multiplexed assays. Multiplexed particle-based immunoassays are now common place, and new assays to study genes, protein function, and molecular assembly. Numerous efforts are underway to extend the multiplexing capabilities of microparticle-based assays through new approaches to particle encoding and analyte reporting. The impact of these developments will be seen in the basic research and clinical laboratories, as well as in drug development. PMID:16604537

Evaluation of the platelet counting by Abbott CELL-DYN SAPPHIRE haematology analyser compared with flow cytometry.

PubMed

Grimaldi, E; Del Vecchio, L; Scopacasa, F; Lo Pardo, C; Capone, F; Pariante, S; Scalia, G; De Caterina, M

2009-04-01

The Abbot Cell-Dyn Sapphire is a new generation haematology analyser. The system uses optical/fluorescence flow cytometry in combination with electronic impedance to produce a full blood count. Optical and impedance are the default methods for platelet counting while automated CD61-immunoplatelet analysis can be run as selectable test. The aim of this study was to determine the platelet count performance of the three counting methods available on the instrument and to compare the results with those provided by Becton Dickinson FACSCalibur flow cytometer used as reference method. A lipid interference experiment was also performed. Linearity, carryover and precision were good, and satisfactory agreement with reference method was found for the impedance, optical and CD61-immunoplatelet analysis, although this latter provided the closest results in comparison with flow cytometry. In the lipid interference experiment, a moderate inaccuracy of optical and immunoplatelet counts was observed starting from a very high lipid value.

Microflow1, a sheathless fiber-optic flow cytometry biomedical platform: demonstration onboard the international space station.

PubMed

Dubeau-Laramée, Geneviève; Rivière, Christophe; Jean, Isabelle; Mermut, Ozzy; Cohen, Luchino Y

2014-04-01

A fiber-optic based flow cytometry platform was designed to build a portable and robust instrument for space applications. At the core of the Microflow1 is a unique fiber-optic flow cell fitted to a fluidic system and fiber coupled to the source and detection channels. A Microflow1 engineering unit was first tested and benchmarked against a commercial flow cytometer as a reference in a standard laboratory environment. Testing in parabolic flight campaigns was performed to establish Microflow1's performance in weightlessness, before operating the new platform on the International Space Station. Microflow1 had comparable performances to commercial systems, and operated remarkably and robustly in weightlessness (microgravity). Microflow1 supported immunophenotyping as well as microbead-based multiplexed cytokine assays in the space environment and independently of gravity levels. Results presented here provide evidence that this fiber-optic cytometer technology is inherently compatible with the space environment with negligible compromise to analytical performance. © 2013 International Society for Advancement of Cytometry.

Long-term clinical and immunological effects of p53-SLP® vaccine in patients with ovarian cancer.

PubMed

Leffers, Ninke; Vermeij, Renee; Hoogeboom, Baukje-Nynke; Schulze, Ute R; Wolf, Rinze; Hamming, Ineke E; van der Zee, Ate G; Melief, Kees J; van der Burg, Sjoerd H; Daemen, Toos; Nijman, Hans W

2012-01-01

Vaccine-induced p53-specific immune responses were previously reported to be associated with improved response to secondary chemotherapy in patients with small cell lung cancer. We investigated long-term clinical and immunological effects of the p53-synthetic long peptide (p53-SLP®) vaccine in patients with recurrent ovarian cancer. Twenty patients were immunized with the p53-SLP® vaccine between July 2006 and August 2007. Follow-up information on patients was obtained. Clinical responses to secondary chemotherapy after p53-SLP® immunizations were determined by computerized tomography and/or tumor marker levels (CA125). Disease-specific survival was compared to a matched historical control group. Immune responses were analyzed by flow cytometry, proliferation assay, interferon gamma (IFN-γ) ELISPOT and/or cytokine bead array. Lymphocytes cultured from skin biopsy were analyzed by flow cytometry and proliferation assay. Of 20 patients treated with the p53-SLP® vaccine, 17 were subsequently treated with chemotherapy. Eight of these patients volunteered another blood sample. No differences in clinical response rates to secondary chemotherapy or disease-specific survival were observed between immunized patients and historical controls (p = 0.925, resp. p = 0.601). p53-specific proliferative responses were observed in 5/8 patients and IFN-γ production in 2/7 patients. Lymphocytes cultured from a prior injection site showing inflammation during chemotherapy did not recognize p53-SLP®. Thus, treatment with the p53-SLP® vaccine does not affect responses to secondary chemotherapy or survival, although p53-specific T-cells do survive chemotherapy. Copyright © 2011 UICC.

Stable Phenotype Of B-Cell Subsets Following Cryopreservation and Thawing of Normal Human Lymphocytes Stored in a Tissue Biobank.

PubMed

Rasmussen, Simon Mylius; Bilgrau, Anders Ellern; Schmitz, Alexander; Falgreen, Steffen; Bergkvist, Kim Steve; Tramm, Anette Mai; Baech, John; Jacobsen, Chris Ladefoged; Gaihede, Michael; Kjeldsen, Malene Krag; Bødker, Julie Støve; Dybkaer, Karen; Bøgsted, Martin; Johnsen, Hans Erik

2014-09-20

Background Cryopreservation is an acknowledged procedure to store vital cells for future biomarker analyses. Few studies, however, have analyzed the impact of the cryopreservation on phenotyping. Methods We have performed a controlled comparison of cryopreserved and fresh cellular aliquots prepared from individual healthy donors. We studied circulating B-cell subset membrane markers and global gene expression, respectively by multiparametric flow cytometry and microarray data. Extensive statistical analysis of the generated data tested the concept that "overall, there are phenotypic differences between cryopreserved and fresh B-cell subsets". Subsequently, we performed a consecutive uncontrolled comparison of tonsil tissue samples. Results By multiparametric flow analysis, we documented no significant changes following cryopreservation of subset frequencies or membrane intensity for the differentiation markers CD19, CD20, CD22, CD27, CD38, CD45, and CD200. By gene expression profiling following cryopreservation, across all samples, only 16 out of 18708 genes were significantly up or down regulated, including FOSB, KLF4, RBP7, ANXA1 or CLC, DEFA3, respectively. Implementation of cryopreserved tissue in our research program allowed us to present a performance analysis, by comparing cryopreserved and fresh tonsil tissue. As expected, phenotypic differences were identified, but to an extent that did not affect the performance of the cryopreserved tissue to generate specific B-cell subset associated gene signatures and assign subset phenotypes to independent tissue samples. Conclusions We have confirmed our working concept and illustrated the usefulness of vital cryopreserved cell suspensions for phenotypic studies of the normal B-cell hierarchy; however, storage procedures need to be delineated by tissue specific comparative analysis. © 2014 Clinical Cytometry Society. Copyright © 2014 Clinical Cytometry Society.

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

Rapid Identification of Micro-Organisms.

DTIC Science & Technology

1985-08-26

mixed cell populations to which this technology has been applied, although many similarities exist as well. In most applications of flow cytometry, it...specific nucleic acid sequences detectable with DNA probes, are applicable only to organisms previously know to and available to the laboratory workers...peak of phycoerythrin, and the 585/593 nm yellow emission from He-Ne lasers now in development is well suited for excitation of phycocyanin . Any of the

Managing Multi-center Flow Cytometry Data for Immune Monitoring

PubMed Central

White, Scott; Laske, Karoline; Welters, Marij JP; Bidmon, Nicole; van der Burg, Sjoerd H; Britten, Cedrik M; Enzor, Jennifer; Staats, Janet; Weinhold, Kent J; Gouttefangeas, Cécile; Chan, Cliburn

2014-01-01

With the recent results of promising cancer vaccines and immunotherapy1–5, immune monitoring has become increasingly relevant for measuring treatment-induced effects on T cells, and an essential tool for shedding light on the mechanisms responsible for a successful treatment. Flow cytometry is the canonical multi-parameter assay for the fine characterization of single cells in solution, and is ubiquitously used in pre-clinical tumor immunology and in cancer immunotherapy trials. Current state-of-the-art polychromatic flow cytometry involves multi-step, multi-reagent assays followed by sample acquisition on sophisticated instruments capable of capturing up to 20 parameters per cell at a rate of tens of thousands of cells per second. Given the complexity of flow cytometry assays, reproducibility is a major concern, especially for multi-center studies. A promising approach for improving reproducibility is the use of automated analysis borrowing from statistics, machine learning and information visualization21–23, as these methods directly address the subjectivity, operator-dependence, labor-intensive and low fidelity of manual analysis. However, it is quite time-consuming to investigate and test new automated analysis techniques on large data sets without some centralized information management system. For large-scale automated analysis to be practical, the presence of consistent and high-quality data linked to the raw FCS files is indispensable. In particular, the use of machine-readable standard vocabularies to characterize channel metadata is essential when constructing analytic pipelines to avoid errors in processing, analysis and interpretation of results. For automation, this high-quality metadata needs to be programmatically accessible, implying the need for a consistent Application Programming Interface (API). In this manuscript, we propose that upfront time spent normalizing flow cytometry data to conform to carefully designed data models enables automated analysis, potentially saving time in the long run. The ReFlow informatics framework was developed to address these data management challenges. PMID:26085786

Monitoring non-immediate allergic reactions to iodine contrast media

PubMed Central

Torres, M J; Mayorga, C; Cornejo-Garcia, J A; Lopez, S; Chaves, P; Rondon, C; Fernandez, T; Blanca, M

2008-01-01

Non-immediate reactions to iodine contrast media (ICM) affect 2–5% of patients receiving these agents. We studied the immunological mechanisms involved in patients with a confirmed non-immediate reaction, maculopapular exanthema, after administration of ICM. The diagnosis was carried out by skin testing or drug provocation test. The immunological study was performed in sequential peripheral blood mononuclear cells taken from the onset of the reaction by flow cytometry and in skin biopsy by immunohistochemistry, with specific recognition by the lymphocyte transformation test (LTT) with different ICM. Flow cytometry showed an increase in the different activation markers [CD69, CD25 and human leucocyte antigen D-related (HLA-DR)] and the skin homing receptor [cutaneous lymphocyte-associated antigen (CLA)] in CD4 lymphocytes, whereas perforin was higher in the CD8 lymphocytes. The skin biopsy showed a perivascular mononuclear infiltrate composed of CD4 lymphocytes, expressing CD25, HLA-DR and CLA, with eosinophils. Intradermal skin tests and the LTT were positive to several ICM, including the culprit agent in four and three patients, respectively, with negative results in all 10 tolerant controls. We showed that a specific immunological mechanism was implicated in patients with non-immediate reactions to ICM. Moreover, the positive results in skin tests and lymphocyte proliferation tests indicated that an important cross-reactivity exists. PMID:18341616

A novel anti-CD22 scFv-apoptin fusion protein induces apoptosis in malignant B-cells.

PubMed

Agha Amiri, Solmaz; Shahhosseini, Soraya; Zarei, Najmeh; Khorasanizadeh, Dorsa; Aminollahi, Elahe; Rezaie, Faegheh; Zargari, Mehryar; Azizi, Mohammad; Khalaj, Vahid

2017-12-01

CD22 marker is a highly internalizing antigen which is located on the surface of B-cells and is being used as a promising target for treatment of B cell malignancies. Monoclonal antibodies targeting CD22 have been introduced and some are currently under investigation in clinical trials. Building on the success of antibody drug conjugates, we developed a fusion protein consisting of a novel anti-CD22 scFv and apoptin and tested binding and therapeutic effects in lymphoma cells. The recombinant protein was expressed in E. coli and successfully purified and refolded. In vitro binding analysis by immunofluorescence and flow cytometry demonstrated that the recombinant protein specifically binds to CD22 positive Raji cells but not to CD22 negative Jurkat cells. The cytotoxic properties of scFv-apoptin were assessed by an MTT assay and Annexin V/PI flow cytometry analysis and showed that the recombinant protein induced apoptosis preferentially in Raji cells with no detectable effects in Jurkat cells. Our findings indicated that the recombinant anti-CD22 scFv-apoptin fusion protein could successfully cross the cell membrane and induce apoptosis with high specificity, make it as a promising molecule for immunotherapy of B-cell malignancies.

CyTOF workflow: differential discovery in high-throughput high-dimensional cytometry datasets

PubMed Central

Nowicka, Malgorzata; Krieg, Carsten; Weber, Lukas M.; Hartmann, Felix J.; Guglietta, Silvia; Becher, Burkhard; Levesque, Mitchell P.; Robinson, Mark D.

2017-01-01

High dimensional mass and flow cytometry (HDCyto) experiments have become a method of choice for high throughput interrogation and characterization of cell populations.Here, we present an R-based pipeline for differential analyses of HDCyto data, largely based on Bioconductor packages. We computationally define cell populations using FlowSOM clustering, and facilitate an optional but reproducible strategy for manual merging of algorithm-generated clusters. Our workflow offers different analysis paths, including association of cell type abundance with a phenotype or changes in signaling markers within specific subpopulations, or differential analyses of aggregated signals. Importantly, the differential analyses we show are based on regression frameworks where the HDCyto data is the response; thus, we are able to model arbitrary experimental designs, such as those with batch effects, paired designs and so on. In particular, we apply generalized linear mixed models to analyses of cell population abundance or cell-population-specific analyses of signaling markers, allowing overdispersion in cell count or aggregated signals across samples to be appropriately modeled. To support the formal statistical analyses, we encourage exploratory data analysis at every step, including quality control (e.g. multi-dimensional scaling plots), reporting of clustering results (dimensionality reduction, heatmaps with dendrograms) and differential analyses (e.g. plots of aggregated signals). PMID:28663787

H2Mab-77 is a Sensitive and Specific Anti-HER2 Monoclonal Antibody Against Breast Cancer.

PubMed

Itai, Shunsuke; Fujii, Yuki; Kaneko, Mika K; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Chang, Yao-Wen; Handa, Saori; Takahashi, Maki; Suzuki, Hiroyoshi; Harada, Hiroyuki; Kato, Yukinari

2017-08-01

Human epidermal growth factor receptor 2 (HER2) plays a critical role in the progression of breast cancers, and HER2 overexpression is associated with poor clinical outcomes. Trastuzumab is an anti-HER2 humanized antibody that leads to significant survival benefits in patients with HER2-positive metastatic breast cancers. In this study, we developed novel anti-HER2 monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. Initially, we expressed the full length or ectodomain of HER2 in LN229 glioblastoma cells and then immunized mice with ectodomain of HER2 or LN229/HER2, and performed the first screening by enzyme-linked immunosorbent assays using ectodomain of HER2. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical analyses (fourth screening). Among 100 mAb clones, only three mAbs reacted with HER2 in Western blot, and clone H 2 Mab-77 (IgG 1 , kappa) was selected. Finally, immunohistochemical analyses with H 2 Mab-77 showed sensitive and specific reactions against breast cancer cells, warranting the use of H 2 Mab-77 to detect HER2 in pathological analyses of breast cancers.

An inter-laboratory comparison of PNH clone detection by high-sensitivity flow cytometry in a Russian cohort.

PubMed

Sipol, Alexandra A; Babenko, Elena V; Borisov, Vyacheslav I; Naumova, Elena V; Boyakova, Elena V; Yakunin, Dimitry I; Glazanova, Tatyana V; Chubukina, Zhanna V; Pronkina, Natalya V; Popov, Alexander M; Saveliev, Leonid I; Lugovskaya, Svetlana A; Lisukov, Igor A; Kulagin, Alexander D; Illingworth, Andrea J

2015-01-01

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal stem cell disorder characterized by partial or absolute deficiency of glycophosphatidyl-inositol (GPI) anchor-linked surface proteins on blood cells. A lack of precise diagnostic standards for flow cytometry has hampered useful comparisons of data between laboratories. We report data from the first study evaluating the reproducibility of high-sensitivity flow cytometry for PNH in Russia. PNH clone sizes were determined at diagnosis in PNH patients at a central laboratory and compared with follow-up measurements in six laboratories across the country. Analyses in each laboratory were performed according to recommendations from the International Clinical Cytometry Society (ICCS) and the more recent 'practical guidelines'. Follow-up measurements were compared with each other and with the values determined at diagnosis. PNH clone size measurements were determined in seven diagnosed PNH patients (five females, two males: mean age 37 years); five had a history of aplastic anemia and three (one with and two without aplastic anemia) had severe hemolytic PNH and elevated plasma lactate dehydrogenase. PNH clone sizes at diagnosis were low in patients with less severe clinical symptoms (0.41-9.7% of granulocytes) and high in patients with severe symptoms (58-99%). There were only minimal differences in the follow-up clone size measurement for each patient between the six laboratories, particularly in those with high values at diagnosis. The ICCS-recommended high-sensitivity flow cytometry protocol was effective for detecting major and minor PNH clones in Russian PNH patients, and showed high reproducibility between laboratories.

Development of an unbiased, semi-automated approach for classifying plasma cell immunophenotype following multicolor flow cytometry of bone marrow aspirates.

PubMed

Post, Steven R; Post, Ginell R; Nikolic, Dejan; Owens, Rebecca; Insuasti-Beltran, Giovanni

2018-03-24

Despite increased usage of multiparameter flow cytometry (MFC) to assess diagnosis, prognosis, and therapeutic efficacy (minimal residual disease, MRD) in plasma cell neoplasms (PCNs), standardization of methodology and data analysis is suboptimal. We investigated the utility of using the mean and median fluorescence intensities (FI) obtained from MFC to objectively describe parameters that distinguish plasma cell (PC) phenotypes. In this retrospective study, flow cytometry results from bone marrow aspirate specimens from 570 patients referred to the Myeloma Institute at UAMS were evaluated. Mean and median FI data were obtained from 8-color MFC of non-neoplastic, malignant, and mixed PC populations using antibodies to CD38, CD138, CD19, CD20, CD27, CD45, CD56, and CD81. Of 570 cases, 252 cases showed only non-neoplastic PCs, 168 showed only malignant PCs, and 150 showed mixed PC populations. Statistical analysis of median FI data for each CD marker showed no difference in expression intensity on non-neoplastic and malignant PCs, between pure and mixed PC populations. ROC analysis of the median FI of CD expression in non-neoplastic and malignant PCs was used to develop an algorithm to convert quantitative FI values to qualitative assessments including "negative," "positive," "dim," and "heterogeneous" expression. FI data derived from 8-color MFC can be used to define marker expression on PCs. Translation of FI data from Infinicyt software to an Excel worksheet streamlines workflow and eliminates transcriptional errors when generating flow reports. © 2018 International Clinical Cytometry Society. © 2018 International Clinical Cytometry Society.

Modeling of cytometry data in logarithmic space: When is a bimodal distribution not bimodal?

PubMed

Erez, Amir; Vogel, Robert; Mugler, Andrew; Belmonte, Andrew; Altan-Bonnet, Grégoire

2018-02-16

Recent efforts in systems immunology lead researchers to build quantitative models of cell activation and differentiation. One goal is to account for the distributions of proteins from single-cell measurements by flow cytometry or mass cytometry as readout of biological regulation. In that context, large cell-to-cell variability is often observed in biological quantities. We show here that these readouts, viewed in logarithmic scale may result in two easily-distinguishable modes, while the underlying distribution (in linear scale) is unimodal. We introduce a simple mathematical test to highlight this mismatch. We then dissect the flow of influence of cell-to-cell variability proposing a graphical model which motivates higher-dimensional analysis of the data. Finally we show how acquiring additional biological information can be used to reduce uncertainty introduced by cell-to-cell variability, helping to clarify whether the data is uni- or bimodal. This communication has cautionary implications for manual and automatic gating strategies, as well as clustering and modeling of single-cell measurements. © 2018 International Society for Advancement of Cytometry. © 2018 International Society for Advancement of Cytometry.

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.

Label-free in vivo flow cytometry in zebrafish using two-photon autofluorescence imaging.

PubMed

Zeng, Yan; Xu, Jin; Li, Dong; Li, Li; Wen, Zilong; Qu, Jianan Y

2012-07-01

We demonstrate a label-free in vivo flow cytometry in zebrafish blood vessels based on two-photon excited autofluorescence imaging. The major discovery in this work is the strong autofluorescence emission from the plasma in zebrafish blood. The plasma autofluorescence provides excellent contrast for visualizing blood vessels and counting blood cells. In addition, the cellular nicotinamide adenine dinucleotide autofluorescence enables in vivo imaging and counting of white blood cells (neutrophils).

Flow Cytometry Techniques in Radiation Biology

DTIC Science & Technology

1988-06-01

Henidtopoietic stem cells SUMMARY Hematopoietic stem cells ( HSC ) are present in the marrow at a concentration of approximately 2-3 HSC per 1000 nucleated marrow...cells. In the past, only clonogenic assays requiring 8-13 days and ten irradiated recipient rodents were available for assaying HSC . Because of the...importance of HSC in the postirradiation syndrome, we have developed a new rapid method based on flow cytometry not only to assay but also to purify and

Flow cytometry on the stromal-vascular fraction of white adipose tissue.

PubMed

Brake, Danett K; Smith, C Wayne

2008-01-01

Adipose tissue contains cell types other than adipocytes that may contribute to complications linked to obesity. For example, macrophages have been shown to infiltrate adipose tissue in response to a high-fat diet. Isolation of the stromal-vascular fraction of adipose tissue allows one to use flow cytometry to analyze cell surface markers on leukocytes. Here, we present a technical approach to identify subsets of leukocytes that differentially express cell surface markers.

Screening of Compounds Toxicity against Human Monocytic cell line-THP-1 by Flow Cytometry

PubMed Central

Pick, Neora; Cameron, Scott; Arad, Dorit

2004-01-01

The worldwide rapid increase in bacterial resistance to numerous antibiotics requires on-going development of new drugs to enter the market. As the development of new antibiotics is lengthy and costly, early monitoring of compound's toxicity is essential in the development of novel agents. Our interest is in a rapid, simple, high throughput screening method to assess cytotoxicity induced by potential agents. Some intracellular pathogens, such as Mycobacterium tuberculosis primary site of infection is human alveolar macrophages. Thus, evaluation of candidate drugs for macrophage toxicity is crucial. Protocols for high throughput drug toxicity screening of macrophages using flow cytometry are lacking in the literature. For this application we modified a preexisting technique, propidium iodide (PI) exclusion staining and utilized it for rapid toxicity tests. Samples were prepared in 96 well plates and analyzed by flow cytometry, which allowed for rapid, inexpensive and precise assessment of compound's toxicity associated with cell death. PMID:15472722

A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes using flow cytometry

PubMed Central

Alves, L.P.S.; Almeida, A.T.; Cruz, L.M.; Pedrosa, F.O.; de Souza, E.M.; Chubatsu, L.S.; Müller-Santos, M.; Valdameri, G.

2017-01-01

The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC) is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB) production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i) cell permeabilization, ii) Nile red staining, and iii) analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2=0.99) compared to a standard method based on methanolysis and GC. This method was successfully applied to the quantification of PHB in epiphytic bacteria isolated from rice roots. PMID:28099582

An improved method for differentiating cell-bound from internalized particles by imaging flow cytometry.

PubMed

Smirnov, Asya; Solga, Michael D; Lannigan, Joanne; Criss, Alison K

2015-08-01

Recognition, binding, internalization, and elimination of pathogens and cell debris are important functions of professional as well as non-professional phagocytes. However, high-throughput methods for quantifying cell-associated particles and discriminating bound from internalized particles have been lacking. Here we describe a protocol for using imaging flow cytometry to quantify the attached and phagocytosed particles that are associated with a population of cells. Cells were exposed to fluorescent particles, fixed, and exposed to an antibody of a different fluorophore that recognizes the particles. The antibody is added without cell permeabilization, such that the antibody only binds extracellular particles. Cells with and without associated particles were identified by imaging flow cytometry. For each cell with associated particles, a spot count algorithm was employed to quantify the number of extracellular (double fluorescent) and intracellular (single fluorescent) particles per cell, from which the percent particle internalization was determined. The spot count algorithm was empirically validated by examining the fluorescence and phase contrast images acquired by the flow cytometer. We used this protocol to measure binding and internalization of the bacterium Neisseria gonorrhoeae by primary human neutrophils, using different bacterial variants and under different cellular conditions. The results acquired using imaging flow cytometry agreed with findings that were previously obtained using conventional immunofluorescence microscopy. This protocol provides a rapid, powerful method for measuring the association and internalization of any particle by any cell type. Copyright © 2015 Elsevier B.V. All rights reserved.

A flow cytometry-based strategy to identify and express IgM from VH1-69+ clonal peripheral B cells.

PubMed

Charles, Edgar D; Orloff, Michael I M; Dustin, Lynn B

2011-01-05

Pathologic rheumatoid factor (RF) levels are hallmarks of several human diseases. Production of monoclonal RF in vitro is essential for studies of the antigenic specificities of RF, as well as for a dissection of the mechanisms of aberrant RF+ B cell activation. We have expanded upon previous methods to develop a flow cytometry-based method to efficiently clone monoclonal antibodies (mAbs) from humans with expansions of RF-like, immunoglobulin heavy chain variable region (IgVH) 1-69 gene segment-containing B cells. The cloned variable regions are expressed as IgM and produced during culture at concentrations between 5 and 20 μg/ml. Using this system, we show that clonal Igs from patients with HCV-related mixed cryoglobulinemia, when expressed as IgM, have RF activity. We anticipate that this system will be useful for the cloning and expression of mAbs partially encoded by VH1-69 and for determination of the reactivity patterns of polyspecific, low-affinity IgMs of human pathogenic importance. Copyright © 2010 Elsevier B.V. All rights reserved.

The characterization of exosome from blood plasma of patients with colorectal cancer

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

Yunusova, N. V., E-mail: Bochkarevanv@oncology.tomsk.ru; Siberian State Medical University, Moskovsky Trakt 2, Tomsk, 634050; Tamkovich, S. N., E-mail: s.tamk@niboch.nsc.ru

Exosomes are extracellular membrane structures involved in many physiological and pathological processes including cancerogenesis and metastasis. The clarification of the criteria for exosome isolating and identifying is the purpose of this study. Exosome samples from the plasma of patients with colorectal cancer and healthy donors were examined using transmission electron microscopy and flow cytometry in accordance with the minimum requirements of “International Society for Extracellular Vesicles”. The choice of the method for isolation of exosomes from the blood plasma by ultrafiltration and ultracentrifugation allowed obtaining highly purified samples of exosomes, in which all the structural components were clearly seen. Themore » results obtained with flow cytometry suggest that exosomes of blood plasma from patients with colorectal cancer can be produced by epithelial cells. Moreover, cells produce different types of exosomes, which correspond to different mechanisms in sorting macromolecules in the membrane of multivesicular bodies. Determination of significant differences in the expression of specific exosomal proteins from colorectal cancer patients compared to healthy donors suggests a high diagnostic potential significance of circulating exosomes.« less

Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis

PubMed Central

Dragovic, Rebecca A.; Gardiner, Christopher; Brooks, Alexandra S.; Tannetta, Dionne S.; Ferguson, David J.P.; Hole, Patrick; Carr, Bob; Redman, Christopher W.G.; Harris, Adrian L.; Dobson, Peter J.; Harrison, Paul; Sargent, Ian L.

2011-01-01

Cellular microvesicles and nanovesicles (exosomes) are involved in many disease processes and have major potential as biomarkers. However, developments in this area are constrained by limitations in the technology available for their measurement. Here we report on the use of fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles. In this system vesicles are visualized by light scattering using a light microscope. A video is taken, and the NTA software tracks the brownian motion of individual vesicles and calculates their size and total concentration. Using human placental vesicles and plasma, we have demonstrated that NTA can measure cellular vesicles as small as ∼50 nm and is far more sensitive than conventional flow cytometry (lower limit ∼300 nm). By combining NTA with fluorescence measurement we have demonstrated that vesicles can be labeled with specific antibody-conjugated quantum dots, allowing their phenotype to be determined. From the Clinical Editor The authors of this study utilized fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles, demonstrating that NTA is far more sensitive than conventional flow cytometry. PMID:21601655

Physiological analysis of yeast cells by flow cytometry during serial-repitching of low-malt beer fermentation.

PubMed

Kobayashi, Michiko; Shimizu, Hiroshi; Shioya, Suteaki

2007-05-01

At the end of beer brewing fermentation, yeast cells are collected and repitched for economical reasons. Although it is generally accepted that the physiological state of inoculated yeast cells affects their subsequent fermentation performance, the effect of serial-repitching on the physiological state of such yeast cells has not been well clarified. In this study, the fermentation performance of yeast cells during serial-repitching was investigated. After multiple repitchings, the specific growth rate and maximum optical density (OD(660)) decreased, and increases in isoamyl alcohol, which causes an undesirable flavor, and residual free amino acid nitrogen (FAN) concentrations were observed. The physiological state of individual cells before inoculation was characterized by flow cytometry using the fluorescent dyes dehydrorhodamine 123 (DHR) and bis-(1,3-dibutylbarbituric acid) trimethine oxonol (OXN). The fluorescence intensities of DHR, an indicator of reactive oxygen species (ROSs), and OXN, which indicates membrane potential, gradually increased as the number of serial-repitching cycles increased. Fluorescence intensity correlated strongly with cell growth. The subsequent fermentation performance can be predicted from this correlation.

Establishment of Novel Monoclonal Antibody PMab-32 Against Rabbit Podoplanin.

PubMed

Honma, Ryusuke; Fujii, Yuki; Ogasawara, Satoshi; Oki, Hiroharu; Liu, Xing; Nakamura, Takuro; Kaneko, Mika K; Takagi, Michiaki; Kato, Yukinari

2016-02-01

Podoplanin (PDPN) is a type I transmembrane O-glycoprotein, which is known as a specific lymphatic marker. PDPN activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelet. PDPN is also expressed in several normal tissues, including podocytes and type I alveolar cells. Although many monoclonal antibodies (MAbs) against human PDPN (hPDPN), mouse PDPN (mPDPN), and rat PDPN (rPDPN) have been established, useful antibodies against rabbit PDPN (rabPDPN) have not been developed. In this study, we immunized mice with the recombinant proteins of rabPDPN, and developed a novel anti-rabPDPN MAb, named PMab-32. PMab-32 could detect endogenous and exogenous rabPDPN in flow cytometry and Western blot analysis. The KD of PMab-32 was determined to be 6.2 × 10(-8) M by flow cytometry. Immunohistochemical analysis showed that PMab-32 is useful for detecting podocytes, type I alveolar cells, and lymphatic endothelial cells in normal rabbit tissues. PMab-32 is expected to be useful for various rabbit experiments.

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

Photothermal and photoacoustic Raman cytometry in vitro and in vivo

PubMed Central

Shashkov, Evgeny V.; Galanzha, Ekaterina I.; Zharov, Vladimir P.

2010-01-01

An integrated Raman-based cytometry was developed with photothermal (PT) and photoacoustic (PA) detection of Raman-induced thermal and acoustic signals in biological samples with Raman-active vibrational modes. The two-frequency, spatially and temporally overlapping pump–Stokes excitation in counterpropagating geometry was provided by a nanosecond tunable (420–2300 nm) optical parametric oscillator and a Raman shifter (639 nm) pumped by a double-pulsed Q-switched Nd:YAG laser using microscopic and fiberoptic delivery of laser radiation. The PA and PT Raman detection and imaging technique was tested in vitro with benzene, acetone, olive oil, carbon nanotubes, chylomicron phantom, and cancer cells, and in vivo in single adipocytes in mouse mesentery model. The integration of linear and nonlinear PA and PT Raman scanning and flow cytometry has the potential to enhance its chemical specificity and sensitivity including nanobubble-based amplification (up to 10- fold) for detection of absorbing and nonabsorbing targets that are important for both basic and clinically relevant studies of lymph and blood biochemistry, cancer, and fat distribution at the single-cell level. PMID:20389713

Combination of CD157 and FLAER to Detect Peripheral Blood Eosinophils by Multiparameter Flow Cytometry.

PubMed

Carulli, Giovanni; Marini, Alessandra; Sammuri, Paola; Domenichini, Cristiana; Ottaviano, Virginia; Pacini, Simone; Petrini, Mario

2015-01-01

The identification of eosinophils by flow cytometry is difficult because most of the surface antigens expressed by eosinophils are shared with neutrophils. Some methods have been proposed, generally based on differential light scatter properties, enhanced autofluorescence, lack of CD16 or selective positivity of CD52. Such methods, however, show several limitations. In the present study we report a novel method based on the analysis of glycosylphosphatidylinositol (GPI)-linked molecules. The combination of CD157 and FLAER was used, since FLAER recognizes all GPI-linked molecules, while CD157 is absent on the membrane of eosinophils and expressed by neutrophils. Peripheral blood samples from normal subjects and patients with variable percentages of eosinophils (n = 31), and without any evidence for circulating immature myeloid cells, were stained with the combination of FLAER-Alexa Fluor and CD157-PE. A FascCanto II cytometer was used. Granulocytes were gated after CD33 staining and eosinophils were identified as CD157(-)/FLAER(+) events. Neutrophils were identified as CD157(+)/FLAER(+) events. The percentages of eosinophils detected by this method showed a very significant correlation both with automated counting and with manual counting (r = 0.981 and 0.989, respectively). Sorting assays were carried out by a S3 Cell Sorter: cytospins obtained from CD157(-)/FLAER(+) events consisted of 100% eosinophils, while samples from CD157(+)/FLAER(+) events were represented only by neutrophils. In conclusion, this method shows high sensitivity and specificity in order to distinguish eosinophils from neutrophils by flow cytometry. However, since CD157 is gradually up-regulated throughout bone marrow myeloid maturation, our method cannot be applied to cases characterized by immature myeloid cells.

Differentiation potential of human CD133 positive hematopoietic stem cells into motor neuron- like cells, in vitro.

PubMed

Moghaddam, Sepideh Alavi; Yousefi, Behnam; Sanooghi, Davood; Faghihi, Faezeh; Hayati Roodbari, Nasim; Bana, Nikoo; Joghataei, Mohammad Taghi; Pooyan, Paria; Arjmand, Babak

2017-12-01

Spinal cord injuries and motor neuron-related disorders impact on life of many patients around the world. Since pharmacotherapy and surgical approaches were not efficient to regenerate these types of defects; stem cell therapy as a good strategy to restore the lost cells has become the focus of interest among the scientists. Umbilical cord blood CD133 + hematopoietic stem cells (UCB- CD133 + HSCs) with self- renewal property and neural lineage differentiation capacity are ethically approved cell candidate for use in regenerative medicine. In this regard the aim of this study was to quantitatively evaluate the capability of these cells to differentiate into motor neuron-like cells (MNL), in vitro. CD133 + HSCs were isolated from human UCB using MACS system. After cell characterization using flow cytometry, the cells were treated with a combination of Retinoic acid, Sonic hedgehog, Brain derived neurotrophic factor, and B27 through a 2- step procedure for two weeks. The expression of MN-specific markers was examined using qRT- PCR, flow cytometry and immunocytochemistry. By the end of the two-week differentiation protocol, CD133 + cells acquired unipolar MNL morphology with thin and long neurites. The expression of Isl-1(62.15%), AChE (41.83%), SMI-32 (21.55%) and Nestin (17.46%) was detected using flow cytometry and immunocytochemistry. The analysis of the expression of PAX6, ISL-1, ACHE, CHAT and SMI-32 revealed that MNLs present these neural markers at levels comparable with undifferentiated cells. In Conclusion Human UCB- CD133 + HSCs are remarkably potent cell candidates to transdifferentiate into motor neuron-like cells, in vitro. Copyright © 2017. Published by Elsevier B.V.

Tracking protein aggregation and mislocalization in cells with flow cytometry.

PubMed

Ramdzan, Yasmin M; Polling, Saskia; Chia, Cheryl P Z; Ng, Ivan H W; Ormsby, Angelique R; Croft, Nathan P; Purcell, Anthony W; Bogoyevitch, Marie A; Ng, Dominic C H; Gleeson, Paul A; Hatters, Danny M

2012-03-18

We applied pulse-shape analysis (PulSA) to monitor protein localization changes in mammalian cells by flow cytometry. PulSA enabled high-throughput tracking of protein aggregation, translocation from the cytoplasm to the nucleus and trafficking from the plasma membrane to the Golgi as well as stress-granule formation. Combining PulSA with tetracysteine-based oligomer sensors in a cell model of Huntington's disease enabled further separation of cells enriched with monomers, oligomers and inclusion bodies.

A Stem Cell-Seeded Nanofibrous Scaffold for Auditory Nerve Replacement

DTIC Science & Technology

2013-10-01

the brightest GFP+ cells by flow cytometry and compared these with GFP- cells (Figure 1A-C). The transfected cells showed robust GFP expression even...al., 2011), but no normative data were provided on SGN loss by cochlear turn and, in contrast to our results, those authors reported no impact on...A) Flow cytometry analysis to identify GFP+ and GFP- cells. The large cluster of cells on the left represent the GFP- cells and exhibited similar

Endothelial Progenitor Cells (EPC) Count by Multicolor Flow Cytometry in Healthy Individuals and Diabetes Mellitus (DM) Patients.

PubMed

Falay, Mesude; Aktas, Server

2016-11-01

The present study aimed to determine circulating Endothelial Progenitor Cell (EPC) counts by multicolor flow cytometry in healthy individuals and diabetic subjects by means of forming an analysis procedure using a combination of monoclonal antibodies (moAbs), which would correctly detect the circulating EPC count. The circulating EPC count was detected in 40 healthy individuals (20 Female, 20 Male; age range: 26 - 50 years) and 30 Diabetes Mellitus (DM) patients (15 Female, 15 Male; age range: 42 - 55) by multicolor flow cytometry (FCM) in a single-tube panel consisting of 5 CD45/CD31/CD34/CD309/ SYTO® and 16 monoclonal antibodies. Circulating EPC count was 11.33 (7.89 - 15.25) cells/µL in the healthy control group and 4.80 (0.70 - 10.85) cells/µL in the DM group. EPC counts were significantly lower in DM cases that developed coronary artery disease (53.3%) as compared to those that did not (p < 0.001). In the present study, we describe a method that identifies circulating EPC counts by multicolor flow cytometry in a single tube and determines the circulating EPC count in healthy individuals. This is the first study conducted on EPC count in Turkish population. We think that the EPC count found in the present study will be a guide for future studies.

A comparison of methods to assess the antimicrobial activity of nanoparticle combinations on bacterial cells.

PubMed

Bankier, Claire; Cheong, Yuen; Mahalingam, Suntharavathanan; Edirisinghe, Mohan; Ren, Guogang; Cloutman-Green, Elaine; Ciric, Lena

2018-01-01

Bacterial cell quantification after exposure to antimicrobial compounds varies widely throughout industry and healthcare. Numerous methods are employed to quantify these antimicrobial effects. With increasing demand for new preventative methods for disease control, we aimed to compare and assess common analytical methods used to determine antimicrobial effects of novel nanoparticle combinations on two different pathogens. Plate counts of total viable cells, flow cytometry (LIVE/DEAD BacLight viability assay) and qPCR (viability qPCR) were used to assess the antimicrobial activity of engineered nanoparticle combinations (NPCs) on Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria at different concentrations (0.05, 0.10 and 0.25 w/v%). Results were analysed using linear models to assess the effectiveness of different treatments. Strong antimicrobial effects of the three NPCs (AMNP0-2) on both pathogens could be quantified using the plate count method and flow cytometry. The plate count method showed a high log reduction (>8-log) for bacteria exposed to high NPC concentrations. We found similar antimicrobial results using the flow cytometry live/dead assay. Viability qPCR analysis of antimicrobial activity could not be quantified due to interference of NPCs with qPCR amplification. Flow cytometry was determined to be the best method to measure antimicrobial activity of the novel NPCs due to high-throughput, rapid and quantifiable results.

Enumeration of antigen-specific CD8+ T lymphocytes by single-platform, HLA tetramer-based flow cytometry: a European multicenter evaluation.

PubMed

Heijnen, Ingmar A F M; Barnett, David; Arroz, Maria J; Barry, Simon M; Bonneville, Marc; Brando, Bruno; D'hautcourt, Jean-Luc; Kern, Florian; Tötterman, Thomas H; Marijt, Erik W A; Bossy, David; Preijers, Frank W M B; Rothe, Gregor; Gratama, Jan W

2004-11-01

HLA class I peptide tetramers represent powerful diagnostic tools for detection and monitoring of antigen-specific CD8(+) T cells. The impetus for the current multicenter study is the critical need to standardize tetramer flow cytometry if it is to be implemented as a routine diagnostic assay. Hence, the European Working Group on Clinical Cell Analysis set out to develop and evaluate a single-platform tetramer-based method that used cytomegalovirus (CMV) as the antigenic model. Absolute numbers of CMV-specific CD8(+) T cells were obtained by combining the percentage of tetramer-binding cells with the absolute CD8(+) T-cell count. Six send-outs of stabilized blood from healthy individuals or CMV-carrying donors with CMV-specific CD8(+) T-cell counts of 3 to 10 cells/microl were distributed to 7 to 16 clinical sites. These sites were requested to enumerate CD8(+) T cells and, in the case of CMV-positive donors, CMV-specific subsets on three separate occasions using the standard method. Between-site coefficients of variation of less than 10% (absolute CD8(+) T-cell counts) and approximately 30% (percentage and absolute numbers of CMV-specific CD8(+) T cells) were achieved. Within-site coefficients of variation were approximately 5% (absolute CD8(+) T-cell counts), approximately 9% (percentage CMV-specific CD8(+) T cells), and approximately 17% (absolute CMV-specific CD8(+) T-cell counts). The degree of variation tended to correlate inversely with the proportion of CMV-specific CD8(+) T-cell subsets. The single-platform MHC tetramer-based method for antigen-specific CD8(+) T-cell counting has been evaluated by a European group of laboratories and can be considered a reproducible assay for routine enumeration of antigen-specific CD8(+) T cells. (c) 2004 Wiley-Liss, Inc.

Selection of Aptamers for Mature White Adipocytes by Cell SELEX Using Flow Cytometry

PubMed Central

Kim, Eun Young; Kim, Ji Won; Kim, Won Kon; Han, Baek Soo; Park, Sung Goo; Chung, Bong Hyun; Lee, Sang Chul; Bae, Kwang-Hee

2014-01-01

Background Adipose tissue, mainly composed of adipocytes, plays an important role in metabolism by regulating energy homeostasis. Obesity is primarily caused by an abundance of adipose tissue. Therefore, specific targeting of adipose tissue is critical during the treatment of obesity, and plays a major role in overcoming it. However, the knowledge of cell-surface markers specific to adipocytes is limited. Methods and Results We applied the CELL SELEX (Systematic Evolution of Ligands by EXponential enrichment) method using flow cytometry to isolate molecular probes for specific recognition of adipocytes. The aptamer library, a mixture of FITC-tagged single-stranded random DNAs, is used as a source for acquiring molecular probes. With the increasing number of selection cycles, there was a steady increase in the fluorescence intensity toward mature adipocytes. Through 12 rounds of SELEX, enriched aptamers showing specific recognition toward mature 3T3-L1 adipocyte cells were isolated. Among these, two aptamers (MA-33 and 91) were able to selectively bind to mature adipocytes with an equilibrium dissociation constant (Kd) in the nanomolar range. These aptamers did not bind to preadipocytes or other cell lines (such as HeLa, HEK-293, or C2C12 cells). Additionally, it was confirmed that MA-33 and 91 can distinguish between mature primary white and primary brown adipocytes. Conclusions These selected aptamers have the potential to be applied as markers for detecting mature white adipocytes and monitoring adipogenesis, and could emerge as an important tool in the treatment of obesity. PMID:24844710

Immune responses to retinal autoantigens and peptides in equine recurrent uveitis.

PubMed

Deeg, C A; Kaspers, B; Gerhards, H; Thurau, S R; Wollanke, B; Wildner, G

2001-02-01

To test the hypothesis that autoimmune mechanisms are involved in horses in which equine recurrent uveitis (ERU) develops spontaneously. Material obtained from horses treated for spontaneous disease by therapeutic routine vitrectomy was analyzed for total IgG content and IgG specific for S-Antigen (S-Ag) and interphotoreceptor retinoid-binding protein (IRBP). The cellular infiltrate of the vitreous was analyzed by differential counts of cytospin preparations and flow cytometry using equine lymphocyte-specific antibodies. Antigen-specific proliferation assays were performed comparing peripheral blood lymphocytes (PBLs) with vitreal lymphocytes by stimulation with S-Ag and several S-Ag- and IRBP-derived peptides. The total IgG content of specimens from horses with ERU was very high with great variability among the investigated samples (11.5 +/- 8.0 mg). Autoantibodies to S-Ag or IRBP or both were found in 72% of vitreous specimens from horses with uveitis. The leukocyte infiltrates (up to 2 x 10(8) cells per sample) were dominated by lymphocytes (>90%) in most cases (22/32). Flow cytometry showed that more than 50% of these cells were CD4(+) T cells. In vitro stimulation of vitreal lymphocytes, but not of PBL, showed a strong proliferative response to peptides derived from S-Ag or IRBP in 9 of 12 patients. In the eyes of horses with ERU, IgG antibodies and autoreactive T cells specific for retinal antigens were detected. These results strongly support the hypothesis that ERU is an autoimmune-mediated disease and is highly similar to recurrent uveitis in humans in both clinical and immunologic parameters.

Imaging flow cytometry assays for quantifying pigment grade titanium dioxide particle internalization and interactions with immune cells in whole blood

PubMed Central

Vis, Bradley; Pele, Laetitia C.; Faria, Nuno; Powell, Jonathan J.

2017-01-01

Abstract Pigment grade titanium dioxide is composed of sub‐micron sized particles, including a nanofraction, and is widely utilized in food, cosmetic, pharmaceutical, and biomedical industries. Oral exposure to pigment grade titanium dioxide results in at least some material entering the circulation in humans, although subsequent interactions with blood immune cells are unknown. Pigment grade titanium dioxide is employed for its strong light scattering properties, and this work exploited that attribute to determine whether single cell–particle associations could be determined in immune cells of human whole blood at “real life” concentrations. In vitro assays, initially using isolated peripheral blood mononuclear cells, identified titanium dioxide associated with the surface of, and within, immune cells by darkfield reflectance in imaging flow cytometry. This was confirmed at the population level by side scatter measurements using conventional flow cytometry. Next, it was demonstrated that imaging flow cytometry could quantify titanium dioxide particle‐bearing cells, within the immune cell populations of fresh whole blood, down to titanium dioxide levels of 10 parts per billion, which is in the range anticipated for human blood following titanium dioxide ingestion. Moreover, surface association and internal localization of titanium dioxide particles could be discriminated in the assays. Overall, results showed that in addition to the anticipated activity of blood monocytes internalizing titanium dioxide particles, neutrophil internalization and cell membrane adhesion also occurred, the latter for both phagocytic and nonphagocytic cell types. What happens in vivo and whether this contributes to activation of one or more of these different cells types in blood merits further attention. © 2017 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of ISAC. PMID:28941170

Femtosecond laser fabrication of fiber based optofluidic platform for flow cytometry applications

NASA Astrophysics Data System (ADS)

Serhatlioglu, Murat; Elbuken, Caglar; Ortac, Bulend; Solmaz, Mehmet E.

2017-02-01

Miniaturized optofluidic platforms play an important role in bio-analysis, detection and diagnostic applications. The advantages of such miniaturized devices are extremely low sample requirement, low cost development and rapid analysis capabilities. Fused silica is advantageous for optofluidic systems due to properties such as being chemically inert, mechanically stable, and optically transparent to a wide spectrum of light. As a three dimensional manufacturing method, femtosecond laser scanning followed by chemical etching shows great potential to fabricate glass based optofluidic chips. In this study, we demonstrate fabrication of all-fiber based, optofluidic flow cytometer in fused silica glass by femtosecond laser machining. 3D particle focusing was achieved through a straightforward planar chip design with two separately fabricated fused silica glass slides thermally bonded together. Bioparticles in a fluid stream encounter with optical interrogation region specifically designed to allocate 405nm single mode fiber laser source and two multi-mode collection fibers for forward scattering (FSC) and side scattering (SSC) signals detection. Detected signal data collected with oscilloscope and post processed with MATLAB script file. We were able to count number of events over 4000events/sec, and achieve size distribution for 5.95μm monodisperse polystyrene beads using FSC and SSC signals. Our platform shows promise for optical and fluidic miniaturization of flow cytometry systems.

Study of low speed flow cytometry for diffraction imaging with different chamber and nozzle designs.

PubMed

Sa, Yu; Feng, Yuanming; Jacobs, Kenneth M; Yang, Jun; Pan, Ran; Gkigkitzis, Ioannis; Lu, Jun Q; Hu, Xin-Hua

2013-11-01

Achieving effective hydrodynamic focusing and flow stability at low speed presents a challenging design task in flow cytometry for studying phenomena such as cell adhesion and diffraction imaging of cells with low-cost cameras. We have developed different designs of flow chamber and sheath nozzle to accomplish the above goal. A 3D computational model of the chambers has been established to simulate the fluid dynamics in different chamber designs and measurements have been performed to determine the velocity and size distributions of the core fluid from the nozzle. Comparison of the simulation data with experimental results shows good agreement. With the computational model significant insights were gained for optimization of the chamber design and improvement of the cell positioning accuracy for study of slow moving cells. The benefit of low flow speed has been demonstrated also by reduced blurring in the diffraction images of single cells. Based on these results, we concluded that the new designs of chamber and sheath nozzle produce stable hydrodynamic focusing of the core fluid at low speed and allow detailed study of cellular morphology under various rheological conditions using the diffraction imaging method. © 2013 International Society for Advancement of Cytometry.

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.

Single-Particle Discrimination of Retroviruses from Extracellular Vesicles by Nanoscale Flow Cytometry.

PubMed

Tang, Vera A; Renner, Tyler M; Fritzsche, Anna K; Burger, Dylan; Langlois, Marc-André

2017-12-19

Retroviruses and small EVs overlap in size, buoyant densities, refractive indices and share many cell-derived surface markers making them virtually indistinguishable by standard biochemical methods. This poses a significant challenge when purifying retroviruses for downstream analyses or for phenotypic characterization studies of markers on individual virions given that EVs are a major contaminant of retroviral preparations. Nanoscale flow cytometry (NFC), also called flow virometry, is an adaptation of flow cytometry technology for the analysis of individual nanoparticles such as extracellular vesicles (EVs) and retroviruses. In this study we systematically optimized NFC parameters for the detection of retroviral particles in the range of 115-130 nm, including viral production, sample labeling, laser power and voltage settings. By using the retroviral envelope glycoprotein as a selection marker, and evaluating a number of fluorescent dyes and labeling methods, we demonstrate that it is possible to confidently distinguish retroviruses from small EVs by NFC. Our findings make it now possible to individually phenotype genetically modified retroviral particles that express a fluorescent envelope glycoprotein without removing EV contaminants from the sample.

Cellular vacuolation and mitochondrial-associated factors induced by Clostridium perfringens epsilon toxin detected using acoustic flow cytometry.

PubMed

Ferrarezi, Marina C; Curci, Vera C L M; Cardoso, Tereza C

2013-12-01

Epsilon toxin (ETX) produced by Clostridium perfringens types B and D is a potent toxin that is responsible for fatal enterotoxaemia. In vitro, ETX, which is considered as a pore-forming toxin, forms a heptamer in Madin-Darby canine kidney (MDCK) cell membranes, which is considered to be a pre-pore stage. After binding of the ETX, vacuoles inside cell cytoplasm are produced. ETX causes decreased levels of essential coenzymes required for host cell energy. Here, we optimized and applied acoustic flow cytometry analysis in order to gain further insight into ETX-pathogenesis. Using acoustic flow cytometer analysis, which considered highly sensitive, ETX-exposed MDCK cells revealed mitochondrial membrane decreases followed by 25.48% and 45.45% of the exposed cells expressing the Bax and BCL-2 proteins at a pre-pore stage, respectively. These results together with high cytotoxicity and visualization of cell vacuoles, demonstrates that acoustic flow cytometry analysis potentially represents an effective tool to study ETX pathogenesis. Copyright © 2013. Published by Elsevier Ltd.

A general method for bead-enhanced quantitation by flow cytometry

PubMed Central

Montes, Martin; Jaensson, Elin A.; Orozco, Aaron F.; Lewis, Dorothy E.; Corry, David B.

2009-01-01

Flow cytometry provides accurate relative cellular quantitation (percent abundance) of cells from diverse samples, but technical limitations of most flow cytometers preclude accurate absolute quantitation. Several quantitation standards are now commercially available which, when added to samples, permit absolute quantitation of CD4+ T cells. However, these reagents are limited by their cost, technical complexity, requirement for additional software and/or limited applicability. Moreover, few studies have validated the use of such reagents in complex biological samples, especially for quantitation of non-T cells. Here we show that addition to samples of known quantities of polystyrene fluorescence standardization beads permits accurate quantitation of CD4+ T cells from complex cell samples. This procedure, here termed single bead-enhanced cytofluorimetry (SBEC), was equally capable of enumerating eosinophils as well as subcellular fragments of apoptotic cells, moieties with very different optical and fluorescent characteristics. Relative to other proprietary products, SBEC is simple, inexpensive and requires no special software, suggesting that the method is suitable for the routine quantitation of most cells and other particles by flow cytometry. PMID:17067632

Novel Quantitative Autophagy Analysis by Organelle Flow Cytometry after Cell Sonication

PubMed Central

Degtyarev, Michael; Reichelt, Mike; Lin, Kui

2014-01-01

Autophagy is a dynamic process of bulk degradation of cellular proteins and organelles in lysosomes. Current methods of autophagy measurement include microscopy-based counting of autophagic vacuoles (AVs) in cells. We have developed a novel method to quantitatively analyze individual AVs using flow cytometry. This method, OFACS (organelle flow after cell sonication), takes advantage of efficient cell disruption with a brief sonication, generating cell homogenates with fluorescently labeled AVs that retain their integrity as confirmed with light and electron microscopy analysis. These AVs could be detected directly in the sonicated cell homogenates on a flow cytometer as a distinct population of expected organelle size on a cytometry plot. Treatment of cells with inhibitors of autophagic flux, such as chloroquine or lysosomal protease inhibitors, increased the number of particles in this population under autophagy inducing conditions, while inhibition of autophagy induction with 3-methyladenine or knockdown of ATG proteins prevented this accumulation. This assay can be easily performed in a high-throughput format and opens up previously unexplored avenues for autophagy analysis. PMID:24489953

Distinction between asymptomatic monoclonal B-cell lymphocytosis with cyclin D1 overexpression and mantle cell lymphoma: from molecular profiling to flow cytometry.

PubMed

Espinet, Blanca; Ferrer, Ana; Bellosillo, Beatriz; Nonell, Lara; Salar, Antonio; Fernández-Rodríguez, Concepción; Puigdecanet, Eulàlia; Gimeno, Javier; Garcia-Garcia, Mar; Vela, Maria Carmen; Luño, Elisa; Collado, Rosa; Navarro, José Tomás; de la Banda, Esmeralda; Abrisqueta, Pau; Arenillas, Leonor; Serrano, Cristina; Lloreta, Josep; Miñana, Belén; Cerutti, Andrea; Florensa, Lourdes; Orfao, Alberto; Sanz, Ferran; Solé, Francesc; Dominguez-Sola, David; Serrano, Sergio

2014-02-15

According to current diagnostic criteria, mantle cell lymphoma (MCL) encompasses the usual, aggressive variants and rare, nonnodal cases with monoclonal asymptomatic lymphocytosis, cyclin D1-positive (MALD1). We aimed to understand the biology behind this clinical heterogeneity and to identify markers for adequate identification of MALD1 cases. We compared 17 typical MCL cases with a homogeneous group of 13 untreated MALD1 cases (median follow-up, 71 months). We conducted gene expression profiling with functional analysis in five MCL and five MALD1. Results were validated in 12 MCL and 8 MALD1 additional cases by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and in 24 MCL and 13 MALD1 cases by flow cytometry. Classification and regression trees strategy was used to generate an algorithm based on CD38 and CD200 expression by flow cytometry. We found 171 differentially expressed genes with enrichment of neoplastic behavior and cell proliferation signatures in MCL. Conversely, MALD1 was enriched in gene sets related to immune activation and inflammatory responses. CD38 and CD200 were differentially expressed between MCL and MALD1 and confirmed by flow cytometry (median CD38, 89% vs. 14%; median CD200, 0% vs. 24%, respectively). Assessment of both proteins allowed classifying 85% (11 of 13) of MALD1 cases whereas 15% remained unclassified. SOX11 expression by qRT-PCR was significantly different between MCL and MALD1 groups but did not improve the classification. We show for the first time that MALD1, in contrast to MCL, is characterized by immune activation and driven by inflammatory cues. Assessment of CD38/CD200 by flow cytometry is useful to distinguish most cases of MALD1 from MCL in the clinical setting. MALD1 should be identified and segregated from the current MCL category to avoid overdiagnosis and unnecessary treatment. ©2013 AACR

Distinction between Asymptomatic Monoclonal B-cell Lymphocytosis with Cyclin D1 Overexpression and Mantle Cell Lymphoma: From Molecular Profiling to Flow Cytometry

PubMed Central

Espinet, Blanca; Ferrer, Ana; Bellosillo, Beatriz; Nonell, Lara; Salar, Antonio; Fernández-Rodríguez, Concepción; Puigdecanet, Eulàlia; Gimeno, Javier; Garcia-Garcia, Mar; Carmen Vela, Maria; Luño, Elisa; Collado, Rosa; Navarro, José Tomás; de la Banda, Esmeralda; Abrisqueta, Pau; Arenillas, Leonor; Serrano, Cristina; Lloreta, Josep; Miñana, Belén; Cerutti, Andrea; Florensa, Lourdes; Orfao, Alberto; Sanz, Ferran; Solé, Francesc; Dominguez-Sola, David; Serrano, Sergio

2015-01-01

Purpose According to current diagnostic criteria, mantle cell lymphoma (MCL) encompasses the usual, aggressive variants and rare, nonnodal cases with monoclonal asymptomatic lymphocytosis, cyclin D1–positive (MALD1). We aimed to understand the biology behind this clinical heterogeneity and to identify markers for adequate identification of MALD1 cases. Experimental Design We compared 17 typical MCL cases with a homogeneous group of 13 untreated MALD1 cases (median follow-up, 71 months). We conducted gene expression profiling with functional analysis in five MCL and five MALD1. Results were validated in 12 MCL and 8 MALD1 additional cases by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and in 24 MCL and 13 MALD1 cases by flow cytometry. Classification and regression trees strategy was used to generate an algorithm based on CD38 and CD200 expression by flow cytometry. Results We found 171 differentially expressed genes with enrichment of neoplastic behavior and cell proliferation signatures in MCL. Conversely, MALD1 was enriched in gene sets related to immune activation and inflammatory responses. CD38 and CD200 were differentially expressed between MCL and MALD1 and confirmed by flow cytometry (median CD38, 89% vs. 14%; median CD200, 0% vs. 24%, respectively). Assessment of both proteins allowed classifying 85% (11 of 13) of MALD1 cases whereas 15% remained unclassified. SOX11 expression by qRT-PCR was significantly different between MCL and MALD1 groups but did not improve the classification. Conclusion We show for the first time that MALD1, in contrast to MCL, is characterized by immune activation and driven by inflammatory cues. Assessment of CD38/CD200 by flow cytometry is useful to distinguish most cases of MALD1 from MCL in the clinical setting. MALD1 should be identified and segregated from the current MCL category to avoid overdiagnosis and unnecessary treatment. PMID:24352646

Novel Peptide Sequence (“IQ-tag”) with High Affinity for NIR Fluorochromes Allows Protein and Cell Specific Labeling for In Vivo Imaging

PubMed Central

McCarthy, Jason R.; Weissleder, Ralph

2007-01-01

Background Probes that allow site-specific protein labeling have become critical tools for visualizing biological processes. Methods Here we used phage display to identify a novel peptide sequence with nanomolar affinity for near infrared (NIR) (benz)indolium fluorochromes. The developed peptide sequence (“IQ-tag”) allows detection of NIR dyes in a wide range of assays including ELISA, flow cytometry, high throughput screens, microscopy, and optical in vivo imaging. Significance The described method is expected to have broad utility in numerous applications, namely site-specific protein imaging, target identification, cell tracking, and drug development. PMID:17653285

Aptamer-Functionalized Fluorescent Silica Nanoparticles for Highly Sensitive Detection of Leukemia Cells

NASA Astrophysics Data System (ADS)

Tan, Juntao; Yang, Nuo; Hu, Zixi; Su, Jing; Zhong, Jianhong; Yang, Yang; Yu, Yating; Zhu, Jianmeng; Xue, Dabin; Huang, Yingying; Lai, Zongqiang; Huang, Yong; Lu, Xiaoling; Zhao, Yongxiang

2016-06-01

A simple, highly sensitive method to detect leukemia cells has been developed based on aptamer-modified fluorescent silica nanoparticles (FSNPs). In this strategy, the amine-labeled Sgc8 aptamer was conjugated to carboxyl-modified FSNPs via amide coupling between amino and carboxyl groups. Sensitivity and specificity of Sgc8-FSNPs were assessed using flow cytometry and fluorescence microscopy. These results showed that Sgc8-FSNPs detected leukemia cells with high sensitivity and specificity. Aptamer-modified FSNPs hold promise for sensitive and specific detection of leukemia cells. Changing the aptamer may allow the FSNPs to detect other types of cancer cells.

Development and validation of receptor occupancy pharmacodynamic assays used in the clinical development of the monoclonal antibody vedolizumab.

PubMed

Wyant, Tim; Estevam, Jose; Yang, Lili; Rosario, Maria

2016-03-01

Vedolizumab is a monoclonal antibody approved for use in ulcerative colitis and Crohn's disease. By specifically binding to α4 β7 integrin, vedolizumab prevents trafficking of lymphocytes to the gut, thereby interfering with disease pathology. During the clinical development program, the pharmacodynamic effect of vedolizumab was evaluated by 2 flow cytometry receptor occupancy assays: act-1 (ACT-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Here we describe the development and validation of these assays. The ACT-1 assay is a receptor occupancy free-site assay that uses a monoclonal antibody with the same binding epitope as vedolizumab to detect free (unbound) sites on α4 β7 integrin. The MAdCAM-1 assay used a soluble version of the natural ligand for α4 β7 integrin to detect free sites. The assays were validated using a fit-for-purpose approach throughout the clinical development of vedolizumab. Both the ACT-1 assay and the MAdCAM-1 assay demonstrated acceptable reproducibility and repeatability. The assays were sufficiently stable to allow for clinical use. During clinical testing the assays demonstrated that vedolizumab was able to saturate peripheral cells at all doses tested. Two pharmacodynamic receptor occupancy assays were developed and validated to assess the effect of vedolizumab on peripheral blood cells. The results of these assays demonstrated the practical use of flow cytometry to examine pharmacodynamic response in clinical trials. © 2015 International Clinical Cytometry Society.

Toward low-cost affinity reagents: lyophilized yeast-scFv probes specific for pathogen antigens.

PubMed

Gray, Sean A; Weigel, Kris M; Ali, Ibne K M; Lakey, Annie A; Capalungan, Jeremy; Domingo, Gonzalo J; Cangelosi, Gerard A

2012-01-01

The generation of affinity reagents, usually monoclonal antibodies, remains a critical bottleneck in biomedical research and diagnostic test development. Recombinant antibody-like proteins such as scFv have yet to replace traditional monoclonal antibodies in antigen detection applications, in large part because of poor performance of scFv in solution. To address this limitation, we have developed assays that use whole yeast cells expressing scFv on their surfaces (yeast-scFv) in place of soluble purified scFv or traditional monoclonal antibodies. In this study, a nonimmune library of human scFv displayed on the surfaces of yeast cells was screened for clones that bind to recombinant cyst proteins of Entamoeba histolytica, an enteric pathogen of humans. Selected yeast-scFv clones were stabilized by lyophilization and used in detection assay formats in which the yeast-scFv served as solid support-bound monoclonal antibodies. Specific binding of antigen to the yeast-scFv was detected by staining with rabbit polyclonal antibodies. In flow cytometry-based assays, lyophilized yeast-scFv reagents retained full binding activity and specificity for their cognate antigens after 4 weeks of storage at room temperature in the absence of desiccants or stabilizers. Because flow cytometry is not available to all potential assay users, an immunofluorescence assay was also developed that detects antigen with similar sensitivity and specificity. Antigen-specific whole-cell yeast-scFv reagents can be selected from nonimmune libraries in 2-3 weeks, produced in vast quantities, and packaged in lyophilized form for extended shelf life. Lyophilized yeast-scFv show promise as low cost, renewable alternatives to monoclonal antibodies for diagnosis and research.

A CLIPS expert system for clinical flow cytometry data analysis

NASA Technical Reports Server (NTRS)

Salzman, G. C.; Duque, R. E.; Braylan, R. C.; Stewart, C. C.

1990-01-01

An expert system is being developed using CLIPS to assist clinicians in the analysis of multivariate flow cytometry data from cancer patients. Cluster analysis is used to find subpopulations representing various cell types in multiple datasets each consisting of four to five measurements on each of 5000 cells. CLIPS facts are derived from results of the clustering. CLIPS rules are based on the expertise of Drs. Stewart, Duque, and Braylan. The rules incorporate certainty factors based on case histories.

Microfabrication and Test of a Three-Dimensional Polymer Hydro-focusing Unit for Flow Cytometry Applications

NASA Technical Reports Server (NTRS)

Yang, Ren; Feeback, Daniel L.; Wang, Wanjun

2004-01-01

This paper details a novel three-dimensional (3D) hydro-focusing micro cell sorter for micro flow cytometry applications. The unit was microfabricated by means of SU-8 3D lithography. The 3D microstructure for coaxial sheathing was designed, microfabricated, and tested. Three-dimensional hydro-focusing capability was demonstrated with an experiment to sort labeled tanned sheep erythrocytes (red blood cells). This polymer hydro-focusing microstructure is easily microfabricated and integrated with other polymer microfluidic structures.

Microfabrication and Test of a Three-Dimensional Polymer Hydro-Focusing Unit for Flow Cytometry Applications

NASA Technical Reports Server (NTRS)

Yang, Ren; Feedback, Daniel L.; Wang, Wanjun

2004-01-01

This paper details a novel three-dimensional (3D) hydro-focusing micro cell sorter for micro flow cytometry applications. The unit was micro-fabricated by means of SU-8 3D lithography. The 3D microstructure for coaxial sheathing was designed, micro-fabricated, and tested. Three-dimensional hydrofocusing capability was demonstrated with an experiment to sort labeled tanned sheep erythrocytes (red blood cells). This polymer hydro-focusing microstructure is easily micro-fabricated and integrated with other polymer microfluidic structures.

Multiparameter Flow Cytometry For Clinical Applications

NASA Astrophysics Data System (ADS)

Stewart, Carleton C.

1989-06-01

Flow Cytometry facilities are well established and provide immunophenotyping and DNA content measurement services. The application of immunophenotyping has been primarily in monitoring therapy and in providing further information to aid in the definitive diagnosis of immunological and neoplastic disease such as: immunodeficiency disease, auto immune disease, organ transplantation, and leukemia and lymphoma. DNA content measurements have been particularly important in determining the fraction of cycling cells and presence of aneuploid cells in neoplasia. This information has been useful in the management of patients with solid tumors.

Application of the flow cytometry for determination of the amount of DNA in Yersinia pestis cells under the influence of serotonin (5-hydroxytryptamine)

NASA Astrophysics Data System (ADS)

Korsukov, Vladimir N.; Shchukovskaya, Tatyana N.; Kravtsov, Alexander L.; Popov, Youri A.

2002-07-01

Using flow cytometry a low DNA content in inoculated Yersinia pestis EV cells have been shown at the beginning of culture in Hottinger broth pH 7.2. The dependence serotonin action of its concentration on DNA content have been demonstrated. Serotonin accelerated Yersinia pestis culture growth during cultivation in Hottinger broth pH 7.2 both at 28 degrees C and 37 degrees C at concentration of 10-5 M.

A novel flow cytometry-based method of analyzing Heinz bodies.

PubMed

Palasuwan, D; Palasuwan, A; Charoensappakit, A; Noulsri, E

2017-02-01

Heinz bodies are important to diagnosing and managing patients. However, microscopic examination of Heinz bodies has several disadvantages, demonstrating the need for a better method. We explored the potential use of flow cytometry to examine Heinz bodies. Whole-blood samples were collected from patients deficient in G6PD and healthy volunteers. Acetylphenylhydrazine was used to induce formation of Heinz bodies in red blood cells (RBCs). Then, RBCs positive for Heinz bodies were examined using a FACSCanto II cytometer. RBCs treated with acetylphenylhydrazine formed Heinz bodies and emitted a broad spectrum of fluorescence that could be detected by flow cytometry. The maximum emission of fluorescence was observed at 45 min after the incubation with acetylphenylhydrazine. In addition, the fluorescence emitted was stable for at least 72 h. The flow cytometer could detect the RBCs positive for Heinz bodies even if they made up as little as 0.1% of the total RBC population. Furthermore, the percentage and number, respectively, of RBCs positive for Heinz bodies in G6PD-deficient patients and normal donors exhibited a mean ± standard deviation (SD) of 68.9 ± 27.5 vs. 50.9 ± 28.6 and 96 014 ±35 732 cells/μL vs. 74 688 ± 36 514 cells/μL. Heinz bodies induced by acetylphenylhydrazine emit fluorescence, and this fluorescence could be examined using flow cytometry. Our study suggests the potential use of the developed method to investigate the formation of Heinz bodies in clinical samples. © 2016 John Wiley & Sons Ltd.

Coexpression of multidrug resistance involve proteins: a flow cytometric analysis.

PubMed

Boutonnat, J; Bonnefoix, T; Mousseau, M; Seigneurin, D; Ronot, X

1998-01-01

Cross resistance to multiple natural cytotoxic products represents a major obstacle in myeloblastic acute leukaemia (AML). Multidrug resistance (MDR) often involves overexpression of plasma membrane drug transporter P-glycoprotein (PGP) or the resistance associated protein (MRP). Recently, a protein overexpressed in a non-PGP MDR lung cancer cell line and termed lung resistance related protein (LRP) was identified. These proteins are known to be associated with a bad prognosis in AML. We have developed a triple indirect labelling analysed by flow cytometry to detect the coexpression of these proteins. Since no cell line expressing all three antigens is known, we mixed K562 cells (resistant to Adriblastine, PGP+, MRP-, LRP-) with GLC4 cells (resistant to Adriblastine, PGP-, MRP+, LRP+) to create a model system to test the method. The antibodies used were UIC2 for PGP, MRPm6 for MRP and LRP56 for LRP. They were revealed by Fab'2 coupled with Fluoresceine-isothiocyanate, Phycoerythrin or Tricolor with isotype specificity. Cells were fixed and permeabilized after PGP labelling because MRPm6 and LRP56 recognize intracellular epitopes. PGP and LRP were easily detected. MRP is expressed at relatively low levels and was more difficult to detect because in the triple labelling the non specific staining was higher than in a single labelling. Despite the increased background in the triple labelling we were able to detect coexpression of PGP, MRP, LRP by flow cytometry. This method appears to be very useful to detect coexpression of markers in AML. Such coexpression could modify the therapeutic approach with revertants.

Flow cytofluorometric assay of human whole blood leukocyte DNA degradation in response to Yersinia pestis and Staphylococcus aureus

NASA Astrophysics Data System (ADS)

Kravtsov, Alexander L.; Grebenyukova, Tatyana P.; Bobyleva, Elena V.; Golovko, Elena M.; Malyukova, Tatyana A.; Lyapin, Mikhail N.; Kostyukova, Tatyana A.; Yezhov, Igor N.; Kuznetsov, Oleg S.

2001-05-01

Human leukocytes containing less than 2C DNA per cell (damaged or dead cells) were detected and quantified by flow cytometry and DNA-specific staining with ethidium bromide and mithramycin in whole blood infected with Staphylococcus aureus or Yersinia pestis. Addition of live S. aureus to the blood (100 microbe cells per one leukocyte) resulted in rapid degradation of leukocyte DNA within 3 to 6 hours of incubation at 37 degree(s)C. However, only about 50 percent cells were damaged and the leukocytes with the intact genetic apparatus could be found in the blood for a period up to 24 hours. The leukocyte injury was preceded by an increase of DNA per cell content (as compared to the normal one) that was likely to be connected with the active phagocytosis of S. aureus by granulocytes (2C DNA of diploid phagocytes plus the all bacterial DNA absorbed). In response to the same dose of actively growing (at 37 degree(s)C) virulent Y. pestis cells, no increase in DNA content per cell could be observed in the human blood leukocytes. The process of the leukocyte DNA degradation started after a 6-hour incubation, and between 18 to 24 hours of incubation about 90 percent leukocytes (phagocytes and lymphocytes) lost their specific DNA fluorescence. These results demonstrated a high potential of flow cytometry in comparative analysis in vitro of the leukocyte DNA degradation process in human blood in response to bacteria with various pathogenic properties. They agree with the modern idea of an apoptotic mechanism of immunosuppression in plague.

Hyperspectral cytometry.

PubMed

Grégori, Gérald; Rajwa, Bartek; Patsekin, Valery; Jones, James; Furuki, Motohiro; Yamamoto, Masanobu; Paul Robinson, J

2014-01-01

Hyperspectral cytometry is an emerging technology for single-cell analysis that combines ultrafast optical spectroscopy and flow cytometry. Spectral cytometry systems utilize diffraction gratings or prism-based monochromators to disperse fluorescence signals from multiple labels (organic dyes, nanoparticles, or fluorescent proteins) present in each analyzed bioparticle onto linear detector arrays such as multianode photomultipliers or charge-coupled device sensors. The resultant data, consisting of a series of characterizing every analyzed cell, are not compensated by employing the traditional cytometry approach, but rather are spectrally unmixed utilizing algorithms such as constrained Poisson regression or non-negative matrix factorization. Although implementations of spectral cytometry were envisioned as early as the 1980s, only recently has the development of highly sensitive photomultiplier tube arrays led to design and construction of functional prototypes and subsequently to introduction of commercially available systems. This chapter summarizes the historical efforts and work in the field of spectral cytometry performed at Purdue University Cytometry Laboratories and describes the technology developed by Sony Corporation that resulted in release of the first commercial spectral cytometry system-the Sony SP6800. A brief introduction to spectral data analysis is also provided, with emphasis on the differences between traditional polychromatic and spectral cytometry approaches.

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.

Novel ligands for cancer diagnosis: selection of peptide ligands for identification and isolation of B-cell lymphomas.

PubMed

McGuire, Michael J; Samli, Kausar N; Chang, Ya-Ching; Brown, Kathlynn C

2006-04-01

Lymphoma and leukemia account for nearly 8% of cancer fatalities each year. Present treatments do not differentiate between normal and malignant cells. New reagents that distinguish malignant cells and enable the isolation of these cells from the normal background will enhance the molecular characterization of disease and specificity of treatment. Peptide ligands were selected from a phage-displayed peptide library by biopanning on the B-cell lymphoma line, A20. The isolated peptides were assessed as reagents for identification and isolation of lymphoma cells by flow cytometry and cell capture with magnetic beads. Two novel peptides and one obtained previously on cardiomyocytes were selected. A20 cells bind phage displaying these peptides 250- to 450-fold over control phage. These phage bind to other bone marrow-derived cancel lines including some macrophage and T cells but do not bind to normal splenocytes. Synthetic constructs of these peptides have binding affinities comparable to B-cell-specific antibodies. Similar to antibodies, these peptides can be used in flow cytometry and magnetic bead capture to distinguish lymphoma cells from normal splenocytes. Bone marrow-derived malignant cells express cell surface markers that can be used to distinguish them from normal cells. These results demonstrate the ability to use an unbiased screen to rapidly generate high-affinity peptide ligands for identification and isolation of lymphoma cells.

Novel image cytometric method for detection of physiological and metabolic changes in Saccharomyces cerevisiae.

PubMed

Chan, Leo L; Kury, Alexandria; Wilkinson, Alisha; Berkes, Charlotte; Pirani, Alnoor

2012-11-01

The studying and monitoring of physiological and metabolic changes in Saccharomyces cerevisiae (S. cerevisiae) has been a key research area for the brewing, baking, and biofuels industries, which rely on these economically important yeasts to produce their products. Specifically for breweries, physiological and metabolic parameters such as viability, vitality, glycogen, neutral lipid, and trehalose content can be measured to better understand the status of S. cerevisiae during fermentation. Traditionally, these physiological and metabolic changes can be qualitatively observed using fluorescence microscopy or flow cytometry for quantitative fluorescence analysis of fluorescently labeled cellular components associated with each parameter. However, both methods pose known challenges to the end-users. Specifically, conventional fluorescent microscopes lack automation and fluorescence analysis capabilities to quantitatively analyze large numbers of cells. Although flow cytometry is suitable for quantitative analysis of tens of thousands of fluorescently labeled cells, the instruments require a considerable amount of maintenance, highly trained technicians, and the system is relatively expensive to both purchase and maintain. In this work, we demonstrate the first use of Cellometer Vision for the kinetic detection and analysis of vitality, glycogen, neutral lipid, and trehalose content of S. cerevisiae. This method provides an important research tool for large and small breweries to study and monitor these physiological behaviors during production, which can improve fermentation conditions to produce consistent and higher-quality products.

In Vivo Biochemistry: Single-Cell Dynamics of Cyclic Di-GMP in Escherichia coli in Response to Zinc Overload.

PubMed

Yeo, Jongchan; Dippel, Andrew B; Wang, Xin C; Hammond, Ming C

2018-01-09

Intracellular signaling enzymes drive critical changes in cellular physiology and gene expression, but their endogenous activities in vivo remain highly challenging to study in real time and for individual cells. Here we show that flow cytometry can be performed in complex media to monitor single-cell population distributions and dynamics of cyclic di-GMP signaling, which controls the bacterial colonization program. These in vivo biochemistry experiments are enabled by our second-generation RNA-based fluorescent (RBF) biosensors, which exhibit high fluorescence turn-on in response to cyclic di-GMP. Specifically, we demonstrate that intracellular levels of cyclic di-GMP in Escherichia coli are repressed with excess zinc, but not with other divalent metals. Furthermore, in both flow cytometry and fluorescence microscopy setups, we monitor the dynamic increase in cellular cyclic di-GMP levels upon zinc depletion and show that this response is due to de-repression of the endogenous diguanylate cyclase DgcZ. In the presence of zinc, cells exhibit enhanced cell motility and increased sensitivity to antibiotics due to inhibited biofilm formation. Taken together, these results showcase the application of RBF biosensors in visualizing single-cell dynamic changes in cyclic di-GMP signaling in direct response to environmental cues such as zinc and highlight our ability to assess whether observed phenotypes are related to specific signaling enzymes and pathways.

Anti-tumor effects and apoptosis induction by Realgar bioleaching solution in Sarcoma-180 cells in vitro and transplanted tumors in mice in vivo.

PubMed

Xie, Qin-Jian; Cao, Xin-Li; Bai, Lu; Wu, Zheng-Rong; Ma, Ying-Ping; Li, Hong-Yu

2014-01-01

Realgar which contains arsenic components has been used in traditional Chinese medicine (TCM) as an anticancer drug. However, neither Realgar nor its formula are soluble in water. As a result, high dose of Realgar has to be administered to achieve an effective blood medicine concentration, and this is associated with adverse side effects. The objective of the present study was to increase the solubility of a formula using hydrometallurgy technology as well as investigating its effects on in vitro and in vivo cell proliferation and apoptosis in Sarcoma-180 cell line. Antiproliferative activity of Realgar Bioleaching Solution (RBS) was evaluated by MTT assay. Further, effects of RBS on cell proliferation and apoptosis were studied using flow cytometry and transmission electron microscopy. Kunming mice were administered RBS in vivo, where arsenic specifically targeted solid tumors. The results indicated that RBS extract potently inhibited the tumor growth of Sarcoma-180 cell line in a dose-dependent manner. Flow cytometry and transmission electron microscopy further indicated that RBS significantly induced cell apoptosis through the inhibition of cell cycle pathway in a dose-dependent manner. Further, on RBS administration to mice, arsenic was specifically targeted to solid tumors RBS could substitute for traditional Realgar or its formula to work as a potent tool in cancer treatment.

A simple method for purification of vestibular hair cells and non-sensory cells, and application for proteomic analysis.

PubMed

Herget, Meike; Scheibinger, Mirko; Guo, Zhaohua; Jan, Taha A; Adams, Christopher M; Cheng, Alan G; Heller, Stefan

2013-01-01

Mechanosensitive hair cells and supporting cells comprise the sensory epithelia of the inner ear. The paucity of both cell types has hampered molecular and cell biological studies, which often require large quantities of purified cells. Here, we report a strategy allowing the enrichment of relatively pure populations of vestibular hair cells and non-sensory cells including supporting cells. We utilized specific uptake of fluorescent styryl dyes for labeling of hair cells. Enzymatic isolation and flow cytometry was used to generate pure populations of sensory hair cells and non-sensory cells. We applied mass spectrometry to perform a qualitative high-resolution analysis of the proteomic makeup of both the hair cell and non-sensory cell populations. Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level. Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells. Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins. Our results demonstrate that enzymatic dissociation of styryl dye-labeled sensory hair cells and non-sensory cells is a valid method to generate pure enough cell populations for flow cytometry and subsequent molecular analyses.

Establishment of CMab-43, a Sensitive and Specific Anti-CD133 Monoclonal Antibody, for Immunohistochemistry.

PubMed

Itai, Shunsuke; Fujii, Yuki; Nakamura, Takuro; Chang, Yao-Wen; Yanaka, Miyuki; Saidoh, Noriko; Handa, Saori; Suzuki, Hiroyoshi; Harada, Hiroyuki; Yamada, Shinji; Kaneko, Mika K; Kato, Yukinari

2017-10-01

CD133, also known as prominin-1, was first described as a cell surface marker on early progenitor and hematopoietic stem cells. It is a five-domain transmembrane protein composed of an N-terminal extracellular tail, two small cytoplasmic loops, two large extracellular loops containing seven potential glycosylation sites, and a short C-terminal intracellular tail. CD133 has been used as a marker to identify cancer stem cells derived from primary solid tumors and as a prognostic marker of gliomas. Herein, we developed novel anti-CD133 monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. We expressed the full length of CD133 in LN229 glioblastoma cells, immunized mice with LN229/CD133 cells, and performed the first screening using flow cytometry. After limiting dilution, we established 100 anti-CD133 mAbs, reacting with LN229/CD133 cells but not with LN229 cells. Subsequently, we performed the second and third screening with Western blot and immunohistochemical analyses, respectively. Among 100 mAbs, 11 strongly reacted with CD133 in Western blot analysis. One of 11 clones, CMab-43 (IgG 2a , kappa), showed a sensitive and specific reaction against colon cancer cells, warranting the use of CMab-43 in detecting CD133 in pathological analyses of CD133-expressing cancers.

ZAP-70 staining in chronic lymphocytic leukemia.

PubMed

Villamor, Neus

2005-05-01

Chronic lymphocytic leukemia (CLL) is the most common chronic leukemia in Western countries. The disease has an extremely variable clinical course, and several prognostic features have been identified to assess individual risk. The configuration of the immunoglobulin variable heavy-chain gene (IgV(H)) is a strong predictor of the outcome. CLL patients with unmutated IgV(H) status have an aggressive clinical course and a short survival. Unfortunately, analysis of IgV(H) gene configuration is not available in most clinical laboratories. A small number of genes are differentially expressed between unmutated IgV(H) and mutated IgV(H) clinical forms of CLL. One of these genes is ZAP-70, which is detected in leukemic cells from patients with the unmutated IgV(H) form of CLL. Flow cytometry presents advantages over other methods to detect ZAP-70, and its quantification by flow cytometry has proved its predictive value. This unit focuses on protocols to quantify ZAP-70 by flow cytometry in CLL.

Elucidation of the critical epitope of an anti-EGFR monoclonal antibody EMab-134.

PubMed

Kaneko, Mika K; Yamada, Shinji; Itai, Shunsuke; Chang, Yao-Wen; Nakamura, Takuro; Yanaka, Miyuki; Kato, Yukinari

2018-07-01

The epidermal growth factor receptor (EGFR) is a type-1 transmembrane receptor tyrosine kinase, which activates the downstream signaling cascades in many tumors, such as oral and lung cancers. We previously developed EMab-134, a novel anti-EGFR monoclonal antibody (mAb), which reacts with endogenous EGFR-expressing cancer cell lines and normal cells independent of glycosylation in Western blotting, flow cytometry, and immunohistochemical analysis. EMab-134 showed very high sensitivity (94.7%) to oral squamous cell carcinomas in immunohistochemical analysis. In this study, we performed enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemical analysis to determine the epitope of EMab-134. A blocking peptide (375-394 amino acids of EGFR) neutralized the EMab-134 reaction against oral cancer cells in flow cytometry and immunohistochemistry. The minimum epitope of EMab-134 was found to be the 377- RGDSFTHTPP -386 sequence. Our findings can be applied for the production of more functional anti-EGFR mAbs that in turn can be used for antitumor treatments.

Determination of critical epitope of PcMab-47 against human podocalyxin.

PubMed

Itai, Shunsuke; Yamada, Shinji; Kaneko, Mika K; Kato, Yukinari

2018-07-01

Podocalyxin (PODXL) is a type I transmembrane protein, which is highly glycosylated. PODXL is expressed in some types of human cancer tissues including oral, breast, and lung cancer tissues and may promote tumor growth, invasion, and metastasis. We previously produced PcMab-47, a novel anti-PODXL monoclonal antibody (mAb) which reacts with endogenous PODXL-expressing cancer cell lines and normal cells independently of glycosylation in Western blot, flow cytometry, and immunohistochemical analysis. In this study, we used enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemical analysis to determine the epitope of PcMab-47. The minimum epitope of PcMab-47 was found to be Asp207, His208, Leu209, and Met210. A blocking peptide containing this minimum epitope completely neutralized PcMab-47 reaction against oral cancer cells by flow cytometry and immunohistochemical analysis. These findings could lead to the production of more functional anti-PODXL mAbs, which are advantageous for antitumor activities.

Improved signal recovery for flow cytometry based on ‘spatially modulated emission’

NASA Astrophysics Data System (ADS)

Quint, S.; Wittek, J.; Spang, P.; Levanon, N.; Walther, T.; Baßler, M.

2017-09-01

Recently, the technique of ‘spatially modulated emission’ has been introduced (Baßler et al 2008 US Patent 0080181827A1; Kiesel et al 2009 Appl. Phys. Lett. 94 041107; Kiesel et al 2011 Cytometry A 79A 317-24) improving the signal-to-noise ratio (SNR) for detecting bio-particles in the field of flow cytometry. Based on this concept, we developed two advanced signal processing methods which further enhance the SNR and selectivity for cell detection. The improvements are achieved by adapting digital filtering methods from RADAR technology and mainly address inherent offset elimination, increased signal dynamics and moreover reduction of erroneous detections due to processing artifacts. We present a comprehensive theory on SNR gain and provide experimental results of our concepts.

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.

Capture of Fluorescence Decay Times by Flow Cytometry

PubMed Central

Naivar, Mark A.; Jenkins, Patrick; Freyer, James P.

2012-01-01

In flow cytometry, the fluorescence decay time of an excitable species has been largely underutilized and is not likely found as a standard parameter on any imaging cytometer, sorting, or analyzing system. Most cytometers lack fluorescence lifetime hardware mainly owing to two central issues. Foremost, research and development with lifetime techniques has lacked proper exploitation of modern laser systems, data acquisition boards, and signal processing techniques. Secondly, a lack of enthusiasm for fluorescence lifetime applications in cells and with bead-based assays has persisted among the greater cytometry community. In this unit, we describe new approaches that address these issues and demonstrate the simplicity of digitally acquiring fluorescence relaxation rates in flow. The unit is divided into protocol and commentary sections in order to provide a most comprehensive discourse on acquiring the fluorescence lifetime with frequency-domain methods. The unit covers (i) standard fluorescence lifetime acquisition (protocol-based) with frequency-modulated laser excitation, (ii) digital frequency-domain cytometry analyses, and (iii) interfacing fluorescence lifetime measurements onto sorting systems. Within the unit is also a discussion on how digital methods are used for aliasing in order to harness higher frequency ranges. Also, a final discussion is provided on heterodyning and processing of waveforms for multi-exponential decay extraction. PMID:25419263

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.

Flow cytometry immunophenotyping in integrated diagnostics of patients with newly diagnosed cytopenia: one tube 10-color 14-antibody screening panel and 3-tube extensive panel for detection of MDS-related features.

PubMed

Porwit, A; Rajab, A

2015-05-01

Acute leukemia, myelodysplastic syndromes (MDS), myeloproliferative neoplasms and lymphomas are the most prevalent diagnoses in adults presenting with new onset cytopenia. Here, we describe two 10-color panels of surface markers (screening and comprehensive panel) applied at the Flow Cytometry Laboratory, University Health Network, Toronto, ON, Canada. A 10-color flow cytometry is applied using the stain-lyse-wash sample preparation method. In patients with <10% blasts and no clear involvement by hematological malignancy based on cytomorphological evaluation of bone marrow (BM) smear, the recently published one-tube 10-color 14-antibody screening panel is applied. This panel allows detection of major B- and T-cell abnormalities, enumeration of cells in blast region (CD45 dim), and gives insight into myeloid BM compartment, including calculation of four-parameter score for MDS-related abnormalities. In patients who present with ≥10 - <20% blasts in blood or BM smears, a comprehensive three-tube panel of surface markers is used up front. The analysis is focused on the detection of abnormal antigen expression patterns not seen in normal/reactive BM, according to the guidelines developed by International/European LeukemiaNet Working Group for Flow Cytometry in MDS. In patients with ≥20% blasts, an additional tube is added to allow the detection of cytoplasmic markers necessary to diagnose mixed phenotype acute leukemia. © 2015 John Wiley & Sons Ltd.

EPIFLUORESCENCE MICROSCOPY AND SOLID PHASE CYTOMETRY AS CONFIRMATORY METHODS FOR THE ENUMERATION OF PROTOZOA BY FLOW CYTOMETRY

EPA Science Inventory

The detection of infective protozoan parasites contained in large volume environmental samples represents a unique challenge in environmental parasitology. Compounding this problem is the fact that infective stages of many protozoan parasites do not readily replicate in media or ...

Opto-fluidics based microscopy and flow cytometry on a cell phone for blood analysis.

PubMed

Zhu, Hongying; Ozcan, Aydogan

2015-01-01

Blood analysis is one of the most important clinical tests for medical diagnosis. Flow cytometry and optical microscopy are widely used techniques to perform blood analysis and therefore cost-effective translation of these technologies to resource limited settings is critical for various global health as well as telemedicine applications. In this chapter, we review our recent progress on the integration of imaging flow cytometry and fluorescent microscopy on a cell phone using compact, light-weight and cost-effective opto-fluidic attachments integrated onto the camera module of a smartphone. In our cell-phone based opto-fluidic imaging cytometry design, fluorescently labeled cells are delivered into the imaging area using a disposable micro-fluidic chip that is positioned above the existing camera unit of the cell phone. Battery powered light-emitting diodes (LEDs) are butt-coupled to the sides of this micro-fluidic chip without any lenses, which effectively acts as a multimode slab waveguide, where the excitation light is guided to excite the fluorescent targets within the micro-fluidic chip. Since the excitation light propagates perpendicular to the detection path, an inexpensive plastic absorption filter is able to reject most of the scattered light and create a decent dark-field background for fluorescent imaging. With this excitation geometry, the cell-phone camera can record fluorescent movies of the particles/cells as they are flowing through the microchannel. The digital frames of these fluorescent movies are then rapidly processed to quantify the count and the density of the labeled particles/cells within the solution under test. With a similar opto-fluidic design, we have recently demonstrated imaging and automated counting of stationary blood cells (e.g., labeled white blood cells or unlabeled red blood cells) loaded within a disposable cell counting chamber. We tested the performance of this cell-phone based imaging cytometry and blood analysis platform by measuring the density of red and white blood cells as well as hemoglobin concentration in human blood samples, which showed a good match to our measurement results obtained using a commercially available hematology analyzer. Such a cell-phone enabled opto-fluidics microscopy, flow cytometry, and blood analysis platform could be especially useful for various telemedicine applications in remote and resource-limited settings.

Quantifying CD4 receptor protein in two human CD4+ lymphocyte preparations for quantitative flow cytometry.

PubMed

Wang, Meiyao; Misakian, Martin; He, Hua-Jun; Bajcsy, Peter; Abbasi, Fatima; Davis, Jeffrey M; Cole, Kenneth D; Turko, Illarion V; Wang, Lili

2014-01-01

In our previous study that characterized different human CD4+ lymphocyte preparations, it was found that both commercially available cryopreserved peripheral blood mononuclear cells (PBMC) and a commercially available lyophilized PBMC (Cyto-Trol™) preparation fulfilled a set of criteria for serving as biological calibrators for quantitative flow cytometry. However, the biomarker CD4 protein expression level measured for T helper cells from Cyto-Trol was about 16% lower than those for cryopreserved PBMC and fresh whole blood using flow cytometry and mass cytometry. A primary reason was hypothesized to be due to steric interference in anti- CD4 antibody binding to the smaller sized lyophilized control cells. Targeted multiple reaction monitoring (MRM) mass spectrometry (MS) is used to quantify the copy number of CD4 receptor protein per CD4+ lymphocyte. Scanning electron microscopy (SEM) is utilized to assist searching the underlying reasons for the observed difference in CD4 receptor copy number per cell determined by MRM MS and CD4 expression measured previously by flow cytometry. The copy number of CD4 receptor proteins on the surface of the CD4+ lymphocyte in cryopreserved PBMCs and in lyophilized control cells is determined to be (1.45 ± 0.09) × 10(5) and (0.85 ± 0.11) × 10(5), respectively, averaged over four signature peptides using MRM MS. In comparison with cryopreserved PBMCs, there are more variations in the CD4 copy number in lyophilized control cells determined based on each signature peptide. SEM images of CD4+ lymphocytes from lyophilized control cells are very different when compared to the CD4+ T cells from whole blood and cryopreserved PBMC. Because of the lyophilization process applied to Cyto-Trol control cells, a lower CD4 density value, defined as the copy number of CD4 receptors per CD4+ lymphocyte, averaged over three different production lots is most likely explained by the loss of the CD4 receptors on damaged and/or broken microvilli where CD4 receptors reside. Steric hindrance of antibody binding and the association of CD4 receptors with other biomolecules likely contribute significantly to the nearly 50% lower CD4 receptor density value for cryopreserved PBMC determined from flow cytometry compared to the value obtained from MRM MS.

In vivo photoacoustic flow cytometry for early malaria diagnosis.

PubMed

Cai, Chengzhong; Carey, Kai A; Nedosekin, Dmitry A; Menyaev, Yulian A; Sarimollaoglu, Mustafa; Galanzha, Ekaterina I; Stumhofer, Jason S; Zharov, Vladimir P

2016-06-01

In vivo photoacoustic (PA) flow cytometry (PAFC) has already demonstrated a great potential for the diagnosis of deadly diseases through ultrasensitive detection of rare disease-associated circulating markers in whole blood volume. Here, we demonstrate the first application of this powerful technique for early diagnosis of malaria through label-free detection of malaria parasite-produced hemozoin in infected red blood cells (iRBCs) as high-contrast PA agent. The existing malaria tests using blood smears can detect the disease at 0.001-0.1% of parasitemia. On the contrary, linear PAFC showed a potential for noninvasive malaria diagnosis at an extremely low level of parasitemia of 0.0000001%, which is ∼10(3) times better than the existing tests. Multicolor time-of-flight PAFC with high-pulse repetition rate lasers at wavelengths of 532, 671, and 820 nm demonstrated rapid spectral and spatial identification and quantitative enumeration of individual iRBCs. Integration of PAFC with fluorescence flow cytometry (FFC) provided real-time simultaneous detection of single iRBCs and parasites expressing green fluorescence proteins, respectively. A combination of linear and nonlinear nanobubble-based multicolor PAFC showed capability to real-time control therapy efficiency by counting of iRBCs before, during, and after treatment. Our results suggest that high-sensitivity, high-resolution ultrafast PAFC-FFC platform represents a powerful research tool to provide the insight on malaria progression through dynamic study of parasite-cell interactions directly in bloodstream, whereas portable hand-worn PAFC device could be broadly used in humans for early malaria diagnosis. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

Photoacoustic-fluorescence in vitro flow cytometry for quantification of absorption, scattering and fluorescence properties of the cells

NASA Astrophysics Data System (ADS)

Nedosekin, D. A.; Sarimollaoglu, M.; Foster, S.; Galanzha, E. I.; Zharov, V. P.

2013-03-01

Fluorescence flow cytometry is a well-established analytical tool that provides quantification of multiple biological parameters of cells at molecular levels, including their functional states, morphology, composition, proliferation, and protein expression. However, only the fluorescence and scattering parameters of the cells or labels are available for detection. Cell pigmentation, presence of non-fluorescent dyes or nanoparticles cannot be reliably quantified. Herewith, we present a novel photoacoustic (PA) flow cytometry design for simple integration of absorbance measurements into schematics of conventional in vitro flow cytometers. The integrated system allow simultaneous measurements of light absorbance, scattering and of multicolor fluorescence from single cells in the flow at rates up to 2 m/s. We compared various combinations of excitation laser sources for multicolor detection, including simultaneous excitation of PA and fluorescence using a single 500 kHz pulsed nanosecond laser. Multichannel detection scheme allows simultaneous detection of up to 8 labels, including 4 fluorescent tags and 4 PA colors. In vitro PA-fluorescence flow cytometer was used for studies of nanoparticles uptake and for the analysis of cell line pigmentation, including genetically encoded melanin expression in breast cancer cell line. We demonstrate that this system can be used for direct nanotoxicity studies with simultaneous quantification of nanoparticles content and assessment of cell viability using a conventional fluorescent apoptosis assays.

Quantification of proteins by flow cytometry: Quantification of human hepatic transporter P-gp and OATP1B1 using flow cytometry and mass spectrometry.

PubMed

Hogg, Karen; Thomas, Jerry; Ashford, David; Cartwright, Jared; Coldwell, Ruth; Weston, Daniel J; Pillmoor, John; Surry, Dominic; O'Toole, Peter

2015-07-01

Flow cytometry is a powerful tool for the quantitation of fluorescence and is proven to be able to correlate the fluorescence intensity to the number of protein on cells surface. Mass spectroscopy can also be used to determine the number of proteins per cell. Here we have developed two methods, using flow cytometry and mass spectroscopy to quantify number of transporters in human cells. These two approaches were then used to analyse the same samples so that a direct comparison could be made. Transporters have a major impact on the behaviour of a diverse number of drugs in human systems. While active uptake studies by transmembrane protein transporters using model substrates are routinely undertaken in human cell lines and hepatocytes as part of drug discovery and development, the interpretation of these results is currently limited by the inability to quantify the number of transporters present in the test samples. Here we provide a flow cytometric method for accurate quantification of transporter levels both on the cell surface and within the cell, and compare this to a quantitative mass spectrometric approach. Two transporters were selected for the study: OATP1B1 (also known as SLCO1B1, LST-1, OATP-C, OATP2) due to its important role in hepatic drug uptake and elimination; P-gp (also known as P-glycoprotein, MDR1, ABCB1) as a well characterised system and due to its potential impact on oral bioavailability, biliary and renal clearance, and brain penetration of drugs that are substrates for this transporter. In all cases the mass spectrometric method gave higher levels than the flow cytometry method. However, the two methods showed very similar trends in the relative ratios of both transporters in the hepatocyte samples investigated. The P-gp antibody allowed quantitative discrimination between externally facing transporters located in the cytoplasmic membrane and the total number of transporters on and in the cell. The proportion of externally facing transporter varied considerably in the four hepatocyte samples analysed, ranging from only 6% to 35% of intact and viable cells. The sample with only 6% externally facing transporter was further analysed by confocal microscopy which qualitatively confirmed the low level of transporter in the membrane and the large internal population. Here we prove that flow cytometry is an important tool for future protein analysis as it can not only quantify the number of proteins that a cell express but also identify the number of proteins on the surface and it is easy to apply for routine assays. Copyright © 2015 Elsevier Inc. All rights reserved.

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.

Hydrostatic Pressure Enhances Vital Staining with Carboxyfluorescein or Carboxydichlorofluorescein in Saccharomyces cerevisiae: Efficient Detection of Labeled Yeasts by Flow Cytometry

PubMed Central

Abe, Fumiyoshi

1998-01-01

The extent of intracellular accumulation of the fluorescent dye carboxyfluorescein or carboxydichlorofluorescein (CDCF) in Saccharomyces cerevisiae was found to be increased 5- to 10-fold under a nonlethal hydrostatic pressure of 30 to 50 MPa. This observation was confirmed by analysis of individual labeled cells by flow cytometry. The pressure-induced enhancement of staining with CDCF required d-glucose and was markedly inhibited by 2-deoxy-d-glucose, suggesting that glucose metabolism has a role in the process. PMID:9501452

Augmenting Trastuzumab Therapy Against Breast Cancer Through Selective Activation of NK Cells

DTIC Science & Technology

2013-10-01

selection and assessed for purity (>90% purity as defined by CD3-CD56+ flow cytometry ) and activation (>50% expression of CD137). Breast cancer cell lines...at a ratio of 1:1. After 24 hours, NK cells were isolated by negative selection and assessed for purity (>90% purity as defined by CD3-CD56+ flow ... cytometry ) and activation (>50% expression of CD137). Chromium-labeled breast cancer cell lines including MCF7 (A), BT474M1 (B), HER18 (C), and SKBR3

Treatment-Induced Autophagy Associated with Tumor Dormancy and Relapse

DTIC Science & Technology

2017-07-01

immunogenic apoptosis, autophagy and senescence in MMC and SKBR3 tumor cell lines. We determined that ADR, but not radiation , induced what appears to...hours after WT and BPTF KD 4T1 cells were treated with 5 Gy γ- radiation exposure. (D) γH2AX was measured by flow cytometry 30 minutes after WT and...BPTF KD 4T1 cells were treated with 6Gy γ- radiation . (E) Flow cytometry measurement of 1 ug/ml ADR accumulation after 24 hours exposure to WT and BPTF

Preparation and flow cytometry of uniform silica-fluorescent dye microspheres.

PubMed

Bele, Marjan; Siiman, Olavi; Matijević, Egon

2002-10-15

Uniform fluorescent silica-dye microspheres have been prepared by coating preformed monodispersed silica particles with silica layers containing rhodamine 6G or acridine orange. The resulting dispersions exhibit intense fluorescent emission between 500 and 600 nm, over a broad excitation wavelength range of 460 to 550 nm, even with exceedingly small amounts of dyes incorporated into the silica particles (10-30 ppm, expressed as weight of dye relative to weight of dry particles). The fluorescent particles can be prepared in micrometer diameters suitable for analyses using flow cytometry with 488-nm laser excitation.

A Rare Case of Pure Erythroid Sarcoma in a Pediatric Patient: Case Report and Literature Review

PubMed Central

Tarín, Fabián; Niveiro, María; Tasso, María; Alda, Olga; Verdú, José J.; De Paz, Francisco; López, Silvia; Del Cañizo, María; Such, Esperanza; Barragán, Eva; Martirena, Fernanda

2017-01-01

We describe an exceptional case of erythroid sarcoma in a pediatric patient as a growing orbital mass with no evidence of morphologic bone marrow involvement, who was finally diagnosed of pure erythroid sarcoma based on histopathology and flow cytometry criteria. We discuss the contribution of standardized eight-color flow cytometry as a rapid and reliable diagnostic method. The use of normal bone marrow databases allowed us to identify small aberrant populations in bone marrow and later confirm the diagnosis in the neoplastic tissue. PMID:29261159

A Rare Case of Pure Erythroid Sarcoma in a Pediatric Patient: Case Report and Literature Review.

PubMed

Manresa, Pablo; Tarín, Fabián; Niveiro, María; Tasso, María; Alda, Olga; López, Francisco; Sarmiento, Héctor; Verdú, José J; De Paz, Francisco; López, Silvia; Del Cañizo, María; Such, Esperanza; Barragán, Eva; Martirena, Fernanda

2017-12-20

We describe an exceptional case of erythroid sarcoma in a pediatric patient as a growing orbital mass with no evidence of morphologic bone marrow involvement, who was finally diagnosed of pure erythroid sarcoma based on histopathology and flow cytometry criteria. We discuss the contribution of standardized eight-color flow cytometry as a rapid and reliable diagnostic method. The use of normal bone marrow databases allowed us to identify small aberrant populations in bone marrow and later confirm the diagnosis in the neoplastic tissue.

Microfabrication and Test of a Three-Dimensional Polymer Hydro-focusing Unit for Flow Cytometry Applications

NASA Technical Reports Server (NTRS)

Yang, Ren; Feeback, Daniel L.; Wang, Wan-Jun

2005-01-01

This paper details a novel three-dimensional (3D) hydro-focusing micro cell sorter for micro flow cytometry applications. The unit was microfabricated by means of SU-8 3D lithography. The 3D microstructure for coaxial sheathing was designed, microfabricated, and tested. Three-dimensional hydrofocusing capability was demonstrated with an experiment to sort labeled tanned sheep erythrocytes (red blood cells). This polymer hydro-focusing microstructure is easily microfabricated and integrated with other polymer microfluidic structures. Keywords: SU-8, three-dimensional hydro-focusing, microfluidic, microchannel, cytometer

Microfluidics and photonics for Bio-System-on-a-Chip: a review of advancements in technology towards a microfluidic flow cytometry chip.

PubMed

Godin, Jessica; Chen, Chun-Hao; Cho, Sung Hwan; Qiao, Wen; Tsai, Frank; Lo, Yu-Hwa

2008-10-01

Microfluidics and photonics come together to form a field commonly referred to as 'optofluidics'. Flow cytometry provides the field with a technology base from which both microfluidic and photonic components be developed and integrated into a useful device. This article reviews some of the more recent developments to familiarize a reader with the current state of the technologies and also highlights the requirements of the device and how researchers are working to meet these needs.

Purification of Cardiomyocytes from Differentiating Pluripotent Stem Cells using Molecular Beacons Targeting Cardiomyocyte-Specific mRNA

PubMed Central

Kim, Sangsung; Park, Hun-Jun; Byun, Jaemin; Cho, Kyu-Won; Saafir, Talib; Song, Ming-Ke; Yu, Shan Ping; Wagner, Mary; Bao, Gang; Yoon, Young-Sup

2013-01-01

Background While methods for generating cardiomyocytes (CMs) from pluripotent stem cells (PSCs) have been reported, current methods produce heterogeneous mixtures of CMs and non-CM cells. Here, we report an entirely novel system in which PSC-derived CMs are purified by CM-specific molecular beacons (MBs). MBs are nano-scale probes that emit a fluorescence signal when hybridized to target mRNAs. Method and Results Five MBs targeting mRNAs of either cardiac troponin T or myosin heavy chain 6/7 were generated. Among five MBs, a MB targeting myosin heavy chain 6/7 mRNA (MHC1-MB) identified up to 99% of HL-1 CMs, a mouse CM cell line, but < 3% of four non-CM cell types in flow cytometry analysis, indicating that MHC1-MB is specific for identifying CMs. We delivered MHC1-MB into cardiomyogenically differentiated PSCs through nucleofection. The detection rate of CMs was similar to the percentages of cardiac troponin T (TNNT2) or cardiac troponin I (TNNI3)-positive CMs, supporting the specificity of MBs. Finally, MHC1-MB-positive cells were FACS-sorted from mouse and human PSC differentiating cultures and ~97% cells expressed TNNT2- or TNNI3 determined by flow cytometry. These MB-based sorted cells maintained their CM characteristics verified by spontaneous beating, electrophysiologic studies, and expression of cardiac proteins. When transplanted in a myocardial infarction model, MB-based purified CMs improved cardiac function and demonstrated significant engraftment for 4 weeks without forming tumors. Conclusions We developed a novel CM selection system that allows production of highly purified CMs. These purified CMs and this system can be valuable for cell therapy and drug discovery. PMID:23995537

Purification of cardiomyocytes from differentiating pluripotent stem cells using molecular beacons that target cardiomyocyte-specific mRNA.

PubMed

Ban, Kiwon; Wile, Brian; Kim, Sangsung; Park, Hun-Jun; Byun, Jaemin; Cho, Kyu-Won; Saafir, Talib; Song, Ming-Ke; Yu, Shan Ping; Wagner, Mary; Bao, Gang; Yoon, Young-Sup

2013-10-22

Although methods for generating cardiomyocytes from pluripotent stem cells have been reported, current methods produce heterogeneous mixtures of cardiomyocytes and noncardiomyocyte cells. Here, we report an entirely novel system in which pluripotent stem cell-derived cardiomyocytes are purified by cardiomyocyte-specific molecular beacons (MBs). MBs are nanoscale probes that emit a fluorescence signal when hybridized to target mRNAs. Five MBs targeting mRNAs of either cardiac troponin T or myosin heavy chain 6/7 were generated. Among 5 MBs, an MB that targeted myosin heavy chain 6/7 mRNA (MHC1-MB) identified up to 99% of HL-1 cardiomyocytes, a mouse cardiomyocyte cell line, but <3% of 4 noncardiomyocyte cell types in flow cytometry analysis, which indicates that MHC1-MB is specific for identifying cardiomyocytes. We delivered MHC1-MB into cardiomyogenically differentiated pluripotent stem cells through nucleofection. The detection rate of cardiomyocytes was similar to the percentages of cardiac troponin T- or cardiac troponin I-positive cardiomyocytes, which supports the specificity of MBs. Finally, MHC1-MB-positive cells were sorted by fluorescence-activated cell sorter from mouse and human pluripotent stem cell differentiating cultures, and ≈97% cells expressed cardiac troponin T or cardiac troponin I as determined by flow cytometry. These MB-based sorted cells maintained their cardiomyocyte characteristics, which was verified by spontaneous beating, electrophysiological studies, and expression of cardiac proteins. When transplanted in a myocardial infarction model, MB-based purified cardiomyocytes improved cardiac function and demonstrated significant engraftment for 4 weeks without forming tumors. We developed a novel cardiomyocyte selection system that allows production of highly purified cardiomyocytes. These purified cardiomyocytes and this system can be valuable for cell therapy and drug discovery.

Streak Imaging Flow Cytometer for Rare Cell Analysis.

PubMed

Balsam, Joshua; Bruck, Hugh Alan; Ossandon, Miguel; Prickril, Ben; Rasooly, Avraham

2017-01-01

There is a need for simple and affordable techniques for cytology for clinical applications, especially for point-of-care (POC) medical diagnostics in resource-poor settings. However, this often requires adapting expensive and complex laboratory-based techniques that often require significant power and are too massive to transport easily. One such technique is flow cytometry, which has great potential for modification due to the simplicity of the principle of optical tracking of cells. However, it is limited in that regard due to the flow focusing technique used to isolate cells for optical detection. This technique inherently reduces the flow rate and is therefore unsuitable for rapid detection of rare cells which require large volume for analysis.To address these limitations, we developed a low-cost, mobile flow cytometer based on streak imaging. In our new configuration we utilize a simple webcam for optical detection over a large area associated with a wide-field flow cell. The new flow cell is capable of larger volume and higher throughput fluorescence detection of rare cells than the flow cells with hydrodynamic focusing used in conventional flow cytometry. The webcam is an inexpensive, commercially available system, and for fluorescence analysis we use a 1 W 450 nm blue laser to excite Syto-9 stained cells with emission at 535 nm. We were able to detect low concentrations of stained cells at high flow rates of 10 mL/min, which is suitable for rapidly analyzing larger specimen volumes to detect rare cells at appropriate concentration levels. The new rapid detection capabilities, combined with the simplicity and low cost of this device, suggest a potential for clinical POC flow cytometry in resource-poor settings associated with global health.

Leptomeningeal metastases: a RANO proposal for response criteria

PubMed Central

Junck, Larry; Brandsma, Dieta; Soffietti, Riccardo; Rudà, Roberta; Raizer, Jeffrey; Boogerd, Willem; Taillibert, Sophie; Groves, Morris D.; Rhun, Emilie Le; Walker, Julie; van den Bent, Martin; Wen, Patrick Y.; Jaeckle, Kurt A.

2017-01-01

Abstract Leptomeningeal metastases (LM) currently lack standardization with respect to response assessment. A Response Assessment in Neuro-Oncology (RANO) working group with expertise in LM developed a consensus proposal for evaluating patients treated for this disease. Three basic elements in assessing response in LM are proposed: a standardized neurological examination, cerebral spinal fluid (CSF) cytology or flow cytometry, and radiographic evaluation. The group recommends that all patients enrolling in clinical trials undergo CSF analysis (cytology in all cancers; flow cytometry in hematologic cancers), complete contrast-enhanced neuraxis MRI, and in instances of planned intra-CSF therapy, radioisotope CSF flow studies. In conjunction with the RANO Neurological Assessment working group, a standardized instrument was created for assessing the neurological exam in patients with LM. Considering that most lesions in LM are nonmeasurable and that assessment of neuroimaging in LM is subjective, neuroimaging is graded as stable, progressive, or improved using a novel radiological LM response scorecard. Radiographic disease progression in isolation (ie, negative CSF cytology/flow cytometry and stable neurological assessment) would be defined as LM disease progression. The RANO LM working group has proposed a method of response evaluation for patients with LM that will require further testing, validation, and likely refinement with use. PMID:28039364

The effects of simulated hypogravity on murine bone marrow cells

NASA Technical Reports Server (NTRS)

Lawless, Desales

1989-01-01

Mouse bone marrow cells grown in complete medium at unit gravity were compared with a similar population cultured in conditions that mimic some aspects of microgravity. After the cells adjusted to the conditions that simulated microgravity, they proliferated as fetal or oncogenic populations; their numbers doubled in twelve hour periods. Differentiated subpopulations were depleted from the heterogeneous mixture with time and the undifferentiated hematopoietic stem cells increased in numbers. The cells in the control groups in unit gravity and those in the bioreactors in conditions of microgravity were monitored under a number of parameters. Each were phenotyped as to cell surface antigens using a panel of monoclonal antibodies and flow cytometry. Other parameters compared included: pH, glucose uptake, oxygen consumption and carbon-dioxide production. Nuclear DNA was monitored by flow cytometry. Functional responses were studied by mitogenic stimulation by various lectins. The importance of these findings should have relevance to the space program. Cells should behave predictably in zero gravity; specific populations can be eliminated from diverse populations and other populations isolated. The availability of stem cell populations will enhance both bone marrow and gene transplant programs. Stem cells will permit developmental biologists study the paths of hematopoiesis.

Study and selection of in vivo protein interactions by coupling bimolecular fluorescence complementation and flow cytometry.

PubMed

Morell, Montse; Espargaro, Alba; Aviles, Francesc Xavier; Ventura, Salvador

2008-01-01

We present a high-throughput approach to study weak protein-protein interactions by coupling bimolecular fluorescent complementation (BiFC) to flow cytometry (FC). In BiFC, the interaction partners (bait and prey) are fused to two rationally designed fragments of a fluorescent protein, which recovers its function upon the binding of the interacting proteins. For weak protein-protein interactions, the detected fluorescence is proportional to the interaction strength, thereby allowing in vivo discrimination between closely related binders with different affinity for the bait protein. FC provides a method for high-speed multiparametric data acquisition and analysis; the assay is simple, thousands of cells can be analyzed in seconds and, if required, selected using fluorescence-activated cell sorting (FACS). The combination of both methods (BiFC-FC) provides a technically straightforward, fast and highly sensitive method to validate weak protein interactions and to screen and identify optimal ligands in biologically synthesized libraries. Once plasmids encoding the protein fusions have been obtained, the evaluation of a specific interaction, the generation of a library and selection of active partners using BiFC-FC can be accomplished in 5 weeks.

Direct quantification of bacterial biomass in influent, effluent and activated sludge of wastewater treatment plants by using flow cytometry.

PubMed

Foladori, P; Bruni, L; Tamburini, S; Ziglio, G

2010-07-01

A rapid multi-step procedure, potentially amenable to automation, was proposed for quantifying viable and active bacterial cells, estimating their biovolume using flow cytometry (FCM) and to calculate their biomass within the main stages of a wastewater treatment plant: raw wastewater, settled wastewater, activated sludge and effluent. Fluorescent staining of bacteria using SYBR-Green I + Propidium Iodide (to discriminate cell integrity or permeabilisation) and BCECF-AM (to identify enzymatic activity) was applied to count bacterial cells by FCM. A recently developed specific procedure was applied to convert Forward Angle Light Scatter measured by FCM into the corresponding bacterial biovolume. This conversion permits the calculation of the viable and active bacterial biomass in wastewater, activated sludge and effluent, expressed as Volatile Suspended Solids (VSS) or particulate Chemical Oxygen Demand (COD). Viable bacterial biomass represented only a small part of particulate COD in raw wastewater (4.8 +/- 2.4%), settled wastewater (10.7 +/- 3.1%), activated sludge (11.1 +/- 2.1%) and effluent (3.2 +/- 2.2%). Active bacterial biomass counted for a percentage of 30-47% of the viable bacterial biomass within the stages of the wastewater treatment plant. Copyright 2010 Elsevier Ltd. All rights reserved.

DNA-based probes for flow cytometry analysis of endocytosis and recycling.

PubMed

Dumont, Claire; Czuba, Ewa; Chen, Moore; Villadangos, Jose A; Johnston, Angus P R; Mintern, Justine D

2017-04-01

The internalization of proteins plays a key role in cell development, cell signaling and immunity. We have previously developed a specific hybridization internalization probe (SHIP) to quantitate the internalization of proteins and particles into cells. Herein, we extend the utility of SHIP to examine both the endocytosis and recycling of surface receptors using flow cytometry. SHIP was used to monitor endocytosis of membrane-bound transferrin receptor (TFR) and its soluble ligand transferrin (TF). SHIP enabled measurements of the proportion of surface molecules internalized, the internalization kinetics and the proportion and rate of internalized molecules that recycle to the cell surface with time. Using this method, we have demonstrated the internalization and recycling of holo-TF and an antibody against the TFR behave differently. This assay therefore highlights the implications of receptor internalization and recycling, where the internalization of the receptor-antibody complex behaves differently to the receptor-ligand complex. In addition, we observe distinct internalization patterns for these molecules expressed by different subpopulations of primary cells. SHIP provides a convenient and high throughput technique for analysis of trafficking parameters for both cell surface receptors and their ligands. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Extracting archaeal populations from iron oxidizing systems

NASA Astrophysics Data System (ADS)

Whitmore, L. M.; Hutchison, J.; Chrisler, W.; Jay, Z.; Moran, J.; Inskeep, W.; Kreuzer, H.

2013-12-01

Unique environments in Yellowstone National Park offer exceptional conditions for studying microorganisms in extreme and constrained systems. However, samples from some extreme systems often contain inorganic components that pose complications during microbial and molecular analysis. Several archaeal species are found in acidic, geothermal ferric-oxyhydroxide mats; these species have been shown to adhere to mineral surfaces in flocculated colonies. For optimal microbial analysis, (microscopy, flow cytometry, genomic extractions, proteomic analysis, stable isotope analysis, and others), improved techniques are needed to better facilitate cell detachment and separation from mineral surfaces. As a requirement, these techniques must preserve cell structure while simultaneously minimizing organic carryover to downstream analysis. Several methods have been developed for removing sediments from mixed prokaryotic populations, including ultra-centrifugation, nycodenz gradient, sucrose cushions, and cell straining. In this study we conduct a comparative analysis of mechanisms used to detach archaeal cell populations from the mineral interface. Specifically, we evaluated mechanical and chemical approaches for cell separation and homogenization. Methods were compared using confocal microscopy, flow cytometry analyses, and real-time PCR detection. The methodology and approaches identified will be used to optimize biomass collection from environmental specimens or isolates grown with solid phases.

Reticulocyte analysis using flow cytometry.

PubMed

Corberand, J X

1996-12-01

Automation of the reticulocyte count by means of flow cytometry has considerably improved the quality of this investigation. This article deals firstly with the reasons for the poor performance of the microscopic technique and with the physiological principles underlying identification and classification of reticulocytes using RNA labeling. It then outlines the automated methods currently on the market, which can be classified in three categories: a) "general-purpose" cytofluorometers, which in clinical laboratories usually deal with lymphocyte immunophenotyping; b) the only commercially available cytofluorometer dedicated to the reticulocyte count; this automat has the advantage of requiring no human intervention as it merely needs to be fed with samples; c) hematology analyzers with specific modules for automatic counting of reticulocytes previously incubated with a non-fluorescent dye. Of the various fluorescent markers available, thiazole orange, DEQTC iodide and auramine are most often used for this basic hematology test. The quality of the count, the availability of new reticulocyte indices (maturation index, percentage of young reticulocytes) and rapidity of the count give this test renewed value in the practical approach to the diagnosis of anemia, and also open new perspectives in the surveillance of aplastic anemia after chemotherapy or bone marrow grafting.

Identification and immunophenotypic characterization of normal and pathological mast cells.

PubMed

Morgado, José Mário; Sánchez-Muñoz, Laura; Teodósio, Cristina; Escribano, Luís

2014-01-01

Mast cells (MCs) are secretory cells that are central players in human allergic disease and immune responses. With the exception of a few pathological situations, MCs are usually present at relatively low frequencies in most tissues. Since their first description, MCs in tissues were identified mostly using their morphological characteristics and their typical coloration when stained with aniline dyes. However, increasing availability of highly specific antibodies now permits the use of fluorescence-based flow cytometry as the method of choice for the quantification, characterization, and purification of cells in suspension. This technique allows for a rapid analysis of thousands of events and for the identification of cells present at frequencies as low as one event in 10(6) unwanted cells. This method also permits for simultaneous characterization of multiple antigens at a single-cell level, which is ideal in order to study rare populations of cells like MCs. Here we describe the basis of flow cytometry-based immunophenotyping applied to the study of MC. The protocol focuses on the study of human MCs present in body fluids (mainly bone marrow) but can easily be adapted to study MCs from other tissues and species.

In utero transplantation of human bone marrow-derived multipotent mesenchymal stem cells in mice.

PubMed

Chou, Shiu-Huey; Kuo, Tom K; Liu, Ming; Lee, Oscar K

2006-03-01

Mesenchymal stem cells (MSCs) are multipotent cells that can be isolated from human bone marrow and possess the potential to differentiate into progenies of embryonic mesoderm. However, current evidence is based predominantly on in vitro experiments. We used a murine model of in utero transplantation (IUT) to study the engraftment capabilities of human MSCs. MSCs were obtained from bone marrow by negative immunoselection and limiting dilution, and were characterized by flow cytometry and by in vitro differentiation into osteoblasts, chondrocytes, and adipocytes. MSCs were transplanted into fetal mice at a gestational age of 14 days. Engraftment of human MSCs was determined by flow cytometry, polymerase chain reaction, and fluorescence in situ hybridization (FISH). MSCs engrafted into tissues originating from all three germ layers and persisted for up to 4 months or more after delivery, as evidenced by the expression of the human-specific beta-2 microglobulin gene and by FISH for donor-derived cells. Donor-derived CD45+ cells were detectable in the peripheral blood of recipients, suggesting the participation of MSCs in hematopoiesis at the fetal stage. This model can further serve to evaluate possible applications of MSCs. Copyright 2006 Orthopaedic Research Society.

Expression of decay-accelerating factor (CD55), membrane cofactor protein (CD46) and CD59 in the human astroglioma cell line, D54-MG, and primary rat astrocytes.

PubMed

Yang, C; Jones, J L; Barnum, S R

1993-09-01

In this report, we have shown the expression of the complement regulatory proteins decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46) and CD59 on human D54-MG astroglioma cells by several methods, including immunofluorescence, flow cytometry and Western blotting and Northern blot analysis. These studies demonstrate that all three proteins are structurally and antigenically similar to their counterparts expressed on HepG2 and SW480 cells (hepatocyte and epithelial cell lines, respectively). D54-MG cells express mRNA for all three proteins of the appropriate size(s). The phosphatidylinositol-specific enzyme, PIPLC, cleaved DAF from the surface of D54-MG cells, demonstrating that DAF is linked by a glycophospholipid anchor as has been shown for other cell types. Flow cytometry demonstrates that primary rat astrocytes also constitutively express all three regulatory proteins. These data are the first to demonstrate the expression of CD59 on astrocytes, and the presence of all three regulatory proteins on astrocytes suggests that regulation of complement activation in the central nervous system is important in neural host defense mechanisms.

A simple clot based assay for detection of procoagulant cell-derived microparticles.

PubMed

Patil, Rucha; Ghosh, Kanjaksha; Shetty, Shrimati

2016-05-01

Cell-derived microparticles (MPs) are important biomarkers in many facets of medicine. However, the MP detection methods used till date are costly and time consuming. The main aim of this study was to standardize an in-house clot based screening method for MP detection which would not only be specific and sensitive, but also inexpensive. Four different methods of MP assessment were performed and the results correlated. Using the flow cytometry technique as the gold standard, 25 samples with normal phosphatidylserine (PS) expressing MP levels and 25 samples with elevated levels were selected, which was cross checked by the commercial STA Procoag PPL clotting time (CT) assay. A simple recalcification time and an in-house clot assay were the remaining two tests. The in-house test measures the CT after the addition of calcium chloride to MP rich plasma, following incubation with Russell viper venom and phospholipid free plasma. The CT obtained by the in-house assay significantly correlated with the results obtained by flow cytometry (R2=0.87, p<0.01). Though preliminary, the in-house assay seems to be efficient, inexpensive and promising. It could definitely be utilized routinely for procoagulant MP assessment in various clinical settings.

SB203580 enhances the RV-induced loss of mitochondrial membrane potential and apoptosis in A549 cells

NASA Astrophysics Data System (ADS)

Li, Hai-yang; Zhuang, Cai-ping; Wang, Xiao-ping; Chen, Tong-sheng

2012-03-01

Resveratrol (RV), a naturally occurring phytoalexin, is known to possess a wide spectrum of chemopreventive and chemotherapeutic effects in various stages of human tumors. p38, a member of the mitogen-activated protein kinase (MAPK) superfamily, is always activated by some extracellular stimulus to regulate many cellular signal transduction pathways, such as apoptosis, proliferation, and inflammation and so on. In this report, we assessed the effect of SB203580, a specific inhibitor of p38 MAPK signaling pathway, on the RV-induced apoptosis in human lung adenocarcinoma (A549) cells. CCK-8 assay showed that pretreatment with SB203580 significantly enhanced the cytotoxicity of RV, which was further verified by analyzing the phosphatidylserine externalization using flow cytometry. In order to further confirm whether SB203580 accelerated apoptosis via the intrinsic apoptosis pathway, we analyzed the dysfunction of mitochondrial membrane potential (Δψm) of cells stained with rhodamine 123 by using flow cytometry after treatment with RV in the absence and presence of SB203580. Our data for the first time reported that p38 inhibitor SB203580 enhanced the RV-induced apoptosis via a mitochondrial pathway.

Establishment and Comparison of Two Different Diagnostic Platforms for Detection of DENV1 NS1 Protein

PubMed Central

Tang, Yin-Liang; Chiu, Chien-Yu; Lin, Chun-Yu; Huang, Chung-Hao; Chen, Yen-Hsu; Destura, Raul V.; Chao, Day-Yu; Wu, Han-Chung

2015-01-01

Dengue virus (DENV) infection is currently at pandemic levels, with populations in tropical and subtropical regions at greatest risk of infection. Early diagnosis and management remain the cornerstone for good clinical outcomes, thus efficient and accurate diagnostic technology in the early stage of the disease is urgently needed. Serotype-specific monoclonal antibodies (mAbs) against the DENV1 nonstructural protein 1 (NS1), DA12-4, DA13-2, and DA15-3, which were recently generated using the hybridoma technique, are suitable for use in diagnostic platforms. Immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA) and Western blot analysis further confirmed the serotype specificity of these three monoclonal antibodies. The ELISA-based diagnostic platform was established using the combination of two highly sensitive mAbs (DA15-3 and DB20-6). The same combination was also used for the flow cytometry-based diagnostic platform. We report here the detection limits of flow cytometry-based and ELISA-based diagnostic platforms using these mAbs to be 0.1 and 1 ng/mL, respectively. The collected clinical patient serum samples were also assayed by these two serotyping diagnostic platforms. The sensitivity and specificity for detecting NS1 protein of DENV1 are 90% and 96%, respectively. The accuracy of our platform for testing clinical samples is more advanced than that of the two commercial NS1 diagnostic platforms. In conclusion, our platforms are suitable for the early detection of NS1 protein in DENV1 infected patients. PMID:26610481

Surface Glycosylation Profiles of Urine Extracellular Vesicles

PubMed Central

Gerlach, Jared Q.; Krüger, Anja; Gallogly, Susan; Hanley, Shirley A.; Hogan, Marie C.; Ward, Christopher J.

2013-01-01

Urinary extracellular vesicles (uEVs) are released by cells throughout the nephron and contain biomolecules from their cells of origin. Although uEV-associated proteins and RNA have been studied in detail, little information exists regarding uEV glycosylation characteristics. Surface glycosylation profiling by flow cytometry and lectin microarray was applied to uEVs enriched from urine of healthy adults by ultracentrifugation and centrifugal filtration. The carbohydrate specificity of lectin microarray profiles was confirmed by competitive sugar inhibition and carbohydrate-specific enzyme hydrolysis. Glycosylation profiles of uEVs and purified Tamm Horsfall protein were compared. In both flow cytometry and lectin microarray assays, uEVs demonstrated surface binding, at low to moderate intensities, of a broad range of lectins whether prepared by ultracentrifugation or centrifugal filtration. In general, ultracentrifugation-prepared uEVs demonstrated higher lectin binding intensities than centrifugal filtration-prepared uEVs consistent with lesser amounts of co-purified non-vesicular proteins. The surface glycosylation profiles of uEVs showed little inter-individual variation and were distinct from those of Tamm Horsfall protein, which bound a limited number of lectins. In a pilot study, lectin microarray was used to compare uEVs from individuals with autosomal dominant polycystic kidney disease to those of age-matched controls. The lectin microarray profiles of polycystic kidney disease and healthy uEVs showed differences in binding intensity of 6/43 lectins. Our results reveal a complex surface glycosylation profile of uEVs that is accessible to lectin-based analysis following multiple uEV enrichment techniques, is distinct from co-purified Tamm Horsfall protein and may demonstrate disease-specific modifications. PMID:24069349

The differentiation of hepatocyte-like cells from monkey embryonic stem cells.

PubMed

Ma, Xiaocui; Duan, Yuyou; Jung, Christine J; Wu, Jian; VandeVoort, Catherine A; Zern, Mark A

2008-12-01

Embryonic stem cells (ESC) hold great potential for the treatment of liver diseases. Here, we report the differentiation of rhesus macaque ESC along a hepatocyte lineage. The undifferentiated monkey ESC line, ORMES-6, was cultured in an optimal culture condition in an effort to differentiate them into hepatocyte-like cells in vitro. The functional efficacy of the differentiated hepatic cells was evaluated using RT-PCR for the expression of hepatocyte specific genes, and Western blot analysis and immunocytochemistry for hepatic proteins such as alpha-fetoprotein (AFP), albumin and alpha1-antitrypsin (alpha1-AT). Functional assays were performed using the periodic acid schiff (PAS) reaction and ELISA. The final yield of ESC-derived hepatocyte-like cells was measured by flow cytometry for cells that were transduced with a liver-specific lentivirus vector containing the alpha1-AT promoter driving the expression of green fluorescence protein (GFP). The treatment of monkey ESC with an optimal culture condition yielded hepatocyte-like cells that expressed albumin, alpha1-AT, AFP, hepatocyte nuclear factor 3beta, glucose-6-phophatase, and cytochrome P450 genes and proteins as determined by RT-PCR and Western blot analysis. Immunofluorescent staining showed the cells positive for albumin, AFP, and alpha1-AT. PAS staining demonstrated that the differentiated cells showed hepatocyte functional activity. Albumin could be detected in the medium after 20 days of differentiation. Flow cytometry data showed that 6.5 +/- 1.0% of the total differentiated cells were positive for GFP. These results suggest that by using a specific, empirically determined, culture condition, we were able to direct monkey ESC toward a hepatocyte lineage.

A CD133-expressing murine liver oval cell population with bilineage potential.

PubMed

Rountree, C Bart; Barsky, Lora; Ge, Shundi; Zhu, Judy; Senadheera, Shantha; Crooks, Gay M

2007-10-01

Although oval cells are postulated to be adult liver stem cells, a well-defined phenotype of a bipotent liver stem cell remains elusive. The heterogeneity of cells within the oval cell fraction has hindered lineage potential studies. Our goal was to identify an enriched population of bipotent oval cells using a combination of flow cytometry and single cell gene expression in conjunction with lineage-specific liver injury models. Expression of cell surface markers on nonparenchymal, nonhematopoietic (CD45-) cells were characterized. Cell populations were isolated by flow cytometry for gene expression studies. 3,5-Diethoxycarbonyl-1,4-dihydrocollidine toxic injury induced cell cycling and expansion specifically in the subpopulation of oval cells in the periportal zone that express CD133. CD133+CD45- cells expressed hepatoblast and stem cell-associated genes, and single cells coexpressed both hepatocyte and cholangiocyte-associated genes, indicating bilineage potential. CD133+CD45- cells proliferated in response to liver injury. Following toxic hepatocyte damage, CD133+CD45- cells demonstrated upregulated expression of the hepatocyte gene Albumin. In contrast, toxic cholangiocyte injury resulted in upregulation of the cholangiocyte gene Ck19. After 21-28 days in culture, CD133+CD45- cells continued to generate cells of both hepatocyte and cholangiocyte lineages. Thus, CD133 expression identifies a population of oval cells in adult murine liver with the gene expression profile and function of primitive, bipotent liver stem cells. In response to lineage-specific injury, these cells demonstrate a lineage-appropriate genetic response. Disclosure of potential conflicts of interest is found at the end of this article.

Reference ranges of lymphocyte subsets balanced for age and gender from a population of healthy adults in Chongqing District of China.

PubMed

Zhang, Kejun; Wang, Feng; Zhang, Mingxu; Cao, Xinglu; Yang, Shaojun; Jia, Shuangrong; Wang, Lixin; Luo, Jie; Deng, Shaoli; Chen, Ming

2016-11-01

The enumeration of lymphocyte subsets plays an essential role in the monitoring of immunological disorders. Immunophenotyping values have been found to be influenced by race, age, gender, and environmental conditions. Therefore, it is important to establish reference ranges for healthy adults from the local population for clinical decision-making. The current study aimed to establish a normal reference range for peripheral blood lymphocyte subsets in healthy adults from the Chongqing District of China by using single-platform flow cytometry. Age- and gender-specific reference ranges were established in 268 healthy adult males and females between 21 and 60 years of age. The CD8+ cell counts decreased with age, CD4+ cell percentages and counts increased with age, and total T cell percentages were higher in the female population. Our results are similar to those reported from other parts of China but different from some results reported from other countries; this further stresses the need to establish local reference ranges by region. Our results will help in the management of patients with human immunodeficiency virus and other immunological disorders in Chongqing District. © 2015 International Clinical Cytometry Society. © 2015 International Clinical Cytometry Society.

Cetuximab-activated natural killer (NK) and dendritic cells (DC) collaborate to trigger tumor antigen-specific T cell immunity in head and neck cancer patients

PubMed Central

Srivastava, Raghvendra M.; Lee, Steve C.; Filho, Pedro A. Andrade; Lord, Christopher A.; Jie, Hyun-bae; Davidson, H. Carter; López-Albaitero, Andrés; Gibson, Sandra P.; Gooding, William E.; Ferrone, Soldano; Ferris, Robert L.

2013-01-01

Purpose Tumor antigen (TA)-specific monoclonal antibodies (mAb) block oncogenic signaling and induce Fcγ receptor (FcγR)-mediated cytotoxicity. However, the role of CD8+ cytotoxic T lymphocyte (CTL) and FcγR in initiating innate and adaptive immune responses in mAb-treated human cancer patients is still emerging. Experimental Design FcγRIIIa codon 158 polymorphism was correlated with survival in 107 cetuximab-treated head and neck cancer (HNC) patients. Flow cytometry was performed to quantify EGFR-specific T cells in cetuximab-treated HNC patients. The effect of cetuximab on NK cell, dendritic cell (DC), and T cell activation was measured using IFN-γ release assays and flow cytometry. Results FcγR IIIa polymorphism did not predict clinical outcome in cetuximab-treated HNC patients, however elevated circulating EGFR -specific CD8+ 853-861 T cells were found in cetuximab-treated HNC patients (p<0.005). Cetuximab promoted EGFR-specific cellular immunity through the interaction of EGFR+ tumor cells and FcγRIIIa on NK cells, but not on the polymorphism per se. Cetuximab-activated NK cells induced IFN-γ dependent expression of DC maturation markers, antigen presentation machinery (APM) components such as TAP-1/2, and Th1 chemokines through NKG2D/MICA binding. Cetuximab initiated adaptive immune responses via NK-cell induced DC maturation, which enhanced cross-presentation to CTL specific for EGFR as well as another TA, MAGE-3. Conclusion Cetuximab-activated NK cells promote DC maturation and CD8+ T cell priming, leading to TA spreading and Th1 cytokine release through ‘NK-DC cross-talk.’ FcγRIIIa polymorphism did not predict clinical response to cetuximab, but was necessary for NK-DC interaction and mAb induced cross-presentation. EGFR-specific T cells in cetuximab treated HNC patients may contribute to clinical response. PMID:23444227

Dynamic thermoregulation of the sample in flow cytometry.

PubMed

Graves, Steven W; Habbersett, Robert C; Nolan, John P

2002-05-01

Fine control of temperature is an important capability for any analytical platform. A circulating water bath has been the traditional means of maintaining constant temperature in the sample chamber of a flow cytometer, but this approach does not permit rapid changes in sample temperature. This unit explains the use of Peltier modules for regulation of sample temperature. The heat pumping generated by the passage of current through properly matched semiconductors, known as the Peltier effect, makes it possible for these thermoelectric modules to both heat and cool. The authors describe the construction of a Peltier module based thermoregulation unit in step-by-step detail and present a demonstration of flow cytometry measurements as a function of temperature.

Assessment of amphotericin B susceptibility in Leishmania infantum promastigotes by flow cytometric membrane potential assay.

PubMed

Azas, N; Di Giorgio, C; Delmas, F; Gasquet, M; Timon-David, P

1997-06-01

Flow cytometry was used for measuring the effects of amphotericin B on the membrane of Leishmania infantum strains. The technique was adapted from the rapid flow cytometric membrane potential assay developed by Ordonez and Wehman (Cytometry 22:154-157, 1995) for evaluating antibiotic-susceptibility of Candida species. The study consisted of measuring membrane potential changes induced by amphotericin B in 3 initial strains and 12 laboratory-generated variants adapted to grow with amphotericin B. Results showed that, after 3 h of incubation, amphotericin B induced a dose-related decrease of membrane potential that reached its maximal level at the same concentrations that inhibited parasite growth. These results suggest that the flow cytometric membrane potential assay could be used to assess the susceptibility of Leishmania promastigotes to amphotericin B.

Validation of a single-platform, volumetric, flow cytometry for CD4 T cell count monitoring in therapeutic mobile unit

PubMed Central

2012-01-01

Background A mobile health unit may be useful to follow up adult and pediatric patients on antiretroviral treatment and living in remote areas devoid of laboratory facilities. The study evaluated the use of the simplified, robust, single-plateform, volumetric, pan-leucogating Auto40 flow cytometer (Apogee Flow Systems Ltd, Hemel Hempstead, UK) for CD4 T cell numeration in a mobile unit, compared against a reference flow cytometry method. Methods The therapeutic mobile unit of the Laboratoire National de Santé Hygiène Mobile, Yaoundé, Cameroon, was equipped with the Auto40. A FACSCalibur flow cytometer (Becton Dickinson Immuno-cytometry System, San Jose, CA, USA) was used as reference method. EDTA-blood samples from volunteers were first subjected to CD4 T cell count in the mobile unit, and an aliquot was sent within 4 hours to Centre International de Référence Chantal Biya, Yaoundé, for FACSCalibur assay. Results Two HIV screening campaigns with the mobile unit were organised in December 2009 and January 2010. The campaign in the suburb of Yaoundé which was 20 km from the reference laboratory included 188 volunteers comprising 93 children less than 5 years old. The campaign in Ambang Bikok (53 km far from Yaoundé) included 69 adult volunteers. In Yaoundé suburb, mean ± standard deviation (SD) CD4 T cell count was 996 ± 874 cells/μl by Auto40, and 989 ± 883 cells/μl by FACSCalibur; in Ambang Bikok, mean ± SD CD4 T cell count was 1041 ± 317 cells/μl by Auto40, and 1032 ± 294 cells/μl by FACSCalibur. Results by Auto40 and FACSCalibur were highly correlated in Yaoundé (r2 = 0.982) as in Ambang Bikok (r2 = 0.921). Bland-Altman analysis showed a close agreement between Auto40 and FACSCalibur results expressed in absolute count as in percentage in Yaoundé and Ambang Bikok. When pooling the 257 CD4 T cell count measurements, the Auto40 yielded a mean difference of +7.6 CD4 T cells/μl higher than by reference flow cytometry; and the sensitivity and specificity of Auto40 in enumerating absolute CD4 T cell counts of less than 200 cells/μl were 87% and 99%, respectively, and in enumerating absolute CD4 T cell counts of less than 350 cells/μl were 87% and 98%, respectively. The intrarun and interun precisions of the Auto40 assay assessed in the mobile unit were 5.5% and 7.9%, respectively. Conclusions The Auto40 flow cytometer installed in a therapeutic mobile unit and operated far from its reference laboratory gave a perfect correlation with the reference method, and could be useful in carrying out immunological monitoring of HIV-infected patients living in areas without access to laboratory facilities. PMID:22309994

Continuous flow microfluidic separation and processing of rare cells and bioparticles found in blood - A review.

PubMed

Antfolk, Maria; Laurell, Thomas

2017-05-01

Rare cells in blood, such as circulating tumor cells or fetal cells in the maternal circulation, posses a great prognostic or diagnostic value, or for the development of personalized medicine, where the study of rare cells could provide information to more specifically targeted treatments. When conventional cell separation methods, such as flow cytometry or magnetic activated cell sorting, have fallen short other methods are desperately sought for. Microfluidics have been extensively used towards isolating and processing rare cells as it offers possibilities not present in the conventional systems. Furthermore, microfluidic methods offer new possibilities for cell separation as they often rely on non-traditional biomarkers and intrinsic cell properties. This offers the possibility to isolate cell populations that would otherwise not be targeted using conventional methods. Here, we provide an extensive review of the latest advances in continuous flow microfluidic rare cell separation and processing with each cell's specific characteristics and separation challenges as a point of view. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

DAFi: A directed recursive data filtering and clustering approach for improving and interpreting data clustering identification of cell populations from polychromatic flow cytometry data.

PubMed

Lee, Alexandra J; Chang, Ivan; Burel, Julie G; Lindestam Arlehamn, Cecilia S; Mandava, Aishwarya; Weiskopf, Daniela; Peters, Bjoern; Sette, Alessandro; Scheuermann, Richard H; Qian, Yu

2018-04-17

Computational methods for identification of cell populations from polychromatic flow cytometry data are changing the paradigm of cytometry bioinformatics. Data clustering is the most common computational approach to unsupervised identification of cell populations from multidimensional cytometry data. However, interpretation of the identified data clusters is labor-intensive. Certain types of user-defined cell populations are also difficult to identify by fully automated data clustering analysis. Both are roadblocks before a cytometry lab can adopt the data clustering approach for cell population identification in routine use. We found that combining recursive data filtering and clustering with constraints converted from the user manual gating strategy can effectively address these two issues. We named this new approach DAFi: Directed Automated Filtering and Identification of cell populations. Design of DAFi preserves the data-driven characteristics of unsupervised clustering for identifying novel cell subsets, but also makes the results interpretable to experimental scientists through mapping and merging the multidimensional data clusters into the user-defined two-dimensional gating hierarchy. The recursive data filtering process in DAFi helped identify small data clusters which are otherwise difficult to resolve by a single run of the data clustering method due to the statistical interference of the irrelevant major clusters. Our experiment results showed that the proportions of the cell populations identified by DAFi, while being consistent with those by expert centralized manual gating, have smaller technical variances across samples than those from individual manual gating analysis and the nonrecursive data clustering analysis. Compared with manual gating segregation, DAFi-identified cell populations avoided the abrupt cut-offs on the boundaries. DAFi has been implemented to be used with multiple data clustering methods including K-means, FLOCK, FlowSOM, and the ClusterR package. For cell population identification, DAFi supports multiple options including clustering, bisecting, slope-based gating, and reversed filtering to meet various autogating needs from different scientific use cases. © 2018 International Society for Advancement of Cytometry. © 2018 International Society for Advancement of Cytometry.

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.

Ultraviolet 320 nm laser excitation for flow cytometry.

PubMed

Telford, William; Stickland, Lynn; Koschorreck, Marco

2017-04-01

Although multiple lasers and high-dimensional analysis capability are now standard on advanced flow cytometers, ultraviolet (UV) lasers (usually 325-365 nm) remain an uncommon excitation source for cytometry. This is primarily due to their cost, and the small number of applications that require this wavelength. The development of the Brilliant Ultraviolet (BUV fluorochromes, however, has increased the importance of this formerly niche excitation wavelength. Historically, UV excitation was usually provided by water-cooled argon- and krypton-ion lasers. Modern flow cytometers primary rely on diode pumped solid state lasers emitting at 355 nm. While useful for all UV-excited applications, DPSS UV lasers are still large by modern solid state laser standards, and remain very expensive. Smaller and cheaper near UV laser diodes (NUVLDs) emitting at 375 nm make adequate substitutes for 355 nm sources in many situations, but do not work as well with very short wavelength probes like the fluorescent calcium chelator indo-1. In this study, we evaluate a newly available UV 320 nm laser for flow cytometry. While shorter in wavelength that conventional UV lasers, 320 is close to the 325 nm helium-cadmium wavelength used in the past on early benchtop cytometers. A UV 320 nm laser was found to excite almost all Brilliant Ultraviolet dyes to nearly the same level as 355 nm sources. Both 320 nm and 355 nm sources worked equally well for Hoechst and DyeCycle Violet side population analysis of stem cells in mouse hematopoetic tissue. The shorter wavelength UV source also showed excellent excitation of indo-1, a probe that is not compatible with NUVLD 375 nm sources. In summary, a 320 nm laser module made a suitable substitute for conventional 355 nm sources. This laser technology is available in a smaller form factor than current 355 nm units, making it useful for small cytometers with space constraints. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Microbubble Sizing and Shell Characterization Using Flow Cytometry

PubMed Central

Tu, Juan; Swalwell, Jarred E.; Giraud, David; Cui, Weicheng; Chen, Weizhong; Matula, Thomas J.

2015-01-01

Experiments were performed to size, count, and obtain shell parameters for individual ultrasound contrast microbubbles using a modified flow cytometer. Light scattering was modeled using Mie theory, and applied to calibration beads to calibrate the system. The size distribution and population were measured directly from the flow cytometer. The shell parameters (shear modulus and shear viscosity) were quantified at different acoustic pressures (from 95 to 333 kPa) by fitting microbubble response data to a bubble dynamics model. The size distribution of the contrast agent microbubbles is consistent with manufacturer specifications. The shell shear viscosity increases with increasing equilibrium microbubble size, and decreases with increasing shear rate. The observed trends are independent of driving pressure amplitude. The shell elasticity does not vary with microbubble size. The results suggest that a modified flow cytometer can be an effective tool to characterize the physical properties of microbubbles, including size distribution, population, and shell parameters. PMID:21622051

Flow Cytometry in Diagnosis of Myelomatous Pleural Effusion: A Case Report.

PubMed

Arora, Parul; Gupta, Sanjeev Kumar; Mallik, Nabhajit; Mittal, Reena; Sharma, Om Dutt; Kumar, Lalit

2016-06-01

Plasma cell myeloma is a multifocal plasma cell neoplasm associated with increased monoclonal protein in serum and/or urine. Pleural effusions in patients with myeloma are uncommon (6 %). However, effusions due to direct infiltration of the pleura by plasma cells (myelomatous pleural effusion) are extremely rare (<1 %) and usually seen with IgA myeloma. The diagnosis of such cases requires pleural fluid cytology, electrophoresis or pleural biopsy. We present a case of myelomatous pleural effusion diagnosed using flow cytometry immunophenotyping in addition to the pleural fluid cytology. A 45 year old female was diagnosed as plasma cell myeloma (IgG kappa) in 2007. She received multiple lines of therapy during the course of her treatment including thalidomide, dexamethasone, lenalidomide, bortezomib, and doxorubicin based regimens. However, the patient had progressive extramedullary disease and developed pleural effusion in 2014. Cytological examination of the pleural fluid showed degenerative changes. Few preserved areas showed mononuclear cells including morphologically abnormal plasma cells. Immunophenotyping of these cells by flow cytometry revealed a pattern indicating neoplastic plasma cells. There was expression of CD38, CD138, and CD56, with absence of CD19, CD10 and CD45. This confirmed the diagnosis of myelomatous pleural effusion. Subsequently, the patient was offered a dexamethasone, cyclophosphamide, etoposide and cisplatin based regimen but, she declined further treatment and succumbed to her disease 3 months later. Myelomatous pleural effusion is a rare complication of plasma cell myeloma. Flow cytometry can be used as an adjunctive technique in its diagnosis particularly in cases with equivocal cytology and electrophoresis findings.

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

Comparison of cell counting methods in rodent pulmonary toxicity studies: automated and manual protocols and considerations for experimental design

PubMed Central

Zeidler-Erdely, Patti C.; Antonini, James M.; Meighan, Terence G.; Young, Shih-Houng; Eye, Tracy J.; Hammer, Mary Ann; Erdely, Aaron

2016-01-01

Pulmonary toxicity studies often use bronchoalveolar lavage (BAL) to investigate potential adverse lung responses to a particulate exposure. The BAL cellular fraction is counted, using automated (i.e. Coulter Counter®), flow cytometry or manual (i.e. hemocytometer) methods, to determine inflammatory cell influx. The goal of the study was to compare the different counting methods to determine which is optimal for examining BAL cell influx after exposure by inhalation or intratracheal instillation (ITI) to different particles with varying inherent pulmonary toxicities in both rat and mouse models. General findings indicate that total BAL cell counts using the automated and manual methods tended to agree after inhalation or ITI exposure to particle samples that are relatively nontoxic or at later time points after exposure to a pneumotoxic particle when the response resolves. However, when the initial lung inflammation and cytotoxicity was high after exposure to a pneumotoxic particle, significant differences were observed when comparing cell counts from the automated, flow cytometry and manual methods. When using total BAL cell count for differential calculations from the automated method, depending on the cell diameter size range cutoff, the data suggest that the number of lung polymorphonuclear leukocytes (PMN) varies. Importantly, the automated counts, regardless of the size cutoff, still indicated a greater number of total lung PMN when compared with the manual method, which agreed more closely with flow cytometry. The results suggest that either the manual method or flow cytometry would be better suited for BAL studies where cytotoxicity is an unknown variable. PMID:27251196

Comparison of cell counting methods in rodent pulmonary toxicity studies: automated and manual protocols and considerations for experimental design.

PubMed

Zeidler-Erdely, Patti C; Antonini, James M; Meighan, Terence G; Young, Shih-Houng; Eye, Tracy J; Hammer, Mary Ann; Erdely, Aaron

2016-08-01

Pulmonary toxicity studies often use bronchoalveolar lavage (BAL) to investigate potential adverse lung responses to a particulate exposure. The BAL cellular fraction is counted, using automated (i.e. Coulter Counter®), flow cytometry or manual (i.e. hemocytometer) methods, to determine inflammatory cell influx. The goal of the study was to compare the different counting methods to determine which is optimal for examining BAL cell influx after exposure by inhalation or intratracheal instillation (ITI) to different particles with varying inherent pulmonary toxicities in both rat and mouse models. General findings indicate that total BAL cell counts using the automated and manual methods tended to agree after inhalation or ITI exposure to particle samples that are relatively nontoxic or at later time points after exposure to a pneumotoxic particle when the response resolves. However, when the initial lung inflammation and cytotoxicity was high after exposure to a pneumotoxic particle, significant differences were observed when comparing cell counts from the automated, flow cytometry and manual methods. When using total BAL cell count for differential calculations from the automated method, depending on the cell diameter size range cutoff, the data suggest that the number of lung polymorphonuclear leukocytes (PMN) varies. Importantly, the automated counts, regardless of the size cutoff, still indicated a greater number of total lung PMN when compared with the manual method, which agreed more closely with flow cytometry. The results suggest that either the manual method or flow cytometry would be better suited for BAL studies where cytotoxicity is an unknown variable.

Expression of {beta}{sub 1} integrins in human endometrial stromal and decidual cells

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

Shiokawa, Shigetatsu; Yoshimura, Yasunori; Nakamura, Yukio

The present study was undertaken to investigate the expression of {beta}{sub 1} integrins in human endometrium and decidua using flow cytometry, immunohistochemistry, and immunoprecipitation. Fluorescence-activated flow cytometry demonstrated the greater expression of the {beta}{sub 1}, {alpha}{sub 1}, {alpha}{sub 2}, and {alpha}{sub 5} subunits of the {beta}{sub 1} integrin family in cultured stromal cells from the midsecretory phase, than in those of the early proliferative phase. The addition of estradiol (E{sub 2}) and progesterone (P) to cultured stromal cells in the early proliferative phase increased the expression of {beta}{sub 1} integrins in vitro. Flow cytometry also demonstrated the expression of themore » {beta}{sub 1}, {alpha}{sub 1}, {alpha}{sub 2}, {alpha}{sub 3}, {alpha}{sub 5}, and {alpha}{sub 6} subunits of {beta}{sub 1} integrin family in cultured decidual cells, and the enriched-fraction of prolactin (PRL)-producing decidual cells isolated by Percoll gradients showed high levels of {beta}{sub 1} integrins expression. Immunohistochemistry confirmed the {beta}{sub 1} integrin cell surface phenotypes in cultured decidual cells observed by flow cytometry. In summary, the present study demonstrated that endometrial stromal and decidual cells expressed {beta}{sub 1} integrin subunits at their surfaces. The expression exhibited a variability throughout the menstrual cycles, being predominantly detected in the secretory phase, and was maintained highly in the decidua. Thus, {beta}{sub 1} integrins in human endometrium and decidua may be important in mediating the organization of extracellular matrix proteins derived from embryos during the early stage of implantation. 43 refs., 7 figs., 2 tabs.« less

Interference of flavonoids with fluorescent intracellular probes: methodological implications in the evaluation of the oxidative burst by flow cytometry.

PubMed

Peluso, Ilaria; Manafikhi, Husseen; Reggi, Raffaella; Palmery, Maura

2014-08-01

The evaluation of oxidative burst is particularly relevant in many pathological and subclinical conditions. Flow cytometry provides quick and accurate measures of the reactive oxygen species production by leukocytes in most situations. However, spurious results, related to probes' efflux may be observed in several instances. Many factors affect the evaluation of the oxidative burst with fluorescent probes that require intracellular deacetylation and could be substrate of the multidrug resistance proteins (MDR). After discussing the implications of the efflux of fluorophores in the normalization strategies in flow cytometry assays, we have pointed out the possible interference of flavonoids with fluorescet probes' staining and signal. We have also reviewed the results from human intervention studies regarding the evaluation of oxidative burst with these probes. In vitro, at concentrations close to post-ingestion circulating levels, some flavonoids and their metabolites could interfere with probes' staining and fluorescence signal through different mechanisms, such as the inhibition of esterases, the modulation of the MDR-mediate efflux of probe and the inhibition of the oxidation of probe. These effects may explain the contrasting results obtained by human intervention studies. Finally, also inflammatory state or the use of drugs substrate of MDR proteins could affect the evaluation of the oxidative burst with intracellular probes. © 2014 International Society for Advancement of Cytometry.

Determination of viability of Aeromonas hydrophila in increasing concentrations of sodium chloride at different temperatures by flow cytometry and plate count technique.

PubMed

Pianetti, Anna; Manti, Anita; Boi, Paola; Citterio, Barbara; Sabatini, Luigia; Papa, Stefano; Rocchi, Marco Bruno Luigi; Bruscolini, Francesca

2008-10-31

Aeromonads in waters and foods can represent a risk to human health. Factors such as sodium chloride concentration and temperature can affect growth and viability of several food and water-borne pathogens. The behaviour of an Aeromonas hydrophila strain in the presence of 1.7%, 3.4% and 6% NaCl concentrations at 24 degrees C and 4 degrees C was studied over a 188 day period. Viability and membrane potential were assessed by flow cytometry; growth was evaluated by plate count technique. Flow cytometry evidenced that A. hydrophila retained viability over the period although varying according to temperature and salt concentrations. Colony Forming Units were generally lower in number than viable cells especially in the presence of 6% NaCl, indicating the occurrence of stressed cells which maintain metabolic activity yet are not able to grow on agar plates. In conclusion, A. hydrophila showed a long-term halotolerance even at elevated (6%) NaCl concentrations and a lesser sensitivity to salt at low temperature; therefore, low temperature and salt, which are two important factors limiting bacterial growth, do not assure safety in the case of high initial contamination. Finally, cytometry appears a valid tool for the rapid detection of the viability of pathogenic bacteria in food and environmental matrices to control and prevent health risks.

Preparation of Proper Immunogen by Cloning and Stable Expression of cDNA coding for Human Hematopoietic Stem Cell Marker CD34 in NIH-3T3 Mouse Fibroblast Cell Line

PubMed Central

Shafaghat, Farzaneh; Abbasi-Kenarsari, Hajar; Majidi, Jafar; Movassaghpour, Ali Akbar; Shanehbandi, Dariush; Kazemi, Tohid

2015-01-01

Purpose: Transmembrane CD34 glycoprotein is the most important marker for identification, isolation and enumeration of hematopoietic stem cells (HSCs). We aimed in this study to clone the cDNA coding for human CD34 from KG1a cell line and stably express in mouse fibroblast cell line NIH-3T3. Such artificial cell line could be useful as proper immunogen for production of mouse monoclonal antibodies. Methods: CD34 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy TA-cloning vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. Results: 1158 bp specific band was aligned completely to reference sequence in NCBI database corresponding to long isoform of human CD34. Transient and stable expression of human CD34 on transfected NIH-3T3 mouse fibroblast cells was achieved (25% and 95%, respectively) as shown by flow cytometry. Conclusion: Cloning and stable expression of human CD34 cDNA was successfully performed and validated by standard flow cytometric analysis. Due to murine origin of NIH-3T3 cell line, CD34-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD34. This approach could bypass the need for purification of recombinant proteins produced in eukaryotic expression systems. PMID:25789221

Flow cytometry analysis of hormone receptors on human peripheral blood mononuclear cells to identify stress-induced neuroendocrine effects

NASA Technical Reports Server (NTRS)

Meehan, R. T.

1986-01-01

Understanding the role of circulating peptide hormones in the pathogenesis of space-flight induced disorders would be greatly facilitated by a method which monitors chronic levels of hormones and their effects upon in vivo cell physiology. Single and simultaneous multiparameter flow cytometry analysis was employed to identify subpopulations of mononuclear cells bearing receptors for ACTH, Endorphin, and Somatomedin-C using monoclonal antibodies and monospecific antisera with indirect immunofluorescence. Blood samples were obtained from normal donors and subjects participating in decompression chamber studies (acute stress), medical student academic examination (chronic stress), and a drug study (Dexamethasone). Preliminary results indicate most ACTH and Endorphin receptor positive cells are monocytes and B-cells, exhibit little diurnal variation but the relative percentages of receptor positive cells are influenced by exposure to various stressors and ACTH inhibition. This study demonstrates the capability of flow cytometry analysis to study cell surface hormone receptor regulation which should allow insight into neuroendocrine modulation of the immune and other cellular systems during exposure to stress or microgravity.

Detection of internal structure by scattered light intensity: Application to kidney cell sorting

NASA Technical Reports Server (NTRS)

Goolsby, C. L.; Kunze, M. E.

1985-01-01

Scattered light measurements in flow cytometry were sucessfully used to distinguish cells on the basis of differing morphology and internal structure. Differences in scattered light patterns due to changes in internal structure would be expected to occur at large scattering angles. Practically, the results of these calculations suggest that in experimental situations an array of detectors would be useful. Although in general the detection of the scattered light intensity at several intervals within the 10 to 60 region would be sufficient, there are many examples where increased sensitivity could be acheived at other angles. The ability to measure at many different angular intervals would allow the experimenter to empirically select the optimum intervals for the varying conditions of cell size, N/C ratio, granule size and internal structure from sample to sample. The feasibility of making scattered light measurements at many different intervals in flow cytometry was demonstrated. The implementation of simplified versions of these techniques in conjunction with independant measurements of cell size could potentially improve the usefulness of flow cytometry in the study of the internal structure of cells.

A flow cytometry-optimized assay using an SOS-green fluorescent protein (SOS-GFP) whole-cell biosensor for the detection of genotoxins in complex environments.

PubMed

Norman, Anders; Hansen, Lars Hestbjerg; Sørensen, Søren J

2006-02-28

Whole-cell biosensors have become popular tools for detection of ecotoxic compounds in environmental samples. We have developed an assay optimized for flow cytometry with detection of genotoxic compounds in mind. The assay features extended pre-incubation and a cell density of only 10(6)-10(7) cells/mL, and proved far more sensitive than a previously published assay using the same biosensor strain. By applying the SOS-green fluorescent protein (GFP) whole-cell biosensor directly to soil microcosms we were also able to evaluate both the applicability and sensitivity of a biosensor based on SOS-induction in whole soil samples. Soil microcosms were spiked with a dilution-series of crude broth extract from the mitomycin C-producing streptomycete Streptomyces caespitosus. Biosensors extracted from these microcosms after 1 day of incubation at 30 degrees C were easily distinguished from extracts of non-contaminated soil particles when using flow cytometry, and induction of the biosensor by mitomycin C was detectable at concentrations as low as 2.5 ng/g of soil.

Real Time Detection of Protein Trafficking with High Throughput Flow Cytometry (HTFC) and Fluorogen Activating Protein (FAP) Base Biosensor

PubMed Central

Wu, Yang; Tapia, Phillip H.; Jarvik, Jonathan; Waggoner, Alan S.; Sklar, Larry A.

2014-01-01

We combined fluorogen activating protein (FAP) technology with high-throughput flow cytometry to detect real-time protein trafficking to and from the plasma membrane in living cells. The hybrid platform allows drug discovery for trafficking receptors, such as G-protein coupled receptors, receptor tyrosine kinases and ion channels, that were previously not suitable for high throughput screening by flow cytometry.. The system has been validated using the β2-adrenergic receptor (β2AR) system and extended to other GPCRs. When a chemical library containing ~1,200 off-patent drugs was screened against cells expressing FAP tagged β2AR, all known β2AR active ligands in the library were successfully identified, together with a few compounds that were later confirmed to regulate receptor internalization in a non-traditional manner. The unexpected discovery of new ligands by this approach indicates the potential of using this protocol for GPCR de-orphanization. In addition, screens of multiplexed targets promise improved efficiency with minor protocol modification. PMID:24510772

[Evaluation of transfection effectiveness using fluorescein-labelled oligonucleotides and entraster-R siRNA transfection into Plasmodium falciparum].

PubMed

Zhou, Hong-Chang; Gao, Yu-Hui; Shao, Sheng-Wen; Zhang, Hui; Zhang, Ting

2013-12-01

The cultured Plasmodium falciparum parasites were synchronized twice by 5% sorbitol treatment twice (8-hour window), and then incubated at 37 degrees C for 16 h. Parasites were transfected with fluorescein-labelled oligonucleotides (group A) or fluorescein-labelled oligonucleotides+Entranster-R siRNA transfection reagent (group B). After 5 h a part of parasites was evaluated by fluorescence microscopy and flow cytometry. The rest of parasites were washed with RPMI 1640 medium, and then incubated with 500 microl new medium containing 2% fresh erythrocytes for another 12 h, and detected by flow cytometry. The fluorescein-labelled oligonucleotides were localized in erythrocytes in group B, but nearly no fluorescence was observed for group A. Flow cytometry analysis indicated that the transfection efficiency of group B [(47.40 +/- 3.39)%] was higher than that of group A [(0.60 +/- 0.27)%]. In the second cell cycle, the transfection efficiency in group B was (26.85 +/- 2.90)%, while that of group A was nearly zero. The results indicated that Entranster-R siRNA transfection reagent may increase the oligonucleotides transfection efficiency.

An oral Sindbis virus replicon-based DNA vaccine containing VP2 gene of canine parvovirus delivered by Escherichia coli elicits immune responses in dogs.

PubMed

Dahiya, S S; Saini, M; Kumar, P; Gupta, P K

2011-01-01

A Sindbis virus replicon-based DNA vaccine containing VP2 gene of canine parvovirus (CPV) was delivered by Escherichia coli to elicit immune responses. The orally immunized dogs developed CPV-specific serum IgG and virus neutralizing antibody responses. The cellular immune responses analyzed using lymphocyte proliferation test and flow cytometry indicated CPV-specific sensitization of both CD3+CD4+ and CD3+CD8+ lymphocytes. This study demonstrated that the oral CPV DNA vaccine delivered by E. coli can be considered as a promising approach for vaccination of dogs against CPV.

NIST/ISAC standardization study: variability in assignment of intensity values to fluorescence standard beads and in cross calibration of standard beads to hard dyed beads.

PubMed

Hoffman, Robert A; Wang, Lili; Bigos, Martin; Nolan, John P

2012-09-01

Results from a standardization study cosponsored by the International Society for Advancement of Cytometry (ISAC) and the US National Institute of Standards and Technology (NIST) are reported. The study evaluated the variability of assigning intensity values to fluorophore standard beads by bead manufacturers and the variability of cross calibrating the standard beads to stained polymer beads (hard-dyed beads) using different flow cytometers. Hard dyed beads are generally not spectrally matched to the fluorophores used to stain cells, and spectral response varies among flow cytometers. Thus if hard dyed beads are used as fluorescence calibrators, one expects calibration for specific fluorophores (e.g., FITC or PE) to vary among different instruments. Using standard beads surface-stained with specific fluorophores (FITC, PE, APC, and Pacific Blue™), the study compared the measured intensity of fluorophore standard beads to that of hard dyed beads through cross calibration on 133 different flow cytometers. Using robust CV as a measure of variability, the variation of cross calibrated values was typically 20% or more for a particular hard dyed bead in a specific detection channel. The variation across different instrument models was often greater than the variation within a particular instrument model. As a separate part of the study, NIST and four bead manufacturers used a NIST supplied protocol and calibrated fluorophore solution standards to assign intensity values to the fluorophore beads. Values assigned to the reference beads by different groups varied by orders of magnitude in most cases, reflecting differences in instrumentation used to perform the calibration. The study concluded that the use of any spectrally unmatched hard dyed bead as a general fluorescence calibrator must be verified and characterized for every particular instrument model. Close interaction between bead manufacturers and NIST is recommended to have reliable and uniformly assigned fluorescence standard beads. Copyright © 2012 International Society for Advancement of Cytometry.

CytometryML with DICOM and FCS

NASA Astrophysics Data System (ADS)

Leif, Robert C.

2018-02-01

Abstract: Flow Cytometry Standard, FCS, and Digital Imaging and Communications in Medicine standard, DICOM, are based on extensive, superb domain knowledge, However, they are isolated systems, do not take advantage of data structures, require special programs to read and write the data, lack the capability to interoperate or work with other standards and FCS lacks many of the datatypes necessary for clinical laboratory data. The large overlap between imaging and flow cytometry provides strong evidence that both modalities should be covered by the same standard. Method: The XML Schema Definition Language, XSD 1.1 was used to translate FCS and/or DICOM objects. A MIFlowCyt file was tested with published values. Results: Previously, a significant part of an XML standard based upon a combination of FCS and DICOM has been implemented and validated with MIFlowCyt data. Strongly typed translations of FCS keywords have been constructed in XML. These keywords contain links to their DICOM and FCS equivalents.

Genistein induces apoptosis by the inactivation of the IGF-1R/p-Akt signaling pathway in MCF-7 human breast cancer cells.

PubMed

Chen, Jun; Duan, Yuxin; Zhang, Xing; Ye, Yu; Ge, Bo; Chen, Jian

2015-03-01

Genistein is an estrogenic soy-derived compound belonging to the isoflavone class and shows anti-cancer effects. However, the specific cell apoptosis mechanisms of genistein have not been fully understood. In this study, we investigated the specific cell apoptosis mechanisms of genistein and the potential involvement of the IGF1R-Akt-Bcl-2 and Bax-mediated pathways in human breast cancer cells in vitro. MCF-7 human breast cancer cells were treated with various concentrations of genistein, and cell proliferation was evaluated by the MTT assay. Morphological changes in treated cells were examined by Hoechst 33258 staining, and treated cells were examined by flow cytometry. The levels of IGF-1R, p-Akt, Bcl-2, and Bax protein expression and Bcl-2 and Bax mRNA expression were evaluated by western blot and RT-PCR, respectively. Genistein inhibited the proliferation of MCF-7 cells and induced cell apoptosis, as determined by Hoechst staining and flow cytometry analysis. Furthermore, genistein induced the inactivation of IGF-1R and p-Akt and downregulated the Bcl-2/Bax protein ratio. These results suggest that genistein inhibited cell proliferation by inactivating the IGF-1R-PI3 K/Akt pathway and decreasing the Bcl-2/Bax mRNA and protein expressions. Our findings help elucidate the mechanisms by which genistein may contribute to the prevention of breast cancer carcinogenesis.

Oriented conjugates of monoclonal and single-domain antibodies with quantum dots for flow cytometry and immunohistochemistry diagnostic applications

NASA Astrophysics Data System (ADS)

Sukhanova, Alyona; Even-Desrumeaux, Klervi; Millot, Jean-Marc; Chames, Patrick; Baty, Daniel; Artemyev, Mikhail; Oleinikov, Vladimir; Cohen, Jacques H. M.; Nabiev, Igor

2012-03-01

Ideal diagnostic nanoprobes should not exceed 15 nm in size and should contain high-affinity homogeneously oriented capture molecules on their surface. An advanced procedure for antibody (Ab) reduction was used to cleave each Ab into two functional half-Abs, 75-kDa heavy-light chain fragments, each containing an intact antigen-binding site. Affinity purification of half-Abs followed by their linkage to quantum dots (QDs) yielded oriented QD-Ab conjugates whose functionality was considerably improved compared to those obtained using the standard protocols. Ultrasmall diagnostic nanoprobes were engineered through oriented conjugation of QDs with 13-kDa single-domain Abs (sdAbs) derived from llama IgG. sdAbs were tagged with QDs via an additional cysteine residue specifically integrated into the C-terminal region of sdAb using genetic engineering. This approach made it possible to obtain sdAb-QD nanoprobes <12 nm in diameter comprising four copies of sdAbs linked to the same QD in an oriented manner. sdAb-QD conjugates against carcinoembryonic antigen (CEA) and HER2 exhibited an extremely high specificity in flow cytometry; the quality of immunohistochemical labeling of biopsy samples was found to be superior to that of labeling according to the current "gold standard" protocols of anatomo-pathological practice. The nano-bioengineering approaches developed can be extended to oriented conjugation of Abs and sdAbs with different semiconductor, noble metal, or magnetic nanoparticles.

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.

Immunophenotype Discovery, Hierarchical Organization, and Template-Based Classification of Flow Cytometry Samples

DOE PAGES

Azad, Ariful; Rajwa, Bartek; Pothen, Alex

2016-08-31

We describe algorithms for discovering immunophenotypes from large collections of flow cytometry samples and using them to organize the samples into a hierarchy based on phenotypic similarity. The hierarchical organization is helpful for effective and robust cytometry data mining, including the creation of collections of cell populations’ characteristic of different classes of samples, robust classification, and anomaly detection. We summarize a set of samples belonging to a biological class or category with a statistically derived template for the class. Whereas individual samples are represented in terms of their cell populations (clusters), a template consists of generic meta-populations (a group ofmore » homogeneous cell populations obtained from the samples in a class) that describe key phenotypes shared among all those samples. We organize an FC data collection in a hierarchical data structure that supports the identification of immunophenotypes relevant to clinical diagnosis. A robust template-based classification scheme is also developed, but our primary focus is in the discovery of phenotypic signatures and inter-sample relationships in an FC data collection. This collective analysis approach is more efficient and robust since templates describe phenotypic signatures common to cell populations in several samples while ignoring noise and small sample-specific variations. We have applied the template-based scheme to analyze several datasets, including one representing a healthy immune system and one of acute myeloid leukemia (AML) samples. The last task is challenging due to the phenotypic heterogeneity of the several subtypes of AML. However, we identified thirteen immunophenotypes corresponding to subtypes of AML and were able to distinguish acute promyelocytic leukemia (APL) samples with the markers provided. Clinically, this is helpful since APL has a different treatment regimen from other subtypes of AML. Core algorithms used in our data analysis are available in the flowMatch package at www.bioconductor.org. It has been downloaded nearly 6,000 times since 2014.« less

Polysaccharopeptides derived from Coriolus versicolor potentiate the S-phase specific cytotoxicity of Camptothecin (CPT) on human leukemia HL-60 cells

PubMed Central

2010-01-01

Background Polysaccharopeptide (PSP) from Coriolus versicolor (Yunzhi) is used as a supplementary cancer treatment in Asia. The present study aims to investigate whether PSP pre-treatment can increase the response of the human leukemia HL-60 cells to apoptosis induction by Camptothecin (CPT). Methods We used bivariate bromodeoxyuridine/propidium iodide (BrdUrd/PI) flow cytometry analysis to measure the relative movement (RM) of the BrdUrd positively labeled cells and DNA synthesis time (Ts) on the HL-60 cell line. We used annexin V/PI flow cytometry analysis to quantify the viable, necrotic and apoptotic cells. The expression of cyclin E and cyclin B1 was determined with annexin V/PI flow cytometry and western blotting. Human peripheral blood mononuclear cells were used to test the cytotoxicity of PSP and CPT. Results PSP reduced cellular proliferation; inhibited cells progression through both S and G2 phase, reduced 3H-thymidine uptake and prolonged DNA synthesis time (Ts) in HL-60 cells. PSP-pretreated cells enhanced the cytotoxicity of CPT. The sensitivity of cells to the cytotoxic effects of CPT was seen to be the highest in the S-phase and to a small extent of the G2 phase of the cell cycle. On the other hand, no cell death (measured by annexin V/PI) was evident with the normal human peripheral blood mononuclear cells with treatment of either PSP or CPT. Conclusion The present study shows that PSP increases the sensitization of the HL-60 cells to undergo effective apoptotic cell death induced by CPT. The pattern of sensitivity of cancer cells is similar to that of HL-60 cells. PSP rapidly arrests and/or kills cells in S-phase and did not interfere with the anticancer action of CPT. PSP is a potential adjuvant to treat human leukemia as rapidly proliferating tumors is characterized by a high proportion of S-phase cells. PMID:20423495

Polysaccharopeptides derived from Coriolus versicolor potentiate the S-phase specific cytotoxicity of Camptothecin (CPT) on human leukemia HL-60 cells.

PubMed

Wan, Jennifer Man-Fan; Sit, Wai-Hung; Yang, Xiaotong; Jiang, Pingping; Wong, Leo Lap-Yan

2010-04-27

Polysaccharopeptide (PSP) from Coriolus versicolor (Yunzhi) is used as a supplementary cancer treatment in Asia. The present study aims to investigate whether PSP pre-treatment can increase the response of the human leukemia HL-60 cells to apoptosis induction by Camptothecin (CPT). We used bivariate bromodeoxyuridine/propidium iodide (BrdUrd/PI) flow cytometry analysis to measure the relative movement (RM) of the BrdUrd positively labeled cells and DNA synthesis time (Ts) on the HL-60 cell line. We used annexin V/PI flow cytometry analysis to quantify the viable, necrotic and apoptotic cells. The expression of cyclin E and cyclin B1 was determined with annexin V/PI flow cytometry and western blotting. Human peripheral blood mononuclear cells were used to test the cytotoxicity of PSP and CPT. PSP reduced cellular proliferation; inhibited cells progression through both S and G2 phase, reduced 3H-thymidine uptake and prolonged DNA synthesis time (Ts) in HL-60 cells. PSP-pretreated cells enhanced the cytotoxicity of CPT. The sensitivity of cells to the cytotoxic effects of CPT was seen to be the highest in the S-phase and to a small extent of the G2 phase of the cell cycle. On the other hand, no cell death (measured by annexin V/PI) was evident with the normal human peripheral blood mononuclear cells with treatment of either PSP or CPT. The present study shows that PSP increases the sensitization of the HL-60 cells to undergo effective apoptotic cell death induced by CPT. The pattern of sensitivity of cancer cells is similar to that of HL-60 cells. PSP rapidly arrests and/or kills cells in S-phase and did not interfere with the anticancer action of CPT. PSP is a potential adjuvant to treat human leukemia as rapidly proliferating tumors is characterized by a high proportion of S-phase cells.

Immunophenotype Discovery, Hierarchical Organization, and Template-Based Classification of Flow Cytometry Samples

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

Azad, Ariful; Rajwa, Bartek; Pothen, Alex

We describe algorithms for discovering immunophenotypes from large collections of flow cytometry samples and using them to organize the samples into a hierarchy based on phenotypic similarity. The hierarchical organization is helpful for effective and robust cytometry data mining, including the creation of collections of cell populations’ characteristic of different classes of samples, robust classification, and anomaly detection. We summarize a set of samples belonging to a biological class or category with a statistically derived template for the class. Whereas individual samples are represented in terms of their cell populations (clusters), a template consists of generic meta-populations (a group ofmore » homogeneous cell populations obtained from the samples in a class) that describe key phenotypes shared among all those samples. We organize an FC data collection in a hierarchical data structure that supports the identification of immunophenotypes relevant to clinical diagnosis. A robust template-based classification scheme is also developed, but our primary focus is in the discovery of phenotypic signatures and inter-sample relationships in an FC data collection. This collective analysis approach is more efficient and robust since templates describe phenotypic signatures common to cell populations in several samples while ignoring noise and small sample-specific variations. We have applied the template-based scheme to analyze several datasets, including one representing a healthy immune system and one of acute myeloid leukemia (AML) samples. The last task is challenging due to the phenotypic heterogeneity of the several subtypes of AML. However, we identified thirteen immunophenotypes corresponding to subtypes of AML and were able to distinguish acute promyelocytic leukemia (APL) samples with the markers provided. Clinically, this is helpful since APL has a different treatment regimen from other subtypes of AML. Core algorithms used in our data analysis are available in the flowMatch package at www.bioconductor.org. It has been downloaded nearly 6,000 times since 2014.« less

Immunologic monitoring of cancer vaccine therapy: results of a workshop sponsored by the Society for Biological Therapy.

PubMed

Keilholz, Ulrich; Weber, Jeffrey; Finke, James H; Gabrilovich, Dmitry I; Kast, W Martin; Disis, Mary L; Kirkwood, John M; Scheibenbogen, Carmen; Schlom, Jeff; Maino, Vernon C; Lyerly, H Kim; Lee, Peter P; Storkus, Walter; Marincola, Franceso; Worobec, Alexandra; Atkins, Michael B

2002-01-01

The Society for Biological Therapy held a Workshop last fall devoted to immune monitoring for cancer immunotherapy trials. Participants included members of the academic and pharmaceutical communities as well as the National Cancer Institute and the Food and Drug Administration. Discussion focused on the relative merits and appropriate use of various immune monitoring tools. Six breakout groups dealt with assays of T-cell function, serologic and proliferation assays to assess B cell and T helper cell activity, and enzyme-linked immunospot assay, tetramer, cytokine flow cytometry, and reverse transcription polymerase chain reaction assays of T-cell immunity. General conclusions included: (1) future vaccine studies should be designed to determine whether T-cell dysfunction (tumor-specific and nonspecific) correlated with clinical outcome; (2) tetramer-based assays yield quantitative but not functional data (3) enzyme-linked immunospot assays have the lowest limit of detection (4) cytokine flow cytometry have a higher limit of detection than enzyme-linked immunospot assay, but offer the advantages of speed and the ability to identify subsets of reactive cells; (5) antibody tests are simple and accurate and should be incorporated to a greater extent in monitoring plans; (6) proliferation assays are imprecise and should not be emphasized in future studies; (7) the reverse transcription polymerase chain reaction assay is a promising research approach that is not ready for widespread application; and (8)there is a critical need to validate these assays as surrogates for vaccine potency and clinical effect. Current data and opinion support the use of a functional assay like the enzyme-linked immunospot assay or cytokine flow cytometry in combination with a quantitative assay like tetramers for immune monitoring. At present, assays appear to be most useful as measures of vaccine potency. Careful immune monitoring in association with larger scale clinical trials ultimately may enable the correlation of monitoring results with clinical benefit.