Routine detection of Epstein-Barr virus specific T-cells in the peripheral blood by flow cytometry

NASA Technical Reports Server (NTRS)

Crucian, B. E.; Stowe, R. P.; Pierson, D. L.; Sams, C. F.

2001-01-01

The ability to detect cytomegalovirus-specific T-cells (CD4(+)) in the peripheral blood by flow cytometry has been recently described by Picker et al. In this method, cells are incubated with viral antigen and responding (cytokine producing) T-cells are then identified by flow cytometry. To date, this technique has not been reliably used to detect Epstein-Barr virus (EBV)-specific T-cells primarily due to the superantigen/mitogenic properties of the virus which non-specifically activate T-cells. By modifying culture conditions under which the antigens are presented, we have overcome this limitation and developed an assay to detect and quantitate EBV-specific T-cells. The detection of cytokine producing T-cells by flow cytometry requires an extremely strong signal (such as culture in the presence of PMA and ionomycin). Our data indicate that in modified culture conditions (early removal of viral antigen) the non-specific activation of T-cells by EBV is reduced, but antigen presentation will continue uninhibited. Using this method, EBV-specific T-cells may be legitimately detected using flow cytometry. No reduction in the numbers of antigen-specific T-cells was observed by the early removal of target antigen when verified using cytomegalovirus antigen (a virus with no non-specific T-cell activation properties). In EBV-seropositive individuals, the phenotype of the EBV-specific cytokine producing T-cells was evaluated using four-color flow cytometry and found to be CD45(+), CD3(+), CD4(+), CD45RA(-), CD69(+), CD25(-). This phenotype indicates the stimulation of circulating previously unactivated memory T-cells. No cytokine production was observed in CD4(+) T-cells from EBV-seronegative individuals, confirming the specificity of this assay. In addition, the use of four color cytometry (CD45, CD3, CD69, IFNgamma/IL-2) allows the total quantitative assessment of EBV-specific T-cells while monitoring the interference of EBV non-specific mitogenic activity. This method may have significant utility for the monitoring of the immune response to latent virus infection/reactivation.

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.

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.

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

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).

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

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

Multiplex polymerase chain reaction on FTA cards vs. flow cytometry for B-lymphocyte clonality.

PubMed

Dictor, Michael; Skogvall, Ingela; Warenholt, Janina; Rambech, Eva

2007-01-01

Two-colour flow cytometry was compared with multiplex PCR with capillary electrophoresis for clonality determination in specific categories of B-cell lymphoma. FTA cards were evaluated for preserving DNA from node imprints and expediting molecular analysis. A single-tube multiplex PCR targeted IGH and lymphoma-specific translocations in DNA extracted from 180 frozen lymphoid tissues and DNA bound to FTA cards from 192 fresh tissues and 137 aspirates. PCR results were compared with flow cytometry in the extracted and aspirated samples. Overall, single-tube multiplex PCR sensitivity was equivalent in the sample groups (intergroup range 79%-91%). False negatives were associated with tumour origin in the follicle centre. Multiplex PCR and flow cytometry were equally sensitive and together detected 98% of B-cell lymphomas. Additional two-tube targeting of IGK suggested an overall molecular sensitivity >90%. False positive (pseudoclonal) single-tube multiplex PCR was associated with necrosis and sparse lymphocytes. Multiplex PCR using template DNA bound to an FTA card effectively detects B-lymphocyte clonality, obviates DNA extraction and refrigeration, and can be used without diminished sensitivity in fine needle aspirates or node imprints as a replacement for or complement to flow cytometry at any point in the diagnostic work-up.

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),...

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.

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...

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

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

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.

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.

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

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.

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.

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.

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.

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...

Fountain Flow cytometry, a new technique for the rapid detection and enumeration of microorganisms in aqueous samples.

PubMed

Johnson, Paul E; Deromedi, Anthony J; Lebaron, Philippe; Catala, Philippe; Cash, Jennifer

2006-12-01

Pathogenic microorganisms are known to cause widespread waterborne disease worldwide. There is an urgent need to develop a technique for the real-time detection of pathogens in environmental samples at low concentrations, <10 microorganisms/ml, in large sample volumes, > or =100 ml. A novel method, Fountain Flowtrade mark cytometry, for the rapid and sensitive detection of individual microorganisms in aqueous samples is presented. Each sample is first incubated with a fluorescent label and then passed as a stream in front of a laser, which excites the label. The fluorescence is detected with a CCD imager as the sample flows toward the imager along its optical axis. The feasibility of Fountain Flow cytometry (FFC) is demonstrated by the detection of Escherichia coli labeled with ChemChrome CV6 and SYBR Gold in buffer and natural river water. Detections of labeled E. coli were made in aqueous suspensions with an efficiency of 96% +/- 14% down to a concentration approximately 200 bacteria/ml. The feasibility of FFC is demonstrated by the detection of E. coli in buffer and natural river water. FFC should apply to the detection of a wide range of pathogenic microorganisms including amoebae.

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

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

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.

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.

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.

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.

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

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

Webcam-based flow cytometer using wide-field imaging for low cell number detection at high throughput.

PubMed

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

2014-09-07

Here we describe a novel low-cost flow cytometer based on a webcam capable of low cell number detection in a large volume which may overcome the limitations of current flow cytometry. Several key elements have been combined to yield both high throughput and high sensitivity. The first element is a commercially available webcam capable of 187 frames per second video capture at a resolution of 320 × 240 pixels. The second element in this design is a 1 W 450 nm laser module for area-excitation, which combined with the webcam allows for rapid interrogation of a flow field. The final element is a 2D flow-cell which overcomes the flow limitation of hydrodynamic focusing and allows for higher sample throughput in a wider flow field. This cell allows for the linear velocity of target cells to be lower than in a conventional "1D" hydrodynamic focusing flow-cells typically used in cytometry at similar volumetric flow rates. It also allows cells to be imaged at the full frame rate of the webcam. Using this webcam-based flow cytometer with wide-field imaging, it was confirmed that the detection of fluorescently tagged 5 μm polystyrene beads in "1D" hydrodynamic focusing flow-cells was not practical for low cell number detection due to streaking from the motion of the beads, which did not occur with the 2D flow-cell design. The sensitivity and throughput of this webcam-based flow cytometer was then investigated using THP-1 human monocytes stained with SYTO-9 florescent dye in the 2D flow-cell. The flow cytometer was found to be capable of detecting fluorescently tagged cells at concentrations as low as 1 cell per mL at flow rates of 500 μL min(-1) in buffer and in blood. The effectiveness of detection was concentration dependent: at 100 cells per mL 84% of the cells were detected compared to microscopy, 10 cells per mL 79% detected and 1 cell per mL 59% of the cells were detected. With the blood samples spiked to 100 cells per mL, the average concentration for all samples was 91.4 cells per mL, with a 95% confidence interval of 86-97 cells per mL. These low cell concentrations and the large volume capabilities of the system may overcome the limitations of current cytometry, and are applicable to rare cell (such as circulating tumor cell) detection The simplicity and low cost of this device suggests that it may have a potential use in developing point-of-care clinical flow cytometry for resource-poor settings associated with global health.

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.

Noninvasive and label-free detection of circulating melanoma cells by in vivo photoacoustic flow cytometry

NASA Astrophysics Data System (ADS)

Yang, Ping; Liu, Rongrong; Niu, Zhenyu; Suo, Yuanzhen; He, Hao; Wei, Xunbin

2015-03-01

Melanoma is a malignant tumor of melanocytes. Circulating melanoma cell has high light absorption due to melanin highly contained in melanoma cells. This property is employed for the detection of circulating melanoma cell by in vivo photoacoustic flow cytometry (PAFC). PAFC is based on photoacoustic effect. Compared to in vivo flow cytometry based on fluorescence, PAFC can employ high melanin content of melanoma cells as endogenous biomarkers to detect circulating melanoma cells in vivo. In our research, we developed in vitro experiments to prove the ability of PAFC system of detecting PA signals from melanoma cells. For in vivo experiments, we constructed a model of melanoma tumor bearing mice by inoculating highly metastatic murine melanoma cancer cells B16F10 with subcutaneous injection. PA signals were detected in the blood vessels of mouse ears in vivo. By counting circulating melanoma cells termly, we obtained the number variation of circulating melanoma cells as melanoma metastasized. Those results show that PAFC is a noninvasive and label-free method to detect melanoma metastases in blood or lymph circulation. Our PAFC system is an efficient tool to monitor melanoma metastases, cancer recurrence and therapeutic efficacy.

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.

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.

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.

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.

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.

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.

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.

Mouse monoclonal antibodies against human c-Mpl and characterization for flow cytometry applications.

PubMed

Abbott, Christina; Huang, Guo; Ellison, Aaron R; Chen, Ching; Arora, Taruna; Szilvassy, Stephen J; Wei, Ping

2010-04-01

Mouse monoclonal antibodies (MAbs) against human c-Mpl, the cognate receptor for thrombopoietin (TPO), were generated using hybridoma technology and characterized by various assays to demonstrate their specificity and affinity. Two such MAbs, 1.6 and 1.75, were determined to be superior for flow cytometry studies and exhibited double-digit picomolar (pM) affinities to soluble human c-Mpl protein. Both MAbs specifically bound to cells engineered to overexpress human c-Mpl protein, immortalized human hematopoietic cell lines that express endogenous c-Mpl, primary human bone marrow and peripheral blood-derived CD34(+) cells, and purified human platelets. No binding was detected on cell lines that did not express c-Mpl. Receptor competition and siRNA knock-down studies further confirmed the specificity of antibodies 1.6 and 1.75 for human c-Mpl. In contrast to these newly generated MAbs, none of eight commercially available anti-c-Mpl antibodies tested were found to bind specifically to human c-Mpl and were thus shown to be unsuitable for flow cytometry studies. Monoclonal antibodies 1.6 and 1.75 will therefore be useful flow cytometry reagents to detect cell surface c-Mpl expression.

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.

[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.

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.

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.

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.

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.

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.

Characterization and use of a rabbit-anti-mouse VPAC1 antibody by flow cytometry

PubMed Central

Hermann, Rebecca J.; Van der Steen, Travis; Vomhof-DeKrey, Emilie E.; Benton, Keith D.; Failing, Jarrett J.; Haring, Jodie S.; Dorsam, Glenn P.

2011-01-01

Vasoactive intestinal peptide receptor – 1 signaling in lymphocytes has been shown to regulate chemotaxis, proliferation, apoptosis and differentiation. During T cell activation, VPAC1 mRNA is downregulated, but the effect on its protein levels is less clear. A small number of studies have reported measurement of human VPAC1 by flow cytometry, but murine VPAC1 reagents are unavailable. Therefore, we set out to generate a reliable and highly specific α-mouse VPAC1 polyclonal antibody for use with flow cytometry. After successfully generating a rabbit α-VPAC1 polyclonal antibody (α-mVPAC1 pAb), we characterized its cross-reactivity and showed that it does not recognize other family receptors (mouse VPAC2 and PAC1, human VPAC1, VPAC2 and PAC1) by flow cytometry. Partial purification of the rabbit α-VPAC1 sera increased the specific-activity of the α-mVPAC1 pAb by 20-fold, and immunofluorescence microscopy (IF) confirmed a plasma membrane subcellular localization for mouse VPAC1 protein. To test the usefulness of this specific α-mVPAC1 pAb, we showed that primary, resting mouse T cells express detectable levels of VPAC1 protein, with little detectable signal from activated T cells, or CD19 B cells. These data support our previously published data showing a downregulation of VPAC1 mRNA during T cell activation. Collectively, we have established a well-characterized, and highly species specific α-mVPAC1 pAb for VPAC1 surface measurement by IF and flow cytometry. PMID:22079255

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.

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.

A Noninvasive and Real-Time Method for Circulating Tumor Cell Detection by In Vivo Flow Cytometry.

PubMed

Wei, Xunbin; Zhou, Jian; Zhu, Xi; Yang, Xinrong; Yang, Ping; Wang, Qiyan

2017-01-01

The quantification of circulating tumor cells (CTCs) has been considered a potentially powerful tool in cancer diagnosis and prognosis, as CTCs have been shown to appear very early in cancer development. Great efforts have been made to develop methods that were less invasive and more sensitive to detect CTCs earlier. There is growing evidence that CTC clusters have greater metastatic potential than single CTCs. Therefore, the detection of CTC clusters is also important. This chapter is aimed to introduce a noninvasive technique for CTCs detection named in vivo flow cytometry (IVFC), which has been demonstrated to be capable of monitoring CTCs dynamics continuously. Furthermore, IVFC could be helpful for CTC cluster enumeration.

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.

Ferroptosis and Cell Death Analysis by Flow Cytometry.

PubMed

Chen, Daishi; Eyupoglu, Ilker Y; Savaskan, Nicolai

2017-01-01

Cell death and its recently discovered regulated form ferroptosis are characterized by distinct morphological, electrophysiological, and pharmacological features. In particular ferroptosis can be induced by experimental compounds and clinical drugs (i.e., erastin, sulfasalazine, sorafenib, and artesunate) in various cell types and cancer cells. Pharmacologically, this cell death process can be inhibited by iron chelators and lipid peroxidation inhibitors. Relevance of this specific cell death form has been found in different pathological conditions such as cancer, neurotoxicity, neurodegeneration, and ischemia. Distinguishing cell viability and cell death is essential for experimental and clinical applications and a key component in flow cytometry experiments. Dead cells can compromise the integrity of the data by nonspecific binding of antibodies and dyes. Therefore it is essential that dead cells are robustly and reproducibly identified and characterized by means of cytometry application. Here we describe a procedure to detect and quantify cell death and its specific form ferroptosis based on standard flow cytometry techniques.

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.

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.

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

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.

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

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.

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 ...

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

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.

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.

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.

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.

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.

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.

The role of multiparametric flow cytometry in the detection of minimal residual disease in acute leukaemia.

PubMed

Lee, Denise; Grigoriadis, George; Westerman, David

2015-12-01

Flow cytometry is the most accessible method for minimal residual disease (MRD) detection due to its availability in most haematological centres. Using a precise combination of different antibodies, immunophenotypic detection of MRD in acute leukaemia can be performed by identifying abnormal combinations or expressions of antigens on malignant cells at diagnosis, during and post treatment. These abnormal phenotypes, referred to as leukaemia-associated immunophenotypes (LAIPs) are either absent or expressed at low frequency in normal bone marrow (BM) cells and are used to monitor the behaviour and quantitate the amount of residual disease following treatment. In paediatric acute lymphoblastic leukaemia (ALL), the level of MRD by multiparametric flow cytometry (MPFC) during therapy is recognised as an important predictor of outcome. Although less extensively studied, adult ALL and adult and paediatric acute myeloid leukaemia (AML) have also demonstrated similar findings. The challenge now is incorporating this information for risk-stratification so that therapy can be tailored individually and ultimately improve outcome while also limiting treatment-related toxicity. In this review we will elaborate on the current and future role of MPFC in MRD in acute leukaemia while also addressing its limitations.

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.

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.

In vivo flow cytometry of circulating clots using negative photothermal and photoacoustic contrasts.

PubMed

Galanzha, Ekaterina I; Sarimollaoglu, Mustafa; Nedosekin, Dmitry A; Keyrouz, Salah G; Mehta, Jawahar L; Zharov, Vladimir P

2011-10-01

Conventional photothermal (PT) and photoacousic (PA) imaging, spectroscopy, and cytometry are preferentially based on positive PT/PA effects, when signals are above background. Here, we introduce PT/PA technique based on detection of negative signals below background. Among various new applications, we propose label-free in vivo flow cytometry of circulating clots. No method has been developed for the early detection of clots of different compositions as a source of thromboembolism including ischemia at strokes and myocardial infarction. When a low-absorbing, platelet-rich clot passes a laser-irradiated vessel volume, a transient decrease in local absorption results in an ultrasharp negative PA hole in blood background. Using this phenomenon alone or in combination with positive contrasts, we demonstrated identification of white, red, and mixed clots on a mouse model of myocardial infarction and human blood. The concentration and size of clots were measured with threshold down to few clots in the entire circulation with size as low as 20 μm. This multiparameter diagnostic platform using portable personal high-speed flow cytometer with negative dynamic contrast mode has potential to real-time defining risk factors for cardiovascular diseases, and for prognosis and prevention of stroke or use clot count as a marker of therapy efficacy. Possibility for label-free detection of platelets, leukocytes, tumor cells or targeting themby negative PA probes (e.g., nonabsorbing beads or bubbles) is also highlighted. Copyright © 2011 International Society for Advancement of Cytometry.

Flow cytometry combined with viSNE for the analysis of microbial biofilms and detection of microplastics

PubMed Central

Sgier, Linn; Freimann, Remo; Zupanic, Anze; Kroll, Alexandra

2016-01-01

Biofilms serve essential ecosystem functions and are used in different technical applications. Studies from stream ecology and waste-water treatment have shown that biofilm functionality depends to a great extent on community structure. Here we present a fast and easy-to-use method for individual cell-based analysis of stream biofilms, based on stain-free flow cytometry and visualization of the high-dimensional data by viSNE. The method allows the combined assessment of community structure, decay of phototrophic organisms and presence of abiotic particles. In laboratory experiments, it allows quantification of cellular decay and detection of survival of larger cells after temperature stress, while in the field it enables detection of community structure changes that correlate with known environmental drivers (flow conditions, dissolved organic carbon, calcium) and detection of microplastic contamination. The method can potentially be applied to other biofilm types, for example, for inferring community structure for environmental and industrial research and monitoring. PMID:27188265

Detection and capture of breast cancer cells with photoacoustic flow cytometry

NASA Astrophysics Data System (ADS)

Bhattacharyya, Kiran; Goldschmidt, Benjamin S.; Viator, John A.

2016-08-01

According to the Centers for Disease Control and Prevention, breast cancer is the most common cancer and the second leading cause of cancer related deaths among women. Metastasis-the presence of secondary tumors caused by the spread of cancer cells via the circulatory or lymphatic systems-significantly worsens the prognosis of any breast cancer patient. A technique is developed to detect circulating breast cancer cells in human blood using a photoacoustic flow cytometry method. A Q-switched laser is used to interrogate thousands of blood cells with one pulse as they flow through the beam path. Cells that are optically absorbing, either naturally or artificially, emit an ultrasound wave as a result of the photoacoustic (PA) effect. Breast cancer cells are targeted with chromophores through immunochemistry in order to enhance optical absorption. After which, the PA cytometry device is calibrated to demonstrate the ability to detect single cells. Cultured breast cancer cells are added to whole blood to reach a biologically relevant concentration of about 25 to 45 breast cancer cells per 1 mL of blood. An in vitro PA flow cytometer is used to detect and isolate these cells followed by capture with the use of a micromanipulator. This method can not only be used to determine the disease state of the patient and the response to therapy but also it can be used for genetic testing and in vitro drug trials since the circulating cell can be captured and studied.

Two-photon in vivo flow cytometry using a fiber probe

NASA Astrophysics Data System (ADS)

Chang, Yu-Chung; Ye, Jing Yong; Thomas, Thommey P.; Cao, Zhengyi; Kotlyar, Alina; Tkaczyk, Eric R.; Baker, James R., Jr.; Norris, Theodore B.

2009-02-01

We have demonstrated the use of a double-clad fiber probe to conduct two-photon excited flow cytometry in vitro and in vivo. We conducted two-channel detection to measure fluorescence at two distinct wavelengths simultaneously. Because the scattering and absorption problems from whole blood were circumvented by the fiber probe, the detected signal strength from the cells were found to be similar in PBS and in whole blood. We achieved the same detection efficiency of the membrane-binding lipophilic dye DiD labeled cells in PBS and in whole blood. High detection efficiency of green fluorescent protein (GFP)-expressing cells in whole blood was demonstrated. DiD-labeled untransfected and GFP-transfected cells were injected into live mice and the circulation dynamics of the externally injected cells were monitored. The detection efficiency of GFP-expressing cells in vivo was consistent with that observed in whole blood.

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.

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.

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

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.

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.

Characterisation of the immune response to type I collagen in scleroderma

PubMed Central

Warrington, Kenneth J; Nair, Usha; Carbone, Laura D; Kang, Andrew H; Postlethwaite, Arnold E

2006-01-01

This study was conducted to examine the frequency, phenotype, and functional profile of T lymphocytes that proliferate in response to type I collagen (CI) in patients with scleroderma (SSc). Peripheral blood mononuclear cells (PBMCs) from SSc patients, healthy controls, and rheumatoid arthritis disease controls were labeled with carboxy-fluorescein diacetate, succinimidyl ester (CFSE), cultured with or without antigen (bovine CI) for 14 days, and analysed by flow cytometry. Surface markers of proliferating cells were identified by multi-color flow cytometry. T-cell lines were derived after sorting for proliferating T cells (CFSElow). Cytokine expression in CI-responsive T cells was detected by intracellular staining/flow cytometry and by multiplex cytokine bead assay (Bio-Plex). A T-cell proliferative response to CI was detected in 8 of 25 (32%) SSc patients, but was infrequent in healthy or disease controls (3.6%; p = 0.009). The proliferating T cells expressed a CD4+, activated (CD25+), memory (CD45RO+) phenotype. Proliferation to CI did not correlate with disease duration or extent of skin involvement. T-cell lines were generated using in vitro CI stimulation to study the functional profile of these cells. Following activation of CI-reactive T cells, we detected intracellular interferon (IFN)-γ but not interleukin (IL)-4 by flow cytometry. Supernatants from the T-cell lines generated in vitro contained IL-2, IFN-γ, GM-CSF (granulocyte macrophage-colony-stimulating factor), and tumour necrosis factor-α, but little or no IL-4 and IL-10, suggesting that CI-responsive T cells express a predominantly Th1 cytokine pattern. In conclusion, circulating memory CD4 T cells that proliferate to CI are present in a subset of patients with SSc, but are infrequent in healthy or disease controls. PMID:16879746

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.

Multi-channel imaging cytometry with a single detector

NASA Astrophysics Data System (ADS)

Locknar, Sarah; Barton, John; Entwistle, Mark; Carver, Gary; Johnson, Robert

2018-02-01

Multi-channel microscopy and multi-channel flow cytometry generate high bit data streams. Multiple channels (both spectral and spatial) are important in diagnosing diseased tissue and identifying individual cells. Omega Optical has developed techniques for mapping multiple channels into the time domain for detection by a single high gain, high bandwidth detector. This approach is based on pulsed laser excitation and a serial array of optical fibers coated with spectral reflectors such that up to 15 wavelength bins are sequentially detected by a single-element detector within 2.5 μs. Our multichannel microscopy system uses firmware running on dedicated DSP and FPGA chips to synchronize the laser, scanning mirrors, and sampling clock. The signals are digitized by an NI board into 14 bits at 60MHz - allowing for 232 by 174 pixel fields in up to 15 channels with 10x over sampling. Our multi-channel imaging cytometry design adds channels for forward scattering and back scattering to the fluorescence spectral channels. All channels are detected within the 2.5 μs - which is compatible with fast cytometry. Going forward, we plan to digitize at 16 bits with an A-toD chip attached to a custom board. Processing these digital signals in custom firmware would allow an on-board graphics processing unit to display imaging flow cytometry data over configurable scanning line lengths. The scatter channels can be used to trigger data buffering when a cell is present in the beam. This approach enables a low cost mechanically robust imaging cytometer.

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.

Detection of Silver Nanoparticles in Cells by Flow Cytometry Using Light Scattering and Far-red Fluorescence

EPA Science Inventory

The cellular uptake of different sized silver nanoparticles (l0 nm, 50 nm, and 75nm) coated with polyvinylpyrrolidone (PVP) or citrate in ARPE-19 cells following 24 hour incubation was detected by side scatter through the use of a flow cytometer. A large far red fluorescence sign...

OpenCyto: An Open Source Infrastructure for Scalable, Robust, Reproducible, and Automated, End-to-End Flow Cytometry Data Analysis

PubMed Central

Finak, Greg; Frelinger, Jacob; Jiang, Wenxin; Newell, Evan W.; Ramey, John; Davis, Mark M.; Kalams, Spyros A.; De Rosa, Stephen C.; Gottardo, Raphael

2014-01-01

Flow cytometry is used increasingly in clinical research for cancer, immunology and vaccines. Technological advances in cytometry instrumentation are increasing the size and dimensionality of data sets, posing a challenge for traditional data management and analysis. Automated analysis methods, despite a general consensus of their importance to the future of the field, have been slow to gain widespread adoption. Here we present OpenCyto, a new BioConductor infrastructure and data analysis framework designed to lower the barrier of entry to automated flow data analysis algorithms by addressing key areas that we believe have held back wider adoption of automated approaches. OpenCyto supports end-to-end data analysis that is robust and reproducible while generating results that are easy to interpret. We have improved the existing, widely used core BioConductor flow cytometry infrastructure by allowing analysis to scale in a memory efficient manner to the large flow data sets that arise in clinical trials, and integrating domain-specific knowledge as part of the pipeline through the hierarchical relationships among cell populations. Pipelines are defined through a text-based csv file, limiting the need to write data-specific code, and are data agnostic to simplify repetitive analysis for core facilities. We demonstrate how to analyze two large cytometry data sets: an intracellular cytokine staining (ICS) data set from a published HIV vaccine trial focused on detecting rare, antigen-specific T-cell populations, where we identify a new subset of CD8 T-cells with a vaccine-regimen specific response that could not be identified through manual analysis, and a CyTOF T-cell phenotyping data set where a large staining panel and many cell populations are a challenge for traditional analysis. The substantial improvements to the core BioConductor flow cytometry packages give OpenCyto the potential for wide adoption. It can rapidly leverage new developments in computational cytometry and facilitate reproducible analysis in a unified environment. PMID:25167361

OpenCyto: an open source infrastructure for scalable, robust, reproducible, and automated, end-to-end flow cytometry data analysis.

PubMed

Finak, Greg; Frelinger, Jacob; Jiang, Wenxin; Newell, Evan W; Ramey, John; Davis, Mark M; Kalams, Spyros A; De Rosa, Stephen C; Gottardo, Raphael

2014-08-01

Flow cytometry is used increasingly in clinical research for cancer, immunology and vaccines. Technological advances in cytometry instrumentation are increasing the size and dimensionality of data sets, posing a challenge for traditional data management and analysis. Automated analysis methods, despite a general consensus of their importance to the future of the field, have been slow to gain widespread adoption. Here we present OpenCyto, a new BioConductor infrastructure and data analysis framework designed to lower the barrier of entry to automated flow data analysis algorithms by addressing key areas that we believe have held back wider adoption of automated approaches. OpenCyto supports end-to-end data analysis that is robust and reproducible while generating results that are easy to interpret. We have improved the existing, widely used core BioConductor flow cytometry infrastructure by allowing analysis to scale in a memory efficient manner to the large flow data sets that arise in clinical trials, and integrating domain-specific knowledge as part of the pipeline through the hierarchical relationships among cell populations. Pipelines are defined through a text-based csv file, limiting the need to write data-specific code, and are data agnostic to simplify repetitive analysis for core facilities. We demonstrate how to analyze two large cytometry data sets: an intracellular cytokine staining (ICS) data set from a published HIV vaccine trial focused on detecting rare, antigen-specific T-cell populations, where we identify a new subset of CD8 T-cells with a vaccine-regimen specific response that could not be identified through manual analysis, and a CyTOF T-cell phenotyping data set where a large staining panel and many cell populations are a challenge for traditional analysis. The substantial improvements to the core BioConductor flow cytometry packages give OpenCyto the potential for wide adoption. It can rapidly leverage new developments in computational cytometry and facilitate reproducible analysis in a unified environment.

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

USGS Publications Warehouse

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

1992-01-01

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

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

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.

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.

Improved Diffuse Fluorescence Flow Cytometer Prototype for High Sensitivity Detection of Rare Circulating Cells In Vivo

NASA Astrophysics Data System (ADS)

Pestana, Noah Benjamin

Accurate quantification of circulating cell populations is important in many areas of pre-clinical and clinical biomedical research, for example, in the study of cancer metastasis or the immune response following tissue and organ transplants. Normally this is done "ex-vivo" by drawing and purifying a small volume of blood and then analyzing it with flow cytometry, hemocytometry or microfludic devices, but the sensitivity of these techniques are poor and the process of handling samples has been shown to affect cell viability and behavior. More recently "in vivo flow cytometry" (IVFC) techniques have been developed where fluorescently-labeled cells flowing in a small blood vessel in the ear or retina are analyzed, but the sensitivity is generally poor due to the small sampling volume. To address this, our group recently developed a method known as "Diffuse Fluorescence Flow Cytometry" (DFFC) that allows detection and counting of rare circulating cells with diffuse photons, offering extremely high single cell counting sensitivity. In this thesis, an improved DFFC prototype was designed and validated. The chief improvements were three-fold, i) improved optical collection efficiency, ii) improved detection electronics, and iii) development of a method to mitigate motion artifacts during in vivo measurements. In combination, these improvements yielded an overall instrument detection sensitivity better than 1 cell/mL in vivo, which is the most sensitive IVFC system reported to date. Second, development and validation of a low-cost microfluidic device reader for analysis of ocular fluids is described. We demonstrate that this device has equivalent or better sensitivity and accuracy compared a fluorescence microscope, but at an order-of-magnitude reduced cost with simplified operation. Future improvements to both instruments are also discussed.

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

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

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.

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

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.

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.

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.

Utilization of microparticles in next-generation assays for microflow cytometers.

PubMed

Kim, Jason S; Ligler, Frances S

2010-11-01

Micron-sized particles have primarily been used in microfabricated flow cytometers for calibration purposes and proof-of-concept experiments. With increasing frequency, microparticles are serving as a platform for assays measured in these small analytical devices. Light scattering has been used to measure the agglomeration of antibody-coated particles in the presence of an antigen. Impedance detection is another technology being integrated into microflow cytometers for microparticle-based assays. Fluorescence is the most popular detection method in flow cytometry, enabling highly sensitive multiplexed assays. Finally, magnetic particles have also been used to measure antigen levels using a magnetophoretic micro-device. We review the progress of microparticle-based assays in microflow cytometry in terms of the advantages and limitations of each approach.

Enumeration of Circulating Tumor Cells and Disseminated Tumor Cells in Blood and Bone Marrow by Immunomagnetic Enrichment and Flow Cytometry (IE/FC).

PubMed

Magbanua, Mark Jesus M; Solanki, Tulasi I; Ordonez, Andrea D; Hsiao, Feng; Park, John W

2017-01-01

Enumerating circulating tumor cells (CTCs) in blood and disseminated tumor cells (DTCs) in bone marrow has shown to be clinically useful, as elevated numbers of these cells predict poor clinical outcomes. Accurate detection and quantification is, however, difficult and technically challenging because CTCs and DTCs are extremely rare. We have developed a novel quantitative detection method for enumeration of CTCs and DTCs. Our approach consists of two steps: (1) EPCAM-based immunomagnetic enrichment followed by (2) flow cytometry (IE/FC). The assay takes approximately 2 h to complete. In addition to tumor cell enumeration, IE/FC offers opportunities for direct isolation of highly pure tumor cells for downstream molecular characterization.

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.

Dynamics of DNA replication during premeiosis and early meiosis in wheat.

PubMed

Rey, María-Dolores; Prieto, Pilar

2014-01-01

Meiosis is a specialised cell division that involves chromosome replication, two rounds of chromosome segregation and results in the formation of the gametes. Meiotic DNA replication generally precedes chromosome pairing, recombination and synapsis in sexually developing eukaryotes. In this work, replication has been studied during premeiosis and early meiosis in wheat using flow cytometry, which has allowed the quantification of the amount of DNA in wheat anther in each phase of the cell cycle during premeiosis and each stage of early meiosis. Flow cytometry has been revealed as a suitable and user-friendly tool to detect and quantify DNA replication during early meiosis in wheat. Chromosome replication was detected in wheat during premeiosis and early meiosis until the stage of pachytene, when chromosomes are associated in pairs to further recombine and correctly segregate in the gametes. In addition, the effect of the Ph1 locus, which controls chromosome pairing and affects replication in wheat, was also studied by flow cytometry. Here we showed that the Ph1 locus plays an important role on the length of meiotic DNA replication in wheat, particularly affecting the rate of replication during early meiosis in wheat.

Dynamics of DNA Replication during Premeiosis and Early Meiosis in Wheat

PubMed Central

Rey, María-Dolores; Prieto, Pilar

2014-01-01

Meiosis is a specialised cell division that involves chromosome replication, two rounds of chromosome segregation and results in the formation of the gametes. Meiotic DNA replication generally precedes chromosome pairing, recombination and synapsis in sexually developing eukaryotes. In this work, replication has been studied during premeiosis and early meiosis in wheat using flow cytometry, which has allowed the quantification of the amount of DNA in wheat anther in each phase of the cell cycle during premeiosis and each stage of early meiosis. Flow cytometry has been revealed as a suitable and user-friendly tool to detect and quantify DNA replication during early meiosis in wheat. Chromosome replication was detected in wheat during premeiosis and early meiosis until the stage of pachytene, when chromosomes are associated in pairs to further recombine and correctly segregate in the gametes. In addition, the effect of the Ph1 locus, which controls chromosome pairing and affects replication in wheat, was also studied by flow cytometry. Here we showed that the Ph1 locus plays an important role on the length of meiotic DNA replication in wheat, particularly affecting the rate of replication during early meiosis in wheat. PMID:25275307

Performance of computer vision in vivo flow cytometry with low fluorescence contrast

NASA Astrophysics Data System (ADS)

Markovic, Stacey; Li, Siyuan; Niedre, Mark

2015-03-01

Detection and enumeration of circulating cells in the bloodstream of small animals are important in many areas of preclinical biomedical research, including cancer metastasis, immunology, and reproductive medicine. Optical in vivo flow cytometry (IVFC) represents a class of technologies that allow noninvasive and continuous enumeration of circulating cells without drawing blood samples. We recently developed a technique termed computer vision in vivo flow cytometry (CV-IVFC) that uses a high-sensitivity fluorescence camera and an automated computer vision algorithm to interrogate relatively large circulating blood volumes in the ear of a mouse. We detected circulating cells at concentrations as low as 20 cells/mL. In the present work, we characterized the performance of CV-IVFC with low-contrast imaging conditions with (1) weak cell fluorescent labeling using cell-simulating fluorescent microspheres with varying brightness and (2) high background tissue autofluorescence by varying autofluorescence properties of optical phantoms. Our analysis indicates that CV-IVFC can robustly track and enumerate circulating cells with at least 50% sensitivity even in conditions with two orders of magnitude degraded contrast than our previous in vivo work. These results support the significant potential utility of CV-IVFC in a wide range of in vivo biological models.

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

NASA Astrophysics Data System (ADS)

Wang, Xiaoling; Yang, Ping; Liu, Rongrong; Niu, Zhenyu; Suo, Yuanzhen; He, Hao; Gao, Wenyuan; Tang, Shuo; Wei, Xunbin

2016-03-01

Melanoma is a malignant tumor of melanocytes. Melanoma cells have high light absorption due to melanin highly contained in melanoma cells. This property is employed for the detection of circulating melanoma cell by in vivo photoacoustic flow cytometry (PAFC), which is based on photoacoustic effect. Compared to in vivo flow cytometry based on fluorescence, PAFC can employ high melanin content of melanoma cells as endogenous biomarkers to detect circulating melanoma cells in vivo. We have developed in vitro experiments to prove the ability of PAFC system of detecting photoacoustic signals from melanoma cells. For in vivo experiments, we have constructed a model of melanoma tumor bearing mice by inoculating highly metastatic murine melanoma cancer cells, B16F10 with subcutaneous injection. PA signals are detected in the blood vessels of mouse ears in vivo. The raw signal detected from target cells often contains some noise caused by electronic devices, such as background noise and thermal noise. We choose the Wavelet denoising method to effectively distinguish the target signal from background noise. Processing in time domain and frequency domain would be combined to analyze the signal after denoising. This algorithm contains time domain filter and frequency transformation. The frequency spectrum image of the signal contains distinctive features that can be used to analyze the property of target cells or particles. The processing methods have a great potential for analyzing signals accurately and rapidly. By counting circulating melanoma cells termly, we obtain the number variation of circulating melanoma cells as melanoma metastasized. Those results show that PAFC is a noninvasive and label-free method to detect melanoma metastases in blood or lymph circulation.

Detection and quantification of Epstein-Barr virus EBER1 in EBV-infected cells by fluorescent in situ hybridization and flow cytometry

NASA Technical Reports Server (NTRS)

Stowe, R. P.; Cubbage, M. L.; Sams, C. F.; Pierson, D. L.; Barrett, A. D.

1998-01-01

A rapid and highly sensitive fluorescent in situ hybridization (FISH) assay was developed to detect Epstein Barr virus (EBV)-infected cells in peripheral blood. Multiple fluorescein-labeled antisense oligonucleotide probes were designed to hybridize to the EBER1 transcript, which is highly expressed in latently infected cells. After a rapid (30 min) hybridization, the cells were analyzed by flow cytometry. EBER1 was detected in several positive control cell lines that have variable numbers of EBV genome copies. No EBER1 was detected in two known EBV-negative cell lines. Northern blot analyses confirmed the presence and quantity of EBER1 transcripts in each cell line. This method was used to quantify the number of EBV-infected cells in peripheral blood from a patient with chronic mononucleosis. These results indicate that EBV-infected cells can be detected at the single cell level, and that this assay can be used to quantify the number of EBV-infected cells in clinical samples.

Optimized signal detection and analysis methods for in vivo photoacoustic flow cytometry

NASA Astrophysics Data System (ADS)

Wang, Qiyan; Zhou, Quanyu; Yang, Ping; Wang, Xiaoling; Niu, Zhenyu; Suo, Yuanzhen; He, Hao; Gao, Wenyuan; Tang, Shuo; Wei, Xunbin

2017-02-01

Melanoma is known as a malignant tumor of melanocytes, which usually appear in the blood circulation at the metastasis stage of cancer. Thus the detection of circulating melanoma cells is useful for early diagnosis and therapy of cancer. Here we have developed an in vivo photoacoustic flow cytometry (PAFC) based on the photoacoustic effect to detect melanoma cells. However, the raw signals we obtain from the target cells contain noises such as environmental sonic noises and electronic noises. Therefore we apply correlation comparison and feature separation methods to the detection and verification of the in vivo signals. Due to similar shape and structure of cells, the photoacoustic signals usually have similar vibration mode. By analyzing the correlations and the signal features in time domain and frequency domain, we are able to provide a method for separating photoacoustic signals generated by target cells from background noises. The method introduced here has proved to optimize the signal acquisition and signal processing, which can improve the detection accuracy in PAFC.

Autofluorescence detection of arbuscular mycorrhizal fungal structures in palm roots: an underestimated experimental method.

PubMed

Dreyer, Beatriz; Morte, Asunción; Pérez-Gilabert, Manuela; Honrubia, Mario

2006-08-01

The aim of this study was to reassess the use of autofluorescence for evaluating AM colonization in mycorrhizal roots in the light of criticisms of this method that affirmed that only metabolically inactive arbuscules autofluoresce. It was also investigated whether other mycorrhizal structures, such as hyphae, vesicles and spores, could be detected by autofluorescence, and whether the autofluorescence pattern of AM fungal structures could be exploited methodologically, for example, in the detection and sorting of spores by flow cytometry. Mycorrhizal roots of the palm species Brahea armata, Chamaerops humilis, Phoenix canariensis and Phoenix dactylifera were sectioned and observed by means of fluorescence microscopy. In addition, fungal structures isolated from mycorrhizal roots of P. dactylifera were examined. The same root sections and isolated fungal structures were subjected to vital staining with nitro blue tetrazolium to determine their metabolic state (active or inactive). Moreover, spores of Glomus intraradices, and Glomus clarum were studied by epifluorescence and flow cytometry. Mycorrhizal whole roots of Medicago sativa were also assessed by autofluorescence detection. In contrast to previous reports, the results presented in this paper clearly demonstrate that all fungal structures, both intra- and extraradical, autofluoresced under blue light excitation, regardless of their state (dead or alive). Some arbuscules isolated from roots and mature spores showed further autofluorescence under green light excitation. The source of the autofluorescence was localized in the fungal cell wall. It was shown that AM spores can be detected by flow cytometry. The results support the use of autofluorescence for the evaluation of AM colonization, at least in palm species, and refute previous criticisms of the method.

MRP8/14 induces autophagy to eliminate intracellular Mycobacterium bovis BCG.

PubMed

Wang, Jinli; Huang, Chunyu; Wu, Minhao; Zhong, Qiu; Yang, Kun; Li, Miao; Zhan, Xiaoxia; Wen, Jinsheng; Zhou, Lin; Huang, Xi

2015-04-01

To explore the role of myeloid-related protein 8/14 in mycobacterial infection. The mRNA and protein expression levels of MRP8 or MRP14 were measured by real-time PCR and flow cytometry, respectively. Role of MRP8/14 was tested by overexpression or RNA interference assays. Flow cytometry and colony forming unit were used to test the phagocytosis and the survival of intracellular Mycobacterium bovis BCG (BCG), respectively. Autophagy mediated by MRP8/14 was detected by Western blot and immunofluorescence. The colocalization of BCG phagosomes with autophagosomes or lysosomes was by detected by confocal microscopy. ROS production was detected by flow cytometry. MRP8/14 expressions were up-regulated in human monocytic THP1 cells and primary macrophages after mycobacterial challenge. Silencing of MRP8/14 suppressed bacterial killing, but had no influence on the phagocytosis of BCG. Importantly, silencing MRP8/14 decreased autophagy and BCG phagosome maturation in THP1-derived macrophages, thereby increasing the BCG survival. Additionally, we demonstrated that MRP8/14 promoted autophagy in a ROS-dependent manner. The present study revealed a novel role of MRP8/14 in the autophagy-mediated elimination of intracellular BCG by promoting ROS generation, which may provide a promising therapeutic target for tuberculosis and other intracellular bacterial infectious diseases. Copyright © 2014 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Improving label-free detection of circulating melanoma cells by photoacoustic flow cytometry

NASA Astrophysics Data System (ADS)

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

2018-02-01

Melanoma is a kind of a malignant tumor of melanocytes with the properties of high mortality and high metastasis rate. The circulating melanoma cells with the high content of melanin can be detected by light absorption to diagnose and treat cancer at an early stage. Compared with conventional detection methods such as in vivo flow cytometry (IVFC) based on fluorescence, the in vivo photoacoustic flow cytometry (PAFC) utilizes melanin cells as biomarkers to collect the photoacoustic (PA) signals without toxic fluorescent dyes labeling in a non-invasive way. The information of target tumor cells is helpful for data analysis and cell counting. However, the raw signals in PAFC system contain numerous noises such as environmental noise, device noise and in vivo motion noise. Conventional denoising algorithms such as wavelet denoising (WD) method and means filter (MF) method are based on the local information to extract the data of clinical interest, which remove the subtle feature and leave many noises. To address the above questions, the nonlocal means (NLM) method based on nonlocal data has been proposed to suppress the noise in PA signals. Extensive experiments on in vivo PA signals from the mice with the injection of B16F10 cells in caudal vein have been conducted. All the results indicate that the NLM method has superior noise reduction performance and subtle information reservation.

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.

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.

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.

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.

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.

Tolerance Induction of Temperature and Starvation with Tricalcium Phosphate on Preservation and Sporulation in Bacillus amyloliquefaciens Detected by Flow Cytometry.

PubMed

Shahrokh Esfahani, Samaneh; Emtiazi, Giti; Shafiei, Rasoul; Ghorbani, Najmeh; Zarkesh Esfahani, Seyed Hamid

2016-09-01

The Bacillus species have many applications in the preparation of various enzymes, probiotic, biofertilizer, and biomarkers for which the survival of resting cells and spore formation under different conditions are important. In this study, water and saline along with different mineral substances such as calcium carbonate, calcium phosphate, and silica were used for the detection of survival and preservation of Bacillus amyloliquefaciens. The results showed intensive death of resting cells at 8 °C, but significant survival at 28 °C after one month. However, preservation by minerals significantly decreased the rate of death and induced sporulation at both the temperatures. The resting cells were maintained at room temperature (about 60 % of the initial population survived after a month) in the presence of tricalcium phosphate. The results showed that temperature has more effect on sporulation compare with starvation. The sporulation in normal saline at 28 °C was 70 times more than that at 8 °C; meanwhile, addition of tricalcium phosphate increases sporulation by 90 times. Also, the FTIR data showed the interaction of tricalcium phosphate with spores and resting cells. The discrimination of sporulation from non-sporulation state was performed by nucleic acid staining with thiazole orange and detected by flow cytometry. The flow cytometric studies confirmed that the rates of sporulation in pure water were significantly more at 28 °C. This is the first report on the detection of bacterial spore with thiazole orange by flow cytometry and also on the interaction of tricalcium phosphate with spores by FTIR analyses.

Detection of mitogen-induced lymphocyte proliferation by bromodeoxyuridine (BrdU) incorporation in the chicken.

PubMed

Motobu, Maki; El-Abasy, Moshira; Na, Ki-Jeong; Hirota, Yoshikazu

2002-04-01

3H-thymidine (3H-TdR) incorporation assay has been generally used to measure lymphocyte proliferation in the chicken. Disadvantages of this assay are that radioisotope is biological hazard to the person and environment and that it can not measure which subset of lymphocytes proliferates. In this study, bromodeoxyuridine (BrdU) incorporation assay by flow cytometry was compared with 3H-TdR incorporation assay. As a result, BrdU incorporation assay showed a strong correlation with 3H-TdR incorporation assay, and it could be applied simultaneously to detect BrdU incorporation and expression of cell surface marker antigens. These results suggest that the BrdU incorporation assay by flow cytometry is useful to analyze lymphocyte proliferation in detail.

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

Novel full-spectral flow cytometry with multiple spectrally-adjacent fluorescent proteins and fluorochromes and visualization of in vivo cellular movement.

PubMed

Futamura, Koji; Sekino, Masashi; Hata, Akihiro; Ikebuchi, Ryoyo; Nakanishi, Yasutaka; Egawa, Gyohei; Kabashima, Kenji; Watanabe, Takeshi; Furuki, Motohiro; Tomura, Michio

2015-09-01

Flow cytometric analysis with multicolor fluoroprobes is an essential method for detecting biological signatures of cells. Here, we present a new full-spectral flow cytometer (spectral-FCM). Unlike conventional flow cytometer, this spectral-FCM acquires the emitted fluorescence for all probes across the full-spectrum from each cell with 32 channels sequential PMT unit after dispersion with prism, and extracts the signals of each fluoroprobe based on the spectral shape of each fluoroprobe using unique algorithm in high speed, high sensitive, accurate, automatic and real-time. The spectral-FCM detects the continuous changes in emission spectra from green to red of the photoconvertible protein, KikGR with high-spectral resolution and separates spectrally-adjacent fluoroprobes, such as FITC (Emission peak (Em) 519 nm) and EGFP (Em 507 nm). Moreover, the spectral-FCM can measure and subtract autofluorescence of each cell providing increased signal-to-noise ratios and improved resolution of dim samples, which leads to a transformative technology for investigation of single cell state and function. These advances make it possible to perform 11-color fluorescence analysis to visualize movement of multilinage immune cells by using KikGR-expressing mice. Thus, the novel spectral flow cytometry improves the combinational use of spectrally-adjacent various FPs and multicolor fluorochromes in metabolically active cell for the investigation of not only the immune system but also other research and clinical fields of use. © 2015 International Society for Advancement of Cytometry.

Detecting endotoxin with a flow cytometry-based magnetic aptasensor.

PubMed

Zuo, Ming-Yan; Chen, Li-Juan; Jiang, Hao; Tan, Lin; Luo, Zhao-Feng; Wang, Yan-Mei

2014-12-01

Endotoxin, which is also known as lipopolysaccharide (LPS), is a marker for intruding gram-negative pathogens. It is essential to detect endotoxin quickly and sensitively in a complex milieu. A new flow cytometry (FCM)-based magnetic aptasensor assay that employs two endotoxin-binding aptamers and magnetic beads has been developed to detect endotoxin. The endotoxin-conjugated sandwich complex on magnetic beads was observed by scanning confocal laser microscopy. The resulting magnetic aptasensor rapidly detected (<1 min) endotoxin within a broad dynamic detection range of 10(-8) to 10(0)mg/ml in the presence of bovine serum albumin (BSA), RNA, sucrose, and glucose, which are most likely to coexist with endotoxin in the majority of biological liquids. Only 2 μl of magnetic aptasensor was required to quantify the endotoxin solution. Furthermore, the magnetic aptasensor could be regenerated seven times and still presented an outstanding response to the endotoxin solution. Therefore, the magnetic aptasensor exhibited high sensitivity, selectivity, and reproducibility, thereby serving as a powerful tool for the quality control and high-throughput detection of endotoxin in the food and pharmaceutical industries. Copyright © 2014 Elsevier Inc. All rights reserved.

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.

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

Red and near-infrared fluorophores inspired by chlorophylls: consideration of practical brightness in multicolor flow cytometry and biomedical sciences

NASA Astrophysics Data System (ADS)

Taniguchi, Masahiko; Hu, Gongfang; Liu, Rui; Du, Hai; Lindsey, Jonathan S.

2018-02-01

Demands in flow cytometry for increased multiplexing (for detection of multiple antigens) and brightness (for detection of rare entities) require new fluorophores (i.e., "colors") with spectrally distinct fluorescence outside the relatively congested visible spectral region. Flow cytometry fluorophores typically must function in aqueous solution upon bioconjugation and ideally should exhibit a host of photophysical features: (i) strong absorption, (ii) sizable Stokes shift, (iii) modest if not strong fluorescence, and (iv) narrow fluorescence band. Tandem dyes have long been pursued to achieve a large effective Stokes shift, increased brightness, and better control over the excitation and emission wavelengths. Here, the attractive photophysical features of chlorophylls and bacteriochlorophylls - Nature's chosen photoactive pigments for photosynthesis - are described with regards to use in flow cytometry. A chlorophyll (or bacteriochlorophyll) constitutes an intrinsic tandem dye given the red (or near-infrared) fluorescence upon excitation in the higher energy ultraviolet (UV) or visible absorption bands (due to rapid internal conversion to the lowest energy state). Synthetic (bacterio)chlorins are available with strong absorption (near-UV molar absorption coefficient ɛ(λexc) 105 M-1cm-1), modest fluorescence quantum yield (Φf = 0.05-0.30), and narrow fluorescence band (10-25 nm) tunable from 600-900 nm depending on synthetic design. The "relative practical brightness" is given by intrinsic brightness [ɛ(λexc) x Φf] times ηf, the fraction of the fluorescence band that is captured by an emission filter in a multicolor experiment. The spectroscopic features of (bacterio)chlorins are evaluated quantitatively to illustrate practical brightness for this novel class of fluorophores in a prospective 8-color panel.

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.

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.

Avoiding false positive antigen detection by flow cytometry on blood cell derived microparticles: the importance of an appropriate negative control.

PubMed

Crompot, Emerence; Van Damme, Michael; Duvillier, Hugues; Pieters, Karlien; Vermeesch, Marjorie; Perez-Morga, David; Meuleman, Nathalie; Mineur, Philippe; Bron, Dominique; Lagneaux, Laurence; Stamatopoulos, Basile

2015-01-01

Microparticles (MPs), also called microvesicles (MVs) are plasma membrane-derived fragments with sizes ranging from 0.1 to 1μm. Characterization of these MPs is often performed by flow cytometry but there is no consensus on the appropriate negative control to use that can lead to false positive results. We analyzed MPs from platelets, B-cells, T-cells, NK-cells, monocytes, and chronic lymphocytic leukemia (CLL) B-cells. Cells were purified by positive magnetic-separation and cultured for 48h. Cells and MPs were characterized using the following monoclonal antibodies (CD19,20 for B-cells, CD3,8,5,27 for T-cells, CD16,56 for NK-cells, CD14,11c for monocytes, CD41,61 for platelets). Isolated MPs were stained with annexin-V-FITC and gated between 300nm and 900nm. The latex bead technique was then performed for easy detection of MPs. Samples were analyzed by Transmission (TEM) and Scanning Electron microscopy (SEM). Annexin-V positive events within a gate of 300-900nm were detected and defined as MPs. Our results confirmed that the characteristic antigens CD41/CD61 were found on platelet-derived-MPs validating our technique. However, for MPs derived from other cell types, we were unable to detect any antigen, although they were clearly expressed on the MP-producing cells in the contrary of several data published in the literature. Using the latex bead technique, we confirmed detection of CD41,61. However, the apparent expression of other antigens (already deemed positive in several studies) was determined to be false positive, indicated by negative controls (same labeling was used on MPs from different origins). We observed that mother cell antigens were not always detected on corresponding MPs by direct flow cytometry or latex bead cytometry. Our data highlighted that false positive results could be generated due to antibody aspecificity and that phenotypic characterization of MPs is a difficult field requiring the use of several negative controls.

Avoiding False Positive Antigen Detection by Flow Cytometry on Blood Cell Derived Microparticles: The Importance of an Appropriate Negative Control

PubMed Central

Crompot, Emerence; Van Damme, Michael; Duvillier, Hugues; Pieters, Karlien; Vermeesch, Marjorie; Perez-Morga, David; Meuleman, Nathalie; Mineur, Philippe; Bron, Dominique; Lagneaux, Laurence; Stamatopoulos, Basile

2015-01-01

Background Microparticles (MPs), also called microvesicles (MVs) are plasma membrane-derived fragments with sizes ranging from 0.1 to 1μm. Characterization of these MPs is often performed by flow cytometry but there is no consensus on the appropriate negative control to use that can lead to false positive results. Materials and Methods We analyzed MPs from platelets, B-cells, T-cells, NK-cells, monocytes, and chronic lymphocytic leukemia (CLL) B-cells. Cells were purified by positive magnetic-separation and cultured for 48h. Cells and MPs were characterized using the following monoclonal antibodies (CD19,20 for B-cells, CD3,8,5,27 for T-cells, CD16,56 for NK-cells, CD14,11c for monocytes, CD41,61 for platelets). Isolated MPs were stained with annexin-V-FITC and gated between 300nm and 900nm. The latex bead technique was then performed for easy detection of MPs. Samples were analyzed by Transmission (TEM) and Scanning Electron microscopy (SEM). Results Annexin-V positive events within a gate of 300-900nm were detected and defined as MPs. Our results confirmed that the characteristic antigens CD41/CD61 were found on platelet-derived-MPs validating our technique. However, for MPs derived from other cell types, we were unable to detect any antigen, although they were clearly expressed on the MP-producing cells in the contrary of several data published in the literature. Using the latex bead technique, we confirmed detection of CD41,61. However, the apparent expression of other antigens (already deemed positive in several studies) was determined to be false positive, indicated by negative controls (same labeling was used on MPs from different origins). Conclusion We observed that mother cell antigens were not always detected on corresponding MPs by direct flow cytometry or latex bead cytometry. Our data highlighted that false positive results could be generated due to antibody aspecificity and that phenotypic characterization of MPs is a difficult field requiring the use of several negative controls. PMID:25978814

Receptor interacting protein 3-induced RGC-5 cell necroptosis following oxygen glucose deprivation.

PubMed

Ding, Wei; Shang, Lei; Huang, Ju-Fang; Li, Na; Chen, Dan; Xue, Li-Xiang; Xiong, Kun

2015-08-04

Necroptosis is a type of regulated form of cell death that has been implicated in the pathogenesis of various diseases. Receptor-interacting protein 3 (RIP3), a member of the RIP family of proteins, has been reported as an important necroptotic pathway mediator in regulating a variety of human diseases, such as myocardial ischemia, inflammatory bowel disease, and ischemic brain injury. Our previous study showed that RIP3 was expressed in rat retinal ganglion cells (RGCs), where it was significantly upregulated during the early stage of acute high intraocular pressure. Furthermore, RIP3 expression was co-localized with propidium iodide (PI)-positive staining (necrotic cells). These results suggested that RIP3 up-regulation might be involved in the necrosis of injured RGCs. In this study, we aimed to reveal the possible involvement of RIP3 in oxygen glucose deprivation (OGD)-induced retinal ganglion cell-5 (RGC-5) necroptosis. RGC-5 cells were cultured in Dulbecco's-modified essential medium and necroptosis was induced by 8 h OGD. PI staining and flow cytometry were performed to detect RGC-5 necrosis. RIP3 expression was detected by western blot and flow cytometry was used to detect the effect of RIP3 on RGC-5 necroptosis following OGD in rip3 knockdown cells. Malondialdehyde (MDA) lipid peroxidation assay was performed to determine the degree of oxidative stress. PI staining showed that necrosis was present in the early stage of OGD-induced RGC-5 cell death. The presence of RGC-5 necroptosis after OGD was detected by flow cytometry using necrostatin-1, a necroptosis inhibitor. Western blot demonstrated that RIP3 up-regulation may be involved in RGC-5 necroptosis. Flow cytometry revealed that the number of OGD-induced necrotic RGC-5 cells was reduced after rip3 knockdown. Furthermore, MDA levels in the normal RGC-5 cells were much higher than in the rip3-knockdown cells after OGD. Our findings suggest that RGC-5 cell necroptosis following OGD is mediated by a RIP3-induced increase in oxidative stress.

Performance of computer vision in vivo flow cytometry with low fluorescence contrast

PubMed Central

Markovic, Stacey; Li, Siyuan; Niedre, Mark

2015-01-01

Abstract. Detection and enumeration of circulating cells in the bloodstream of small animals are important in many areas of preclinical biomedical research, including cancer metastasis, immunology, and reproductive medicine. Optical in vivo flow cytometry (IVFC) represents a class of technologies that allow noninvasive and continuous enumeration of circulating cells without drawing blood samples. We recently developed a technique termed computer vision in vivo flow cytometry (CV-IVFC) that uses a high-sensitivity fluorescence camera and an automated computer vision algorithm to interrogate relatively large circulating blood volumes in the ear of a mouse. We detected circulating cells at concentrations as low as 20  cells/mL. In the present work, we characterized the performance of CV-IVFC with low-contrast imaging conditions with (1) weak cell fluorescent labeling using cell-simulating fluorescent microspheres with varying brightness and (2) high background tissue autofluorescence by varying autofluorescence properties of optical phantoms. Our analysis indicates that CV-IVFC can robustly track and enumerate circulating cells with at least 50% sensitivity even in conditions with two orders of magnitude degraded contrast than our previous in vivo work. These results support the significant potential utility of CV-IVFC in a wide range of in vivo biological models. PMID:25822954

Prognostic value of bone marrow involvement by clonal immunoglobulin gene rearrangements in follicular lymphoma

PubMed Central

Berget, Ellen; Helgeland, Lars; Liseth, Knut; Løkeland, Turid; Molven, Anders; Vintermyr, Olav Karsten

2014-01-01

Aims We aimed to evaluate the prognostic value of routine use of PCR amplification of immunoglobulin gene rearrangements in bone marrow (BM) staging in patients with follicular lymphoma (FL). Methods Clonal rearrangements were assessed by immunoglobulin heavy and light-chain gene rearrangement analysis in BM aspirates from 96 patients diagnosed with FL and related to morphological detection of BM involvement in biopsies. In 71 patients, results were also compared with concurrent flow cytometry analysis. Results BM involvement was detected by PCR in 34.4% (33/96) of patients. The presence of clonal rearrangements by PCR was associated with advanced clinical stage (I–III vs IV; p<0.001), high FL International Prognostic Index (FLIPI) score (0–1, 2 vs ≥3; p=0.003), and detection of BM involvement by morphology and flow cytometry analysis (p<0.001 for both). PCR-positive patients had a significantly poorer survival than PCR-negative patients (p=0.001, log-rank test). Thirteen patients positive by PCR but without morphologically detectable BM involvement, had significantly poorer survival than patients with negative morphology and negative PCR result (p=0.002). The poor survival associated with BM involvement by PCR was independent of the FLIPI score (p=0.007, Cox regression). BM involvement by morphology or flow cytometry did not show a significant impact on survival. Conclusions Our results showed that routine use of PCR-based clonality analysis significantly improved the prognostic impact of BM staging in patients with FL. BM involvement by PCR was also an independent adverse prognostic factor. PMID:25233852

Detection of microparticles from human red blood cells by multiparametric flow cytometry

PubMed Central

Grisendi, Giulia; Finetti, Elena; Manganaro, Daniele; Cordova, Nicoletta; Montagnani, Giuliano; Spano, Carlotta; Prapa, Malvina; Guarneri, Valentina; Otsuru, Satoru; Horwitz, Edwin M.; Mari, Giorgio; Dominici, Massimo

2015-01-01

Background During storage, red blood cells (RBC) undergo chemical and biochemical changes referred to as “storage lesions”. These events determine the loss of RBC integrity, resulting in lysis and release of microparticles. There is growing evidence of the clinical importance of microparticles and their role in blood transfusion-related side effects and pathogen transmission. Flow cytometry is currently one of the most common techniques used to quantify and characterise microparticles. Here we propose multiparametric staining to monitor and quantify the dynamic release of microparticles by stored human RBC. Material and methods RBC units (n=10) were stored under blood bank conditions for up to 42 days. Samples were tested at different time points to detect microparticles and determine the haemolysis rate (HR%). Microparticles were identified by flow cytometry combining carboxyfluorescein diacetate succinimidyl ester (CFSE) dye, annexin V and anti-glycophorin A antibody. Results We demonstrated that CFSE can be successfully used to label closed vesicles with an intact membrane. The combination of CFSE and glycophorin A antibody was effective for monitoring and quantifying the dynamic release of microparticles from RBC during storage. Double staining with CFSE/glycophorin A was a more precise approach, increasing vesicle detection up to 4.7-fold vs the use of glycophorin A/annexin V alone. Moreover, at all the time points tested, we found a robust correlation (R=0.625; p=0.0001) between HR% and number of microparticles detected. Discussion Multiparametric staining, based on a combination of CFSE, glycophorin A antibody and annexin V, was able to detect, characterise and monitor the release of microparticles from RBC units during storage, providing a sensitive approach to labelling and identifying microparticles for transfusion medicine and, more broadly, for cell-based therapies. PMID:25369588

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

PubMed

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

2018-05-10

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

Combining Cell Type-Restricted Adenoviral Targeting with Immunostaining and Flow Cytometry to Identify Cells-of-Origin of Lung Cancer.

PubMed

Best, Sarah A; Kersbergen, Ariena; Asselin-Labat, Marie-Liesse; Sutherland, Kate D

2018-01-01

Lung cancers display considerable intertumoral heterogeneity, leading to the classification of distinct tumor subtypes. Our understanding of the genetic aberrations that underlie tumor subtypes has been greatly enhanced by recent genomic sequencing studies and state-of-the-art gene targeting technologies, highlighting evidence that distinct lung cancer subtypes may be derived from different "cells-of-origin". Here, we describe the intra-tracheal delivery of cell type-restricted Ad5-Cre viruses into the lungs of adult mice, combined with immunohistochemical and flow cytometry strategies for the detection of lung cancer-initiating cells in vivo.

[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.

[The mechanism of docetaxel-induced apoptosis in human lung cancer cells].

PubMed

Li, Y; Shi, T; Zhao, W

2000-05-01

To study the mechanism of docetaxel-induced apoptosis. Morphological study, DNA gel electrophoresis, flow cytometry and fluorescin labeled Annexin V to detect apoptosis, RT-PCR to detect the gene related with apoptosis. Human lung cancer A549 cells treated with docetaxel induced cell cycle arrest at G2M phase, leading to apoptosis. The morphology of A549 showed nuclear chromatine condensation and fragmentation. Typical ladder pattern of DNA fragmentation was observed. Sub-G1 peak was found by flow cytometry. Transcription of Fas gene was enhanced, while no change in c-myc and bcl-2 genes. Annexin labeling results revealed the co-existence of cell apoptosis and necrosis in docetaxel-treated A549 cells. Docetaxel induces apoptosis and necrosis of human lung cancer. The induction of apoptosis may be related to expression of Fas.

In vivo flow cytometry of circulating clots using negative phototothermal and photoacoustic contrasts

PubMed Central

Galanzha, Ekaterina I.; Sarimollaoglu, Mustafa; Nedosekin, Dmitry A.; Keyrouz, Salah G.; Mehta, Jawahar L.; Zharov, Vladimir P.

2012-01-01

Conventional photothermal (PT) and photoacousic (PA) imaging, spectroscopy, and cytometry are preferentially based on positive PT/PA effects, when signals are above background. Here, we introduce PT/PA technique based on detection of negative signals below background. Among various new applications, we propose label-free in vivo flow cytometry of circulating clots. No method has been developed for the early detection of clots of different compositions as a source of severe thromboembolisms including ischemia at strokes and myocardial dysfunction at heart attack. When a low-absorbing, platelet-rich clot passes a laser-irradiated vessel volume, a transient decrease in local absorption results in an ultrasharp negative PA hole in blood background. Using this phenomenon alone or in combination with positive contrasts, we demonstrated identification of white, red and mixed clots on a mouse model of myocardial infarction and human blood. The concentration and size of clots were measured with threshold down to few clots in the entire circulation with size as low as 20 µm. This multiparameter diagnostic platform using portable personal high-speed flow cytometer with negative dynamic contrast mode has potential to real-time defining risk factors for cardiovascular diseases, and for prognosis and prevention of stroke or use clot count as a marker of therapy efficacy. Possibility for label-free detection of platelets, leukocytes, tumor cells or targeting them by negative PA probes (e.g., nonabsorbing beads or bubbles) is also highlighted. PMID:21976458

Combination of automated high throughput platforms, flow cytometry, and hierarchical clustering to detect cell state.

PubMed

Kitsos, Christine M; Bhamidipati, Phani; Melnikova, Irena; Cash, Ethan P; McNulty, Chris; Furman, Julia; Cima, Michael J; Levinson, Douglas

2007-01-01

This study examined whether hierarchical clustering could be used to detect cell states induced by treatment combinations that were generated through automation and high-throughput (HT) technology. Data-mining techniques were used to analyze the large experimental data sets to determine whether nonlinear, non-obvious responses could be extracted from the data. Unary, binary, and ternary combinations of pharmacological factors (examples of stimuli) were used to induce differentiation of HL-60 cells using a HT automated approach. Cell profiles were analyzed by incorporating hierarchical clustering methods on data collected by flow cytometry. Data-mining techniques were used to explore the combinatorial space for nonlinear, unexpected events. Additional small-scale, follow-up experiments were performed on cellular profiles of interest. Multiple, distinct cellular profiles were detected using hierarchical clustering of expressed cell-surface antigens. Data-mining of this large, complex data set retrieved cases of both factor dominance and cooperativity, as well as atypical cellular profiles. Follow-up experiments found that treatment combinations producing "atypical cell types" made those cells more susceptible to apoptosis. CONCLUSIONS Hierarchical clustering and other data-mining techniques were applied to analyze large data sets from HT flow cytometry. From each sample, the data set was filtered and used to define discrete, usable states that were then related back to their original formulations. Analysis of resultant cell populations induced by a multitude of treatments identified unexpected phenotypes and nonlinear response profiles.

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.

Inhibition of calpain on oxygen glucose deprivation-induced RGC-5 necroptosis.

PubMed

Chen, Shuang; Yan, Jie; Deng, Hai-Xiao; Long, Ling-Ling; Hu, Yong-Jun; Wang, Mi; Shang, Lei; Chen, Dan; Huang, Ju-Fang; Xiong, Kun

2016-10-01

The purpose of this study was to investigate the effect of inhibition of calpain on retinal ganglion cell-5 (RGC-5) necroptosis following oxygen glucose deprivation (OGD). RGC-5 cells were cultured in Dulbecco's-modified essential medium and necroptosis was induced by 8-h OGD. PI staining and flow cytometry were performed to detect RGC-5 necrosis. The calpain expression was detected by Western blotting and immunofluorescence staining. The calpain activity was tested by activity detection kit. Flow cytometry was used to detect the effect of calpain on RGC-5 necroptosis following OGD with or without N-acetyl-leucyl-leucyl-norleucinal (ALLN) pre-treatment. Western blot was used to detect the protein level of truncated apoptosis inducing factor (tAIF) in RGC-5 cells following OGD. The results showed that there was an up-regulation of the calpain expression and activity following OGD. Upon adding ALLN, the calpain activity was inhibited and tAIF was reduced following OGD along with the decreased number of RGC-5 necroptosis. In conclusion, calpain was involved in OGD-induced RGC-5 necroptosis with the increased expression of its downstream molecule tAIF.

Detection of Sirtuin-1 protein expression in peripheral blood leukocytes in dogs.

PubMed

Yoshimura, Kuniko; Matsuu, Aya; Sasaki, Kai; Momoi, Yasuyuki

2018-05-11

Sirtuin-1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD + )-dependent histone deacetylase with a large number of protein substrates. It has attracted a lot of attention in association with extending lifespan. The objective of this study was to enable the evaluation of SIRT1 expression in peripheral blood mononuclear cells (PBMCs) from dogs by flow cytometry. Three transcript variants were amplified from PBMCs by reverse transcription PCR and the nucleotide sequences were analyzed. On the basis deduced amino acid sequence, a monoclonal antibody against human SIRT1, 1F3, was selected to detect canine SIRT1. Canine SIRT1 in peripheral blood mononuclear cells was successfully detected by western blotting using this antibody. Intracellular canine SIRT1 was also detected in permeabilized 293T cells transfected with a canine SIRT1 expression plasmid by flow cytometry using this antibody. SIRT1 was detected in all leukocyte subsets including lymphocytes, granulocytes and monocytes. The expression level was markedly different among individual dogs. These results indicated that the method applied in this study is useful for evaluating canine SIRT1 levels in PBMCs from dogs.

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.

DETECTION AND IDENTIFICATION OF PATHOGENIC CANDIDA SPECIES IN WATER USING FLOW CYTOMETRY COUPLED WITH TAQMAN PCR

EPA Science Inventory

As the incidence of human fungal infection increases, the ability to detect and identify pathogenic fungi in potential environmental reservoirs becomes increasingly important for disease control. PCR based assays are widely used for diagnostic purposes, but may be inadequate for...

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

Monitoring circulating prostate tumor cells after tumor resection by in vivo flow cytometry

NASA Astrophysics Data System (ADS)

Ding, Nan; Zhu, Xi; Xie, Chengying; Wei, Dan; Yang, Zhangru; Suo, Yuanzhen; Wei, Xunbin

2018-02-01

Prostate cancer has already become the biggest threat among all cancer types for male people and many people died because of its bone metastases. Circulating tumor cells (CTCs) can be used as early metastasis marker so that the detection of CTCs in blood is meaningful for early diagnosis and treatment. However, the relationship between these therapies and metastasis has not been fully clarified yet. Hence, we built PC3 subcutaneous tumor model and developed in vivo flow cytometer (IVFC) platform to record the dynamics of CTC before and after tumor resection. We found out that tumor resection can reduce CTC quantities instantaneously while having a good control of metastasis. CTC re-occurred 7 days after surgery, which might be correlated with early disseminated and deposited tumors. In conclusion, in vivo flow cytometry (IVFC) is capable of detecting CTC dynamics in prostate subcutaneous tumor model and this method could facilitate further research about relationship between other cancer therapies and circulating tumor cells.

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

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

PubMed

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

2011-01-01

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

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.

Novel full‐spectral flow cytometry with multiple spectrally‐adjacent fluorescent proteins and fluorochromes and visualization of in vivo cellular movement

PubMed Central

Futamura, Koji; Sekino, Masashi; Hata, Akihiro; Ikebuchi, Ryoyo; Nakanishi, Yasutaka; Egawa, Gyohei; Kabashima, Kenji; Watanabe, Takeshi; Furuki, Motohiro

2015-01-01

Abstract Flow cytometric analysis with multicolor fluoroprobes is an essential method for detecting biological signatures of cells. Here, we present a new full‐spectral flow cytometer (spectral‐FCM). Unlike conventional flow cytometer, this spectral‐FCM acquires the emitted fluorescence for all probes across the full‐spectrum from each cell with 32 channels sequential PMT unit after dispersion with prism, and extracts the signals of each fluoroprobe based on the spectral shape of each fluoroprobe using unique algorithm in high speed, high sensitive, accurate, automatic and real‐time. The spectral‐FCM detects the continuous changes in emission spectra from green to red of the photoconvertible protein, KikGR with high‐spectral resolution and separates spectrally‐adjacent fluoroprobes, such as FITC (Emission peak (Em) 519 nm) and EGFP (Em 507 nm). Moreover, the spectral‐FCM can measure and subtract autofluorescence of each cell providing increased signal‐to‐noise ratios and improved resolution of dim samples, which leads to a transformative technology for investigation of single cell state and function. These advances make it possible to perform 11‐color fluorescence analysis to visualize movement of multilinage immune cells by using KikGR‐expressing mice. Thus, the novel spectral flow cytometry improves the combinational use of spectrally‐adjacent various FPs and multicolor fluorochromes in metabolically active cell for the investigation of not only the immune system but also other research and clinical fields of use. © 2015 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of ISAC PMID:26217952

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

PubMed

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

1996-03-01

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

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.

Polychromatic flow cytometry is more sensitive than microscopy in detecting small monoclonal plasma cell populations.

PubMed

Tran, Daniel N; Smith, Sandy A B C; Brown, David A; Parker, Andrew J C; Joseph, Joanne E; Armstrong, Nicola; Sewell, William A

2017-03-01

There is an emerging role for flow cytometry (FC) in the assessment of small populations of plasma cells (PC). However, FC's utility has been questioned due to consistent underestimation of the percentage of PC compared to microscopy. A retrospective study was performed on bone marrow samples analysed by 8-colour FC. Plasma cell populations were classified as polyclonal or monoclonal based on FC analysis. FC findings were compared with microscopy of aspirates, histology and immunohistochemistry of trephine biopsies, and immunofixation (IFX) of serum and/or urine. FC underestimated PC compared to aspirate and trephine microscopy. The 10% diagnostic cutoff for MM on aspirate microscopy corresponded to a 3.5% cutoff on FC. Abnormal plasma cell morphology by aspirate microscopy and clonality by FC correlated in 229 of 294 cases (78%). However, in 50 cases, FC demonstrated a monoclonal population but microscopy reported no abnormality. In 15 cases, abnormalities were reported by microscopy but not by FC. Clonality assessment by trephine microscopy and FC agreed in 251/280 cases (90%), but all 29 discordant cases were monoclonal by FC and not monoclonal by microscopy. These cases had fewer PC and proportionally more polyclonal PC, and when IFX detected a paraprotein, it had the same light chain as in the PC determined by FC. FC was more sensitive in detecting monoclonal populations that were small or accompanied by polyclonal PC. This study supports the inclusion of FC in the evaluation of PC, especially in the assessment of small populations. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

FlowCam: Quantification and Classification of Phytoplankton by Imaging Flow Cytometry.

PubMed

Poulton, Nicole J

2016-01-01

The ability to enumerate, classify, and determine biomass of phytoplankton from environmental samples is essential for determining ecosystem function and their role in the aquatic community and microbial food web. Traditional micro-phytoplankton quantification methods using microscopic techniques require preservation and are slow, tedious and very laborious. The availability of more automated imaging microscopy platforms has revolutionized the way particles and cells are detected within their natural environment. The ability to examine cells unaltered and without preservation is key to providing more accurate cell concentration estimates and overall phytoplankton biomass. The FlowCam(®) is an imaging cytometry tool that was originally developed for use in aquatic sciences and provides a more rapid and unbiased method for enumerating and classifying phytoplankton within diverse aquatic environments.

Identification of residual leukemic cells by flow cytometry in childhood B-cell precursor acute lymphoblastic leukemia: verification of leukemic state by flow-sorting and molecular/cytogenetic methods.

PubMed

Øbro, Nina F; Ryder, Lars P; Madsen, Hans O; Andersen, Mette K; Lausen, Birgitte; Hasle, Henrik; Schmiegelow, Kjeld; Marquart, Hanne V

2012-01-01

Reduction in minimal residual disease, measured by real-time quantitative PCR or flow cytometry, predicts prognosis in childhood B-cell precursor acute lymphoblastic leukemia. We explored whether cells reported as minimal residual disease by flow cytometry represent the malignant clone harboring clone-specific genomic markers (53 follow-up bone marrow samples from 28 children with B-cell precursor acute lymphoblastic leukemia). Cell populations (presumed leukemic and non-leukemic) were flow-sorted during standard flow cytometry-based minimal residual disease monitoring and explored by PCR and/or fluorescence in situ hybridization. We found good concordance between flow cytometry and genomic analyses in the individual flow-sorted leukemic (93% true positive) and normal (93% true negative) cell populations. Four cases with discrepant results had plausible explanations (e.g. partly informative immunophenotype and antigen modulation) that highlight important methodological pitfalls. These findings demonstrate that with sufficient experience, flow cytometry is reliable for minimal residual disease monitoring in B-cell precursor acute lymphoblastic leukemia, although rare cases require supplementary PCR-based monitoring.

Antisperm antibodies in prepubertal boys and their reactivity with antigenic determinants on differentiated spermatozoa.

PubMed

Domagała, A; Kamieniczna, M; Kowalczyk, D; Kurpisz, M

1998-09-01

Antisperm antibodies induced in prepubertal boys with testicular failures were characterized by using four techniques of antibody detection. The reactivity of circulating antisperm antibodies in prepubertal boys and the reactivity of antibodies in sera samples of adult fertile and infertile males were compared against the same sperm antigenic pools (live or fixed spermatozoa, or sperm antigenic extracts). The incidence of antisperm antibodies in sera samples of 69 prepubertal boys with testicular failures and 21 samples obtained from adult, male individuals was assessed by indirect immunobead binding test (IDIBT), flow cytometry measurement, enzyme-linked immunosorbent assay, and Western blotting. Immunoblot analysis was performed by using sperm extracts of glycosylated and deglycosylated solubilized membrane antigens. Sera samples were studied in a group composed of healthy prepubertal boys (n = 7) and prepubertal boys with testicular failures (n = 69). Applied tests of antibody detection revealed striking differences in a group of boys with testicular pathology. With IDIBT, 7% of the sera samples were found positive, whereas with flow cytometry measurement, 48% of the sera samples were positive. Immunosorbent assay (fixed sperm) indicated 32% positive cases in the same group. The sera samples were found to be positive in 65% of immunoblotting reactions with glycosylated antigens and in 70% of immunoblotting reactions with deglycosylated antigens. All applied detection assays were clearly negative on sera samples from fertile, adult males. Western immunoblotting indicated an immunodominant antigenic determinant of 58 kDa. Tests of antibody detection with the use of live sperm (IDIBT and flow cytometry measurements) presented low sensitivity (8% and 48%, respectively) in a group of prepubertal boys. This observation underlines the difficulties in assigning the prospective prognosis of future fertility status in prepubertal boys with antisperm antibodies.

Detection of Silver and TiO2 Nanoparticles using Light Scatter by Flow Cytometry and Darkfield Microscopy

EPA Science Inventory

Titanium Dioxide (Ti02) and Silver (Ag) nanoparticles are used in many domestic applications, including sunscreens and paints. Evaluation of the potential hazard of manmade nanomaterials has been hampered by a limited ability to detect and measure nanoparticles in cells. In the p...

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

A flow-cytometry-based method to simplify the analysis and quantification of protein association to chromatin in mammalian cells

PubMed Central

Forment, Josep V.; Jackson, Stephen P.

2016-01-01

Protein accumulation on chromatin has traditionally been studied using immunofluorescence microscopy or biochemical cellular fractionation followed by western immunoblot analysis. As a way to improve the reproducibility of this kind of analysis, make it easier to quantify and allow a stream-lined application in high-throughput screens, we recently combined a classical immunofluorescence microscopy detection technique with flow cytometry1. In addition to the features described above, and by combining it with detection of both DNA content and DNA replication, this method allows unequivocal and direct assignment of cell-cycle distribution of protein association to chromatin without the need for cell culture synchronization. Furthermore, it is relatively quick (no more than a working day from sample collection to quantification), requires less starting material compared to standard biochemical fractionation methods and overcomes the need for flat, adherent cell types that are required for immunofluorescence microscopy. PMID:26226461

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

PubMed

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

2018-01-01

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

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.

Lab-on-Chip Cytometry Based on Magnetoresistive Sensors for Bacteria Detection in Milk

PubMed Central

Fernandes, Ana C.; Duarte, Carla M.; Cardoso, Filipe A.; Bexiga, Ricardo.; Cardoso, Susana.; Freitas, Paulo P.

2014-01-01

Flow cytometers have been optimized for use in portable platforms, where cell separation, identification and counting can be achieved in a compact and modular format. This feature can be combined with magnetic detection, where magnetoresistive sensors can be integrated within microfluidic channels to detect magnetically labelled cells. This work describes a platform for in-flow detection of magnetically labelled cells with a magneto-resistive based cell cytometer. In particular, we present an example for the validation of the platform as a magnetic counter that identifies and quantifies Streptococcus agalactiae in milk. PMID:25196163

Lab-on-chip cytometry based on magnetoresistive sensors for bacteria detection in milk.

PubMed

Fernandes, Ana C; Duarte, Carla M; Cardoso, Filipe A; Bexiga, Ricardo; Cardoso, Susana; Freitas, Paulo P

2014-08-21

Flow cytometers have been optimized for use in portable platforms, where cell separation, identification and counting can be achieved in a compact and modular format. This feature can be combined with magnetic detection, where magnetoresistive sensors can be integrated within microfluidic channels to detect magnetically labelled cells. This work describes a platform for in-flow detection of magnetically labelled cells with a magneto-resistive based cell cytometer. In particular, we present an example for the validation of the platform as a magnetic counter that identifies and quantifies Streptococcus agalactiae in milk.

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.

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.

[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.

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

Optofluidic time-stretch microscopy: recent advances

NASA Astrophysics Data System (ADS)

Lei, Cheng; Nitta, Nao; Ozeki, Yasuyuki; Goda, Keisuke

2018-06-01

Flow cytometry is an indispensable method for valuable applications in numerous fields such as immunology, pathology, pharmacology, molecular biology, and marine biology. Optofluidic time-stretch microscopy is superior to conventional flow cytometry methods for its capability to acquire high-quality images of single cells at a high-throughput exceeding 10,000 cells per second. This makes it possible to extract copious information from cellular images for accurate cell detection and analysis with the assistance of machine learning. Optofluidic time-stretch microscopy has proven its effectivity in various applications, including microalga-based biofuel production, evaluation of thrombotic disorders, as well as drug screening and discovery. In this review, we discuss the principles and recent advances of optofluidic time-stretch microscopy.

Optofluidic time-stretch microscopy: recent advances

NASA Astrophysics Data System (ADS)

Lei, Cheng; Nitta, Nao; Ozeki, Yasuyuki; Goda, Keisuke

2018-04-01

Flow cytometry is an indispensable method for valuable applications in numerous fields such as immunology, pathology, pharmacology, molecular biology, and marine biology. Optofluidic time-stretch microscopy is superior to conventional flow cytometry methods for its capability to acquire high-quality images of single cells at a high-throughput exceeding 10,000 cells per second. This makes it possible to extract copious information from cellular images for accurate cell detection and analysis with the assistance of machine learning. Optofluidic time-stretch microscopy has proven its effectivity in various applications, including microalga-based biofuel production, evaluation of thrombotic disorders, as well as drug screening and discovery. In this review, we discuss the principles and recent advances of optofluidic time-stretch microscopy.

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.

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.

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.

Comparative exploration of multidimensional flow cytometry software: a model approach evaluating T cell polyfunctional behavior.

PubMed

Spear, Timothy T; Nishimura, Michael I; Simms, Patricia E

2017-08-01

Advancement in flow cytometry reagents and instrumentation has allowed for simultaneous analysis of large numbers of lineage/functional immune cell markers. Highly complex datasets generated by polychromatic flow cytometry require proper analytical software to answer investigators' questions. A problem among many investigators and flow cytometry Shared Resource Laboratories (SRLs), including our own, is a lack of access to a flow cytometry-knowledgeable bioinformatics team, making it difficult to learn and choose appropriate analysis tool(s). Here, we comparatively assess various multidimensional flow cytometry software packages for their ability to answer a specific biologic question and provide graphical representation output suitable for publication, as well as their ease of use and cost. We assessed polyfunctional potential of TCR-transduced T cells, serving as a model evaluation, using multidimensional flow cytometry to analyze 6 intracellular cytokines and degranulation on a per-cell basis. Analysis of 7 parameters resulted in 128 possible combinations of positivity/negativity, far too complex for basic flow cytometry software to analyze fully. Various software packages were used, analysis methods used in each described, and representative output displayed. Of the tools investigated, automated classification of cellular expression by nonlinear stochastic embedding (ACCENSE) and coupled analysis in Pestle/simplified presentation of incredibly complex evaluations (SPICE) provided the most user-friendly manipulations and readable output, evaluating effects of altered antigen-specific stimulation on T cell polyfunctionality. This detailed approach may serve as a model for other investigators/SRLs in selecting the most appropriate software to analyze complex flow cytometry datasets. Further development and awareness of available tools will help guide proper data analysis to answer difficult biologic questions arising from incredibly complex datasets. © Society for Leukocyte Biology.

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.

Multiparameter FLAER-based flow cytometry for screening of paroxysmal nocturnal hemoglobinuria enhances detection rates in patients with aplastic anemia.

PubMed

Sachdeva, Man Updesh Singh; Varma, Neelam; Chandra, Dinesh; Bose, Parveen; Malhotra, Pankaj; Varma, Subhash

2015-05-01

Flow cytometry is the gold standard methodology for screening of paroxysmal nocturnal hemoglobinuria. In the last few years, proaerolysin conjugated with fluorescein (FLAER) has become an important component of antibody panel used for the detection of paroxysmal nocturnal hemoglobinuria (PNH) clone. This study aimed to compare PNH clone detection by flow cytometry in the pre-FLAER era versus the FLAER era. This was a retrospective analysis of 4 years and included 1004 individuals screened for PNH clone, either presenting as hemolytic anemia or as aplastic anemia. In the pre-FLAER time period, the RBCs and neutrophils were screened with antibodies against CD55 and CD59. With the introduction of FLAER, neutrophils were screened with FLAER/CD24/CD15 and monocytes with FLAER/CD14/CD33 combination. A comparative analysis was done for detection of PNH clone in aplastic anemia patients versus non-aplastic anemia patients, as well as between pre-FLAER and FLAER era. Out of a total of 1004 individuals, 59 (5.8%) were detected to have PNH clone positivity. The frequency of PNH clone detected in aplastic anemia and non-aplastic anemia groups was 12.02 and 3.36%, respectively. The detection rate of PNH clone increased from 4.5% (32/711) in the pre-FLAER era to 9.2% (27/293) with the introduction of FLAER. However, this increase could be attributed to increased detection of PNH clone in the aplastic anemia group, which showed a significant increase from 8.3 to 18.2% after use of FLAER. In the non-aplastic group, PNH clone was detected with similar frequencies before and after use of FLAER (3.2 versus 3.8%, respectively). Mean PNH clone size was lower in the aplastic anemia group when compared with the non-aplastic group. RBCs always showed a lower clone size than neutrophils. PNH clone on neutrophils and monocytes was however similar. Inclusion of FLAER increases the sensitivity of the test which is especially useful in picking up small PNH clones in patients of aplastic anemia.

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

Flow Cytometry Detection of Infectious Rotaviruses in Environmental and Clinical Samples

PubMed Central

Abad, F. Xavier; Pintó, Rosa M.; Bosch, Albert

1998-01-01

A method for the detection of infectious human rotaviruses based on infection of CaCo-2 cells and detection of infected cells by indirect immunofluorescence and flow cytometry (IIF-FC) has been developed. The technique was validated by performing a seminested reverse transcription-PCR assay with sorted cell populations. The efficiency of the procedure has been compared with that of the standard method of infection of MA104 cells and ulterior detection by IIF and optical microscopy (IIF-OM) and with that of infection of MA104 cells and detection by IIF-FC. The limit of sensitivity for the detection of the cell-adapted strain Itor P13, expressed as the most probable number of cytopathogenic units, was established as 200 and 2 for MA104 and CaCo-2 cells, respectively, by the IIF-FC method. The ratio of infectious virus particles to total virus particles for a wild-type rotavirus was determined to be 1/2 × 106 and 1/2 × 104 for IIF-OM with MA104 cells and IIF-FC with CaCo-2 cells, respectively. The use of IIF-FC with CaCo-2 cells was tested with fecal and water samples and proved to be more effective than the standard procedure for rotavirus detection. PMID:9647805

A novel sensitive pathogen detection system based on Microbead Quantum Dot System.

PubMed

Wu, Tzong-Yuan; Su, Yi-Yu; Shu, Wei-Hsien; Mercado, Augustus T; Wang, Shi-Kwun; Hsu, Ling-Yi; Tsai, Yow-Fu; Chen, Chung-Yung

2016-04-15

A fast and accurate detection system for pathogens can provide immediate measurements for the identification of infectious agents. Therefore, the Microbead Quantum-dots Detection System (MQDS) was developed to identify and measure target DNAs of pathogenic microorganisms and eliminated the need of PCR amplifications. This nanomaterial-based technique can detect different microorganisms by flow cytometry measurements. In MQDS, pathogen specific DNA probes were designed to form a hairpin structure and conjugated on microbeads. In the presence of the complementary target DNA sequence, the probes will compete for binding with the reporter probes but will not interfere with the binding between the probe and internal control DNA. To monitor the binding process by flow cytometry, both the reporter probes and internal control probes were conjugated with Quantum dots that fluoresce at different emission wavelengths using the click reaction. When MQDS was used to detect the pathogens in environmental samples, a high correlation coefficient (R=0.994) for Legionella spp., with a detection limit of 0.1 ng of the extracted DNAs and 10 CFU/test, can be achieved. Thus, this newly developed technique can also be applied to detect other pathogens, particularly viruses and other genetic diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

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...

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.

Extended flow cytometry characterization of normal bone marrow progenitor cells by simultaneous detection of aldehyde dehydrogenase and early hematopoietic antigens: implication for erythroid differentiation studies

PubMed Central

Mirabelli, Peppino; Di Noto, Rosa; Lo Pardo, Catia; Morabito, Paolo; Abate, Giovanna; Gorrese, Marisa; Raia, Maddalena; Pascariello, Caterina; Scalia, Giulia; Gemei, Marica; Mariotti, Elisabetta; Del Vecchio, Luigi

2008-01-01

Background Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme highly expressed in hematopoietic precursors from cord blood and granulocyte-colony stimulating factor mobilized peripheral blood, as well as in bone marrow from patients with acute myeloblastic leukemia. As regards human normal bone marrow, detailed characterization of ALDH+ cells has been addressed by one single study (Gentry et al, 2007). The goal of our work was to provide new information about the dissection of normal bone marrow progenitor cells based upon the simultaneous detection by flow cytometry of ALDH and early hematopoietic antigens, with particular attention to the expression of ALDH on erythroid precursors. To this aim, we used three kinds of approach: i) multidimensional analytical flow cytometry, detecting ALDH and early hematopoietic antigens in normal bone marrow; ii) fluorescence activated cell sorting of distinct subpopulations of progenitor cells, followed by in vitro induction of erythroid differentiation; iii) detection of ALDH+ cellular subsets in bone marrow from pure red cell aplasia patients. Results In normal bone marrow, we identified three populations of cells, namely ALDH+CD34+, ALDH-CD34+ and ALDH+CD34- (median percentages were 0.52, 0.53 and 0.57, respectively). As compared to ALDH-CD34+ cells, ALDH+CD34+ cells expressed the phenotypic profile of primitive hematopoietic progenitor cells, with brighter expression of CD117 and CD133, accompanied by lower display of CD38 and CD45RA. Of interest, ALDH+CD34- population disclosed a straightforward erythroid commitment, on the basis of three orders of evidences. First of all, ALDH+CD34- cells showed a CD71bright, CD105+, CD45- phenotype. Secondly, induction of differentiation experiments evidenced a clear-cut expression of glycophorin A (CD235a). Finally, ALDH+CD34- precursors were not detectable in patients with pure red cell aplasia (PRCA). Conclusion Our study, comparing surface antigen expression of ALDH+/CD34+, ALDH-/CD34+ and ALDH+/CD34- progenitor cell subsets in human bone marrow, clearly indicated that ALDH+CD34- cells are mainly committed towards erythropoiesis. To the best of our knowledge this finding is new and could be useful for basic studies about normal erythropoietic differentiation as well as for enabling the employment of ALDH as a red cell marker in polychromatic flow cytometry characterization of bone marrow from patients with aplastic anemia and myelodysplasia. PMID:18510759

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.

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.

Detection, isolation, and capture of circulating breast cancer cells with photoacoustic flow cytometry

NASA Astrophysics Data System (ADS)

Bhattacharyya, Kiran; Njoroge, Martin; Goldschmidt, Benjamin S.; Gaffigan, Brian; Rood, Kyle; Viator, John A.

2013-03-01

According to the CDC, breast cancer is the most common cancer and the second leading cause of cancer related deaths among women. Metastasis, or the presence of secondary tumors caused by the spread of cancer cells via the circulatory or lymphatic systems, significantly worsens the prognosis of any breast cancer patient. In this study, a technique is developed to detect circulating breast cancer cells in human blood using a photoacoustic flow cytometry method. A Q-switched laser with a 5 ns pulse at 532 nm is used to interrogate thousands of cells with one pulse as they flow through the beam path. Cells which are pigmented, either naturally or artificially, emit an ultrasound wave as a result of the photoacoustic (PA) effect. Breast cancer cells are targeted with chromophores through immunochemistry in order to provide pigment. After which, the device is calibrated to demonstrate a single-cell detection limit. Cultured breast cancer cells are added to whole blood to reach a biologically relevant concentration of about 25-45 breast cancer cells per 1 mL of blood. An in vitro photoacoustic flow cytometer is used to detect and isolate these cells followed by capture with the use of a micromanipulator. This method can not only be used to determine the disease state of the patient and the response to therapy, it can also be used for genetic testing and in vitro drug trials since the circulating cell can be captured and studied.

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

PubMed

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

2018-01-01

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

Flow 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

In vivo flow cytometry and time-resolved near-IR angiography and lymphography

NASA Astrophysics Data System (ADS)

Galanzha, Ekaterina I.; Tuchin, Valery V.; Brock, Robert W.; Zharov, Vladimir P.

2007-05-01

Integration of photoacoustic and photothermal techniques with high-speed, high-resolution transmission and fluorescence microscopy shows great potential for in vivo flow cytometry and indocyanine green (ICG) near-infrared (IR) angiography of blood and lymph microvessels. In particular, the capabilities of in vivo flow cytometry using rat mesentery and nude mouse ear models are demonstrated for real-time quantitative detection of circulating and migrating individual blood and cancer cells in skin, mesentery, lymph nodes, liver, kidney; studying vascular dynamics with a focus on lymphatics; monitoring cell traffic between blood and lymph systems; high-speed imaging of cell deformability in flow; and label-free real-time monitoring of single cell extravasation from blood vessel lumen into tissue. As presented, the advantages of ICG IR-angiography include estimation of time resolved dye dynamics (appearance and clearance) in blood and lymph microvessels using fluorescent and photoacoustic modules of the integrated technique. These new approaches are important for monitoring and quantifying metastatic and apoptotic cells; comparative measurements of plasma and cell velocities; analysis of immune responses; monitoring of circulating macromolecules, chylomicrons, bacteria, viruses and nanoparticles; molecular imaging. In the future, we believe that the integrated technique presented will have great potential for translation to early disease diagnoses (e.g. cancer) or assessment of innovative therapeutic interventions in humans.

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.

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

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

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.

A flow-cytometry-based method for detecting simultaneously five allergens in a complex food matrix.

PubMed

Otto, Gaetan; Lamote, Amandine; Deckers, Elise; Dumont, Valery; Delahaut, Philippe; Scippo, Marie-Louise; Pleck, Jessica; Hillairet, Caroline; Gillard, Nathalie

2016-12-01

To avoid carry-over contamination with allergens, food manufacturers implement quality control strategies relying primarily on detection of allergenic proteins by ELISA. Although sensitive and specific, this method allowed detection of only one allergen per analysis and effective control policies were thus based on multiplying the number of tests done in order to cover the whole range of allergens. We present in this work an immunoassay for the simultaneous detection of milk, egg, peanut, mustard and crustaceans in cookies samples. The method was based on a combination of flow cytometry with competitive ELISA where microbeads were used as sorbent surface. The test was able to detect the presence of the five allergens with median inhibitory concentrations (IC50) ranging from 2.5 to 15 mg/kg according to the allergen to be detected. The lowest concentrations of contaminants inducing a significant difference of signal between non-contaminated controls and test samples were 2 mg/kg of peanut, 5 mg/kg of crustaceans, 5 mg/kg of milk, 5 mg/kg of mustard and 10 mg/kg of egg. Assay sensitivity was influenced by the concentration of primary antibodies added to the sample extract for the competition and by the concentration of allergenic proteins bound to the surface of the microbeads.

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.

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.

Liquid Crystal Droplet-Based Amplification of Microvesicles that are Shed by Mammalian Cells

PubMed Central

Tan, Lie Na; Wiepz, Gregory J.; Miller, Daniel S.; Shusta, Eric V.; Abbott, Nicholas L.

2014-01-01

Membrane-derived microvesicles (MVs) shed by cells are being investigated for their role in intercellular communication and as potential biomarkers of disease, but facile and sensitive methods for their analysis do not exist. Here we demonstrate new principles for analysis of MVs that use micrometer-sized droplets of liquid crystals (LCs) to amplify MVs that are selectively captured via antibody-mediated interactions. The influence of the MVs on the micrometer-sized LC droplets is shown to be readily quantified via use of flow cytometry. The methodology was developed using MVs shed by epidermoid carcinoma A431 cells that contain epidermal growth factor receptor (EGFR) as an important and representative example of MVs containing signaling proteins that play a central role in cancer. The LC droplets were found to be sensitive to 106 MVs containing EGFR (relative to controls using isotype control antibody) and to possess a dynamic range of response across several orders of magnitude. Because the 100 nm-sized MVs captured via EGFR generate an optical response in the micrometer-sized LC droplets that can be readily detected by flow cytometry in light scattering mode, the approach possesses significant advantages over direct detection of MVs by flow cytometry. The LC droplets are also substantially more sensitive than techniques such as immunoblotting because the lipid-component of the MVs serves to amplify the antibody-mediated capture of the target proteins in the MVs. Other merits of the approach are defined and discussed in the paper. PMID:24667742

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

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.

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.

Detection and isolation of rare cells by 2-step enrichment high-speed flow cytometry/cell sorting and single cell LEAP laser ablation

NASA Astrophysics Data System (ADS)

Zordan, M. D.; Leary, James F.

2011-02-01

The clonal isolation of rare cells, especially cancer and stem cells, in a population is important to the development of improved medical treatment. We have demonstrated that the Laser-Enabled Analysis and Processing (LEAP, Cyntellect Inc., San Diego, CA) instrument can be used to efficiently produce single cell clones by photoablative dilution. Additionally, we have also shown that cells present at low frequencies can be cloned by photoablative dilution after they are pre-enriched by flow cytometry based cell sorting. Circulating tumor cells were modeled by spiking isolated peripheral blood cells with cells from the lung carcinoma cell line A549. Flow cytometry based cell sorting was used to perform an enrichment sort of A549 cells directly into a 384 well plate. Photoablative dilution was performed with the LEAPTM instrument to remove any contaminating cells, and clonally isolate 1 side population cell per well. We were able to isolate and grow single clones of side population cells using this method at greater than 90% efficiency. We have developed a 2 step method that is able to perform the clonal isolation of rare cells based on a medically relevant functional phenotype.

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

PubMed Central

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

2010-01-01

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

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

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

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

Application of a Short Intracellular pH Method to Flow Cytometry for Determining Saccharomyces cerevisiae Vitality ▿

PubMed Central

Weigert, Claudia; Steffler, Fabian; Kurz, Tomas; Shellhammer, Thomas H.; Methner, Frank-Jürgen

2009-01-01

The measurement of yeast's intracellular pH (ICP) is a proven method for determining yeast vitality. Vitality describes the condition or health of viable cells as opposed to viability, which defines living versus dead cells. In contrast to fluorescence photometric measurements, which show only average ICP values of a population, flow cytometry allows the presentation of an ICP distribution. By examining six repeated propagations with three separate growth phases (lag, exponential, and stationary), the ICP method previously established for photometry was transferred successfully to flow cytometry by using the pH-dependent fluorescent probe 5,6-carboxyfluorescein. The correlation between the two methods was good (r2 = 0.898, n = 18). With both methods it is possible to track the course of growth phases. Although photometry did not yield significant differences between exponentially and stationary phases (P = 0.433), ICP via flow cytometry did (P = 0.012). Yeast in an exponential phase has a unimodal ICP distribution, reflective of a homogeneous population; however, yeast in a stationary phase displays a broader ICP distribution, and subpopulations could be defined by using the flow cytometry method. In conclusion, flow cytometry yielded specific evidence of the heterogeneity in vitality of a yeast population as measured via ICP. In contrast to photometry, flow cytometry increases information about the yeast population's vitality via a short measurement, which is suitable for routine analysis. PMID:19581482

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.

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.

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.

Quantitative Analysis of Protein Translocations by Microfluidic Total Internal Reflection Fluorescence Flow Cytometry

PubMed Central

Wang, Jun; Fei, Bei; Geahlen, Robert L.

2010-01-01

Protein translocation, or the change in a protein’s location between different subcellular compartments, is a critical process by which intracellular proteins carry out their cellular functions. Aberrant translocation events contribute to various diseases ranging from metabolic disorders to cancer. In this study, we demonstrate the use of a newly developed single-cell tool, microfluidic total internal reflection fluorescence flow cytometry (TIRF-FC), for detecting both cytosol to plasma membrane and cytosol to nucleus translocations using the tyrosine kinase Syk and the transcription factor NF-κB as models. This technique detects fluorescent molecules at the plasma membrane and in the membrane-proximal cytosol in single cells. We were able to record quantitatively changes in the fluorescence density in the evanescent field associated with these translocation processes for large cell populations with single cell resolution. We envision that TIRF-FC will provide a new approach to explore the molecular biology and clinical relevance of protein translocations. PMID:20820633

CMOS based image cytometry for detection of phytoplankton in ballast water.

PubMed

Pérez, J M; Jofre, M; Martínez, P; Yáñez, M A; Catalan, V; Parker, A; Veldhuis, M; Pruneri, V

2017-02-01

We introduce an image cytometer (I-CYT) for the analysis of phytoplankton in fresh and marine water environments. A linear quantification of cell numbers was observed covering several orders of magnitude using cultures of Tetraselmis and Nannochloropsis measured by autofluorescence in a laboratory environment. We assessed the functionality of the system outside the laboratory by phytoplankton quantification of samples taken from a marine water environment (Dutch Wadden Sea, The Netherlands) and a fresh water environment (Lake Ijssel, The Netherlands). The I-CYT was also employed to study the effects of two ballast water treatment systems (BWTS), based on chlorine electrolysis and UV sterilization, with the analysis including the vitality of the phytoplankton. For comparative study and benchmarking of the I-CYT, a standard flow cytometer was used. Our results prove a limit of detection (LOD) of 10 cells/ml with an accuracy between 0.7 and 0.5 log, and a correlation of 88.29% in quantification and 96.21% in vitality, with respect to the flow cytometry results.

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

Confirmation of Pig-a mutation in flow cytometry-identified CD48-deficient T-lymphocytes from F344 rats.

PubMed

Revollo, Javier; Pearce, Mason G; Petibone, Dayton M; Mittelstaedt, Roberta A; Dobrovolsky, Vasily N

2015-05-01

The Pig-a assay is used for monitoring somatic cell mutation in laboratory animals and humans. The assay detects haematopoietic cells deficient in glycosylphosphatidylinositol (GPI)-anchored protein surface markers using flow cytometry. However, given that synthesis of the protein markers (and the expression of their genes) is independent of the expression of the X-linked Pig-a gene and the function of its enzyme product, the deficiency of markers at the surface of the cells may be caused by a number of events (e.g. by mutation or epigenetic silencing in the marker gene itself or in any of about two dozen autosomal genes involved in the synthesis of GPI). Here we provide direct evidence that the deficiency of the GPI-anchored surface marker CD48 in rat T-cells is accompanied by mutation in the endogenous X-linked Pig-a gene. We treated male F344 rats with N-ethyl-N-nitrosourea (ENU), and established colonies from flow cytometry-identified and sorted CD48-deficient spleen T-lymphocytes. Molecular analysis confirmed that the expanded sorted cells have mutations in the Pig-a gene. The spectrum of Pig-a mutation in our model was consistent with the spectrum of ENU-induced mutation determined in other in vivo models, mostly base-pair substitutions at A:T with the mutated T on the non-transcribed strand of Pig-a genomic DNA. We also used next generation sequencing to derive a similar mutational spectrum from a pool of 64 clones developed from flow-sorted CD48-deficient lymphocytes. Our findings confirm that Pig-a assays detect what they are designed to detect-gene mutation in the Pig-a gene. © The Author 2015. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

[Which technique should be used in the phenotyping of lymphocytic alveolitis: Immunocytochemistry or flow cytometry].

PubMed

Mlika, Mona; Kasmi, Rihem; Safra, Ines; Braham, Emna; Chebbi, Chokri; Mezni, Faouzi El

2017-10-01

Diffuse interstitial pneumonias are considered as a group of multiple affections characterized by challenging diagnoses because of the lack of specific clinical signs. Radiologic investigations highlight the diagnoses in most of the cases but bronchoalveolar lavage plays a key role in the diagnostic diagram. We aim to compare the immunocytochemical technique and the flow cytometry in the phenotyping of lymphocytic alveolitis. We described a series of 32 lymphocytic alveolitis, which were analyzed using immunocytochemistry and flow cytometry. We found a good reproducibility between the immunocytochemistry performed on smears and cytoblocks (kappa=0.7) and a poor reproducibility between immunocytochemistry and flow cytometry (kappa=0.35). Our study emphasized on the poor reproducibility between immunocytochemistry and flow cytometry. Further studies about the reliability of both techniques are needed especially in discordant cases. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Level 2 validation of a flow cytometric method for detection of Escherichia coli O157:H7 in raw spinach.

PubMed

Williams, Anna J; Cooper, Willie M; Summage-West, Christine V; Sims, Lillie M; Woodruff, Robert; Christman, Jessica; Moskal, Ted J; Ramsaroop, Shawn; Sutherland, John B; Alusta, Pierre; Wilkes, Jon G; Buzatu, Dan A

2015-12-23

The Bacteriological Analytical Manual (BAM) method currently used by the United States Food and Drug Administration (FDA) to detect Escherichia coli O157:H7 in spinach was systematically compared to a new flow cytometry based method. This Food and Drug Administration (FDA) level 2 external laboratory validation study was designed to determine the latter method's sensitivity and speed for analysis of this pathogen in raw spinach. Detection of target cell inoculations with a low cell count is critical, since enterohemorrhagic strains of E. coli require an infective dose of as few as 10 cells (Schmid-Hempel and Frank, 2007). Although, according to the FDA, the infectious dose is unknown (Food and Drug Administration, 1993). Therefore, the inoculation level into the spinach, a total of 2.0±2.6 viable E. coli O157 cells, was specified to yield between 25% and 75% detection by the new method, out of 20 samples (10 positives and 10 negatives). This criterion was met in that the new method detected 60% of the nominally positive samples; the corresponding sensitivity of the reference method was 50%. For both methods the most likely explanation for false negatives was that no viable cells were actually introduced into the sample. In this validation study, the flow cytometry method was equal to the BAM in sensitivity and far superior in speed. Published by Elsevier B.V.

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

miRNA detection at single-cell resolution using microfluidic LNA flow-FISH

DOE PAGES

Wu, Meiye; Piccini, Matthew Ernest; Koh, Chung -Yan; ...

2014-08-20

Flow cytometry in combination with fluorescent in situ hybridization (flow-FISH) is a powerful technique that can be utilized to rapidly detect nucleic acids at single-cell resolution without the need for homogenization or nucleic acid extraction. Here, we describe a microfluidic-based method which enables the detection of microRNAs or miRNAs in single intact cells by flow-FISH using locked nucleic acid (LNA)-containing probes. Our method can be applied to all RNA species including mRNA and small noncoding RNA and is suitable for multiplexing with protein immunostaining in the same cell. For demonstration of our method, this chapter details the detection of miR155more » and CD69 protein in PMA and ionomycin-stimulated Jurkat cells. Here, we also include instructions on how to set up a microfluidic chip sample preparation station to prepare cells for imaging and analysis on a commercial flow cytometer or a custom-built micro-flow cytometer.« less

Visualization of Pulmonary Clearance Mechanisms via Noninvasive Optical Imaging Validated by Near-Infrared Flow Cytometry

PubMed Central

Zhou, Haiying; Gunsten, Sean P.; Zhegalova, Natalia G.; Bloch, Sharon; Achilefu, Samuel; Holley, J. Christopher; Schweppe, Daniel; Akers, Walter; Brody, Steven L.; Eades, William; Berezin, Mikhail Y.

2016-01-01

In vivo optical imaging with near-infrared (NIR) probes is an established method of diagnostics in preclinical and clinical studies. However, the specificities of these probes are difficult to validate ex vivo due to the lack of NIR flow cytometry. To address this limitation, we modified a flow cytometer to include an additional NIR channel using a 752 nm laser line. The flow cytometry system was tested using NIR microspheres and cell lines labeled with a combination of visible range and NIR fluorescent dyes. The approach was verified in vivo in mice evaluated for immune response in lungs after intratracheal delivery of the NIR contrast agent. Flow cytometry of cells obtained from the lung bronchoalveolar lavage demonstrated that the NIR dye was taken up by pulmonary macrophages as early as four-hours post-injection. This combination of optical imaging with NIR flow cytometry extends the capability of imaging and enables complementation of in vivo imaging with cell-specific studies. PMID:25808737

In vivo Raman flow cytometry for real-time detection of carbon nanotube kinetics in lymph, blood, and tissues

PubMed Central

Biris, Alexandru S.; Galanzha, Ekaterina I.; Li, Zhongrui; Mahmood, Meena; Xu, Yang; Zharov, Vladimir P.

2016-01-01

Nanoparticles are intensively being explored as contrast agents for medical diagnostics and therapies using various optical methods. We present the first demonstration of the use of time-resolved Raman spectroscopy for in vivo real-time detection of circulating carbon nanotubes (CNTs) or cancer cells labeled with CNTs in the lymph, blood, and tissues of live animals with fast spectral acquisition times of down to few milliseconds. After intravenously administering CNTs in the tail vein of the rat, this technique provides the ability to detect the circulation of CNTs in the blood microvessels of the intact rat ear. The capability of Raman spectroscopy is also demonstrated to monitor, identify, and image the CNTs during their transportation by lymphatics in the rat ear and mesentery. The strong and specific Raman scattering properties of CNTs make it possible to detect in vitro and in vivo single cancer cells (HeLa) tagged with CNTs. In vivo Raman flow cytometry opens a new avenue for multiparameter analysis of circulating nanoparticles with strong Raman scattering properties and their pharmokinetics in blood and lymph systems. Moreover, this technology has the potential for molecular detection and identification of circulating tumor cells, and infections labeled with CNTs. PMID:19405719

In vivo Raman flow cytometry for real-time detection of carbon nanotube kinetics in lymph, blood, and tissues

NASA Astrophysics Data System (ADS)

Biris, Alexandru S.; Galanzha, Ekaterina I.; Li, Zhongrui; Mahmood, Meena; Xu, Yang; Zharov, Vladimir P.

2009-03-01

Nanoparticles are intensively being explored as contrast agents for medical diagnostics and therapies using various optical methods. We present the first demonstration of the use of time-resolved Raman spectroscopy for in vivo real-time detection of circulating carbon nanotubes (CNTs) or cancer cells labeled with CNTs in the lymph, blood, and tissues of live animals with fast spectral acquisition times of down to few milliseconds. After intravenously administering CNTs in the tail vein of the rat, this technique provides the ability to detect the circulation of CNTs in the blood microvessels of the intact rat ear. The capability of Raman spectroscopy is also demonstrated to monitor, identify, and image the CNTs during their transportation by lymphatics in the rat ear and mesentery. The strong and specific Raman scattering properties of CNTs make it possible to detect in vitro and in vivo single cancer cells (HeLa) tagged with CNTs. In vivo Raman flow cytometry opens a new avenue for multiparameter analysis of circulating nanoparticles with strong Raman scattering properties and their pharmokinetics in blood and lymph systems. Moreover, this technology has the potential for molecular detection and identification of circulating tumor cells, and infections labeled with CNTs.

Detection of circulating breast cancer cells using photoacoustic flow cytometry

NASA Astrophysics Data System (ADS)

Bhattacharyya, Kiran

According to the American Cancer Society, more than 200,000 new cases of breast cancer are expected to be diagnosed this year. Moreover, about 40,000 women died from breast cancer last year alone. As breast cancer progresses in an individual, it can transform from a localized state to a metastatic one with multiple tumors distributed through the body, not necessarily contained within the breast. Metastasis is the spread of cancer through the body by circulating tumor cells (CTCs) which can be found in the blood and lymph of the diagnosed patient. Diagnosis of a metastatic state by the discovery of a secondary tumor can often come too late and hence, significantly reduce the patient's chance of survival. There is a current need for a CTC detection method which would diagnose metastasis before the secondary tumor occurs or reaches a size resolvable by current imaging systems. Since earlier detection would improve prognosis, this study proposes a method of labeling of breast cancer cells for detection with a photoacoustic flow cytometry system as a model for CTC detection in human blood. Gold nanoparticles and fluorescent polystyrene nanoparticles are proposed as contrast agents for T47D, the breast cancer cell line of choice. The labeling, photoacoustic detection limit, and sensitivity are first characterized and then applied to a study to show detection from human blood.

Flow cytometry of HEK 293T cells interacting with polyelectrolyte multilayer capsules containing fluorescein-labeled poly(acrylic acid) as a pH sensor.

PubMed

Reibetanz, Uta; Halozan, David; Brumen, Milan; Donath, Edwin

2007-06-01

Polyelectrolyte multilayer sensor capsules, 5 microm in diameter, which contained fluorescein-labeled poly(acrylic acid) (PAAAF) as pH-sensitive reporter molecules, were fabricated and employed to explore their endocytotic uptake into HEK 293T cells by flow cytometry. The percentage of capsules residing in the endolysosomal compartment was estimated from the fluorescence intensity decrease caused by acidification. Capsules attached to the extracellular surface of the plasma membrane were identified by trypan blue quenching. The number of capsules in the cytoplasm was rather small, being below the detection limit of the method. The advantages of polyelectrolyte multilayer capsules are that the fluorophore is protected from interaction with cellular compartments and that the multilayer can be equipped with additional functions.

Chemo Resistance of Breast Cancer Stem Cells

DTIC Science & Technology

2006-05-01

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

Use of a Novel Fluidics Microbead Trap/Flow-cell Enhances Speed and Sensitivity of Bead-Based Bioassays

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

Ozanich, Rich M.; Antolick, Kathryn C.; Bruckner-Lea, Cindy J.

2007-09-15

Automated devices and methods for biological sample preparation often utilize surface functionalized microbeads (superparamagnetic or non-magnetic) to allow capture, purification and pre-concentration of trace amounts of proteins, cells, or nucleic acids (DNA/RNA) from complex samples. We have developed unique methods and hardware for trapping either magnetic or non-magnetic functionalized beads that allow samples and reagents to be efficiently perfused over a micro-column of beads. This approach yields enhanced mass transport and up to 5-fold improvements in assay sensitivity or speed, dramatically improving assay capability relative to assays conducted in more traditional “batch modes” (i.e., in tubes or microplate wells). Summarymore » results are given that highlight the analytical performance improvements obtained for automated microbead processing systems utilizing novel microbead trap/flow-cells for various applications, including: 1) simultaneous capture of multiple cytokines using an antibody-coupled polystyrene bead assay with subsequent flow cytometry detection; 2) capture of nucleic acids using oligonucleotide coupled polystyrene beads with flow cytometry detection; and 3) capture of Escherichia coli 0157:H7 (E. coli) from 50 mL sample volumes using antibody-coupled superparamagnetic microbeads with subsequent culturing to assess capture efficiency.« less

Microfluidic flow cytometer for quantifying photobleaching of fluorescent proteins in cells

PubMed Central

Lubbeck, Jennifer L.; Dean, Kevin M.; Ma, Hairong; Palmer, Amy E.; Jimenez, Ralph

2012-01-01

Traditional flow cytometers are capable of rapid cellular assays on the basis of fluorescence intensity and light scatter. Microfluidic flow cytometers have largely followed the same path of technological development as their traditional counterparts, however the significantly smaller transport distance and resulting lower cell speeds in microchannels provides for the opportunity to detect novel spectroscopic signatures based on multiple, non-temporally-coincident excitation beams. Here, we characterize the design and operation of a cytometer with a 3-beam, probe/bleach/probe geometry, employing HeLa suspension cells expressing fluorescent proteins. The data collection rate exceeds 20 cells/s under a range of beam intensities (5 kW – 179 kW/cm2). The measured percent photobleaching (ratio of fluorescence intensities excited by the first and third beams: Sbeam3/Sbeam1) partially resolves a mixture of four red fluorescent proteins in mixed samples. Photokinetic simulations are presented and demonstrate that the percent photobleaching reflects a combination of the reversible and irreversible photobleaching kinetics. By introducing a photobleaching optical signature, which complements traditional fluorescence intensity-based detection, this method adds another dimension to multi-channel fluorescence cytometry, and provides a means for flow-cytometry-based screening of directed libraries of fluorescent protein photobleaching. PMID:22424298

Early Detection of NSCLC Using Stromal Markers in Peripheral Blood

DTIC Science & Technology

2015-09-01

post- surgery patients, and COPD patients Subtask 2: Flow cytometry sorting of circulating myeloid cells. Subtask 3: RNA-Sequencing Subtask 4: RNA...recruitment including pre- and post- surgery patients, and COPD patients During this reporting period, we have recruited 23 NSCLC patients and collected

Fluorescent genetic barcoding in mammalian cells for enhanced multiplexing capabilities in flow cytometry.

PubMed

Smurthwaite, Cameron A; Hilton, Brett J; O'Hanlon, Ryan; Stolp, Zachary D; Hancock, Bryan M; Abbadessa, Darin; Stotland, Aleksandr; Sklar, Larry A; Wolkowicz, Roland

2014-01-01

The discovery of the green fluorescent protein from Aequorea victoria has revolutionized the field of cell and molecular biology. Since its discovery a growing panel of fluorescent proteins, fluorophores and fluorescent-coupled staining methodologies, have expanded the analytical capabilities of flow cytometry. Here, we exploit the power of genetic engineering to barcode individual cells with genes encoding fluorescent proteins. For genetic engineering, we utilize retroviral technology, which allows for the expression of ectopic genetic information in a stable manner in mammalian cells. We have genetically barcoded both adherent and nonadherent cells with different fluorescent proteins. Multiplexing power was increased by combining both the number of distinct fluorescent proteins, and the fluorescence intensity in each channel. Moreover, retroviral expression has proven to be stable for at least a 6-month period, which is critical for applications such as biological screens. We have shown the applicability of fluorescent barcoded multiplexing to cell-based assays that rely themselves on genetic barcoding, or on classical staining protocols. Fluorescent genetic barcoding gives the cell an inherited characteristic that distinguishes it from its counterpart. Once cell lines are developed, no further manipulation or staining is required, decreasing time, nonspecific background associated with staining protocols, and cost. The increasing number of discovered and/or engineered fluorescent proteins with unique absorbance/emission spectra, combined with the growing number of detection devices and lasers, increases multiplexing versatility, making fluorescent genetic barcoding a powerful tool for flow cytometry-based analysis. © 2013 International Society for Advancement of Cytometry.

Quantitative Method of Measuring Metastatic Activity

NASA Technical Reports Server (NTRS)

Morrison, Dennis R. (Inventor)

1999-01-01

The metastatic potential of tumors can be evaluated by the quantitative detection of urokinase and DNA. The cell sample selected for examination is analyzed for the presence of high levels of urokinase and abnormal DNA using analytical flow cytometry and digital image analysis. Other factors such as membrane associated uroldnase, increased DNA synthesis rates and certain receptors can be used in the method for detection of potentially invasive tumors.

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

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.

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

PubMed

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

2014-09-01

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

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.

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 of DNA Replication and RNA Synthesis: Historical Perspective and Recent Advances Based on “Click Chemistry”

PubMed Central

Darzynkiewicz, Zbigniew; Traganos, Frank; Zhao, Hong; Halicka, H. Dorota; Li, Jiangwei

2011-01-01

This review covers progress in the development of cytometric methodologies designed to assess DNA replication and RNA synthesis. The early approaches utilizing autoradiography to detect incorporation of 3H- or 14C-labeled thymidine were able to identify the four fundamental phases of the cell cycle G1, S, G2, and M, and by analysis of the fraction of labeled mitosis (FLM), to precisely define the kinetics of cell progression through these phases. Analysis of 3H-uridine incorporation and RNA content provided the means to distinguish quiescent G0 from cycling G1 cells. Subsequent progress in analysis of DNA replication was based on the use of BrdU as a DNA precursor and its detection by the quenching of the fluorescence intensity of DNA-bound fluorochromes such as Hoechst 33358 or acridine orange as measured by flow cytometry. Several variants of this methodology have been designed and used in studies to detect anticancer drug-induced perturbations of cell cycle kinetics. The next phase of method development, which was particularly useful in studies of the cell cycle in vivo, including clinical applications, relied on immunocytochemical detection of incorporated halogenated DNA or RNA precursors. This approach however was hampered by the need for DNA denaturation, which made it difficult to concurrently detect other cell constituents for multiparametric analysis. The recently introduced “click chemistry” approach has no such limitation and is the method of choice for analysis of DNA replication and RNA synthesis. This method is based on the use of 5-ethynyl-2′deoxyuridine (EdU) as a DNA precursor or 5-ethynyluridine (EU) as an RNA precursor and their detection with fluorochrome-tagged azides utilizing a copper (I) catalyzed [3+2] cycloaddition. Several examples are presented that illustrate incorporation of EdU or EU in cells subjected to DNA damage detected as histone H2AX phosphorylation that have been analyzed by flow or laser scanning cytometry. PMID:21425239

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.

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

Cancer Detection Device

NASA Technical Reports Server (NTRS)

1998-01-01

Ratcom, Inc., has joined NASA Johnson Space Center in an active program to develop cytometry capabilities for space station freedom. This agreement results from a cooperative program that NASA entered into with the American Cancer Society to aid in cancer prevention, diagnosis, and treatment. The flow cytometer is used by cancer researchers to make cellular measurements.

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

MicroRNA-126 enhances the sensitivity of osteosarcoma cells to cisplatin and methotrexate

PubMed Central

JIANG, LIANGDONG; HE, AIYONG; HE, XIAOJIE; TAO, CHENG

2015-01-01

The establishment of novel chemotherapy drugs for osteosarcoma is urgently required, and the mechanisms and effects of cisplatin (DDP) and methotrexate (MTX) in the current treatment of osteosarcoma have not been fully elucidated. The present study aimed to observe the effect of DDP, MTX and rapamycin on osteosarcoma cell proliferation and apoptosis, and to investigate the association between miR-126 and the effects of DDP and MTX in osteosarcoma cells. miR-126-overexpressing and -silencing lentiviral vectors were constructed, and MG63 and U-2 OS osteosarcoma cells were infected. An MTT assay was conducted to detect transfected cell proliferation, and the effects of the chemotherapy drugs on transfected cell apoptosis were detected by flow cytometry. The cell cycle of the transfected cells was analyzed via flow cytometry. As the miR-126-overexpressing and -silencing osteosarcoma cell lines were successfully constructed, it was observed that DDP and MTX inhibited osteosarcoma cell proliferation. With the decreased expression of miR-126, the sensitivity of osteosarcoma cells to DDP and MTX was reduced at the same concentration. The flow cytometry suggested that DDP and MTX could promote the apoptosis of osteosarcoma cells with overexpressed miR-126, whereas they could not significantly impact the apoptosis of the miR-126-silenced osteosarcoma cells. Meanwhile, DDP inhibited the cell cycle of the miR-126-overexpressing osteosarcoma cells. In conclusion, DDP and MTX inhibited the proliferation and promoted the apoptosis of the osteosarcoma cells, and these processes were dependent upon the expression of miR-126. PMID:26788206

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

Influence of Macrophages on the Rooster Spermatozoa Quality.

PubMed

Kuzelova, L; Vasicek, J; Chrenek, P

2015-08-01

The goal of this study was to evaluate the occurrence of macrophages in rooster semen and to investigate their impact on the spermatozoa quality. Ross 308 breeder males (n = 30) with no evidence of genital tract infections were used to determine the concentration of macrophages using fluorescently conjugated acetylated low-density lipoprotein (AcLDL). Subsequently, the roosters were divided into two groups on the basis of semen macrophage concentration, and semen quality was compared in two heterospermic samples. We applied computer-assisted semen analysis (CASA) system to determine motility parameters. Fluorescence microscopy and flow cytometry were used to evaluate occurrence of apoptotic and dead spermatozoa. Spermatozoa fertility potential was examined after intravaginal artificial insemination of hens. Eighteen roosters (control group) contained 0.2-3% of macrophages within spermatozoa population and ten roosters (macrophage group) had 10-15% of macrophages. Males from macrophage group had lower (p < 0.05) motility parameters (total and progressive movement, velocity curved line) and increased concentration of dead spermatozoa detected by flow cytometry and fluorescence microscopy (p < 0.001 and p ˂ 0.05, respectively). Differences (p < 0.05) between fluorescent microscopy and flow cytometry in results on spermatozoa apoptosis and viability were observed. No significant difference was found between groups in fertility of spermatozoa. In conclusion, the higher presence of macrophages in rooster semen may have a negative effect on some parameters of rooster spermatozoa evaluated in vitro. Furthermore, our study suggests that flow cytometry allows more precise examination of spermatozoa viability and apoptosis in a very short time compared with the fluorescent microscopy. © 2015 Blackwell Verlag GmbH.

Leukocyte Populations in Human Preterm and Term Breast Milk Identified by Multicolour Flow Cytometry

PubMed Central

Trend, Stephanie; de Jong, Emma; Lloyd, Megan L.; Kok, Chooi Heen; Richmond, Peter; Doherty, Dorota A.; Simmer, Karen; Kakulas, Foteini; Strunk, Tobias; Currie, Andrew

2015-01-01

Background Extremely preterm infants are highly susceptible to bacterial infections but breast milk provides some protection. It is unknown if leukocyte numbers and subsets in milk differ between term and preterm breast milk. This study serially characterised leukocyte populations in breast milk of mothers of preterm and term infants using multicolour flow cytometry methods for extended differential leukocyte counts in blood. Methods Sixty mothers of extremely preterm (<28 weeks gestational age), very preterm (28–31 wk), and moderately preterm (32–36 wk), as well as term (37–41 wk) infants were recruited. Colostrum (d2–5), transitional (d8–12) and mature milk (d26–30) samples were collected, cells isolated, and leukocyte subsets analysed using flow cytometry. Results The major CD45+ leukocyte populations circulating in blood were also detectable in breast milk but at different frequencies. Progression of lactation was associated with decreasing CD45+ leukocyte concentration, as well as increases in the relative frequencies of neutrophils and immature granulocytes, and decreases in the relative frequencies of eosinophils, myeloid and B cell precursors, and CD16- monocytes. No differences were observed between preterm and term breast milk in leukocyte concentration, though minor differences between preterm groups in some leukocyte frequencies were observed. Conclusions Flow cytometry is a useful tool to identify and quantify leukocyte subsets in breast milk. The stage of lactation is associated with major changes in milk leukocyte composition in this population. Fresh preterm breast milk is not deficient in leukocytes, but shorter gestation may be associated with minor differences in leukocyte subset frequencies in preterm compared to term breast milk. PMID:26288195

Human Eosinophils Express Functional CCR7

PubMed Central

Ueki, Shigeharu; Estanislau, Jessica; Weller, Peter F.

2013-01-01

Human eosinophils display directed chemotactic activity toward an array of soluble chemokines. Eosinophils have been observed to migrate to draining lymph nodes in experimental models of allergic inflammation, yet it is unknown whether eosinophils express CCR7, a key chemokine receptor in coordinating leukocyte trafficking to lymph nodes. The purpose of this study is to demonstrate expression of CCR7 by human eosinophils and functional responses to CCL19 and CCL21, the known ligands of CCR7. Human eosinophils were purified by negative selection from healthy donors. CCR7 expression of freshly purified, unstimulated eosinophils and of IL-5–primed eosinophils was determined by flow cytometry and Western blot. Chemotaxis to CCL19 and CCL21 was measured in transwell assays. Shape changes to CCL19 and CCL21 were analyzed by flow cytometry and microscopy. Calcium fluxes of fluo-4 AM–loaded eosinophils were recorded by flow cytometry after chemokine stimulation. ERK phosphorylation of CCL19- and CCL21-stimulated eosinophils was measured by Western blot and Luminex assay. Human eosinophils expressed CCR7 as demonstrated by flow cytometry and Western blots. Eosinophils exhibited detectable cell surface expression of CCR7. IL-5–primed eosinophils exhibited chemotaxis toward CCL19 and CCL21 in a dose-dependent fashion. Upon stimulation with CCL19 or CCL21, IL-5–primed eosinophils demonstrated dose-dependent shape changes with polarization of F-actin and exhibited calcium influxes. Finally, primed eosinophils stimulated with CCL19 or CCL21 exhibited increased phosphorylation of ERK in response to both CCR7 ligands. We demonstrate that human eosinophils express CCR7 and have multipotent responses to the known ligands of CCR7. PMID:23449735

Evaluation of Leishmania species reactivity in human serologic diagnosis of leishmaniasis.

PubMed

Silvestre, Ricardo; Santarém, Nuno; Teixeira, Lúcia; Cunha, Joana; Schallig, Henk; Cordeiro-da-Silva, Anabela

2009-08-01

The sensitivities and specificities of IgG-ELISA and IgG flow cytometry based techniques using different Leishmania species were determined using sera collected from 40 cutaneous or visceral leishmaniasis patients. The flow cytometry technique, using promastigote parasite forms, performed better than total soluble extract IgG-ELISA. At the species level, the use of Leishmania amazonensis and Leishmania major as antigens in enzyme linked immunosorbent assay (ELISA) decreased the overall sensitivity. To assess the specificity of these tests, sera from malaria, toxoplasmosis, amoebiasis, schistosomiasis, and leprosy patients were used. We also included sera from Leishmania non-infected endemic individuals. The cutaneous species displayed a decreased specificity in both assays. Although more sensitive, flow cytometry using promastigote parasite forms generally presented lower levels of specificity when compared with total extract of IgG-ELISA. Overall, the results of the study show the potential of IgG flow cytometry for the diagnosis of leishmaniasis. Although highly sensitive, a refinement of the flow cytometry method should be performed to improve the overall specificity.

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

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

DTIC Science & Technology

2004-09-01

Matrigel plug and sorted by flow cytometry . Sorting of these retrieved cells based on co-expression of the GFP marker and cell- surface endothelial...express the green fluorescent protein (GFP) and clonal MSC populations can be isolated and phenotypically and genotypically analyzed by flow cytometry ...monoclonal populations of these GFP+ murine MSCs and conducted flow cytometry analysis to determine their phenotype. Specifically, we determined if

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.

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.

Improving the signal analysis for in vivo photoacoustic flow cytometry

NASA Astrophysics Data System (ADS)

Niu, Zhenyu; Yang, Ping; Wei, Dan; Tang, Shuo; Wei, Xunbin

2015-03-01

At early stage of cancer, a small number of circulating tumor cells (CTCs) appear in the blood circulation. Thus, early detection of malignant circulating tumor cells has great significance for timely treatment to reduce the cancer death rate. We have developed an in vivo photoacoustic flow cytometry (PAFC) to monitor the metastatic process of CTCs and record the signals from target cells. Information of target cells which is helpful to the early therapy would be obtained through analyzing and processing the signals. The raw signal detected from target cells often contains some noise caused by electronic devices, such as background noise and thermal noise. We choose the Wavelet denoising method to effectively distinguish the target signal from background noise. Processing in time domain and frequency domain would be combined to analyze the signal after denoising. This algorithm contains time domain filter and frequency transformation. The frequency spectrum image of the signal contains distinctive features that can be used to analyze the property of target cells or particles. The PAFC technique can detect signals from circulating tumor cells or other particles. The processing methods have a great potential for analyzing signals accurately and rapidly.

Urine TREM-1 as a marker of urinary tract infection in children.

PubMed

Sierra-Diaz, Erick; Bravo Cuéllar, Alejandro; Ortiz Lazareno, Pablo Cesar; García Gutiérrez, Mariana; Georgina, Hernandez Flores; Anaya Prado, Roberto

2017-04-01

Objective Triggering receptor expressed on myeloid cells (TREM)-1 is a receptor that is thought to improve recognition of patients with true infection. In this study, we investigated whether Triggering receptor expressed on myeloid cells (TREM-1) is present in urine samples from children with urinary tract infection (UTI) and in samples from healthy children. Methods A total of 128 samples met the inclusion criteria for the study. Urine samples were processed for culture and urinalysis as a regular protocol for patients with UTI. Samples were classified according to culture and urinalysis results. TREM-1 protein expression was detected with flow cytometry and sTREM-1 was assessed by ELISA. Results Flow cytometry showed detectable expression of TREM-1 in 100% of samples, UTI and non-UTI groups ( p < 0.001). Mean fluorescence intensity of TREM-1 was different between the groups ( p < 0.001). Levels of sTREM-1 were detected in patients with UTI, but not in non-UTI patients. Conclusions All of our patients (healthy and diseased) showed TREM-1 expression. However, TREM-1 levels in patients with UTI tend to be higher and are associated with increased neutrophils and cytokine activity induced by bacteria.

Urine TREM-1 as a marker of urinary tract infection in children

PubMed Central

Sierra-Diaz, Erick; Ortiz Lazareno, Pablo Cesar; García Gutiérrez, Mariana; Georgina, Hernandez Flores; Anaya Prado, Roberto

2017-01-01

Objective Triggering receptor expressed on myeloid cells (TREM)-1 is a receptor that is thought to improve recognition of patients with true infection. In this study, we investigated whether Triggering receptor expressed on myeloid cells (TREM-1) is present in urine samples from children with urinary tract infection (UTI) and in samples from healthy children. Methods A total of 128 samples met the inclusion criteria for the study. Urine samples were processed for culture and urinalysis as a regular protocol for patients with UTI. Samples were classified according to culture and urinalysis results. TREM-1 protein expression was detected with flow cytometry and sTREM-1 was assessed by ELISA. Results Flow cytometry showed detectable expression of TREM-1 in 100% of samples, UTI and non-UTI groups (p < 0.001). Mean fluorescence intensity of TREM-1 was different between the groups (p < 0.001). Levels of sTREM-1 were detected in patients with UTI, but not in non-UTI patients. Conclusions All of our patients (healthy and diseased) showed TREM-1 expression. However, TREM-1 levels in patients with UTI tend to be higher and are associated with increased neutrophils and cytokine activity induced by bacteria. PMID:28367708

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.

Correlation of DNA Ploidy with Progression of Cervical Cancer

PubMed Central

Singh, M.; Mehrotra, S.; Kalra, N.; Singh, U.; Shukla, Y.

2008-01-01

The majority of squamous cell carcinomas of cervix are preceded by visible changes in the cervix, most often detected by cervical smear. As cervical cancer is preceded by long precancerous stages, identification of the high-risk population through detection of DNA ploidy may be of importance in effective management of this disease. Here we attempted to correlate aneuploid DNA patterns and their influence on biological behavior of flow-cytometry analysis of DNA ploidy which was carried out in cytologically diagnosed cases of mild (79), moderate (36), and severe (12) dysplasia, as well as “atypical squamous cells of unknown significance (ASCUS)” (57) along with controls (69), in order to understand its importance in malignant progression of disease. Cytologically diagnosed dysplasias, which were employed for DNA ploidy studies, 39 mild, 28 moderate, and 11 severe dysplasia cases were found to be aneuploid. Out of the 69 control subjects, 6 cases showed aneuploidy pattern and the rest 63 subjects were diploid. An aneuploidy pattern was observed in 8 out of 57 cases of cytologically evaluated ASCUS. The results of the followup studies showed that aberrant DNA content reliably predicts the occurrence of squamous cell carcinoma in cervical smear. Flow cytometric analysis of DNA ploidy may provide a strategic diagnostic tool for early detection of carcinoma cervix. Therefore, it is a concept of an HPV screening with reflex cytology in combination with DNA flow cytometry to detect progressive lesions with the greatest possible sensitivity and specificity. PMID:20445775

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

PubMed

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

2016-01-01

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

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

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

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.

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.

Quantitative method of measuring cancer cell urokinase and metastatic potential

NASA Technical Reports Server (NTRS)

Morrison, Dennis R. (Inventor)

1993-01-01

The metastatic potential of tumors can be evaluated by the quantitative detection of urokinase and DNA. The cell sample selected for examination is analyzed for the presence of high levels of urokinase and abnormal DNA using analytical flow cytometry and digital image analysis. Other factors such as membrane associated urokinase, increased DNA synthesis rates and certain receptors can be used in the method for detection of potentially invasive tumors.

In Vivo Rat T-Lymphocyte Pig-a Assay: Detection and Expansion of Cells Deficient in the GPI-Anchored CD48 Surface Marker for Analysis of Mutation in the Endogenous Pig-a Gene.

PubMed

Dobrovolsky, Vasily N; Revollo, Javier; Petibone, Dayton M; Heflich, Robert H

2017-01-01

The Pig-a assay is being developed as an in vivo gene mutation assay for regulatory safety assessments. The assay is based on detecting mutation in the endogenous Pig-a gene of treated rats by using flow cytometry to measure changes in cell surface markers of peripheral blood cells. Here we present a methodology for demonstrating that phenotypically mutant rat T-cells identified by flow cytometry contain mutations in the Pig-a gene, an important step for validating the assay. In our approach, the mutant phenotype T-cells are sorted into individual wells of 96-well plates and expanded into clones. Subsequent sequencing of genomic DNA from the expanded clones confirms that the Pig-a assay detects exactly what it claims to detect-cells with mutations in the endogenous Pig-a gene. In addition, determining the spectra of Pig-a mutations provides information for better understanding the mutational mechanism of compounds of interest. Our methodology of combining phenotypic antibody labeling, magnetic enrichment, sorting, and single-cell clonal expansion can be used in genotoxicity/mutagenicity studies and in other general immunotoxicology research requiring identification, isolation, and expansion of extremely rare subpopulations of T-cells.

A rapid method for infectivity titration of Andes hantavirus using flow cytometry.

PubMed

Barriga, Gonzalo P; Martínez-Valdebenito, Constanza; Galeno, Héctor; Ferrés, Marcela; Lozach, Pierre-Yves; Tischler, Nicole D

2013-11-01

The focus assay is currently the most commonly used technique for hantavirus titer determination. This method requires an incubation time of between 5 and 11 days to allow the appearance of foci after several rounds of viral infection. The following work presents a rapid Andes virus (ANDV) titration assay, based on viral nucleocapsid protein (N) detection in infected cells by flow cytometry. To this end, an anti-N monoclonal antibody was used that was developed and characterized previously. ANDV N could be detected as early as 6 h post-infection, while viral release was not observed until 24-48 h post-infection. Given that ANDV detection was performed during its first round of infection, a time reduction for titer determination was possible and provided results in only two days. The viral titer was calculated from the percentage of N positive cells and agreed with focus assay titers. Furthermore, the assay was applied to quantify the inhibition of ANDV cell entry by patient sera and by preventing endosome acidification. This novel hantavirus titration assay is a highly quantitative and sensitive tool that facilitates infectivity titration of virus stocks, rapid screening for antiviral drugs, and may be further used to detect and quantify infectious virus in human samples. Copyright © 2013 Elsevier B.V. All rights reserved.

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.

Identification of CD56 and CD57 by flow cytometry in Ewing's sarcoma or primitive neuroectodermal tumor.

PubMed

Gardner, L J; Polski, J M; Fallon, R; Dunphy, C H

1998-07-01

CD56 and CD57 are commonly considered as natural killer and neuroectodermal markers, but their expression has been identified in a wide spectrum of neoplasms including some cases of Ewing's sarcoma (ES) and primitive neuroectodermal tumor (PNET). We report two cases of small, round blue cell tumor (SRBCT), in which flow cytometry immunophenotyping (FCI) detected strong expression of CD56 and CD57 (one case). Immunohistochemical staining with Leu-19 and Leu-7 confirmed the FI results. Although CD56 and CD57 expression is consistent with ES/PNET, it can be potentially misleading if results of FCI are interpreted in the absence of other findings. These cases suggest the utility of FCI in undifferentiated SRBCT. The literature on CD56 and CD57 expression in ES/PNET is reviewed and discussed.

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

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

Microfluidic flow rate detection based on integrated optical fiber cantilever.

PubMed

Lien, Victor; Vollmer, Frank

2007-10-01

We demonstrate an integrated microfluidic flow sensor with ultra-wide dynamic range, suitable for high throughput applications such as flow cytometry and particle sorting/counting. A fiber-tip cantilever transduces flow rates to optical signal readout, and we demonstrate a dynamic range from 0 to 1500 microL min(-1) for operation in water. Fiber-optic sensor alignment is guided by preformed microfluidic channels, and the dynamic range can be adjusted in a one-step chemical etch. An overall non-linear response is attributed to the far-field angular distribution of single-mode fiber output.

Flow cytometric detection method for DNA samples

DOEpatents

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

2011-07-05

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

Flow cytometric detection method for DNA samples

DOEpatents

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

2006-08-01

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

Flow Cytometry of Extracellular Vesicles: Potential, Pitfalls, and Prospects.

PubMed

Nolan, John P

2015-07-01

Evidence suggests that extracellular vesicles (EVs) can play roles in physiology and pathology, providing impetus to explore their use as diagnostic and therapeutic targets. However, EVs are also small, heterogeneous, and difficult to measure, and so this potential has not yet been realized. The development of improved approaches to EV detection and characterization will be critical to further understanding their roles in physiology and disease. Flow cytometry has been a popular tool for measuring cell-derived EVs, but has often been used in an uncritical manner in which fundamental principles and limitations of the instrument are ignored. Recent efforts to standardize procedures and document the effects of different methodologies have helped to address this shortcoming, but much work remains. In this paper, I address some of the instrument, reagent, and analysis considerations relevant to measurement of individual EVs in flow, with the aim of clarifying a path to quantitative and standardized measurement of these interesting and potentially important biological nanoparticles. Copyright © 2015 John Wiley & Sons, Inc.

Consensus guidelines on plasma cell myeloma minimal residual disease analysis and reporting.

PubMed

Arroz, Maria; Came, Neil; Lin, Pei; Chen, Weina; Yuan, Constance; Lagoo, Anand; Monreal, Mariela; de Tute, Ruth; Vergilio, Jo-Anne; Rawstron, Andy C; Paiva, Bruno

2016-01-01

Major heterogeneity between laboratories in flow cytometry (FC) minimal residual disease (MRD) testing in multiple myeloma (MM) must be overcome. Cytometry societies such as the International Clinical Cytometry Society and the European Society for Clinical Cell Analysis recognize a strong need to establish minimally acceptable requirements and recommendations to perform such complex testing. A group of 11 flow cytometrists currently performing FC testing in MM using different instrumentation, panel designs (≥ 6-color) and analysis software compared the procedures between their respective laboratories and reviewed the literature to propose a consensus guideline on flow-MRD analysis and reporting in MM. Consensus guidelines support i) the use of minimum of five initial gating parameters (CD38, CD138, CD45, forward, and sideward light scatter) within the same aliquot for accurate identification of the total plasma cell compartment; ii) the analysis of potentially aberrant phenotypic markers and to report the antigen expression pattern on neoplastic plasma cells as being reduced, normal or increased, when compared to a normal reference plasma cell immunophenotype (obtained using the same instrument and parameters); and iii) the percentage of total bone marrow plasma cells plus the percentages of both normal and neoplastic plasma cells within the total bone marrow plasma cell compartment, and over total bone marrow cells. Consensus guidelines on minimal current and future MRD analyses should target a lower limit of detection of 0.001%, and ideally a limit of quantification of 0.001%, which requires at least 3 × 10(6) and 5 × 10(6) bone marrow cells to be measured, respectively. © 2015 International Clinical Cytometry Society.

Detection of Ti02 nanoparticles using flow cytometry and optimized darkfield microscopy

EPA Science Inventory

Evaluation of the potential hazard of man made nanomaterials has been hampered by a limited ability to observe and measure nanoparticles in cells. In the present study, TiOz nanoparticles (rutile, 30-40 nm) were suspended in DMEM/F12 with 10% fetal bovine serum, sonicated, and ch...

Hyperspectral Imaging, Flow cytometry and Microscopic Morphology of Silver Nanoparticle within Cells

EPA Science Inventory

The ability to detect and track silver nanoparticles (AgNP) that enter cells is important to understand the potential biological and toxicological actions of AgNP. The uptake and fate in cells of four different types of AgNP was studied in a retinal pigment epithelial cell line ...

[Evolution of paroxysmal nocturnal hemoglobinuria clone during an hemolytic crisis in a patient with aplastic anemia. Flow cytometry study].

PubMed

Canalejo, K; Galassi, N; Riera, N; Bengió, R; Aixalá, M

2001-01-01

The expansion of paroxysmal nocturnal hemoglobinuria (PHN) clone was evaluated in a patient with aplastic anemia (AA) of 18 years of evolution during an hemolytic crisis. On day 0, Ham and Sucrosa tests were positive and hematological parameters were altered. Low hemoglobin (Hb) levels and erythrocyte and leukocyte counts were found and continued decreasing on days 7 and 24 (last day of study). High LDH levels, indirect bilirubin and reticulocyte counts were detected throughout. We evaluated CD55 and CD59 on erythrocytes by flow cytometry. Our results showed low CD55 expression with respect to the normal pattern. Since day 0, CD59 staining detected two red cell populations: PNH I (48%), cells with positive fluorescence similar to normal and PNH III (52%), negative cells (PNH clone). These negative cells increased, reaching 70% on day 24. Other membrane anchored leukocyte proteins were also absent (CD14) or decreased (CD16). We found a good correlation between clinical observations, evolution of the laboratory values and expansion of the PNH clone.

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.

[Effects of methyl tertiary butyl ether on cell cycle and cell apoptosis].

PubMed

Zhou, W; Huang, G; Zhang, H; Ye, S

2000-07-01

To explore the effects of the new gasoline additive, methyl tertiary butyl ether (MTBE) on cell cycle and cell apoptosis. Flow cytometry was used to evaluate the effect of MTBE (1, 2, 4 microl/ml, 24 h) on NIH/3T3 cell cycles; and the effect of MTBE on Hela cell apoptosis was evaluated by detecting cell survival using crystal violet staining. Flow cytometry showed that MTBE could change NIH/3T3 cell cycles, decrease the number of cells in S stage, and arrest cells at G(2) + M stage. The results suggested that MTBE could affect NIH/3T3 cell cycles and induce cell proliferation. This situation existed 48 hours after the treatment, and cell cycles came back normal 96 hours after the treatment. By detecting cell survival using crystal violet staining, we found that MTBE could inhibit the apoptosis of Hela cells which was induced by tumor necrosis factor (TNF)alpha and cycloheximide. MTBE's carcinogenicity to animals may relate to induction of cell proliferation and inhibition of cell apoptosis.

Detection and isolation of cell-derived microparticles are compromised by protein complexes resulting from shared biophysical parameters.

PubMed

György, Bence; Módos, Károly; Pállinger, Eva; Pálóczi, Krisztina; Pásztói, Mária; Misják, Petra; Deli, Mária A; Sipos, Aron; Szalai, Anikó; Voszka, István; Polgár, Anna; Tóth, Kálmán; Csete, Mária; Nagy, György; Gay, Steffen; Falus, András; Kittel, Agnes; Buzás, Edit I

2011-01-27

Numerous diseases, recently reported to associate with elevated microvesicle/microparticle (MP) counts, have also long been known to be characterized by accelerated immune complex (IC) formation. The goal of this study was to investigate the potential overlap between parameters of protein complexes (eg, ICs or avidin-biotin complexes) and MPs, which might perturb detection and/or isolation of MPs. In this work, after comprehensive characterization of MPs by electron microscopy, atomic force microscopy, dynamic light-scattering analysis, and flow cytometry, for the first time, we drive attention to the fact that protein complexes, especially insoluble ICs, overlap in biophysical properties (size, light scattering, and sedimentation) with MPs. This, in turn, affects MP quantification by flow cytometry and purification by differential centrifugation, especially in diseases in which IC formation is common, including not only autoimmune diseases, but also hematologic disorders, infections, and cancer. These data may necessitate reevaluation of certain published data on patient-derived MPs and contribute to correct the clinical laboratory assessment of the presence and biologic functions of MPs in health and disease.

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

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.

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

PubMed

Józwa, Wojciech; Czaczyk, Katarzyna

2012-04-02

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

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

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.

New mononuclear leukocyte-like populations within the granulocyte scatter gate detected by flow cytometry (Conference Presentation)

NASA Astrophysics Data System (ADS)

Melzer, Susanne; Löffler, Markus; Kautzner, Marlene; Tárnok, Attila

2017-02-01

Granulocytes are the major players in innate immunity and are prognostic markers in diseases. An in-depth phenotypic characterization of granulocyte subtypes and correlation with biometry or lifestyle is so far lacking. The reason is, that either preparation of mononuclear cells was analyzed or that cells in the neutrophil window were neglected in the analysis. Here we show for the first time lymphocyte- (LL) and monocyte-like (ML) cells within the granulocyte scatter gate as new, previously unknown cell subpopulation. Immunophenotyping of 905 healthy German adults from the LIFE study [1] was performed by 10-color flow cytometry [2]. Age of men (n=420): 56.5±14.0 years, women (n=485): 56.7±13.6 y (range of 18-81 y). Data analyzed by FlowJo v10.0.6. Values compared by Mann-Whitney-U test: men vs women, young (18-49 y) vs. elderly (50-81 y.) men, and young (19-49 y.) vs. elderly (50-81 y.) women; significance: p<0.05. Within the granulocyte gate four phenotypically distinct cell types were detected (all CD45+, SSCmid-high): LL1 CD3+,CD4+,CD8++,CD16/56+,CD38+,HLA-DR+ LL2 CD3+,CD4low,CD8+,CD38low LL3 CD3+,CD4+,CD8- ML1 CD3-,CD4low,CD14+,CD38+ LL2 counts were increased in men (p=0.042), as well as ML1 counts (p <0.001). Most of the cell counts were not dependent on age, except LL2 in women. In conclusion, new lymphocyte like cell types with the neutrophil scatter characteristics are reported. Counts correlate with age and gender. We plan to sort these new subtypes for further functional characterization and aim to establish them as cellular biomarkers for the early detection of various diseases. [1] BMC Public Health. 2015;15:691; [2] Cytometry A. 2014;85(9):781

Immunophenotyping by slide-based cytometry and by flow cytometry are comparable

NASA Astrophysics Data System (ADS)

Gerstner, Andreas O.; Laffers, Wiebke; Mittag, Anja; Daehnert, Ingo; Lenz, Domnik; Bootz, Friedrich; Bocsi, Jozsef; Tarnok, Attila

2005-03-01

Immunophenotyping of peripheral blood leukocytes (PBLs) is performed by flow cytometry (FCM) as the golden standard. Slide based cytometry systems for example laser scanning cytometer (LSC) can give additional information (repeated staining and scanning, morphology). In order to adequately judge on the clinical usefulness of immunophenotyping by LSC it is obligatory to compare it with the long established FCM assays. We performed this study to systematically compare the two methods, FCM and LSC for immunophenotyping and to test the correlation of the results. Leucocytes were stained with directly labeled monoclonal antibodies with whole blood staining method. Aliquots of the same paraformaldehyde fixed specimens were analyzed in a FACScan (BD-Biosciences) using standard protocols and parallel with LSC (CompuCyte) after placing to glass slide, drying and fixation by aceton and 7-AAD staining. Calculating the percentage distribution of PBLs obtained by LSC and by FCM shows very good correlation with regression coefficients close to 1.0 for the major populations (neutrophils, lymphocytes, and monocytes), as well as for the lymphocyte sub-populations (T-helper-, T-cytotoxic-, B-, NK-cells). LSC can be recommended for immunophenotyping of PBLs especially in cases where only very limited sample volumes are available or where additional analysis of the cells" morphology is important. There are limitations in the detection of rare leucocytes or weak antigens where appropriate amplification steps for immunofluorescence should be engaged.

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...

Elucidation of Seventeen Human Peripheral Blood B cell Subsets and Quantification of the Tetanus Response Using a Density-Based Method for the Automated Identification of Cell Populations in Multidimensional Flow Cytometry Data

PubMed Central

Qian, Yu; Wei, Chungwen; Lee, F. Eun-Hyung; Campbell, John; Halliley, Jessica; Lee, Jamie A.; Cai, Jennifer; Kong, Megan; Sadat, Eva; Thomson, Elizabeth; Dunn, Patrick; Seegmiller, Adam C.; Karandikar, Nitin J.; Tipton, Chris; Mosmann, Tim; Sanz, Iñaki; Scheuermann, Richard H.

2011-01-01

Background Advances in multi-parameter flow cytometry (FCM) now allow for the independent detection of larger numbers of fluorochromes on individual cells, generating data with increasingly higher dimensionality. The increased complexity of these data has made it difficult to identify cell populations from high-dimensional FCM data using traditional manual gating strategies based on single-color or two-color displays. Methods To address this challenge, we developed a novel program, FLOCK (FLOw Clustering without K), that uses a density-based clustering approach to algorithmically identify biologically relevant cell populations from multiple samples in an unbiased fashion, thereby eliminating operator-dependent variability. Results FLOCK was used to objectively identify seventeen distinct B cell subsets in a human peripheral blood sample and to identify and quantify novel plasmablast subsets responding transiently to tetanus and other vaccinations in peripheral blood. FLOCK has been implemented in the publically available Immunology Database and Analysis Portal – ImmPort (http://www.immport.org) for open use by the immunology research community. Conclusions FLOCK is able to identify cell subsets in experiments that use multi-parameter flow cytometry through an objective, automated computational approach. The use of algorithms like FLOCK for FCM data analysis obviates the need for subjective and labor intensive manual gating to identify and quantify cell subsets. Novel populations identified by these computational approaches can serve as hypotheses for further experimental study. PMID:20839340

Flow cytometric characterization of phenotype, DNA indices and p53 gene expression in 55 cases of acute leukemia.

PubMed

Powari, Manish; Varma, Neelam; Varma, Subhash; Marwaha, Ram Kumar; Sandhu, Harpreet; Ganguly, Nirmal Kumar

2002-06-01

To characterize the phenotype of acute leukemia cases using flow cytometry, to detect mixed lineage cases and to use DNA index determination, including S-phase fraction (SPF) and p53 detection, to find if there was any correlation of SPF and p53 expression with outcome. Fifty-five cases of acute leukemia were enrolled in this study. A complete hemogram and routine bone marrow examination, including cytochemistry, was done. Mycloperoxidase-negative cases were evaluated on a flow cytometer using monoclonal antibodies. DNA indices were determined by flow cytometry in all cases, and p53 was detected immunohistochemically using the alkaline phosphatase/antialkaline phosphatase technique. Acute myeloblastic leukemia (AML) was diagnosed in 32 cases; acute lymphoblastic leukemia (ALL) was diagnosed in 18 (14 B lineage and 4 T line age). Four cases showed mixed lineage leukemia, and undifferentiated acute leukemia was diagnosed in one case. The mean/range of SPF for these groups were 3.76/0.33-6.91, 6.25/0.15-21.4, 2.89/0.35-10.64, 2.60/0.72-6.94 and 7.34, respectively. Aneuploidy was detected in two cases of B-lineage ALL and tetraploidy in a case of AML-M7, while all others were diploid p53. Was detected in 6 of 55 cases (10.90%). Follow-up was available for 24 patients. Five patients relapsed, and four had B-cell type ALL and were diploid and expressed no p53 gene. SPF% did not show any correlation with outcome. These data suggest that within acute leukemia subtypes, there is a wide variation in SPF. SPF does not seem to correlate with outcome. Immunophenotyping is essential to determine the lineage in myeloperoxidase-negative cases. It is perhaps the only way to diagnose mixed lineage leukemia and aberrant expression of markers presently. The p53 gene was detected less frequently. However, more studies are required from different centers with longer follow-up to evaluate prognostic significance.

Ovarian stem cells are always accompanied by very small embryonic-like stem cells in adult mammalian ovary.

PubMed

Bhartiya, Deepa

2015-11-05

Existing dogma that a female is born with fixed number of eggs was challenged by the detection of stem cells in adult mammalian ovary. Data has accumulated in support of ovarian stem cells (OSCs) proliferation, maintenance in culture, formation of germ cell nests and differentiation into oocytes and primordial follicle assembly using different strategies. Flow cytometry analysis identified >8 μm OSCs which are DDX1 positive and are considered equivalent to spermatogonial stem cells (SSCs) in testis. Analysis of both ovarian and testicular smears obtained after enzymatic digestion has led to the identification of an additional stem cell population termed very small embryonic-like stem cells (VSELs). VSELs and OSCs/SSCs differ from each other in their size and OCT-4 expression. VSELs express pluripotent markers including nuclear OCT-4 whereas OSCs/SSCs express cytoplasmic OCT-4 suggesting a differentiated state. VSELs can be studied by flow cytometry as small sized cells which are LIN-/CD45-/Sca-1+. We have reported 0.02 ± 0.008, 0.03 ± 0.017 and 0.08 ± 0.03 % of total cells as VSELs in normal, chemoablated and after FSH treatment to chemoablated mouse ovary. VSELs have remained poorly studied till now because of their very small size and rare occurrence. Spinning cells obtained after enzymatic digestion of ovarian tissue at a speed of 1000G (rather than 1200 rpm) throughout processing allows reliable detection of the VSELs by flow cytometry. VSELs exist in aged, chemoablated and non-functional ovary and providing a healthy niche to support their function offers an interesting strategy to manage infertility.

In vivo multispectral photoacoustic and photothermal flow cytometry with multicolor dyes: a potential for real-time assessment of circulation, dye-cell interaction, and blood volume.

PubMed

Proskurnin, Mikhail A; Zhidkova, Tatyana V; Volkov, Dmitry S; Sarimollaoglu, Mustafa; Galanzha, Ekaterina I; Mock, Donald; Nedosekin, Dmitry A; Zharov, Vladimir P

2011-10-01

Recently, photoacoustic (PA) flow cytometry (PAFC) has been developed for in vivo detection of circulating tumor cells and bacteria targeted by nanoparticles. Here, we propose multispectral PAFC with multiple dyes having distinctive absorption spectra as multicolor PA contrast agents. As a first step of our proof-of-concept, we characterized high-speed PAFC capability to monitor the clearance of three dyes (Indocyanine Green [ICG], Methylene Blue [MB], and Trypan Blue [TB]) in an animal model in vivo and in real time. We observed strong dynamic PA signal fluctuations, which can be associated with interactions of dyes with circulating blood cells and plasma proteins. PAFC demonstrated enumeration of circulating red and white blood cells labeled with ICG and MB, respectively, and detection of rare dead cells uptaking TB directly in bloodstream. The possibility for accurate measurements of various dye concentrations including Crystal Violet and Brilliant Green were verified in vitro using complementary to PAFC photothermal (PT) technique and spectrophotometry under batch and flow conditions. We further analyze the potential of integrated PAFC/PT spectroscopy with multiple dyes for rapid and accurate measurements of circulating blood volume without a priori information on hemoglobin content, which is impossible with existing optical techniques. This is important in many medical conditions including surgery and trauma with extensive blood loss, rapid fluid administration, and transfusion of red blood cells. The potential for developing a robust clinical PAFC prototype that is safe for human, and its applications for studying the liver function are further highlighted. Copyright © 2011 International Society for Advancement of Cytometry.

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.

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

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.

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

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

DTIC Science & Technology

2013-02-01

get enough sorted mammary cells for the transplantation experiments. We are currently working with our Flow Cytometry Core to sort Lin-/CD24+/CD49...activity our flow cytometry data suggests t here is a 2-fold increase in the number of FOG+ MEC’s in BATgal animals compared to contro ls which...this populat ion of cells is enriched for stem cell activity. Flow cytometry will determine the percentage of FOG+ cells within pre-neoplastic BATgai

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

Validation of cell-based fluorescence assays: practice guidelines from the ICSH and ICCS - part II - preanalytical issues.

PubMed

Davis, Bruce H; Dasgupta, Amar; Kussick, Steven; Han, Jin-Yeong; Estrellado, Annalee

2013-01-01

Flow cytometry and other technologies of cell-based fluorescence assays are as a matter of good laboratory practice required to validate all assays, which when in clinical practice may pass through regulatory review processes using criteria often defined with a soluble analyte in plasma or serum samples in mind. Recently the U.S. Food and Drug Administration (FDA) has entered into a public dialogue in the U.S. regarding their regulatory interest in laboratory developed tests (LDTs) or so-called "home brew" assays performed in clinical laboratories. The absence of well-defined guidelines for validation of cell-based assays using fluorescence detection has thus become a subject of concern for the International Council for Standardization of Haematology (ICSH) and International Clinical Cytometry Society (ICCS). Accordingly, a group of over 40 international experts in the areas of test development, test validation, and clinical practice of a variety of assay types using flow cytometry and/or morphologic image analysis were invited to develop a set of practical guidelines useful to in vitro diagnostic (IVD) innovators, clinical laboratories, regulatory scientists, and laboratory inspectors. The focus of the group was restricted to fluorescence reporter reagents, although some common principles are shared by immunohistochemistry or immunocytochemistry techniques and noted where appropriate. The work product of this two year effort is the content of this special issue of this journal, which is published as 5 separate articles, this being Validation of Cell-based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS - Part II - Preanalytical issues. © 2013 International Clinical Cytometry Society. © 2013 International Clinical Cytometry Society.

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.

Assessment of the Utility of Cytology and Flow Cytometry of Cerebrospinal Fluid Samples in Clinical Practice.

PubMed

Nam, Anna S; Giorgadze, Tamara; Tam, Wayne; Chadburn, Amy

2018-01-01

We sought to assess the utility and limitations of both flow cytometry (FC) and cytology for the analysis of cerebrospinal fluid (CSF) in a practical clinical setting. A total of 393 consecutive CSF samples from 171 patients submitted for both cytomorphologic and FC assessments were analyzed. Both FC and cytology findings were negative for malignancy in 315/393 samples (80%), and either positive (POS) or suspicious/atypical (SUSP/AT) in 7% of samples. This resulted in high agreement between FC and cytology (87%). Minor discrepancies were present in 4% of the cases. In 28 samples, an abnormal population was detected by FC but not by cytology. FC and cytology are important complementary methods for analyzing CSF samples. In cases where cytology is SUSP/AT and FC is inconclusive or negative, additional specimens should be submitted for immunostaining, cytogenetics, and/or molecular studies. © 2018 S. Karger AG, Basel.

Correspondence of Neutralizing Humoral Immunity and CD4 T Cell Responses in Long Recovered Sudan Virus Survivors

PubMed Central

Sobarzo, Ariel; Stonier, Spencer W.; Herbert, Andrew S.; Ochayon, David E.; Kuehne, Ana I.; Eskira, Yael; Fedida-Metula, Shlomit; Tali, Neta; Lewis, Eli C.; Egesa, Moses; Cose, Stephen; Lutwama, Julius Julian; Yavelsky, Victoria; Dye, John M.; Lobel, Leslie

2016-01-01

Robust humoral and cellular immunity are critical for survival in humans during an ebolavirus infection. However, the interplay between these two arms of immunity is poorly understood. To address this, we examined residual immune responses in survivors of the Sudan virus (SUDV) outbreak in Gulu, Uganda (2000–2001). Cytokine and chemokine expression levels in SUDV stimulated whole blood cultures were assessed by multiplex ELISA and flow cytometry. Antibody and corresponding neutralization titers were also determined. Flow cytometry and multiplex ELISA results demonstrated significantly higher levels of cytokine and chemokine responses in survivors with serological neutralizing activity. This correspondence was not detected in survivors with serum reactivity to SUDV but without neutralization activity. This previously undefined relationship between memory CD4 T cell responses and serological neutralizing capacity in SUDV survivors is key for understanding long lasting immunity in survivors of filovirus infections. PMID:27187443

Correspondence of Neutralizing Humoral Immunity and CD4 T Cell Responses in Long Recovered Sudan Virus Survivors.

PubMed

Sobarzo, Ariel; Stonier, Spencer W; Herbert, Andrew S; Ochayon, David E; Kuehne, Ana I; Eskira, Yael; Fedida-Metula, Shlomit; Tali, Neta; Lewis, Eli C; Egesa, Moses; Cose, Stephen; Lutwama, Julius Julian; Yavelsky, Victoria; Dye, John M; Lobel, Leslie

2016-05-11

Robust humoral and cellular immunity are critical for survival in humans during an ebolavirus infection. However, the interplay between these two arms of immunity is poorly understood. To address this, we examined residual immune responses in survivors of the Sudan virus (SUDV) outbreak in Gulu, Uganda (2000-2001). Cytokine and chemokine expression levels in SUDV stimulated whole blood cultures were assessed by multiplex ELISA and flow cytometry. Antibody and corresponding neutralization titers were also determined. Flow cytometry and multiplex ELISA results demonstrated significantly higher levels of cytokine and chemokine responses in survivors with serological neutralizing activity. This correspondence was not detected in survivors with serum reactivity to SUDV but without neutralization activity. This previously undefined relationship between memory CD4 T cell responses and serological neutralizing capacity in SUDV survivors is key for understanding long lasting immunity in survivors of filovirus infections.

Flow cytometry and immunomorphological characteristics of apoptosis in hepatocytes of white mice during aging.

PubMed

Gujabidze, N; Rukhadze, R

2006-08-01

Apoptosis, sometimes called "programmed cell death", the process that goes on continuously throughout life has received phenomenal attention in the past few years. In the process of aging of organism, most of organs undergo morphological and functional changes at various frequencies. Initially, the role of apoptosis regarding aging was evaluated negatively, however, at present the issue is in the process of reconsideration. The experiments were performed on 74 white mice, distributed in three age groups (juveniles, adults, and senescents). Apoptotic nuclei were detected by immunomorphological and flow cytometry assay. So, the analysis of the data obtained that apoptosis in hepatocytes of white mice decreases with age and afterwards increases in a credible way. The maximum value is reached in the senescent mice. It has been considered, that aging increases the susceptibility of hepatocytes to apoptosis in white mice.

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.

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.

Resetting the T Cell Repertoire in Prostate Cancer Bearing Host

DTIC Science & Technology

2009-03-01

splenocytes were harvested and stained with fluorochrome conjugated antibodies anti CD4 (RM4.5), anti CDS (53 6.7), and anti V(3S.I+S.2 (MRS 2; BD... harvested 20 h later and analyzed by flow cytome~ for the relative abundance of CFSEiow (SV40 TAg peptide) and CFSE i (HSV peptide) populations. Detection...and spleens were harvested S days after the last injection. Cells were stained for flow cytometry analysis. This treatment did not affect either

Stability of nuclear DNA content among divergent and isolated populations of Fraser fir

Treesearch

L.D. Auckland; J.S. Johnston; H.J. Price; F.E. Bridgwater

2001-01-01

Fraser fir (Abies fraseri (Pursh) Poir.) is an endemic species consisting of six major disjunct populations in the Appalachian Mountains, U.S.A. Nuclear DNA content was measured with laser flow cytometry to determine if genome size differences could be detected among the disjunct populations of Fraser fir and its close relatives, balsam fir

The Embryonic Stem Cell Test as Tool to Assess Structure-Dependent Teratogenicity: The Case of Valproic Acid

PubMed Central

Riebeling, Christian; Pirow, Ralph; Becker, Klaus; Buesen, Roland; Eikel, Daniel; Kaltenhäuser, Johanna; Meyer, Frauke; Nau, Heinz; Slawik, Birgitta; Visan, Anke; Volland, Jutta; Spielmann, Horst; Luch, Andreas; Seiler, Andrea

2011-01-01

Teratogenicity can be predicted in vitro using the embryonic stem cell test (EST). The EST, which is based on the morphometric measurement of cardiomyocyte differentiation and cytotoxicity parameters, represents a scientifically validated method for the detection and classification of chemicals according to their teratogenic potency. Furthermore, an abbreviated protocol applying flow cytometry of intracellular marker proteins to determine differentiation into the cardiomyocyte lineage is available. Although valproic acid (VPA) is in worldwide clinical use as antiepileptic drug, it exhibits two severe side effects, i.e., teratogenicity and hepatotoxicity. These limitations have led to extensive research into derivatives of VPA. Here we chose VPA as model compound to test the applicability domain and to further evaluate the reliability of the EST. To this end, we study six closely related congeners of VPA and demonstrate that both the standard and the molecular flow cytometry-based EST are well suited to indicate differences in the teratogenic potency among VPA analogs that differ only in chirality or side chain length. Our data show that identical results can be obtained by using the standard EST or a shortened protocol based on flow cytometry of intracellular marker proteins. Both in vitro protocols enable to reliably determine differentiation of murine stem cells toward the cardiomyocyte lineage and to assess its chemical-mediated inhibition. PMID:21227905

Revelation of Different Nanoparticle-Uptake Behavior in Two Standard Cell Lines NIH/3T3 and A549 by Flow Cytometry and Time-Lapse Imaging

PubMed Central

Jochums, André; Friehs, Elsa; Sambale, Franziska; Lavrentieva, Antonina; Bahnemann, Detlef; Scheper, Thomas

2017-01-01

The uptake of nanomaterials into different cell types is a central pharmacological issue for the determination of nanotoxicity as well as for the development of drug delivery strategies. Most responses of the cells depend on their intracellular interactions with nanoparticles (NPs). Uptake behavior can be precisely investigated in vitro, with sensitive high throughput methods such as flow cytometry. In this study, we investigated two different standard cell lines, human lung carcinoma (A549) and mouse fibroblast (NIH/3T3) cells, regarding their uptake behavior of titanium dioxide NPs. Cells were incubated with different concentrations of TiO2 NPs and samples were taken at certain time points to compare the uptake kinetics of both cell lines. Samples were analyzed with the help of flow cytometry by studying changes in the side and forward scattering signal. To additionally enable a detection via fluorescence, NPs were labeled with the fluorescent dye fluorescein isothiocyanate (FITC) and propidium iodide (PI). We found that NIH/3T3 cells take up the studied NPs more efficiently than A549 cells. These findings were supported by time-lapse microscopic imaging of the cells incubated with TiO2 NPs. Our results confirm that the uptake behavior of individual cell types has to be considered before interpreting any results of nanomaterial studies. PMID:29051447

Selective induction of apoptosis in human gastric cancer cells by Lactobacillus kefiri (PFT), a novel kefir product

PubMed Central

GHONEUM, MAMDOOH; FELO, NOURAN

2015-01-01

The present study was undertaken to evaluate the effect of Lactobacillus kefiri (PFT), a novel kefir product, on apoptosis of gastric cancer cells (AGS), breast cancer cells (4T1), and human peripheral blood mononuclear cells (PBMCs). Cells were cultured with PFT and apoptosis was determined by flow cytometry using 7-AAD dye and cytospin preparation. Mitochondrial dysfunction and expression of Bcl2 were monitored by flow cytometry. Results showed that PFT induced apoptosis in AGS gastric cancer cells in a dose-dependent manner. Apoptosis was detected at a concentration of 0.3 mg/ml (20.8%), increased to 25.8% at 0.6 mg/ml, 37% at 1.2 mg/ml, 53.1% at 2.5 mg/ml, and peaked at 66.3% at 5.0 mg/ml. Apoptosis is associated with the decreased polarization of mitochondrial membrane potential (MMP) and decreased Bcl2 expression. PFT-treated AGS cells manifested membrane blebbing, nuclear condensation, and fragmentation as identified in cytospin cytocentrifuge Giemsa stained preparations. On the other hand, flow cytometry analysis showed that PFT did not induce apoptosis in 4T1 breast cancer cells nor in PBMCs. These results suggest that PFT is safe for white blood cells and selectively induces apoptotic effects in gastric cancer cells. Hence, it may have potential as a therapeutic agent for the treatment of gastric cancers. PMID:26251956

Multiparametric Flow Cytometry Using Near-Infrared Fluorescent Proteins Engineered from Bacterial Phytochromes

PubMed Central

Telford, William G.; Shcherbakova, Daria M.; Buschke, David; Hawley, Teresa S.; Verkhusha, Vladislav V.

2015-01-01

Engineering of fluorescent proteins (FPs) has followed a trend of achieving longer fluorescence wavelengths, with the ultimate goal of producing proteins with both excitation and emission in the near-infrared (NIR) region of the spectrum. Flow cytometers are now almost universally equipped with red lasers, and can now be equipped with NIR lasers as well. Most red-shifted FPs of the GFP-like family are maximally excited by orange lasers (590 to 610 nm) not commonly found on cytometers. This has changed with the development of the iRFP series of NIR FPs from the protein family of bacterial phytochromes. The shortest wavelength variants of this series, iRFP670 and iRFP682 showed maximal excitation with visible red lasers. The longer wavelength variants iRFP702, iRFP713 and iRFP720 could be optimally excited by NIR lasers ranging from 685 to 730 nm. Pairs of iRFPs could be detected simultaneously by using red and NIR lasers. Moreover, a novel spectral cytometry technique, which relies on spectral deconvolution rather than optical filters, allowed spectra of all five iRFPs to be analyzed simultaneously with no spectral overlap. Together, the combination of iRFPs with the advanced flow cytometry will allow to first image tissues expressing iRFPs deep in live animals and then quantify individual cell intensities and sort out the distinct primary cell subpopulations ex vivo. PMID:25811854

Multiparametric flow cytometry using near-infrared fluorescent proteins engineered from bacterial phytochromes.

PubMed

Telford, William G; Shcherbakova, Daria M; Buschke, David; Hawley, Teresa S; Verkhusha, Vladislav V

2015-01-01

Engineering of fluorescent proteins (FPs) has followed a trend of achieving longer fluorescence wavelengths, with the ultimate goal of producing proteins with both excitation and emission in the near-infrared (NIR) region of the spectrum. Flow cytometers are now almost universally equipped with red lasers, and can now be equipped with NIR lasers as well. Most red-shifted FPs of the GFP-like family are maximally excited by orange lasers (590 to 610 nm) not commonly found on cytometers. This has changed with the development of the iRFP series of NIR FPs from the protein family of bacterial phytochromes. The shortest wavelength variants of this series, iRFP670 and iRFP682 showed maximal excitation with visible red lasers. The longer wavelength variants iRFP702, iRFP713 and iRFP720 could be optimally excited by NIR lasers ranging from 685 to 730 nm. Pairs of iRFPs could be detected simultaneously by using red and NIR lasers. Moreover, a novel spectral cytometry technique, which relies on spectral deconvolution rather than optical filters, allowed spectra of all five iRFPs to be analyzed simultaneously with no spectral overlap. Together, the combination of iRFPs with the advanced flow cytometry will allow to first image tissues expressing iRFPs deep in live animals and then quantify individual cell intensities and sort out the distinct primary cell subpopulations ex vivo.

Selective induction of apoptosis in human gastric cancer cells by Lactobacillus kefiri (PFT), a novel kefir product.

PubMed

Ghoneum, Mamdooh; Felo, Nouran

2015-10-01

The present study was undertaken to evaluate the effect of Lactobacillus kefiri (PFT), a novel kefir product, on apoptosis of gastric cancer cells (AGS), breast cancer cells (4T1), and human peripheral blood mononuclear cells (PBMCs). Cells were cultured with PFT and apoptosis was determined by flow cytometry using 7-AAD dye and cytospin preparation. Mitochondrial dysfunction and expression of Bcl2 were monitored by flow cytometry. Results showed that PFT induced apoptosis in AGS gastric cancer cells in a dose-dependent manner. Apoptosis was detected at a concentration of 0.3 mg/ml (20.8%), increased to 25.8% at 0.6 mg/ml, 37% at 1.2 mg/ml, 53.1% at 2.5 mg/ml, and peaked at 66.3% at 5.0 mg/ml. Apoptosis is associated with the decreased polarization of mitochondrial membrane potential (MMP) and decreased Bcl2 expression. PFT-treated AGS cells manifested membrane blebbing, nuclear condensation, and fragmentation as identified in cytospin cytocentrifuge Giemsa stained preparations. On the other hand, flow cytometry analysis showed that PFT did not induce apoptosis in 4T1 breast cancer cells nor in PBMCs. These results suggest that PFT is safe for white blood cells and selectively induces apoptotic effects in gastric cancer cells. Hence, it may have potential as a therapeutic agent for the treatment of gastric cancers.

Morphological observation and analysis using automated image cytometry for the comparison of trypan blue and fluorescence-based viability detection method.

PubMed

Chan, Leo Li-Ying; Kuksin, Dmitry; Laverty, Daniel J; Saldi, Stephanie; Qiu, Jean

2015-05-01

The ability to accurately determine cell viability is essential to performing a well-controlled biological experiment. Typical experiments range from standard cell culturing to advanced cell-based assays that may require cell viability measurement for downstream experiments. The traditional cell viability measurement method has been the trypan blue (TB) exclusion assay. However, since the introduction of fluorescence-based dyes for cell viability measurement using flow or image-based cytometry systems, there have been numerous publications comparing the two detection methods. Although previous studies have shown discrepancies between TB exclusion and fluorescence-based viability measurements, image-based morphological analysis was not performed in order to examine the viability discrepancies. In this work, we compared TB exclusion and fluorescence-based viability detection methods using image cytometry to observe morphological changes due to the effect of TB on dead cells. Imaging results showed that as the viability of a naturally-dying Jurkat cell sample decreased below 70 %, many TB-stained cells began to exhibit non-uniform morphological characteristics. Dead cells with these characteristics may be difficult to count under light microscopy, thus generating an artificially higher viability measurement compared to fluorescence-based method. These morphological observations can potentially explain the differences in viability measurement between the two methods.

Spontaneous anaplasia in pilocytic astrocytoma of cerebellum.

PubMed

Lach, B; Al Shail, E; Patay, Z

2003-06-01

We report a cystic cerebellar astrocytoma with a mural nodule that contained an additional focus of astrocytoma with the histological features of anaplasia, and showed up to 48% of aneuploid and 3% S-phase cells on flow cytometry. This focus was detectable on the enhanced, as well as non-enhanced T1 and T2 images. This appears to be the first case of pilocytic astrocytoma of cerebellum with focal anaplasia detected on histological and radiological studies.

Intracellular Detection of Viral Transcription and Replication Using RNA FISH

DTIC Science & Technology

2016-05-26

sequence and how it can be rapidly performed to minimize time spent in high containment. We have adapted existing protocols for mRNA detection with...We and others have been expanded this technique to use in virus-infected tissue sections, high -throughput imaging, and for flow-cytometry based...free water. Add 1 gram of dextran sulfate and mix for 10 minutes at room temperature . Aliquot and store the hybridization buffer at 4ºC. Wash

Flow Cytometry Based Detection and Isolation of Plasmodium falciparum Liver Stages In Vitro.

PubMed

Dumoulin, Peter C; Trop, Stefanie A; Ma, Jinxia; Zhang, Hao; Sherman, Matthew A; Levitskaya, Jelena

2015-01-01

Malaria, the disease caused by Plasmodium parasites, remains a major global health burden. The liver stage of Plasmodium falciparum infection is a leading target for immunological and pharmacological interventions. Therefore, novel approaches providing specific detection and isolation of live P. falciparum exoerythrocytic forms (EEFs) are warranted. Utilizing a recently generated parasite strain expressing green fluorescent protein (GFP) we established a method which, allows for detection and isolation of developing live P. falciparum liver stages by flow cytometry. Using this technique we compared the susceptibility of five immortalized human hepatocyte cell lines and primary hepatocyte cultures from three donors to infection by P. falciparum sporozoites. Here, we show that EEFs can be detected and isolated from in vitro infected cultures of the HC-04 cell line and primary human hepatocytes. We confirmed the presence of developing parasites in sorted live human hepatocytes and characterized their morphology by fluorescence microscopy. Finally, we validated the practical applications of our approach by re-examining the importance of host ligand CD81 for hepatocyte infection by P. falciparum sporozoites in vitro and assessment of the inhibitory activity of anti-sporozoite antibodies. This methodology provides us with the tools to study both, the basic biology of the P. falciparum liver stage and the effects of host-derived factors on the development of P. falciparum EEFs.

Flow Cytometry Based Detection and Isolation of Plasmodium falciparum Liver Stages In Vitro

PubMed Central

Dumoulin, Peter C.; Trop, Stefanie A.; Ma, Jinxia; Zhang, Hao; Sherman, Matthew A.; Levitskaya, Jelena

2015-01-01

Malaria, the disease caused by Plasmodium parasites, remains a major global health burden. The liver stage of Plasmodium falciparum infection is a leading target for immunological and pharmacological interventions. Therefore, novel approaches providing specific detection and isolation of live P. falciparum exoerythrocytic forms (EEFs) are warranted. Utilizing a recently generated parasite strain expressing green fluorescent protein (GFP) we established a method which, allows for detection and isolation of developing live P. falciparum liver stages by flow cytometry. Using this technique we compared the susceptibility of five immortalized human hepatocyte cell lines and primary hepatocyte cultures from three donors to infection by P. falciparum sporozoites. Here, we show that EEFs can be detected and isolated from in vitro infected cultures of the HC-04 cell line and primary human hepatocytes. We confirmed the presence of developing parasites in sorted live human hepatocytes and characterized their morphology by fluorescence microscopy. Finally, we validated the practical applications of our approach by re-examining the importance of host ligand CD81 for hepatocyte infection by P. falciparum sporozoites in vitro and assessment of the inhibitory activity of anti-sporozoite antibodies. This methodology provides us with the tools to study both, the basic biology of the P. falciparum liver stage and the effects of host-derived factors on the development of P. falciparum EEFs. PMID:26070149

Generation of human-to-pig chimerism to induce tolerance through transcutaneous in utero injection of cord blood-derived mononuclear cells or human bone marrow mesenchymals cells in a preclinical program of liver xenotransplantation: preliminary results.

PubMed

Abellaneda, J M; Ramis, G; Martínez-Alarcón, L; Majado, M J; Quereda, J J; Herrero-Medrano, J M; Mendonça, L; García-Nicolás, O; Reus, M; Insausti, C; Ríos, A; López-Navas, A; González, M R; Pallarés, F J; Munoz, A; Ramírez, P; Parrilla, P

2012-01-01

Using a percutaneous ecoguided injection system to obtain chimeric piglets through a less invasive and traumatic technique than previously reported. The two types of human cells included umbilical cord blood mononuclear elements and mesenchymal stem cells cultured from bone marrow. Four sows at gestational day 50 were anesthetized. A needle was inserted through the skin and uterine wall to reach the peritoneal cavity of the fetuses under continuous ultrasound guidance. Fourteen piglets were injected with various cell concentrations. All sows carried pregnancies to term yielding 69 piglets, among which 67 were alive and two mummified. Two piglets died during the first 48 hours of life. Chimerism was detected using flow cytometry and by quantitative polymerase chain reaction (q-PCR) to detect Alu gene in blood or tissues samples. The analysis detected blood chimerism in 13 piglets (21%) by flow cytometry and the presence of the human Alu gene in 33 (51%) by q-PCR. The results suggest cell trafficking between littermates after in utero injection. Transcutaneous echo-guided injection succeeded to produce chimeric piglets without disadvantages to the sow or the fetuses and avoiding abortions or fetal death. Copyright © 2012 Elsevier Inc. All rights reserved.

A complementary role of multiparameter flow cytometry and high-throughput sequencing for minimal residual disease detection in chronic lymphocytic leukemia: an European Research Initiative on CLL study.

PubMed

Rawstron, A C; Fazi, C; Agathangelidis, A; Villamor, N; Letestu, R; Nomdedeu, J; Palacio, C; Stehlikova, O; Kreuzer, K-A; Liptrot, S; O'Brien, D; de Tute, R M; Marinov, I; Hauwel, M; Spacek, M; Dobber, J; Kater, A P; Gambell, P; Soosapilla, A; Lozanski, G; Brachtl, G; Lin, K; Boysen, J; Hanson, C; Jorgensen, J L; Stetler-Stevenson, M; Yuan, C; Broome, H E; Rassenti, L; Craig, F; Delgado, J; Moreno, C; Bosch, F; Egle, A; Doubek, M; Pospisilova, S; Mulligan, S; Westerman, D; Sanders, C M; Emerson, R; Robins, H S; Kirsch, I; Shanafelt, T; Pettitt, A; Kipps, T J; Wierda, W G; Cymbalista, F; Hallek, M; Hillmen, P; Montserrat, E; Ghia, P

2016-04-01

In chronic lymphocytic leukemia (CLL) the level of minimal residual disease (MRD) after therapy is an independent predictor of outcome. Given the increasing number of new agents being explored for CLL therapy, using MRD as a surrogate could greatly reduce the time necessary to assess their efficacy. In this European Research Initiative on CLL (ERIC) project we have identified and validated a flow-cytometric approach to reliably quantitate CLL cells to the level of 0.0010% (10(-5)). The assay comprises a core panel of six markers (i.e. CD19, CD20, CD5, CD43, CD79b and CD81) with a component specification independent of instrument and reagents, which can be locally re-validated using normal peripheral blood. This method is directly comparable to previous ERIC-designed assays and also provides a backbone for investigation of new markers. A parallel analysis of high-throughput sequencing using the ClonoSEQ assay showed good concordance with flow cytometry results at the 0.010% (10(-4)) level, the MRD threshold defined in the 2008 International Workshop on CLL guidelines, but it also provides good linearity to a detection limit of 1 in a million (10(-6)). The combination of both technologies would permit a highly sensitive approach to MRD detection while providing a reproducible and broadly accessible method to quantify residual disease and optimize treatment in CLL.

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

PubMed Central

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

2015-01-01

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

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…

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

PubMed Central

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

2018-01-01

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

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

PubMed

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

2018-01-01

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

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.

Optofluidic Fluorescent Imaging Cytometry on a Cell Phone

PubMed Central

Zhu, Hongying; Mavandadi, Sam; Coskun, Ahmet F.; Yaglidere, Oguzhan; Ozcan, Aydogan

2012-01-01

Fluorescent microscopy and flow cytometry are widely used tools in biomedical sciences. Cost-effective translation of these technologies to remote and resource-limited environments could create new opportunities especially for telemedicine applications. Toward this direction, here we demonstrate the integration of imaging cytometry and fluorescent microscopy on a cell phone using a compact, lightweight, and cost-effective optofluidic attachment. In this cell-phone-based optofluidic imaging cytometry platform, fluorescently labeled particles or cells of interest are continuously delivered to our imaging volume through a disposable microfluidic channel that is positioned above the existing camera unit of the cell phone. The same microfluidic device also acts as a multilayered optofluidic waveguide and efficiently guides our excitation light, which is butt-coupled from the side facets of our microfluidic channel using inexpensive light-emitting diodes. Since the excitation of the sample volume occurs through guided waves that propagate perpendicular to the detection path, our cell-phone camera can record fluorescent movies of the specimens 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 target solution of interest. We tested the performance of our cell-phone-based imaging cytometer by measuring the density of white blood cells in human blood samples, which provided a decent match to a commercially available hematology analyzer. We further characterized the imaging quality of the same platform to demonstrate a spatial resolution of ~2 μm. This cell-phone-enabled optofluidic imaging flow cytometer could especially be useful for rapid and sensitive imaging of bodily fluids for conducting various cell counts (e.g., toward monitoring of HIV+ patients) or rare cell analysis as well as for screening of water quality in remote and resource-poor settings. PMID:21774454

Optofluidic fluorescent imaging cytometry on a cell phone.

PubMed

Zhu, Hongying; Mavandadi, Sam; Coskun, Ahmet F; Yaglidere, Oguzhan; Ozcan, Aydogan

2011-09-01

Fluorescent microscopy and flow cytometry are widely used tools in biomedical sciences. Cost-effective translation of these technologies to remote and resource-limited environments could create new opportunities especially for telemedicine applications. Toward this direction, here we demonstrate the integration of imaging cytometry and fluorescent microscopy on a cell phone using a compact, lightweight, and cost-effective optofluidic attachment. In this cell-phone-based optofluidic imaging cytometry platform, fluorescently labeled particles or cells of interest are continuously delivered to our imaging volume through a disposable microfluidic channel that is positioned above the existing camera unit of the cell phone. The same microfluidic device also acts as a multilayered optofluidic waveguide and efficiently guides our excitation light, which is butt-coupled from the side facets of our microfluidic channel using inexpensive light-emitting diodes. Since the excitation of the sample volume occurs through guided waves that propagate perpendicular to the detection path, our cell-phone camera can record fluorescent movies of the specimens 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 target solution of interest. We tested the performance of our cell-phone-based imaging cytometer by measuring the density of white blood cells in human blood samples, which provided a decent match to a commercially available hematology analyzer. We further characterized the imaging quality of the same platform to demonstrate a spatial resolution of ~2 μm. This cell-phone-enabled optofluidic imaging flow cytometer could especially be useful for rapid and sensitive imaging of bodily fluids for conducting various cell counts (e.g., toward monitoring of HIV+ patients) or rare cell analysis as well as for screening of water quality in remote and resource-poor settings.

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.

Development of RAP Tag, a Novel Tagging System for Protein Detection and Purification.

PubMed

Fujii, Yuki; Kaneko, Mika K; Ogasawara, Satoshi; Yamada, Shinji; Yanaka, Miyuki; Nakamura, Takuro; Saidoh, Noriko; Yoshida, Kanae; Honma, Ryusuke; Kato, Yukinari

2017-04-01

Affinity tag systems, possessing high affinity and specificity, are useful for protein detection and purification. The most suitable tag for a particular purpose should be selected from many available affinity tag systems. In this study, we developed a novel affinity tag called the "RAP tag" system, which comprises a mouse antirat podoplanin monoclonal antibody (clone PMab-2) and the RAP tag (DMVNPGLEDRIE). This system is useful not only for protein detection in Western blotting, flow cytometry, and sandwich enzyme-linked immunosorbent assay, but also for protein purification.

Aptamer-based microspheres for highly sensitive protein detection using fluorescently-labeled DNA nanostructures.

PubMed

Han, Daehoon; Hong, Jinkee; Kim, Hyun Cheol; Sung, Jong Hwan; Lee, Jong Bum

2013-11-01

Many highly sensitive protein detection techniques have been developed and have played an important role in the analysis of proteins. Herein, we report a novel technique that can detect proteins sensitively and effectively using aptamer-based DNA nanostructures. Thrombin was used as a target protein and aptamer was used to capture fluorescent dye-labeled DNA nanobarcodes or thrombin on a microsphere. The captured DNA nanobarcodes were replaced by a thrombin and aptamer interaction. The detection ability of this approach was confirmed by flow cytometry with different concentrations of thrombin. Our detection method has great potential for rapid and simple protein detection with a variety of aptamers.

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.

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

PubMed

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

1979-01-01

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

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.

Femtosecond laser pulse optimization for multiphoton cytometry and control of fluorescence

NASA Astrophysics Data System (ADS)

Tkaczyk, Eric Robert

This body of work encompasses optimization of near infrared femtosecond laser pulses both for enhancement of flow cytometry as well as adaptive pulse shaping to control fluorescence. A two-photon system for in vivo flow cytometry is demonstrated, which allows noninvasive quantification of circulating cell populations in a single live mouse. We monitor fluorescently-labeled red blood cells for more than two weeks, and are also able to noninvasively measure circulation times of two distinct populations of breast cancer cells simultaneously in a single mouse. We build a custom laser excitation source in the form of an extended cavity mode-locked oscillator, which enables superior detection in whole blood or saline of cell lines expressing fluorescent proteins including the green fluorescent protein (GFP), tdTomato and mPlum. A mathematical model explains unique features of the signals. The ability to distinguish different fluorescent species is central to simultaneous measurement of multiple molecular targets in high throughput applications including the multiphoton flow cytometer. We demonstrate that two dyes which are not distinguishable to one-photon measurements can be differentiated and in fact quantified in mixture via phase-shaped two-photon excitation pulses found by a genetic algorithm. We also selectively enhance or suppress two-photon fluorescence of numerous common dyes with tailored pulse shapes. Using a multiplicative (rather than ratiometric) fitness parameter, we are able to control the fluorescence while maintaining a strong signal. With this method, we control the two-photon fluorescence of the blue fluorescent protein (BFP), which is of particular interest in investigations of protein-protein interactions, and has frustrated previous attempts of control. Implementing an acousto-optic interferometer, we use the same experimental setup to measure two-photon excitation cross-sections of dyes and prove that photon-photon interferences are the predominant mechanism of control. This research establishes the basis for molecularly tailored pulse shaping in multiphoton flow cytometry, which will advance our ability to probe the biology of circulating cells during disease progression and response to therapy.

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.

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

PubMed

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

2014-05-01

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

Rapid multi-wavelength optical assessment of circulating blood volume without a priori data

NASA Astrophysics Data System (ADS)

Loginova, Ekaterina V.; Zhidkova, Tatyana V.; Proskurnin, Mikhail A.; Zharov, Vladimir P.

2016-03-01

The measurement of circulating blood volume (CBV) is crucial in various medical conditions including surgery, iatrogenic problems, rapid fluid administration, transfusion of red blood cells, or trauma with extensive blood loss including battlefield injuries and other emergencies. Currently, available commercial techniques are invasive and time-consuming for trauma situations. Recently, we have proposed high-speed multi-wavelength photoacoustic/photothermal (PA/PT) flow cytometry for in vivo CBV assessment with multiple dyes as PA contrast agents (labels). As the first step, we have characterized the capability of this technique to monitor the clearance of three dyes (indocyanine green, methylene blue, and trypan blue) in an animal model. However, there are strong demands on improvements in PA/PT flow cytometry. As additional verification of our proof-of-concept of this technique, we performed optical photometric CBV measurements in vitro. Three label dyes—methylene blue, crystal violet and, partially, brilliant green—were selected for simultaneous photometric determination of the components of their two-dye mixtures in the circulating blood in vitro without any extra data (like hemoglobin absorption) known a priori. The tests of single dyes and their mixtures in a flow system simulating a blood transfusion system showed a negligible difference between the sensitivities of the determination of these dyes under batch and flow conditions. For individual dyes, the limits of detection of 3×10-6 M‒3×10-6 M in blood were achieved, which provided their continuous determination at a level of 10-5 M for the CBV assessment without a priori data on the matrix. The CBV assessment with errors no higher than 4% were obtained, and the possibility to apply the developed procedure for optical photometric (flow cytometry) with laser sources was shown.

Validation of cell-based fluorescence assays: practice guidelines from the ICSH and ICCS - part I - rationale and aims.

PubMed

Davis, Bruce H; Wood, Brent; Oldaker, Teri; Barnett, David

2013-01-01

Flow cytometry and other technologies of cell-based fluorescence assays are as a matter of good laboratory practice required to validate all assays, which when in clinical practice may pass through regulatory review processes using criteria often defined with a soluble analyte in plasma or serum samples in mind. Recently the U.S. Food and Drug Administration (FDA) has entered into a public dialogue in the U.S. regarding their regulatory interest in laboratory developed tests (LDTs) or so-called "home brew" assays performed in clinical laboratories. The absence of well-defined guidelines for validation of cell-based assays using fluorescence detection has thus become a subject of concern for the International Council for Standardization of Haematology (ICSH) and International Clinical Cytometry Society (ICCS). Accordingly, a group of over 40 international experts in the areas of test development, test validation, and clinical practice of a variety of assay types using flow cytometry and/or morphologic image analysis were invited to develop a set of practical guidelines useful to in vitro diagnostic (IVD) innovators, clinical laboratories, regulatory scientists, and laboratory inspectors. The focus of the group was restricted to fluorescence reporter reagents, although some common principles are shared by immunohistochemistry or immunocytochemistry techniques and noted where appropriate. The work product of this two year effort is the content of this special issue of this journal, which is published as 5 separate articles, this being Validation of Cell-based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS - Part I - Rationale and aims. © 2013 International Clinical Cytometry Society. © 2013 International Clinical Cytometry Society.

Molecular evaluation of microalgal communities in full-scale waste stabilisation ponds.

PubMed

Eland, Lucy E; Davenport, Russell J; Santos, Andre Bezerra Dos; Mota Filho, Cesar R

2018-02-22

Waste stabilisation ponds (WSPs) are widely used across the world as a passive wastewater treatment for domestic wastewaters, but little is known about their ecology, especially their phototrophic communities. This study uses molecular methods and flow cytometry to assess the cyanobacterial and eukaryotic communities longitudinally throughout two systems, one treating domestic wastewater and the other mixed industrial/domestic wastewaters. More variation was seen between the systems than between different stages in the treatment processes for both eukaryotic and cyanobacterial communities. Chlorella species and Planktophrix cyanobacteria dominated both treatment systems. Arthrospira cyanobacteria were detected only in the industrial/domestic system. The balance between non-photosynthetic and photosynthetic organisms is rarely considered, though both play vital roles in WSP functioning. Flow cytometry showed that the facultative and first maturation pond in the industrial system contained a lower proportion of photosynthetic organisms compared to the domestic system. This is reflected in the species richness data and low dissolved oxygen levels detected. All data indicated that both systems are significantly different from one another and that variation longitudinally throughout the systems is lower. A more systematic study is needed to determine if it is the wastewater source rather than the initial inoculum that drives community composition.

Fluorescence triggering: A general strategy for enumerating and phenotyping extracellular vesicles by flow cytometry.

PubMed

Arraud, Nicolas; Gounou, Céline; Turpin, Delphine; Brisson, Alain R

2016-02-01

Plasma contains cell-derived extracellular vesicles (EVs) which participate in various physiopathological processes and have potential biomedical applications. Despite intense research activity, knowledge on EVs is limited mainly due to the difficulty of isolating and characterizing sub-micrometer particles like EVs. We have recently reported that a simple flow cytometry (FCM) approach based on triggering the detection on a fluorescence signal enabled the detection of 50× more Annexin-A5 binding EVs (Anx5+ EVs) in plasma than the conventional FCM approach based on light scattering triggering. Here, we present the application of the fluorescence triggering approach to the enumeration and phenotyping of EVs from platelet free plasma (PFP), focusing on CD41+ and CD235a+ EVs, as well as their sub-populations which bind or do not bind Anx5. Higher EV concentrations were detected by fluorescence triggering as compared to light scattering triggering, namely 40× for Anx5+ EVs, 75× for CD41+ EVs, and 15× for CD235a+ EVs. We found that about 30% of Anx5+ EVs were of platelet origin while only 3% of them were of erythrocyte origin. In addition, a majority of EVs from platelet and erythrocyte origin do not expose PS, in contrast to the classical theory of EV formation. Furthermore, the same PFP samples were analyzed fresh and after freeze-thawing, showing that freeze-thawing processes induce an increase, of about 35%, in the amount of Anx5+ EVs, while the other EV phenotypes remain unchanged. The method of EV detection and phenotyping by fluorescence triggering is simple, sensitive and reliable. We foresee that its application to EV studies will improve our understanding on the formation mechanisms and functions of EVs in health and disease and help the development of EV-based biomarkers. © 2015 International Society for Advancement of Cytometry.

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.

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.

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.

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

Flexible planar microfluidic chip employing a light emitting diode and a PIN-photodiode for portable flow cytometers.

PubMed

Kettlitz, Siegfried W; Valouch, Sebastian; Sittel, Wiebke; Lemmer, Uli

2012-01-07

Detection of fluorescence particles is a key method of flow cytometry. We evaluate the performance of a design for a microfluidic fluorescence particle detection device. Due to the planar design with low layer thicknesses, we avoid optical components such as lenses or dichroic mirrors and substitute them with a shadow mask and colored film filters. A commercially available LED is used as the light source and a PIN-photodiode as detector. This design approach reduces component cost and power consumption and enables supplying the device with power from a standard USB port. From evaluation of this design, we obtain a maximum particle detection frequency of up to 600 particles per second at a sensitivity of better than 4.7 × 10(5) MESF (molecules of equivalent soluble fluorochrome) measured with particles for FITC sensitivity calibration. Lowering the flow rate increases the instrument sensitivity by an order of magnitude enabling the detection of particles with 4.5 × 10(4) MESF.

Rapid Assessment of Genotoxicity by Flow Cytometric Detection of Cell Cycle Alterations.

PubMed

Bihari, Nevenka

2017-01-01

Flow cytometry is a convenient method for the determination of genotoxic effects of environmental pollution and can reveal genotoxic compounds in unknown environmental mixtures. It is especially suitable for the analyses of large numbers of samples during monitoring programs. The speed of detection is one of the advantages of this technique which permits the acquisition of 10 4 -10 5 cells per sample in 5 min. This method can rapidly detect cell cycle alterations resulting from DNA damage. The outcome of such an analysis is a diagram of DNA content across the cell cycle which indicates cell proliferation, G 2 arrests, G 1 delays, apoptosis, and ploidy.Here, we present the flow cytometric procedure for rapid assessment of genotoxicity via detection of cell cycle alterations. The described protocol simplifies the analysis of genotoxic effects in marine environments and is suitable for monitoring purposes. It uses marine mussel cells in the analysis and can be adapted to investigations on a broad range of marine invertebrates.

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.

Towards resolving the Knautia arvensis agg. (Dipsacaceae) puzzle: primary and secondary contact zones and ploidy segregation at landscape and microgeographic scales

PubMed Central

Kolář, Filip; Štech, Milan; Trávníček, Pavel; Rauchová, Jana; Urfus, Tomáš; Vít, Petr; Kubešová, Magdalena; Suda, Jan

2009-01-01

Background and Aims Detailed knowledge of variations in ploidy levels and their geographic distributions is one of the key tasks faced in polyploid research in natural systems. Flow cytometry has greatly facilitated the field of cytogeography by allowing characterization of ploidy levels at both the regional and population scale, and at multiple stages of the life cycle. In the present study, flow cytometry was employed to investigate the patterns and dynamics of ploidy variation in the taxonomically challenging complex Knautia arvensis (Dipsacaceae) and some of its allies (K. dipsacifolia, K. slovaca) in Central Europe. Methods DNA ploidy levels were estimated by DAPI flow cytometry in 5205 adult plants, 228 seedlings and 400 seeds collected from 292 Knautia populations in seven European countries. The flow cytometric data were supplemented with conventional chromosome counts. A subset of 79 accessions was subjected to estimation of the absolute genome size using propidium iodide flow cytometry. Key Results and Conclusions Five different ploidy levels (from 2x to 6x) were found, with triploids of K. arvensis being recorded for the first time. The species also exhibited variation in the monoploid genome size, corresponding to the types of habitats occupied (grassland diploid populations had larger genome sizes than relict and subalpine diploid populations). Disregarding relict populations, the distribution of 2x and 4x cytotypes was largely parapatric, with a diffuse secondary contact zone running along the north-west margin of the Pannonian basin. Spatial segregation of the cytotypes was also observed on regional and microgeographic scales. The newly detected sympatric growth of diploids and tetraploids in isolated relict habitats most likely represents the primary zone of cytotype contact. Ploidy level was found to be a major determinant of the strength of inter-cytotype reproductive barriers. While mixed 2x + 4x populations virtually lacked the intermediate ploidy level at any ontogenetic stage, pentaploid hybrids were common in 4x +6x populations, despite the cytotypes representing different taxonomic entities. PMID:19196717

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

Parallel pulse processing and data acquisition for high speed, low error flow cytometry

DOEpatents

van den Engh, Gerrit J.; Stokdijk, Willem

1992-01-01

A digitally synchronized parallel pulse processing and data acquisition system for a flow cytometer has multiple parallel input channels with independent pulse digitization and FIFO storage buffer. A trigger circuit controls the pulse digitization on all channels. After an event has been stored in each FIFO, a bus controller moves the oldest entry from each FIFO buffer onto a common data bus. The trigger circuit generates an ID number for each FIFO entry, which is checked by an error detection circuit. The system has high speed and low error rate.

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

Assimilable organic carbon (AOC) determination using GFP-tagged Pseudomonas fluorescens P-17 in water by flow cytometry.

PubMed

Tang, Peng; Wu, Jie; Liu, Hou; Liu, Youcai; Zhou, Xingding

2018-01-01

One of the newly developed methods for Assimilable organic carbon (AOC) determination is leveraged on the cell enumeration by flow cytometry (FC) which could provide a rapid and automated solution for AOC measurement. However, cell samples staining with fluorescence dye is indispensable to reduce background and machine noise. This step would bring additional cost and time consuming for this method. In this study, a green fluorescence protein (GFP) tagged strain derived of AOC testing strain Pseudomonas fluorescens P-17 (GFP-P17) was generated using Tn5 transposon mutagenesis. Continuous culture of this mutant GFP-P17 showed stable expression of eGFP signal detected by flow cytometry without staining step. In addition, this GFP-P17 strain displayed faster growth rate and had a wider range of carbon substrate utilization patterns as compared with P17 wild-type. With this strain, the capability of a new FC method with no dye staining was explored in standard acetate solution, which suggests linear correlation of counts with acetate carbon concentration. Furthermore, this FC method with GFP-P17 strain is applicable in monitoring GAC/BAC efficiency and condition as similar trends of AOC level in water treatment process were measured by both FC method and conventional spread plating count method. Therefore, this fast and easily applicable GFP-P17 based FC method could serve as a tool for routine microbiological drinking water monitoring.

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.

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/.

Click Chemistry for Analysis of Cell Proliferation in Flow Cytometry.

PubMed

Clarke, Scott T; Calderon, Veronica; Bradford, Jolene A

2017-10-02

The measurement of cellular proliferation is fundamental to the assessment of cellular health, genotoxicity, and the evaluation of drug efficacy. Labeling, detection, and quantification of cells in the synthesis phase of cell cycle progression are not only important for characterizing basic biology, but also in defining cellular responses to drug treatments. Changes in DNA replication during S-phase can provide valuable insights into mechanisms of cell growth, cell cycle kinetics, and cytotoxicity. A common method for detection of cell proliferation is the incorporation of a thymidine analog during DNA synthesis. This chapter presents a pulse labeling method using the thymidine analog, 5-ethynyl-2'-deoxyuridine (EdU), with subsequent detection by click chemistry. EdU detection using click chemistry is bio-orthogonal to most living systems and does not non-specifically label other biomolecules. Live cells are first pulsed with EdU. After antibody labeling cell surface markers, fixation, and permeabilization, the incorporated EdU is covalently labeled using click chemistry thereby identifying proliferating cells. Improvements in click chemistry allow for labeling in the presence of fluorescent proteins and phycobiliproteins without quenching due to copper. Measuring DNA replication during cell cycle progression has cell health applications in flow cytometry, fluorescence microscopy, and high content imaging. This protocol has been developed and optimized for research use only and is not suitable for use in diagnostic procedures. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

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.

Detection and proteomic characterization of extracellular vesicles in human pancreatic juice.

PubMed

Osteikoetxea, Xabier; Benke, Márton; Rodriguez, Marta; Pálóczi, Krisztina; Sódar, Barbara W; Szvicsek, Zsuzsanna; Szabó-Taylor, Katalin; Vukman, Krisztina V; Kittel, Ágnes; Wiener, Zoltán; Vékey, Károly; Harsányi, László; Szűcs, Ákos; Turiák, Lilla; Buzás, Edit I

2018-04-30

The prognosis of patients with pancreatic cancer has remained virtually unchanged with a high mortality rate compared to other types of cancers. An earlier detection would provide a time window of opportunity for treatment and prevention of deaths. In the present study we investigated extracellular vesicle (EV)-associated potential biomarkers for pancreatic cancer by directly assessing EV size-based subpopulations in pancreatic juice samples of patients with chronic pancreatitis or pancreatic cancer. In addition, we also studied blood plasma and pancreatic cancer cell line-derived EVs. Comparative proteomic analysis was performed of 102 EV preparations from human pancreatic juices, blood, and pancreatic cancer cell lines Capan-1 and MIA PaCa-2. EV preparations were also characterized by electron microscopy, tunable resistive pulse sensing, and flow cytometry. Here we describe the presence of EVs in human pancreatic juice samples. Pancreatic juice EV-associated proteins that we identified as possible candidate markers for pancreatic cancer included mucins, such as MUC1, MUC4, MUC5AC, MUC6 and MUC16, CFTR, and MDR1 proteins. These candidate biomarkers could also be detected by flow cytometry in EVs found in pancreatic juice and those secreted by pancreatic cancer cell lines. Together our data show that detection and characterization of EVs directly in pancreatic juice is feasible and may prove to be a valuable source of potential biomarkers of pancreatic cancer. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Detection and quantification of microparticles from different cellular lineages using flow cytometry. Evaluation of the impact of secreted phospholipase A2 on microparticle assessment.

PubMed

Rousseau, Matthieu; Belleannee, Clemence; Duchez, Anne-Claire; Cloutier, Nathalie; Levesque, Tania; Jacques, Frederic; Perron, Jean; Nigrovic, Peter A; Dieude, Melanie; Hebert, Marie-Josee; Gelb, Michael H; Boilard, Eric

2015-01-01

Microparticles, also called microvesicles, are submicron extracellular vesicles produced by plasma membrane budding and shedding recognized as key actors in numerous physio(patho)logical processes. Since they can be released by virtually any cell lineages and are retrieved in biological fluids, microparticles appear as potent biomarkers. However, the small dimensions of microparticles and soluble factors present in body fluids can considerably impede their quantification. Here, flow cytometry with improved methodology for microparticle resolution was used to detect microparticles of human and mouse species generated from platelets, red blood cells, endothelial cells, apoptotic thymocytes and cells from the male reproductive tract. A family of soluble proteins, the secreted phospholipases A2 (sPLA2), comprises enzymes concomitantly expressed with microparticles in biological fluids and that catalyze the hydrolysis of membrane phospholipids. As sPLA2 can hydrolyze phosphatidylserine, a phospholipid frequently used to assess microparticles, and might even clear microparticles, we further considered the impact of relevant sPLA2 enzymes, sPLA2 group IIA, V and X, on microparticle quantification. We observed that if enriched in fluids, certain sPLA2 enzymes impair the quantification of microparticles depending on the species studied, the source of microparticles and the means of detection employed (surface phosphatidylserine or protein antigen detection). This study provides analytical considerations for appropriate interpretation of microparticle cytofluorometric measurements in biological samples containing sPLA2 enzymes.

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.

Immunophenotypic characterization of bone marrow mast cells in mastocytosis and other mast cell disorders.

PubMed

Sánchez-Muñoz, Laura; Teodósio, Cristina; Morgado, José M; Escribano, Luis

2011-01-01

Mastocytosis is a term used to designate a heterogeneous group of disorders characterized by an abnormal proliferation and accumulation of mast cells (MCs) in one or multiple tissues including skin, bone marrow (BM), liver, spleen, and lymph nodes, among others. Recent advances in our understanding of mast cell biology and disease resulted in the identification of important differences in the expression of mast cell surface antigens between normal and neoplastic mast cells. Most notably, detection of aberrant expression of CD25 and CD2 on the surface of neoplastic mast cells but not on their normal counterparts lead to the inclusion of this immunophenotypic abnormality in the World Health Organization diagnostic criteria for systemic mastocytosis. Aberrant mast cell surface marker expression can be detected in the bone marrow aspirate by flow cytometry, even in patients lacking histopathologically detectable aggregates of mast cells in bone marrow biopsy sections. These aberrant immunophenotypic features are of great relevance for the assessment of tissue involvement in mastocytosis with consequences in the diagnosis, classification, and follow-up of the disease and in its differential diagnosis with other entities. In this chapter, we provide the reader with information for the objective and reproducible identification of pathologic MCs by using quantitative multiparametric flow cytometry, for their phenotypic characterization, and the criteria currently used for correct interpretation of the immunophenotypic results obtained. Copyright © 2011 Elsevier Inc. All rights reserved.

Imaging System and Method for Biomedical Analysis

DTIC Science & Technology

2013-03-11

biological particles and items of interest. Broadly, Padmanabhan et al. utilize the diffraction of a laser light source in flow cytometry to count...spread of light from multiple LED devices over the entire sample surface. Preferably, light source 308 projects a full spectrum white light. Light...for example, red blood cells, white blood cells (which may include lymphocytes which are relatively large and easily detectable), T-helper cells

[Tryptase inhibits cell apoptosis through upregulating PAR-2 and Rho kinase in rheumatoid arthritis synovial fibroblasts].

PubMed

Zheng, Qianqian; Li, Shigang; Jia, Yunli; Liu, Wei; Yu, Lingling; Chen, Xianyong; Wang, Jinling

2016-12-01

Objective To investigate the regulatory effects of tryptase on protease-activated receptors 2 (PAR-2), Rho signal pathway and apoptosis of MH7A rheumatoid arthritis synovial fibroblasts. Methods In MH7A cells, the expression of PAR-2 was measured by flow cytometry; cell apoptosis was examined by annexin V- FITC/PI staining combined with flow cytometry; the expression of Rho kinase was detected by Pull-down assay and Western botting. Results Tryptase upregulated the expression of PAR-2 in MH7A cells, and suppressed Fas-mediated apoptosis of MH7A cells in a dose-dependent manner. Meanwhile, PAR-2 inhibitor, FSLLRY-NH2 significantly reduced anti-apoptotic effects of tryptase in MH7A cells, which was related with the increase of activated Rho kinase expression. Conclusion Tryptase plays a role in resistance to the apoptosis of MH7A cells through raising PAR-2 and activating Rho kinase.

[Pulmonary pathology in fatal human influenza A (H1N1) infection].

PubMed

Duan, Xue-jing; Li, Yong; Gong, En-cong; Wang, Jue; Lü, Fu-dong; Zhang, He-qiu; Sun, Lin; Yue, Zhu-jun; Song, Chen-chao; Zhang, Shi-Jie; Li, Ning; Dai, Jie

2011-12-01

To study the pulmonary pathology in patients died of fatal human influenza A(H1N1) infection. Eight cases of fatal human influenza A (H1N1) infection, including 2 autopsy cases and 6 paramortem needle puncture biopsies, were enrolled into the study. Histologic examination, immunohistochemitry, flow cytometry and Western blotting were carried out. The major pathologic changes included necrotizing bronchiolitis with surrounding inflammation, diffuse alveolar damage and pulmonary hemorrhage. Influenza viral antigen expression was detected in the lung tissue by Western blotting. Immunohistochemical study demonstrated the presence of nuclear protein and hemagglutinin virus antigens in parts of trachea, bronchial epithelium and glands, alveolar epithelium, macrophages and endothelium. Flow cytometry showed that the apoptotic rate of type II pneumocytes (32.15%, 78.15%) was significantly higher than that of the controls (1.93%, 3.77%). Necrotizing bronchiolitis, diffuse alveolar damage and pulmonary hemorrhage followed by pulmonary fibrosis in late stage are the major pathologic changes in fatal human influenza A (H1N1) infection.

Heavy Metals Effect on Cyanobacteria Synechocystis aquatilis Study Using Absorption, Fluorescence, Flow Cytometry, and Photothermal Measurements

NASA Astrophysics Data System (ADS)

Dudkowiak, A.; Olejarz, B.; Łukasiewicz, J.; Banaszek, J.; Sikora, J.; Wiktorowicz, K.

2011-04-01

The toxic effect of six heavy metals on cyanobacteria Synechocystis aquatilis was studied by absorption, fluorescence, flow cytometry, and photothermal measurements. This study indicates that at the concentration used, the cyanobacteria are more sensitive to silver, copper, and mercury than to cadmium, lead, and zinc metals. Disregarding the decrease in the yields of the related radiative processes caused by photochemical processes and/or damage to phycobilisomes, no changes were detected in the efficiency of thermal deactivation processes within a few microseconds, which can indicate the lack of disturbances in the photosynthetic light reaction and the lack of damage to the photosystem caused by the heavy metal ions in the concentrations used. The results demonstrate that the relative values of fluorescence yield as well as promptly generated heat calculated for the metal-affected and unaffected (reference) bacteria are sensitive indicators of environmental pollution with heavy metal ions, whereas the complementary methods proposed could be used as a noninvasive and fast procedure for in vivo assessment of their toxicity.

A rapid, reliable method of evaluating growth and viability of intraerythrocytic protozoan hemoparasites using fluorescence flow cytometry.

PubMed

Davis, W C; Wyatt, C R; Hamilton, M J; Goff, W L

1992-01-01

Fluorescence flow cytometry was employed to assess the potential of a vital dye, hydroethidine, for use in the detection and monitoring of the viability of hemoparasites in infected erythrocytes, using Babesia bovis as a model parasite. The studies demonstrated that hydroethidine is taken up by B. bovis and metabolically converted to the DNA binding fluorochrome, ethidium. Following uptake of the dye, erythrocytes containing viable parasites were readily distinguished and quantitated. Timed studies with the parasiticidal drug, Ganaseg, showed that it is possible to use the fluorochrome assay to monitor the effects of the drug on the rate of replication and viability of B. bovis in culture. The assay provides a rapid method for evaluation of the in vitro effect of drugs on hemoparasites and for analysis of the effect of various components of the immune response, such as lymphokines, monocyte products, antibodies, and effector cells (T, NK, LAK, ADCC) on the growth and viability of intraerythrocytic parasites.

Detection of circulating microparticles by flow cytometry: influence of centrifugation, filtration of buffer, and freezing

PubMed Central

Dey-Hazra, Emily; Hertel, Barbara; Kirsch, Torsten; Woywodt, Alexander; Lovric, Svjetlana; Haller, Hermann; Haubitz, Marion; Erdbruegger, Uta

2010-01-01

The clinical importance of microparticles resulting from vesiculation of platelets and other blood cells is increasingly recognized, although no standardized method exists for their measurement. Only a few studies have examined the analytical and preanalytical steps and variables affecting microparticle detection. We focused our analysis on microparticle detection by flow cytometry. The goal of our study was to analyze the effects of different centrifugation protocols looking at different durations of high and low centrifugation speeds. We also analyzed the effect of filtration of buffer and long-term freezing on microparticle quantification, as well as the role of Annexin V in the detection of microparticles. Absolute and platelet-derived microparticles were 10- to 15-fold higher using initial lower centrifugation speeds at 1500 × g compared with protocols using centrifugation speeds at 5000 × g (P < 0.01). A clear separation between true events and background noise was only achieved using higher centrifugation speeds. Filtration of buffer with a 0.2 μm filter reduced a significant amount of background noise. Storing samples for microparticle detection at −80°C decreased microparticle levels at days 28, 42, and 56 (P < 0.05 for all comparisons with fresh samples). We believe that staining with Annexin V is necessary to distinguish true events from cell debris or precipitates. Buffers should be filtered and fresh samples should be analyzed, or storage periods will have to be standardized. Higher centrifugation speeds should be used to minimize contamination by smaller size platelets. PMID:21191433

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

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

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.

Genetic diversity and population structure of Musa accessions in ex situ conservation

PubMed Central

2013-01-01

Background Banana cultivars are mostly derived from hybridization between wild diploid subspecies of Musa acuminata (A genome) and M. balbisiana (B genome), and they exhibit various levels of ploidy and genomic constitution. The Embrapa ex situ Musa collection contains over 220 accessions, of which only a few have been genetically characterized. Knowledge regarding the genetic relationships and diversity between modern cultivars and wild relatives would assist in conservation and breeding strategies. Our objectives were to determine the genomic constitution based on Internal Transcribed Spacer (ITS) regions polymorphism and the ploidy of all accessions by flow cytometry and to investigate the population structure of the collection using Simple Sequence Repeat (SSR) loci as co-dominant markers based on Structure software, not previously performed in Musa. Results From the 221 accessions analyzed by flow cytometry, the correct ploidy was confirmed or established for 212 (95.9%), whereas digestion of the ITS region confirmed the genomic constitution of 209 (94.6%). Neighbor-joining clustering analysis derived from SSR binary data allowed the detection of two major groups, essentially distinguished by the presence or absence of the B genome, while subgroups were formed according to the genomic composition and commercial classification. The co-dominant nature of SSR was explored to analyze the structure of the population based on a Bayesian approach, detecting 21 subpopulations. Most of the subpopulations were in agreement with the clustering analysis. Conclusions The data generated by flow cytometry, ITS and SSR supported the hypothesis about the occurrence of homeologue recombination between A and B genomes, leading to discrepancies in the number of sets or portions from each parental genome. These phenomenons have been largely disregarded in the evolution of banana, as the “single-step domestication” hypothesis had long predominated. These findings will have an impact in future breeding approaches. Structure analysis enabled the efficient detection of ancestry of recently developed tetraploid hybrids by breeding programs, and for some triploids. However, for the main commercial subgroups, Structure appeared to be less efficient to detect the ancestry in diploid groups, possibly due to sampling restrictions. The possibility of inferring the membership among accessions to correct the effects of genetic structure opens possibilities for its use in marker-assisted selection by association mapping. PMID:23497122

Expression of Foxp3, CD25 and IL-2 in the B16F10 cancer cell line and melanoma is correlated with tumor growth in mice

PubMed Central

MIRANDA-HERNÁNDEZ, D.F.; FRANCO-MOLINA, M.A.; MENDOZA-GAMBOA, E.; ZAPATA-BENAVIDES, P.; SIERRA-RIVERA, C.A.; CORONADO-CERDA, E.E.; ROSAS-TARACO, A.G.; TAMÉZ-GUERRA, R.S.; RODRÍGUEZ-PADILLA, C.

2013-01-01

The forkhead box P3 (Foxp3) transcription factor is one of the most studied markers used to identify CD4+CD25+ regulatory T cells (Tregs), and has been identified as a key regulator in the development and function of Tregs. Foxp3 expression has been reported in a variety of solid human tumors, including melanoma. The aims of the present study were to analyze Foxp3 expression in B16F10 melanoma cells in vitro, to determine whether this expression was affected during tumor growth in a murine melanoma model and to correlate Foxp3 expression with CD25 expression, interleukin (IL)-2 production and tumor weight. Foxp3 expression was analyzed with quantitative (q)PCR, flow cytometry and confocal microscopy. CD25 expression was analyzed by flow cytometry, and cytokine production was measured by ELISA [IL-2, interferon (IFN)-γ, transforming growth factor (TGF)-β and IL-10] and flow cytometry (IL-2, IFN-γ, IL-4 and IL-5). Foxp3 and CD25 expression was detected in the B16F10 cells in culture and in the intratumoral B16F10 cells. An increase in Foxp3 and CD25 expression was observed in a time-dependent manner during tumor growth at 7, 14 and 21 days. The production of the IL-2, IL-10, IFN-γ and TGF-β cytokines was observed in the B16F10 cells and also detected in the tumoral microenvironment during tumor growth (7, 14 and 21 days). An increase in IL-2 and IL-10 production was observed, whereas IFN-γ production decreased in a time-dependent manner. The production of tumor necrosis factor (TNF)-α was not observed in culture, but was detected during tumor growth, whereas the production of IL-4 and IL-5 was not detected. These data showed a positive correlation between the expression of Foxp3, CD25 and IL-2 and tumor weight in murine melanoma. From these data, it may be suggested that Foxp3 participates in melanoma growth, the modulation of the IL-2, IFN-γ and TNF-α cytokines and CD25 expression, and that it also plays a possible role in immunosuppression. PMID:24179494

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.

Facile and quantitative electrochemical detection of yeast cell apoptosis

NASA Astrophysics Data System (ADS)

Yue, Qiulin; Xiong, Shiquan; Cai, Dongqing; Wu, Zhengyan; Zhang, Xin

2014-03-01

An electrochemical method based on square wave anodic stripping voltammetry (SWASV) was developed to detect the apoptosis of yeast cells conveniently and quantitatively through the high affinity between Cu2+ and phosphatidylserine (PS) translocated from the inner to the outer plasma membrane of the apoptotic cells. The combination of negatively charged PS and Cu2+ could decrease the electrochemical response of Cu2+ on the electrode. The results showed that the apoptotic rates of cells could be detected quantitatively through the variations of peak currents of Cu2+ by SWASV, and agreed well with those obtained through traditional flow cytometry detection. This work thus may provide a novel, simple, immediate and accurate detection method for cell apoptosis.

Validation of cell-based fluorescence assays: practice guidelines from the ICSH and ICCS - part IV - postanalytic considerations.

PubMed

Barnett, David; Louzao, Raaul; Gambell, Peter; De, Jitakshi; Oldaker, Teri; Hanson, Curtis A

2013-01-01

Flow cytometry and other technologies of cell-based fluorescence assays are as a matter of good laboratory practice required to validate all assays, which when in clinical practice may pass through regulatory review processes using criteria often defined with a soluble analyte in plasma or serum samples in mind. Recently the U.S. Food and Drug Administration (FDA) has entered into a public dialogue in the U.S. regarding their regulatory interest in laboratory developed tests (LDTs) or so-called home brew assays performed in clinical laboratories. The absence of well-defined guidelines for validation of cell-based assays using fluorescence detection has thus become a subject of concern for the International Council for Standardization of Haematology (ICSH) and International Clinical Cytometry Society (ICCS). Accordingly, a group of over 40 international experts in the areas of test development, test validation, and clinical practice of a variety of assay types using flow cytometry and/or morphologic image analysis were invited to develop a set of practical guidelines useful to in vitro diagnostic (IVD) innovators, clinical laboratories, regulatory scientists, and laboratory inspectors. The focus of the group was restricted to fluorescence reporter reagents, although some common principles are shared by immunohistochemistry or immunocytochemistry techniques and noted where appropriate. The work product of this two year effort is the content of this special issue of this journal, which is published as 5 separate articles, this being Validation of Cell-based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS - Part IV - Postanalytic considerations. © 2013 International Clinical Cytometry Society.

Inertial focusing in a straight channel with asymmetrical expansion-contraction cavity arrays using two secondary flows

NASA Astrophysics Data System (ADS)

Zhang, J.; Li, M.; Li, W. H.; Alici, G.

2013-08-01

The focusing of particles has a variety of applications in industry and biomedicine, including wastewater purification, fermentation filtration, and pathogen detection in flow cytometry, etc. In this paper a novel inertial microfluidic device using two secondary flows to focus particles is presented. The geometry of the proposed microfluidic channel is a simple straight channel with asymmetrically patterned triangular expansion-contraction cavity arrays. Three different focusing patterns were observed under different flow conditions: (1) a single focusing streak on the cavity side; (2) double focusing streaks on both sides; (3) half of the particles were focused on the opposite side of the cavity, while the other particles were trapped by a horizontal vortex in the cavity. The focusing performance was studied comprehensively up to flow rates of 700 µl min-1. The focusing mechanism was investigated by analysing the balance of forces between the inertial lift forces and secondary flow drag in the cross section. The influence of particle size and cavity geometry on the focusing performance was also studied. The experimental results showed that more precise focusing could be obtained with large particles, some of which even showed a single-particle focusing streak in the horizontal plane. Meanwhile, the focusing patterns and their working conditions could be adjusted by the geometry of the cavity. This novel inertial microfluidic device could offer a continuous, sheathless, and high-throughput performance, which can be potentially applied to high-speed flow cytometry or the extraction of blood cells.

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

PubMed

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

2015-04-01

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

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

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.

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.

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.

A critical review of published methods for analysis of red cell antigen-antibody reactions by flow cytometry, and approaches for resolving problems with red cell agglutination.

PubMed

Arndt, Patricia A; Garratty, George

2010-07-01

Flow cytometry operators often apply familiar white blood cell (WBC) methods when studying red blood cell (RBC) antigens and antibodies. Some WBC methods are not appropriate for RBCs, as the analysis of RBCs requires special considerations, for example, avoidance of agglutination. One hundred seventy-six published articles from 88 groups studying RBC interactions were reviewed. Three fourths of groups used at least one unnecessary WBC procedure for RBCs, and about one fourth did not use any method to prevent/disperse RBC agglutination. Flow cytometric studies were performed to determine the effect of RBC agglutination on results and compare different methods of preventing and/or dispersing agglutination. The presence of RBC agglutinates have been shown to be affected by the type of pipette tip used for mixing RBC suspensions, the number of antigen sites/RBC, the type and concentration of primary antibody, and the type of secondary antibody. For quantitation methods, for example, fetal maternal hemorrhage, the presence of agglutinates have been shown to adversely affect results (fewer fetal D+ RBCs detected). Copyright 2010 Elsevier Inc. All rights reserved.

Measuring T cell-mediated cytotoxicity using fluorogenic caspase substrates.

PubMed

Chahroudi, A; Silvestri, G; Feinberg, M B

2003-10-01

Cytotoxic T lymphocytes (CTLs) play a major role in the immune response against viruses and other intracellular pathogens. In addition, CTLs are implicated in the control of tumor cells in certain settings. Accurate measures of CTL function are of critical importance to study the pathogenesis of infectious diseases and to evaluate the efficacy of new vaccines and immunotherapies. To this end, we have recently developed a flow cytometry-based CTL (FCC) assay that measures the CTL-induced caspase activation within target cells using cell permeable fluorogenic caspase substrates. This novel assay reliably detects, by flow cytometry or fluorescence/confocal microscopy, antigen-specific CTLs in a wide variety of human and murine systems, and is safer and more informative than the standard 51Cr-release assay. In addition, the flow cytometric CTL (FCC) assay provides an alternative method that is often more sensitive and physiologically informative when compared to previously described FCC assays, as it measures a biological indicator of apoptosis within the target cell. The FCC assay may thus represent a useful tool to further understand the molecular and cellular mechanisms that underlie CTL-mediated killing during tumorigenesis or following infection with viruses or other intracellular pathogens.

Photoacoustic and photothermal detection of circulating tumor cells, bacteria and nanoparticles in cerebrospinal fluid in vivo and ex vivo.

PubMed

Nedosekin, Dmitry A; Juratli, Mazen A; Sarimollaoglu, Mustafa; Moore, Christopher L; Rusch, Nancy J; Smeltzer, Mark S; Zharov, Vladimir P; Galanzha, Ekaterina I

2013-06-01

Circulating cells, bacteria, proteins, microparticles, and DNA in cerebrospinal fluid (CSF) are excellent biomarkers of many diseases, including cancer and infections. However, the sensitivity of existing methods is limited in their ability to detect rare CSF biomarkers at the treatable, early-stage of diseases. Here, we introduce novel CSF tests based on in vivo photoacoustic flow cytometry (PAFC) and ex vivo photothermal scanning cytometry. In the CSF of tumor-bearing mice, we molecularly detected in vivo circulating tumor cells (CTCs) before the development of breast cancer brain metastasis with 20-times higher sensitivity than with current assays. For the first time, we demonstrated assessing three pathways (i.e., blood, lymphatic, and CSF) of CTC dissemination, tracking nanoparticles in CSF in vivo and their imaging ex vivo. In label-free CSF samples, we counted leukocytes, erythrocytes, melanoma cells, and bacteria and imaged intracellular cytochromes, hemoglobin, melanin, and carotenoids, respectively. Taking into account the safety of PAFC, its translation for use in humans is expected to improve disease diagnosis beyond conventional detection limits. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Asymmetric-detection time-stretch optical microscopy (ATOM) for ultrafast high-contrast cellular imaging in flow

PubMed Central

Wong, Terence T. W.; Lau, Andy K. S.; Ho, Kenneth K. Y.; Tang, Matthew Y. H.; Robles, Joseph D. F.; Wei, Xiaoming; Chan, Antony C. S.; Tang, Anson H. L.; Lam, Edmund Y.; Wong, Kenneth K. Y.; Chan, Godfrey C. F.; Shum, Ho Cheung; Tsia, Kevin K.

2014-01-01

Accelerating imaging speed in optical microscopy is often realized at the expense of image contrast, image resolution, and detection sensitivity – a common predicament for advancing high-speed and high-throughput cellular imaging. We here demonstrate a new imaging approach, called asymmetric-detection time-stretch optical microscopy (ATOM), which can deliver ultrafast label-free high-contrast flow imaging with well delineated cellular morphological resolution and in-line optical image amplification to overcome the compromised imaging sensitivity at high speed. We show that ATOM can separately reveal the enhanced phase-gradient and absorption contrast in microfluidic live-cell imaging at a flow speed as high as ~10 m/s, corresponding to an imaging throughput of ~100,000 cells/sec. ATOM could thus be the enabling platform to meet the pressing need for intercalating optical microscopy in cellular assay, e.g. imaging flow cytometry – permitting high-throughput access to the morphological information of the individual cells simultaneously with a multitude of parameters obtained in the standard assay. PMID:24413677

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

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

Detection of Antineutrophil Autoantibodies by Flow Cytometry: Use of Unfixed Neutrophils as Antigenic Targets

DTIC Science & Technology

1993-01-01

irmuanoglobulins, neutropenia , systemic lupus erythematosus, IL PRICE OO rheumatoid arthritis. 17. SECURITY CLASSIFICATION 18. SECURITY...sera of a variety of neutropenic patients with typical RA had patients with neutropenia and associated au- positive results, 51/244 (21%) of sera from...from patients were used to study the effects of cellular ac- with suspected immune neutropenia were tivation, and increased antibody binding was studied

Assessment of the role of circulating breast cancer cells in tumor formation and metastatic potential using in vivo flow cytometry

NASA Astrophysics Data System (ADS)

Hwu, Derrick; Boutrus, Steven; Greiner, Cherry; Dimeo, Theresa; Kuperwasser, Charlotte; Georgakoudi, Irene

2011-04-01

The identification of breast cancer patients who will ultimately progress to metastatic disease is of significant clinical importance. The quantification and assessment of circulating tumor cells (CTCs) has been proposed as one strategy to monitor treatment effectiveness and disease prognosis. However, CTCs have been an elusive population of cells to study because of their small number and difficulties associated with isolation protocols. In vivo flow cytometry (IVFC) can overcome these limitations and provide insights in the role these cells play during primary and metastatic tumor growth. In this study, we used two-color IVFC to examine, for up to ten weeks following orthotopic implantation, changes in the number of circulating human breast cells expressing GFP and a population of circulating hematopoietic cells with strong autofluorescence. We found that the number of detected CTCs in combination with the number of red autofluorescent cells (650 to 690 nm) during the first seven days following implantation was predictive in development of tumor formation and metastasis eight weeks later. These results suggest that the combined detection of these two cell populations could offer a novel approach in the monitoring and prognosis of breast cancer progression, which in turn could aid significantly in their effective treatment.

Online flow cytometry reveals microbial dynamics influenced by concurrent natural and operational events in groundwater used for drinking water treatment.

PubMed

Besmer, Michael D; Epting, Jannis; Page, Rebecca M; Sigrist, Jürg A; Huggenberger, Peter; Hammes, Frederik

2016-12-07

Detailed measurements of physical, chemical and biological dynamics in groundwater are key to understanding the important processes in place and their influence on water quality - particularly when used for drinking water. Measuring temporal bacterial dynamics at high frequency is challenging due to the limitations in automation of sampling and detection of the conventional, cultivation-based microbial methods. In this study, fully automated online flow cytometry was applied in a groundwater system for the first time in order to monitor microbial dynamics in a groundwater extraction well. Measurements of bacterial concentrations every 15 minutes during 14 days revealed both aperiodic and periodic dynamics that could not be detected previously, resulting in total cell concentration (TCC) fluctuations between 120 and 280 cells μL -1 . The aperiodic dynamic was linked to river water contamination following precipitation events, while the (diurnal) periodic dynamic was attributed to changes in hydrological conditions as a consequence of intermittent groundwater extraction. Based on the high number of measurements, the two patterns could be disentangled and quantified separately. This study i) increases the understanding of system performance, ii) helps to optimize monitoring strategies, and iii) opens the possibility for more sophisticated (quantitative) microbial risk assessment of drinking water treatment systems.

Online flow cytometry reveals microbial dynamics influenced by concurrent natural and operational events in groundwater used for drinking water treatment

PubMed Central

Besmer, Michael D.; Epting, Jannis; Page, Rebecca M.; Sigrist, Jürg A.; Huggenberger, Peter; Hammes, Frederik

2016-01-01

Detailed measurements of physical, chemical and biological dynamics in groundwater are key to understanding the important processes in place and their influence on water quality – particularly when used for drinking water. Measuring temporal bacterial dynamics at high frequency is challenging due to the limitations in automation of sampling and detection of the conventional, cultivation-based microbial methods. In this study, fully automated online flow cytometry was applied in a groundwater system for the first time in order to monitor microbial dynamics in a groundwater extraction well. Measurements of bacterial concentrations every 15 minutes during 14 days revealed both aperiodic and periodic dynamics that could not be detected previously, resulting in total cell concentration (TCC) fluctuations between 120 and 280 cells μL−1. The aperiodic dynamic was linked to river water contamination following precipitation events, while the (diurnal) periodic dynamic was attributed to changes in hydrological conditions as a consequence of intermittent groundwater extraction. Based on the high number of measurements, the two patterns could be disentangled and quantified separately. This study i) increases the understanding of system performance, ii) helps to optimize monitoring strategies, and iii) opens the possibility for more sophisticated (quantitative) microbial risk assessment of drinking water treatment systems. PMID:27924920

Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry

PubMed Central

Gaber, Rok; Majerle, Andreja; Jerala, Roman; Benčina, Mojca

2013-01-01

To effectively fight against the human immunodeficiency virus infection/acquired immunodeficiency syndrome (HIV/AIDS) epidemic, ongoing development of novel HIV protease inhibitors is required. Inexpensive high-throughput screening assays are needed to quickly scan large sets of chemicals for potential inhibitors. We have developed a Förster resonance energy transfer (FRET)-based, HIV protease-sensitive sensor using a combination of a fluorescent protein pair, namely mCerulean and mCitrine. Through extensive in vitro characterization, we show that the FRET-HIV sensor can be used in HIV protease screening assays. Furthermore, we have used the FRET-HIV sensor for intracellular quantitative detection of HIV protease activity in living cells, which more closely resembles an actual viral infection than an in vitro assay. We have developed a high-throughput method that employs a ratiometric flow cytometry for analyzing large populations of cells that express the FRET-HIV sensor. The method enables FRET measurement of single cells with high sensitivity and speed and should be used when subpopulation-specific intracellular activity of HIV protease needs to be estimated. In addition, we have used a confocal microscopy sensitized emission FRET technique to evaluate the usefulness of the FRET-HIV sensor for spatiotemporal detection of intracellular HIV protease activity. PMID:24287545

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

CytometryML and other data formats

NASA Astrophysics Data System (ADS)

Leif, Robert C.

2006-02-01

Cytology automation and research will be enhanced by the creation of a common data format. This data format would provide the pathology and research communities with a uniform way for annotating and exchanging images, flow cytometry, and associated data. This specification and/or standard will include descriptions of the acquisition device, staining, the binary representations of the image and list-mode data, the measurements derived from the image and/or the list-mode data, and descriptors for clinical/pathology and research. An international, vendor-supported, non-proprietary specification will allow pathologists, researchers, and companies to develop and use image capture/analysis software, as well as list-mode analysis software, without worrying about incompatibilities between proprietary vendor formats. Presently, efforts to create specifications and/or descriptions of these formats include the Laboratory Digital Imaging Project (LDIP) Data Exchange Specification; extensions to the Digital Imaging and Communications in Medicine (DICOM); Open Microscopy Environment (OME); Flowcyt, an extension to the present Flow Cytometry Standard (FCS); and CytometryML. The feasibility of creating a common data specification for digital microscopy and flow cytometry in a manner consistent with its use for medical devices and interoperability with both hospital information and picture archiving systems has been demonstrated by the creation of the CytometryML schemas. The feasibility of creating a software system for digital microscopy has been demonstrated by the OME. CytometryML consists of schemas that describe instruments and their measurements. These instruments include digital microscopes and flow cytometers. Optical components including the instruments' excitation and emission parts are described. The description of the measurements made by these instruments includes the tagged molecule, data acquisition subsystem, and the format of the list-mode and/or image data. Many of the CytometryML data-types are based on the Digital Imaging and Communications in Medicine (DICOM). Binary files for images and list-mode data have been created and read.

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.

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

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.

Analysis of Flow Cytometry DNA Damage Response Protein Activation Kinetics Following X-rays and High Energy Iron Nuclei Exposure

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

Universities Space Research Association; Chappell, Lori J.; Whalen, Mary K.

2010-12-15

We developed a mathematical method to analyze flow cytometry data to describe the kinetics of {gamma}H2AX and pATF2 phosphorylations ensuing various qualities of low dose radiation in normal human fibroblast cells. Previously reported flow cytometry kinetic results for these DSB repair phospho-proteins revealed that distributions of intensity were highly skewed, severely limiting the detection of differences in the very low dose range. Distributional analysis reveals significant differences between control and low dose samples when distributions are compared using the Kolmogorov-Smirnov test. Radiation quality differences are found in the distribution shapes and when a nonlinear model is used to relate dosemore » and time to the decay of the mean ratio of phosphoprotein intensities of irradiated samples to controls. We analyzed cell cycle phase and radiation quality dependent characteristic repair times and residual phospho-protein levels with these methods. Characteristic repair times for {gamma}H2AX were higher following Fe nuclei as compared to X-rays in G1 cells (4.5 {+-} 0.46 h vs 3.26 {+-} 0.76 h, respectively), and in S/G2 cells (5.51 {+-} 2.94 h vs 2.87 {+-} 0.45 h, respectively). The RBE in G1 cells for Fe nuclei relative to X-rays for {gamma}H2AX was 2.05 {+-} 0.61 and 5.02 {+-} 3.47, at 2 h and 24-h postirradiation, respectively. For pATF2, a saturation effect is observed with reduced expression at high doses, especially for Fe nuclei, with much slower characteristic repair times (>7 h) compared to X-rays. RBEs for pATF2 were 0.66 {+-} 0.13 and 1.66 {+-} 0.46 at 2 h and 24 h, respectively. Significant differences in {gamma}H2AX and pATF2 levels comparing irradiated samples to control were noted even at the lowest dose analyzed (0.05 Gy) using these methods of analysis. These results reveal that mathematical models can be applied to flow cytometry data to uncover important and subtle differences following exposure to various qualities of low dose radiation.« less

[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.

Parallel pulse processing and data acquisition for high speed, low error flow cytometry

DOEpatents

Engh, G.J. van den; Stokdijk, W.

1992-09-22

A digitally synchronized parallel pulse processing and data acquisition system for a flow cytometer has multiple parallel input channels with independent pulse digitization and FIFO storage buffer. A trigger circuit controls the pulse digitization on all channels. After an event has been stored in each FIFO, a bus controller moves the oldest entry from each FIFO buffer onto a common data bus. The trigger circuit generates an ID number for each FIFO entry, which is checked by an error detection circuit. The system has high speed and low error rate. 17 figs.

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

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.

Comparative analysis of cell wall surface glycan expression in Candida albicans and Saccharomyces cerevisiae yeasts by flow cytometry.

PubMed

Martínez-Esparza, M; Sarazin, A; Jouy, N; Poulain, D; Jouault, T

2006-07-31

The yeast Candida albicans is an opportunistic pathogen, part of the normal human microbial flora that causes infections in immunocompromised individuals with a high morbidity and mortality levels. Recognition of yeasts by host cells is based on components of the yeast cell wall, which are considered part of its virulence attributes. Cell wall glycans play an important role in the continuous interchange that regulates the balance between saprophytism and parasitism, and also between resistance and infection. Some of these molecular entities are expressed both by the pathogenic yeast C. albicans and by Saccharomyces cerevisiae, a related non-pathogenic yeast, involving similar molecular mechanisms and receptors for recognition. In this work we have exploited flow cytometry methods for probing surface glycans of the yeasts. We compared glycan expression by C. albicans and by S. cerevisiae, and studied the effect of culture conditions. Our results show that the expression levels of alpha- and beta-linked mannosides as well as beta-glucans can be successfully evaluated by flow cytometry methods using different antibodies independent of agglutination reactions. We also found that the surface expression pattern of beta-mannosides detected by monoclonal or polyclonal antibodies are differently modulated during the growth course. These data indicate that the yeast beta-mannosides exposed on mannoproteins and/or phospholipomannan are increased in stationary phase, whereas those linked to mannan are not affected by the yeast growth phase. The cytometric method described here represents a useful tool to investigate to what extent C. albicans is able to regulate its glycan surface expression and therefore modify its virulence properties.

Interleukin-12 in patients with cancer is synthesized by peripheral helper T lymphocytes.

PubMed

Michelin, Marcia A; Montes, Leticia; Nomelini, Rosekeila S; Abdalla, Douglas R; Aleixo, Andre A R; Murta, Eddie F C

2015-09-01

The production of cytokines by helper T lymphocytes is a critical event in the immune response, as alterations in the regulation of this process may result in an appropriate immune response, persistent infection or the development of autoimmune disease. Previously, this group has used flow cytometry to demonstrate the expression of interleukin-12 (IL-12) in peripheral blood CD4+ T lymphocytes from patients and mice with advanced cancer. The aim of the present study was to investigate whether CD4+ T lymphocytes from the peripheral blood (PB) of patients with cancer produce IL-12, using molecular approaches, flow cytometry and cellular imaging techniques. CD3+ and CD4+ cells, and cells producing IL-12, were isolated from the PB obtained from patients with cancer, using a cell sorting flow cytometry technique. The positivity of cells for CD3, CD4 and IL-12, which were identified by cell sorting, was visualized using immunofluorescent cellular imaging. Total RNA was extracted from the CD3+CD4+IL-12+ cells, obtained by cell sorting, for confirmation of the presence of IL-12 mRNA, using reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR demonstrated the presence of IL-12 mRNA in all patients (n=14), in contrast to the control group, in whom IL-12 expression was not detected. Immunofluorescent analysis of CD4+ T lymphocytes showed positive intracytoplasmatic IL-12 staining. These results demonstrated that CD3+CD4+ T lymphocytes in the PB of patients with cancer have the capacity to synthesize and express IL-12.

Interleukin-12 in patients with cancer is synthesized by peripheral helper T lymphocytes

PubMed Central

MICHELIN, MARCIA A.; MONTES, LETICIA; NOMELINI, ROSEKEILA S.; ABDALLA, DOUGLAS R.; ALEIXO, ANDRE A. R.; MURTA, EDDIE F. C.

2015-01-01

The production of cytokines by helper T lymphocytes is a critical event in the immune response, as alterations in the regulation of this process may result in an appropriate immune response, persistent infection or the development of autoimmune disease. Previously, this group has used flow cytometry to demonstrate the expression of interleukin-12 (IL-12) in peripheral blood CD4+ T lymphocytes from patients and mice with advanced cancer. The aim of the present study was to investigate whether CD4+ T lymphocytes from the peripheral blood (PB) of patients with cancer produce IL-12, using molecular approaches, flow cytometry and cellular imaging techniques. CD3+ and CD4+ cells, and cells producing IL-12, were isolated from the PB obtained from patients with cancer, using a cell sorting flow cytometry technique. The positivity of cells for CD3, CD4 and IL-12, which were identified by cell sorting, was visualized using immunofluorescent cellular imaging. Total RNA was extracted from the CD3+CD4+IL-12+ cells, obtained by cell sorting, for confirmation of the presence of IL-12 mRNA, using reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR demonstrated the presence of IL-12 mRNA in all patients (n=14), in contrast to the control group, in whom IL-12 expression was not detected. Immunofluorescent analysis of CD4+ T lymphocytes showed positive intracytoplasmatic IL-12 staining. These results demonstrated that CD3+CD4+ T lymphocytes in the PB of patients with cancer have the capacity to synthesize and express IL-12. PMID:26622702

Quantification of Al2O3 nanoparticles in human cell lines applying inductively coupled plasma mass spectrometry (neb-ICP-MS, LA-ICP-MS) and flow cytometry-based methods

NASA Astrophysics Data System (ADS)

Böhme, Steffi; Stärk, Hans-Joachim; Meißner, Tobias; Springer, Armin; Reemtsma, Thorsten; Kühnel, Dana; Busch, Wibke

2014-09-01

In order to quantify and compare the uptake of aluminum oxide nanoparticles of three different sizes into two human cell lines (skin keratinocytes (HaCaT) and lung epithelial cells (A549)), three analytical methods were applied: digestion followed by nebulization inductively coupled plasma mass spectrometry (neb-ICP-MS), direct laser ablation ICP-MS (LA-ICP-MS), and flow cytometry. Light and electron microscopy revealed an accumulation and agglomeration of all particle types within the cell cytoplasm, whereas no particles were detected in the cell nuclei. The internalized Al2O3 particles exerted no toxicity in the two cell lines after 24 h of exposure. The smallest particles with a primary particle size ( x BET) of 14 nm (Alu1) showed the lowest sedimentation velocity within the cell culture media, but were calculated to have settled completely after 20 h. Alu2 ( x BET = 111 nm) and Alu3 ( x BET = 750 nm) were calculated to reach the cell surface after 7 h and 3 min, respectively. The internal concentrations determined with the different methods lay in a comparable range of 2-8 µg Al2O3/cm2 cell layer, indicating the suitability of all methods to quantify the nanoparticle uptake. Nevertheless, particle size limitations of analytical methods using optical devices were demonstrated for LA-ICP-MS and flow cytometry. Furthermore, the consideration and comparison of particle properties as parameters for particle internalization revealed the particle size and the exposure concentration as determining factors for particle uptake.

Flow cytometric quantification of intraperitoneal free tumor cells in patients with peritoneal metastasis.

PubMed

Kitayama, Joji; Emoto, Shigenobu; Yamaguchi, Hironori; Ishigami, Hironori; Kamei, Takao; Yamashita, Hiroharu; Seto, Yasuyuki; Matsuzaki, Keisuke; Watanabe, Toshiaki

2014-01-01

Peritoneal metastasis (PM) is the most life-threatening type of metastasis in abdominal malignancy. To improve the diagnostic accuracy of cytologic detection (CY) of free tumor cells (FTC) in the peritoneal cavity, we tried to quantify the FTC to leukocyte ratio using flow cytometry in patients with peritoneal metastasis. Cells were recovered from ascites or peritoneal lavages from 106 patients who underwent abdominal surgery and additional 89 samples which were obtained from peritoneal catheter or access port in patients with PM (+) gastric cancer. The cells were immunostained with monoclonal antibodies to CD45 and to CD326 (EpCAM). Using flow cytometry, CD326 (+) and CD45 (+) cells were classified as either tumor cells (T) or leukocytes (L) and the T/L ratio (TLR) was calculated. In 106 samples obtained by laparotomy, Median (M) of the TLR of PM (+) patients was 1.39% (0-807.87%) which was significantly higher than PM (-) patients (M=0%, 0-2.14%, P < 0.001). In PM (+) patients, 86 CY (+) samples showed higher TLR than 61 CY (-) samples (M=2.81%, 0.02-1868.44% vs. M=0%, 0-3.45%, p<0.0001). In all of the 24 patients who were monitored for TLR before and after intraperitoneal (IP) chemotherapy, the TLR was reduced which was more dramatic than the results of the change in cytology. TLR measured with FACS is an excellent reflection of the tumor spread in the peritoneal cavity and could be a reliable diagnostic biomarker to determine the severity of PM as well as effectiveness of IP chemotherapy. © 2013 International Clinical Cytometry Society.

Retigeric acid B exerts antifungal effect through enhanced reactive oxygen species and decreased cAMP.

PubMed

Chang, Wen-Qiang; Wu, Xiu-Zhen; Cheng, Ai-Xia; Zhang, Li; Ji, Mei; Lou, Hong-Xiang

2011-05-01

Retigeric acid B (RAB), a triterpene acid isolated from Lobaria kurokawae exerts antifungal effect. The present study was designed to elucidate the underlying mechanisms by which RAB regulates the proliferation and cell death of Candida albicans. We measured the metabolic activity of C. albicans with WST1 Cell Proliferation and Cytotoxicity Assay Kit, analyzed the cell cycle by flow cytometry, visualized the ultrastructure by transmission electron microscopy (TEM) and investigated the apoptosis and necrosis induced by RAB using confocal microscopy. The reactive oxygen species (ROS) accumulation was determined by spectrophotometry, flow cytometry and fluorescent microscopy. The mtΔψ was detected using flow cytometry. And the levels of intracellular cAMP and ATP were measured with cAMP ELISA and ATP Assay Kits, respectively. The proliferation of the yeasts was blocked in G(2)/M phase by a low dose of RAB treatment and in G(1) phase at high concentration. When cultured in phosphate buffered saline (PBS) deprived of energy source, yeasts displayed the phenotype of death caused by accumulated ROS, mtΔψ hyperpolarization and dramatic decrease in ATP level in the presence of high dose of RAB. RAB inhibits the growth of C. albicans by stimulating ROS production and reducing intracellular cAMP. The ROS accumulation, mtΔψ hyperpolarization, ATP depletion and damaged plasma membrane integrity together mediate cell death of C. albicans induced by RAB. Our findings provide a novel molecular mechanism for exploring possible applications of lichen derived metabolites in fighting fungal infection in humans. Copyright © 2011 Elsevier B.V. All rights reserved.

In vivo acoustic and photoacoustic focusing of circulating cells

NASA Astrophysics Data System (ADS)

Galanzha, Ekaterina I.; Viegas, Mark G.; Malinsky, Taras I.; Melerzanov, Alexander V.; Juratli, Mazen A.; Sarimollaoglu, Mustafa; Nedosekin, Dmitry A.; Zharov, Vladimir P.

2016-03-01

In vivo flow cytometry using vessels as natural tubes with native cell flows has revolutionized the study of rare circulating tumor cells in a complex blood background. However, the presence of many blood cells in the detection volume makes it difficult to count each cell in this volume. We introduce method for manipulation of circulating cells in vivo with the use of gradient acoustic forces induced by ultrasound and photoacoustic waves. In a murine model, we demonstrated cell trapping, redirecting and focusing in blood and lymph flow into a tight stream, noninvasive wall-free transportation of blood, and the potential for photoacoustic detection of sickle cells without labeling and of leukocytes targeted by functionalized nanoparticles. Integration of cell focusing with intravital imaging methods may provide a versatile biological tool for single-cell analysis in circulation, with a focus on in vivo needleless blood tests, and preclinical studies of human diseases in animal models.

In vivo acoustic and photoacoustic focusing of circulating cells

PubMed Central

Galanzha, Ekaterina I.; Viegas, Mark G.; Malinsky, Taras I.; Melerzanov, Alexander V.; Juratli, Mazen A.; Sarimollaoglu, Mustafa; Nedosekin, Dmitry A.; Zharov, Vladimir P.

2016-01-01

In vivo flow cytometry using vessels as natural tubes with native cell flows has revolutionized the study of rare circulating tumor cells in a complex blood background. However, the presence of many blood cells in the detection volume makes it difficult to count each cell in this volume. We introduce method for manipulation of circulating cells in vivo with the use of gradient acoustic forces induced by ultrasound and photoacoustic waves. In a murine model, we demonstrated cell trapping, redirecting and focusing in blood and lymph flow into a tight stream, noninvasive wall-free transportation of blood, and the potential for photoacoustic detection of sickle cells without labeling and of leukocytes targeted by functionalized nanoparticles. Integration of cell focusing with intravital imaging methods may provide a versatile biological tool for single-cell analysis in circulation, with a focus on in vivo needleless blood tests, and preclinical studies of human diseases in animal models. PMID:26979811

3D flow focusing for microfluidic flow cytometry with ultrasonics

NASA Astrophysics Data System (ADS)

Gnyawali, Vaskar; Strohm, Eric M.; Daghighi, Yasaman; van de Vondervoort, Mia; Kolios, Michael C.; Tsai, Scott S. H.

2015-11-01

We are developing a flow cytometer that detects unique acoustic signature waves generated from single cells due to interactions between the cells and ultrasound waves. The generated acoustic waves depend on the size and biomechanical properties of the cells and are sufficient for identifying cells in the medium. A microfluidic system capable of focusing cells through a 10 x 10 μm ultrasound beam cross section was developed to facilitate acoustic measurements of single cells. The cells are streamlined in a hydro-dynamically 3D focused flow in a 300 x 300 μm channel made using PDMS. 3D focusing is realized by lateral sheath flows and an inlet needle (inner diameter 100 μm). The accuracy of the 3D flow focusing is measured using a dye and detecting its localization using confocal microscopy. Each flowing cell would be probed by an ultrasound pulse, which has a center frequency of 375 MHz and bandwidth of 250 MHz. The same probe would also be used for recording the scattered waves from the cells, which would be processed to distinguish the physical and biomechanical characteristics of the cells, eventually identifying them. This technique has potential applications in detecting circulating tumor cells, blood cells and blood-related diseases.

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

Binding diversity of antibodies against external and internal epitopes of the multidrug resistance gene product P-glycoprotein

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

Lehne, G.; De Angelis, P.; Clausen, O.P.F.

1995-07-01

P-glycoprotein (Pgp) is a trans-membraneous protein that is associated with multidrug resistance (MDR) in human cancer, including hepatocellular carcinomas and leukemia. There is no consensus regarding methods of choice for analysis of Pgp expression, and development of reliable analytical methods is now essential. We have studied the Pgp expression in human hepatoma and leukemia cell lines using flow cytometry. The aim of the study was to compare binding properties of anti-Pgp antibodies reacting with surface (MRK16, UIC2) and cytoplasmic (C219, JSB-1) epitopes to assess which antibody performed best with respect to fluorescence discrimination. By histogram subtraction the fractions of resistantmore » human hepatoma cells positive for Pgp were 99% (MRK16), 97% (UIC2), 77% (USB-1), and 51% (C219), demonstrating variations in antibody reactivity. The resolution in detecting decreasing levels of Pgp in hepatoma cells was superior for the externally binding antibodies, showing that there is a correlation between antibody reactivity and fluorescence discrimination. Similar results were obtained for parental and resistant KG1a human leukemia cell lines. The Pgp epitopes remained reactive to the anti-Pgp MAbs after methanol fixation and cryopreservation. By dual parameter flow cytometry it was shown that Pgp expression in viable cells may be assessed together with uptake of epirubicin, which was low in cells expressing high levels of Pgp and vice versa. In conclusion, all tested antibodies proved useful for flow cytometric detection of high levels of Pgp, but the externally binding ones were superior in detection of low and variable levels of Pgp. 36 refs., 8 figs., 1 tab.« less

A novel all-fiber optic flow cytometer technology for Point-of Care and Remote Environments

NASA Astrophysics Data System (ADS)

Mermut, Ozzy

Traditional flow cytometry designs tend to be bulky systems with a complex optical-fluidic sub-system and often require trained personnel for operation. This makes them difficult to readily translate to remote site testing applications. A new compact and portable fiber-optic flow cell (FOFC) technology has been developed at INO. We designed and engineered a specialty optical fiber through which a square hole is transversally bored by laser micromachining. A capillary is fitted into that hole to flow analyte within the fiber square cross-section for detection and counting. With demonstrated performance benchmarks potentially comparable to commercial flow cytometers, our FOFC provides several advantages compared to classic free-space con-figurations, e.g., sheathless flow, low cost, reduced number of optical components, no need for alignment (occurring in the fabrication process only), ease-of-use, miniaturization, portability, and robustness. This sheathless configuration, based on a fiber optic flow module, renders this cytometer amenable to space-grade microgravity environments. We present our recent results for an all-fiber approach to achieve a miniature FOFC to translate flow cytometry from bench to a portable, point-of-care device for deployment in remote settings. Our unique fiber approach provides the capability to illuminate a large surface with a uniform intensity distri-bution, independently of the initial shape originating from the light source, and without loss of optical power. The CVs and sensitivities are measured and compared to industry benchmarks. Finally, integration of LEDs enable several advantages in cost, compactness, and wavelength availability.

Effect of cryopreservation on delineation of immune cell subpopulations in tumor specimens as determinated by multiparametric single cell mass cytometry analysis.

PubMed

Kadić, Elma; Moniz, Raymond J; Huo, Ying; Chi, An; Kariv, Ilona

2017-02-02

Comprehensive understanding of cellular immune subsets involved in regulation of tumor progression is central to the development of cancer immunotherapies. Single cell immunophenotyping has historically been accomplished by flow cytometry (FC) analysis, enabling the analysis of up to 18 markers. Recent advancements in mass cytometry (MC) have facilitated detection of over 50 markers, utilizing high resolving power of mass spectrometry (MS). This study examined an analytical and operational feasibility of MC for an in-depth immunophenotyping analysis of the tumor microenvironment, using the commercial CyTOF™ instrument, and further interrogated challenges in managing the integrity of tumor specimens. Initial longitudinal studies with frozen peripheral blood mononuclear cells (PBMCs) showed minimal MC inter-assay variability over nine independent runs. In addition, detection of common leukocyte lineage markers using MC and FC detection confirmed that these methodologies are comparable in cell subset identification. An advanced multiparametric MC analysis of 39 total markers enabled a comprehensive evaluation of cell surface marker expression in fresh and cryopreserved tumor samples. This comparative analysis revealed significant reduction of expression levels of multiple markers upon cryopreservation. Most notably myeloid derived suppressor cells (MDSC), defined by co-expression of CD66b + and CD15 + , HLA-DR dim and CD14 - phenotype, were undetectable in frozen samples. These results suggest that optimization and evaluation of cryopreservation protocols is necessary for accurate biomarker discovery in frozen tumor specimens.

Masks in Imaging Flow Cytometry

PubMed Central

Dominical, Venina; Samsel, Leigh; McCoy, J. Philip

2016-01-01

Data analysis in imaging flow cytometry incorporates elements of flow cytometry together with other aspects of morphological analysis of images. A crucial early step in this analysis is the creation of a mask to distinguish the portion of the image upon which further examination of specified features can be performed. Default masks are provided by the manufacturer of the imaging flow cytometer but additional custom masks can be created by the individual user for specific applications. Flawed or inaccurate masks can have a substantial negative impact on the overall analysis of a sample, thus great care must be taken to ensure the accuracy of masks. Here we discuss various types of masks and cite examples of their use. Furthermore we provide our insight for how to approach selecting and assessing the optimal mask for a specific analysis. PMID:27461256

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

Attenuation of doxorubicin-induced cardiotoxicity by esculetin through modulation of Bmi-1 expression.

PubMed

Xu, Fan; Li, Xiao; Liu, Lanfang; Xiao, Xu; Zhang, Li; Zhang, Shenglin; Lin, Pingping; Wang, Xiaojie; Wang, Yongwei; Li, Qingshan

2017-09-01

The protective effects and mechanisms of esculetin on doxorubicin (DOX)-induced injury of H9c2 cells were investigated. H9c2 cells were cultured and the logarithmic growth phase of the cells was divided into a control group, a DOX group and an esculetin + DOX group. Cell viability was detected by MTT assay. Annexin V-PI (AV-PI) double staining flow cytometry was carried out to detect cell apoptosis. Intracellular reactive oxygen species (ROS) were detected by flow cytometry. Transmission electron microscope (TEM) was used to evaluate cell ultrastructure. Cleaved caspase-3, cleaved PARP, Bcl-2, Bid and Bmi-1 proteins levels were investigated by western blot analysis. Bmi-1 siRNA was used to detect the role of Bmi-1 in the protective effects of esculetin against DOX-induced toxicity in H9c2 cells. The MTT and AV-PI double staining results showed that esculetin significantly increased H9c2 cell viability. Compared with the control group, the levels of cleaved caspase-3, cleaved PARP, Bid and ROS levels were significantly decreased, but the expression of Bcl-2 and Bmi-1 were significantly increased in the esculetin + DOX group. TEM showed that the cell structure of the mitochondria was protected by esculetin. The results of Bmi-1 siRNA showed that esculetin could protect DOX-induced cardiotoxicity by modulating Bmi-1 expression. Esculetin can protect DOX-induced cardiotoxicity and the effects may be attributable to modulation of Bmi-1 expression, provoking intracellular ROS accumulation, protecting the structure of mitochondria and reducing cell apoptosis.

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

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

PubMed

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

2018-05-20

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

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

Analysis of subcellular sized particles. Capillary electrophoresis with post-column laser-induced fluorescence detection versus flow cytometry.

PubMed

Poe, Bobby G; Navratil, Marian; Arriaga, Edgar A

2006-12-29

Flow cytometry (FCM) and more recently capillary electrophoresis with post-column laser-induced fluorescence detection (CE-LIF) have both been used for subcellular particle analysis but their analytical performance has not been compared. In this work, we compare a commercial FCM with an in-house built CE-LIF instrument using fluorescently labeled microspheres and isolated mitochondria. As evidenced by the relative standard deviation (RSD) of the individual fluorescence intensities, FCM is two-fold better than CE-LIF for microspheres with > or =1.5 x 10(6) molecules of equivalent soluble fluorescein (MESF). However, FCM has a comparatively low signal-to-noise ratio (S/N) and high RSD for microspheres with <1.5 x 10(6) MESF. CE-LIF, on the other hand, produces S/N ratios that are >25 times higher than FCM for all the microspheres tested and a lower RSD for microspheres with <1.5 x 10(6) MESF. When 10-N-nonyl acridine orange (NAO)-labeled mitochondria are analyzed, the S/N ratios of both techniques are similar. This appears to result from photobleaching of NAO-labeled mitochondria as they are detected by the LIF detector of the CE-LIF instrument. Both techniques have a niche in subcellular analysis; FCM has the advantage of collecting data for thousands of particles quickly, whereas CE-LIF consumes less than a nanoliter of sample and provides the electrophoretic mobility for individual particles.

Improving the Sensitivity and Functionality of Mobile Webcam-Based Fluorescence Detectors for Point-of-Care Diagnostics in Global Health.

PubMed

Rasooly, Reuven; Bruck, Hugh Alan; Balsam, Joshua; Prickril, Ben; Ossandon, Miguel; Rasooly, Avraham

2016-05-17

Resource-poor countries and regions require effective, low-cost diagnostic devices for accurate identification and diagnosis of health conditions. Optical detection technologies used for many types of biological and clinical analysis can play a significant role in addressing this need, but must be sufficiently affordable and portable for use in global health settings. Most current clinical optical imaging technologies are accurate and sensitive, but also expensive and difficult to adapt for use in these settings. These challenges can be mitigated by taking advantage of affordable consumer electronics mobile devices such as webcams, mobile phones, charge-coupled device (CCD) cameras, lasers, and LEDs. Low-cost, portable multi-wavelength fluorescence plate readers have been developed for many applications including detection of microbial toxins such as C. Botulinum A neurotoxin, Shiga toxin, and S. aureus enterotoxin B (SEB), and flow cytometry has been used to detect very low cell concentrations. However, the relatively low sensitivities of these devices limit their clinical utility. We have developed several approaches to improve their sensitivity presented here for webcam based fluorescence detectors, including (1) image stacking to improve signal-to-noise ratios; (2) lasers to enable fluorescence excitation for flow cytometry; and (3) streak imaging to capture the trajectory of a single cell, enabling imaging sensors with high noise levels to detect rare cell events. These approaches can also help to overcome some of the limitations of other low-cost optical detection technologies such as CCD or phone-based detectors (like high noise levels or low sensitivities), and provide for their use in low-cost medical diagnostics in resource-poor settings.

Improving the Sensitivity and Functionality of Mobile Webcam-Based Fluorescence Detectors for Point-of-Care Diagnostics in Global Health

PubMed Central

Rasooly, Reuven; Bruck, Hugh Alan; Balsam, Joshua; Prickril, Ben; Ossandon, Miguel; Rasooly, Avraham

2016-01-01

Resource-poor countries and regions require effective, low-cost diagnostic devices for accurate identification and diagnosis of health conditions. Optical detection technologies used for many types of biological and clinical analysis can play a significant role in addressing this need, but must be sufficiently affordable and portable for use in global health settings. Most current clinical optical imaging technologies are accurate and sensitive, but also expensive and difficult to adapt for use in these settings. These challenges can be mitigated by taking advantage of affordable consumer electronics mobile devices such as webcams, mobile phones, charge-coupled device (CCD) cameras, lasers, and LEDs. Low-cost, portable multi-wavelength fluorescence plate readers have been developed for many applications including detection of microbial toxins such as C. Botulinum A neurotoxin, Shiga toxin, and S. aureus enterotoxin B (SEB), and flow cytometry has been used to detect very low cell concentrations. However, the relatively low sensitivities of these devices limit their clinical utility. We have developed several approaches to improve their sensitivity presented here for webcam based fluorescence detectors, including (1) image stacking to improve signal-to-noise ratios; (2) lasers to enable fluorescence excitation for flow cytometry; and (3) streak imaging to capture the trajectory of a single cell, enabling imaging sensors with high noise levels to detect rare cell events. These approaches can also help to overcome some of the limitations of other low-cost optical detection technologies such as CCD or phone-based detectors (like high noise levels or low sensitivities), and provide for their use in low-cost medical diagnostics in resource-poor settings. PMID:27196933

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...

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.

Spectroscopic chemical analysis methods and apparatus

NASA Technical Reports Server (NTRS)

Hug, William F. (Inventor); Reid, Ray D. (Inventor)

2009-01-01

Spectroscopic chemical analysis methods and apparatus are disclosed which employ deep ultraviolet (e.g. in the 200 nm to 300 nm spectral range) electron beam pumped wide bandgap semiconductor lasers, incoherent wide bandgap semiconductor light emitting devices, and hollow cathode metal ion lasers to perform non-contact, non-invasive detection of unknown chemical analytes. These deep ultraviolet sources enable dramatic size, weight and power consumption reductions of chemical analysis instruments. Chemical analysis instruments employed in some embodiments include capillary and gel plane electrophoresis, capillary electrochromatography, high performance liquid chromatography, flow cytometry, flow cells for liquids and aerosols, and surface detection instruments. In some embodiments, Raman spectroscopic detection methods and apparatus use ultra-narrow-band angle tuning filters, acousto-optic tuning filters, and temperature tuned filters to enable ultra-miniature analyzers for chemical identification. In some embodiments Raman analysis is conducted simultaneously with native fluorescence spectroscopy to provide high levels of sensitivity and specificity in the same instrument.

Spectroscopic chemical analysis methods and apparatus

NASA Technical Reports Server (NTRS)

Reid, Ray D. (Inventor); Hug, William F. (Inventor)

2010-01-01

Spectroscopic chemical analysis methods and apparatus are disclosed which employ deep ultraviolet (e.g. in the 200 nm to 300 nm spectral range) electron beam pumped wide bandgap semiconductor lasers, incoherent wide bandgap semiconductor light emitting devices, and hollow cathode metal ion lasers to perform non-contact, non-invasive detection of unknown chemical analytes. These deep ultraviolet sources enable dramatic size, weight and power consumption reductions of chemical analysis instruments. Chemical analysis instruments employed in some embodiments include capillary and gel plane electrophoresis, capillary electrochromatography, high performance liquid chromatography, flow cytometry, flow cells for liquids and aerosols, and surface detection instruments. In some embodiments, Raman spectroscopic detection methods and apparatus use ultra-narrow-band angle tuning filters, acousto-optic tuning filters, and temperature tuned filters to enable ultra-miniature analyzers for chemical identification. In some embodiments Raman analysis is conducted simultaneously with native fluorescence spectroscopy to provide high levels of sensitivity and specificity in the same instrument.

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.

Detection of silver nanoparticles in cells by flow cytometry using light scatter and far-red fluorescence.

PubMed

Zucker, R M; Daniel, K M; Massaro, E J; Karafas, S J; Degn, L L; Boyes, W K

2013-10-01

The cellular uptake of different sized silver nanoparticles (AgNP) (10, 50, and 75 nm) coated with polyvinylpyrrolidone (PVP) or citrate on a human derived retinal pigment epithelial cell line (ARPE-19) was detected by flow cytometry following 24-h incubation of the cells with AgNP. A dose dependent increase of side scatter and far red fluorescence was observed with both PVP and citrate-coated 50 nm or 75 nm silver particles. Using five different flow cytometers, a far red fluorescence signal in the 700-800 nm range increased as much as 100 times background as a ratio comparing the intensity measurements of treated sample and controls. The citrate-coated silver nanoparticles (AgNP) revealed slightly more side scatter and far red fluorescence than did the PVP coated silver nanoparticles. This increased far red fluorescence signal was observed with 50 and 75 nm particles, but not with 10 nm particles. Morphological evaluation by dark field microscopy showed silver particles (50 and 75 nm) clumped and concentrated around the nucleus. One possible hypothesis to explain the emission of far red fluorescence from cells incubated with silver nanoparticles is that the silver nanoparticles inside cells agglomerate into small nano clusters that form surface plasmon resonance which interacts with laser light to emit a strong far red fluorescence signal. The results demonstrate that two different parameters (side scatter and far red fluorescence) on standard flow cytometers can be used to detect and observe metallic nanoparticles inside cells. The strength of the far red fluorescence suggests that it may be particularly useful for applications that require high sensitivity. © Published 2013 Wiley-Periodicals, Inc. Published 2013 Wiley‐Periodicals, Inc. This article is a US government work and, as such, is in the public domain in the United States of America.

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

Frequency of Dendritic Cells and Their Expression of Costimulatory Molecules in Children with Autism Spectrum Disorders

ERIC Educational Resources Information Center

Saad, Khaled; Zahran, Asmaa M.; Elsayh, Khalid I.; Abdel-Rahman, Ahmed A.; Al-Atram, Abdulrahman A.; Hussein, Almontaser; El-Gendy, Yasmin G.

2017-01-01

The aim of our study was to evaluate the frequencies of myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) in children with ASD. Subjects were 32 children with ASD and 30 healthy children as controls. The numbers of mDCs and pDCs and the expression of CD86 and CD80 on the entire DCs were detected by flow cytometry. ASD children…

Real-time cytometric assay of nitric oxide and superoxide interaction in peripheral blood monocytes: A no-wash, no-lyse kinetic method.

PubMed

Balaguer, Susana; Diaz, Laura; Gomes, Angela; Herrera, Guadalupe; O'Connor, José-Enrique; Urios, Amparo; Felipo, Vicente; Montoliu, Carmina

2017-05-01

Nitric oxide (NO) and its related reactive nitrogen species (RNS) and reactive oxygen species (ROS) are crucial in monocyte responses against pathogens and also in inflammatory conditions. Central to both processes is the generation of the strong oxidant peroxynitrite (ONOO) by a fast reaction between NO and superoxide anion. ONOO is a biochemical junction for ROS- and RNS cytotoxicity and causes protein nitrosylation. Circulating by-products of protein nitrosylation are early biomarkers of inflammation-based conditions, including minimal hepatic encephalopathy in cirrhotic patients (Montoliu et al., Am J Gastroenterol 2011; 106:1629-1637). In this context, we have designed a novel no-wash, no-lyse real-time flow cytometry assay to detect and follow-up the NO- and superoxide-driven generation of ONOO in peripheral blood monocytes. Whole blood samples were stained with CD45 and CD14 antibodies plus one of a series of fluorescent probes sensitive to RNS, ROS, or glutathione, namely 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, dihydrorhodamine 123, MitoSOX Red, dihydroethidium, and 5-chloromethylfluorescein diacetate. Samples were exposed sequentially to a NO donor and three different superoxide donors, and analyzed in real time by kinetic flow cytometry. Relevant kinetic descriptors, such as the rate of fluorescence change, were calculated from the kinetic plot. The generation of ONOO, which consumes both NO and superoxide, led to a decrease in the intensity of the cellular fluorescence of the probes sensitive to these molecules. This is a fast and simple assay that may be used to monitor the intracellular generation of ONOO in physiological, pathological, and pharmacological contexts. © 2015 International Clinical Cytometry Society. © 2015 International Clinical Cytometry Society.

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.

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.

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.