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Sample records for flow cytometry platforms

  1. Flow cytometry

    NASA Astrophysics Data System (ADS)

    Steinkamp, John A.

    1984-09-01

    Flow cytometry instrumentation developed from early efforts to count cells and particles in liquid suspension as they passed through a sensing device. Since the mid-1960's sophisticated instruments have been designed for analyzing cells based on various cytological, biochemical, and functional properties. These instruments have revolutionized automated cell analysis methods in that measurements are made at high speed, multiparameter data is correlated on each cell, statistical precision is high, and cells are separated in high purity from heterogeneous mixtures for identification and functional analysis. Advanced instruments capable of measuring cell volume, surface area, multicolor fluorescence, fluorescence polarization, light scatter within various angular regions, and axial light loss (extinction) at different wavelengths are being used in biomedical research for analyzing and sorting normal and abnormal cell populations. This article reviews the development of flow cytometers, the conceptual basis of flow measurements, and discusses some of the numerous applications of the technology in biology and medicine.

  2. In vitro flow cytometry-based screening platform for cellulase engineering

    PubMed Central

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

    2016-01-01

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

  3. In vitro flow cytometry-based screening platform for cellulase engineering.

    PubMed

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

    2016-01-01

    Ultrahigh throughput screening (uHTS) plays an essential role in directed evolution for tailoring biocatalysts for industrial applications. Flow cytometry-based uHTS provides an efficient coverage of the generated protein sequence space by analysis of up to 10(7) events per hour. Cell-free enzyme production overcomes the challenge of diversity loss during the transformation of mutant libraries into expression hosts, enables directed evolution of toxic enzymes, and holds the promise to efficiently design enzymes of human or animal origin. The developed uHTS cell-free compartmentalization platform (InVitroFlow) is the first report in which a flow cytometry-based screened system has been combined with compartmentalized cell-free expression for directed cellulase enzyme evolution. InVitroFlow was validated by screening of a random cellulase mutant library employing a novel screening system (based on the substrate fluorescein-di-β-D-cellobioside), and yielded significantly improved cellulase variants (e.g. CelA2-H288F-M1 (N273D/H288F/N468S) with 13.3-fold increased specific activity (220.60 U/mg) compared to CelA2 wildtype: 16.57 U/mg). PMID:27184298

  4. A fluorescent hydrogel-based flow cytometry high-throughput screening platform for hydrolytic enzymes.

    PubMed

    Pitzler, Christian; Wirtz, Georgette; Vojcic, Ljubica; Hiltl, Stephanie; Böker, Alexander; Martinez, Ronny; Schwaneberg, Ulrich

    2014-12-18

    Screening throughput is a key in directed evolution experiments and enzyme discovery. Here, we describe a high-throughput screening platform based on a coupled reaction of glucose oxidase and a hydrolase (Yersinia mollaretii phytase [YmPh]). The coupled reaction produces hydroxyl radicals through Fenton's reaction, acting as initiator of poly(ethyleneglycol)-acrylate-based polymerization incorporating a fluorescent monomer. As a consequence, a fluorescent hydrogel is formed around Escherichia coli cells expressing active YmPh. We achieve five times enrichment of active cell population through flow cytometry analysis and sorting of mixed populations. Finally, we validate the performance of the fluorescent polymer shell (fur-shell) technology by directed phytase evolution that yielded improved variants starting from a library containing 10(7) phytase variants. Thus, fur-shell technology represents a rapid and nonlaborious way of identifying the most active variants from vast populations, as well as a platform for generation of polymer-hybrid cells for biobased interactive materials. PMID:25525992

  5. Photoacoustic flow cytometry

    PubMed Central

    Galanzha, Ekaterina I.; Zharov, Vladimir P.

    2016-01-01

    Conventional flow cytometry using scattering and fluorescent detection methods has been a fundamental tool of biological discoveries for many years. Invasive extraction of cells from a living organism, however, may lead to changes in cell properties and prevents the long-term study of cells in their native environment. Here, we summarize recent advances of new generation flow cytometry for in vivo noninvasive label-free or targeted detection of cells in blood, lymph, bone, cerebral and plant vasculatures using photoacoustic (PA) detection techniques, multispectral high-pulse-repetition-rate lasers, tunable ultrasharp (up to 0.8 nm) rainbow plasmonic nanoprobes, positive and negative PA contrasts, in vivo magnetic enrichment, time-of-flight cell velocity measurement, PA spectral analysis, and integration of PA, photothermal (PT), fluorescent, and Raman methods. Unique applications of this tool are reviewed with a focus on ultrasensitive detection of normal blood cells at different functional states (e.g., apoptotic and necrotic) and rare abnormal cells including circulating tumor cells (CTCs), cancer stem cells, pathogens, clots, sickle cells as well as pharmokinetics of nanoparticles, dyes, microbubbles and drug nanocarriers. Using this tool we discovered that palpation, biopsy, or surgery can enhance CTC release from primary tumors, increasing the risk of metastasis. The novel fluctuation flow cytometry provided the opportunity for the dynamic study of blood rheology including red blood cell aggregation and clot formation in different medical conditions (e.g., blood disorders, cancer, or surgery). Theranostics, as a combination of PA diagnosis and PT nanobubble-amplified multiplex therapy, was used for eradication of CTCs, purging of infected blood, and thrombolysis of clots using PA guidance to control therapy efficiency. In vivo flow cytometry using a portable fiber-based devices can provide a breakthrough platform for early diagnosis of cancer, infection and

  6. 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. PMID:24339248

  7. Flow cytometry of sperm

    SciTech Connect

    Gledhill, B.L.

    1987-09-21

    This brief paper summarizes automated flow cytometric determination of sperm morphology and flow cytometry/sorting of sperm with application to sex preselection. In the latter context, mention is made of results of karyotypic determination of sex chromosome ratios in albumin-processed human sperm. 23 refs., 1 fig., 1 tab.

  8. National Flow Cytometry Resource

    SciTech Connect

    Bell-Prince, C.; Dickson, J.A.; Jett, J.H.; Stevenson, A.P.; Sklar, L.A. )

    1993-01-01

    thee National Flow Cytometry and Sorting Resource (NFCR) was established in 1982 to develop advanced flow cytometric instrumentation and methodology, to provide facilities for using the fruits of the NFCR developments in collaborative projects and to disseminate the results to the cytometry community at large. Achievements of the NFCR for 1992 include: (1) preliminary studies of DNA inactivation in preparation for the development of an optical chromosome sorter; (2) modeling of real-time cytometry data using th ISML software package on a Cray supercomputer; (3) execution of proof-of-principle experiments on a phase sensitive flow cytometer in which cellular fluorescence lifetimes were determined; (4) continued development of the DiDAC data acquisition system to include bit mapped sorting and multi-laser capabilities; (5) development of new display modalities for flow cytometric data using the high level graphics language IDL; (6) development and testing of new approaches to clustering of multivariate data; (7) novel applications of Fourier transform flow cytometry to questions of cell activation and molecular structure.

  9. Prostate extracellular vesicles in patient plasma as a liquid biopsy platform for prostate cancer using nanoscale flow cytometry

    PubMed Central

    Al-Zahrani, Ali A.; Pardhan, Siddika; Brett, Sabine I.; Guo, Qiu Q.; Yang, Jun; Wolf, Philipp; Power, Nicholas E.; Durfee, Paul N.; MacMillan, Connor D.; Townson, Jason L.; Brinker, Jeffrey C.; Fleshner, Neil E.; Izawa, Jonathan I.; Chambers, Ann F.; Chin, Joseph L.; Leong, Hon S.

    2016-01-01

    distinguishing metastatic PCa and localized PCa patients. Nanoscale flow cytometry of PMPs presents an emerging biomarker platform for various stages of prostate cancer. PMID:26814433

  10. Flow cytometry: an introduction.

    PubMed

    Givan, Alice L

    2011-01-01

    A flow cytometer is an instrument that illuminates cells (or other particles) as they flow individually in front of a light source and then detects and correlates the signals from those cells that result from the illumination. In this chapter, each of the aspects of that definition will be described: the characteristics of cells suitable for flow cytometry, methods to illuminate cells, the use of fluidics to guide the cells individually past the illuminating beam, the types of signals emitted by the cells and the detection of those signals, the conversion of light signals to digital data, and the use of computers to correlate and analyze the data after they are stored in a data file. The final section of the chapter will discuss the use of a flow cytometer to sort cells. This chapter can be read as a brief, self-contained survey. It can also be read as a gateway with signposts into the field. Other chapters in this book will provide more details, more references, and even an intriguing view of the future of cytometry. PMID:21116976

  11. Cell Surface Profiling Using High-Throughput Flow Cytometry: A Platform for Biomarker Discovery and Analysis of Cellular Heterogeneity

    PubMed Central

    Gedye, Craig A.; Hussain, Ali; Paterson, Joshua; Smrke, Alannah; Saini, Harleen; Sirskyj, Danylo; Pereira, Keira; Lobo, Nazleen; Stewart, Jocelyn; Go, Christopher; Ho, Jenny; Medrano, Mauricio; Hyatt, Elzbieta; Yuan, Julie; Lauriault, Stevan; Kondratyev, Maria; van den Beucken, Twan; Jewett, Michael; Dirks, Peter; Guidos, Cynthia J.; Danska, Jayne; Wang, Jean; Wouters, Bradly; Neel, Benjamin; Rottapel, Robert; Ailles, Laurie E.

    2014-01-01

    Cell surface proteins have a wide range of biological functions, and are often used as lineage-specific markers. Antibodies that recognize cell surface antigens are widely used as research tools, diagnostic markers, and even therapeutic agents. The ability to obtain broad cell surface protein profiles would thus be of great value in a wide range of fields. There are however currently few available methods for high-throughput analysis of large numbers of cell surface proteins. We describe here a high-throughput flow cytometry (HT-FC) platform for rapid analysis of 363 cell surface antigens. Here we demonstrate that HT-FC provides reproducible results, and use the platform to identify cell surface antigens that are influenced by common cell preparation methods. We show that multiple populations within complex samples such as primary tumors can be simultaneously analyzed by co-staining of cells with lineage-specific antibodies, allowing unprecedented depth of analysis of heterogeneous cell populations. Furthermore, standard informatics methods can be used to visualize, cluster and downsample HT-FC data to reveal novel signatures and biomarkers. We show that the cell surface profile provides sufficient molecular information to classify samples from different cancers and tissue types into biologically relevant clusters using unsupervised hierarchical clustering. Finally, we describe the identification of a candidate lineage marker and its subsequent validation. In summary, HT-FC combines the advantages of a high-throughput screen with a detection method that is sensitive, quantitative, highly reproducible, and allows in-depth analysis of heterogeneous samples. The use of commercially available antibodies means that high quality reagents are immediately available for follow-up studies. HT-FC has a wide range of applications, including biomarker discovery, molecular classification of cancers, or identification of novel lineage specific or stem cell markers. PMID:25170899

  12. GenePattern flow cytometry suite

    PubMed Central

    2013-01-01

    Background Traditional flow cytometry data analysis is largely based on interactive and time consuming analysis of series two dimensional representations of up to 20 dimensional data. Recent technological advances have increased the amount of data generated by the technology and outpaced the development of data analysis approaches. While there are advanced tools available, including many R/BioConductor packages, these are only accessible programmatically and therefore out of reach for most experimentalists. GenePattern is a powerful genomic analysis platform with over 200 tools for analysis of gene expression, proteomics, and other data. A web-based interface provides easy access to these tools and allows the creation of automated analysis pipelines enabling reproducible research. Results In order to bring advanced flow cytometry data analysis tools to experimentalists without programmatic skills, we developed the GenePattern Flow Cytometry Suite. It contains 34 open source GenePattern flow cytometry modules covering methods from basic processing of flow cytometry standard (i.e., FCS) files to advanced algorithms for automated identification of cell populations, normalization and quality assessment. Internally, these modules leverage from functionality developed in R/BioConductor. Using the GenePattern web-based interface, they can be connected to build analytical pipelines. Conclusions GenePattern Flow Cytometry Suite brings advanced flow cytometry data analysis capabilities to users with minimal computer skills. Functionality previously available only to skilled bioinformaticians is now easily accessible from a web browser. PMID:23822732

  13. Two-Photon Flow Cytometry

    NASA Technical Reports Server (NTRS)

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

    2004-01-01

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

  14. Flow cytometry apparatus

    SciTech Connect

    Pinkel, D.

    1991-01-29

    This paper describes an apparatus for orienting cells in a sheath fluid in a otometer/sorter. It comprises: flow chamber; means for flowing the sheath fluid through the flow chamber along a direction of flow; means for obstructing the flow of the sheath fluid in the flow chamber with a first dimension, which extends substantially across the flow chamber and is substantially perpendicular to the direction of flow and with a thickness perpendicular to the first dimension of the obstructing means wherein the sheath fluid flows around the thickness so that the sheath fluid converges in only one dimension at the downstream edge of the means for obstructing; and means for introducing the cells through the means for obstructing the flow to the region where the sheath fluid converges in only one dimension in the sheath fluid to orient the cells, with an aperture wherein as the cells pass from the means for introducing the cells to the region where the sheath fluid converges the cells pass through the aperture with a cross-sectional length substantially less than or equal to the thickness of the means for obstructing the flow.

  15. Analyzing the Tumor Microenvironment by Flow Cytometry.

    PubMed

    Young, Yoon Kow; Bolt, Alicia M; Ahn, Ryuhjin; Mann, Koren K

    2016-01-01

    Flow cytometry is an essential tool for studying the tumor microenvironment. It allows us to quickly quantify and identify multiple cell types in a heterogeneous sample. A brief overview of flow cytometry instrumentation and the appropriate considerations and steps in building a good flow cytometry staining panel are discussed. In addition, a lymphoid tissue and solid tumor leukocyte infiltrate flow cytometry staining protocol and an example of flow cytometry data analysis are presented. PMID:27581017

  16. MXS-Chaining: A Highly Efficient Cloning Platform for Imaging and Flow Cytometry Approaches in Mammalian Systems.

    PubMed

    Sladitschek, Hanna L; Neveu, Pierre A

    2015-01-01

    The continuous improvement of imaging technologies has driven the development of sophisticated reporters to monitor biological processes. Such constructs should ideally be assembled in a flexible enough way to allow for their optimization. Here we describe a highly reliable cloning method to efficiently assemble constructs for imaging or flow cytometry applications in mammalian cell culture systems. We bioinformatically identified a list of restriction enzymes whose sites are rarely found in human and mouse cDNA libraries. From the best candidates, we chose an enzyme combination (MluI, XhoI and SalI: MXS) that enables iterative chaining of individual building blocks. The ligation scar resulting from the compatible XhoI- and SalI-sticky ends can be translated and hence enables easy in-frame cloning of coding sequences. The robustness of the MXS-chaining approach was validated by assembling constructs up to 20 kb long and comprising up to 34 individual building blocks. By assessing the success rate of 400 ligation reactions, we determined cloning efficiency to be 90% on average. Large polycistronic constructs for single-cell imaging or flow cytometry applications were generated to demonstrate the versatility of the MXS-chaining approach. We devised several constructs that fluorescently label subcellular structures, an adapted version of FUCCI (fluorescent, ubiquitination-based cell cycle indicator) optimized to visualize cell cycle progression in mouse embryonic stem cells and an array of artificial promoters enabling dosage of doxycyline-inducible transgene expression. We made publicly available through the Addgene repository a comprehensive set of MXS-building blocks comprising custom vectors, a set of fluorescent proteins, constitutive promoters, polyadenylation signals, selection cassettes and tools for inducible gene expression. Finally, detailed guidelines describe how to chain together prebuilt MXS-building blocks and how to generate new customized MXS

  17. Flow cytometry and cell sorting.

    PubMed

    Ibrahim, Sherrif F; van den Engh, Ger

    2007-01-01

    Flow cytometry and cell sorting are well-established technologies in clinical diagnostics and biomedical research. Heterogeneous mixtures of cells are placed in suspension and passed single file across one or more laser interrogation points. Light signals emitted from the particles are collected and correlated to entities such as cell morphology, surface and intracellular protein expression, gene expression, and cellular physiology. Based on user-defined parameters, individual cells can then be diverted from the fluid stream and collected into viable, homogeneous fractions at exceptionally high speeds and a purity that approaches 100%. As such, the cell sorter becomes the launching point for numerous downstream studies. Flow cytometry is a cornerstone in clinical diagnostics, and cheaper, more versatile machines are finding their way into widespread and varied uses. In addition, advances in computing and optics have led to a new generation of flow cytometers capable of processing cells at orders of magnitudes faster than their predecessors, and with staggering degrees of complexity, making the cytometer a powerful discovery tool in biotechnology. This chapter will begin with a discussion of basic principles of flow cytometry and cell sorting, including a technical description of factors that contribute to the performance of these instruments. The remaining sections will then be divided into clinical- and research-based applications of flow cytometry and cell sorting, highlighting salient studies that illustrate the versatility of this indispensable technology. PMID:17728993

  18. Flow cytometry apparatus

    DOEpatents

    Pinkel, Daniel

    1991-01-01

    An obstruction across the flow chamber creates a one dimensional convergence of a sheath fluid. A passageway in the construction directs flat cells near to the area of one dimensional convergence in the sheath fluid to provide proper orientation of flat cells at fast rates.

  19. Flow cytometry apparatus

    DOEpatents

    Pinkel, D.

    1987-11-30

    An obstruction across the flow chamber creates a one-dimensional convergence of a sheath fluid. A passageway in the obstruction directs flat cells near to the area of one-dimensional convergence in the sheath fluid to provide proper orientation of flat cells at fast rates. 6 figs.

  20. Field evaluation in Chad of community usage of CD4 T lymphocyte counting by alternative single-platform flow cytometry

    PubMed Central

    2013-01-01

    Background Field and community evaluation of the routine usage of CD4 T counting platforms is essential in resource-poor countries for efficient and cost-effective monitoring of HIV-infected adults and children attending health care centers. Methods We herein addressed the principal issues raised by the implementation of the single-platform, volumetric Auto40 flow cytometer (Apogee Flow Systems Ltd, Hemel Hempstead, UK) in 8 community HIV monitoring laboratories of different levels throughout Chad. This is a country with particularly difficult conditions, both in terms of climate and vast geographical territory, making the decentralization of the therapeutic management of HIV-infected patients challenging. Results The routine usage of the Auto40 flow cytometers for a period of 5 years (2008–2013) confirms the reliability and robustness of the analyzer for community-based CD4 T cell enumeration in terms of both absolute numbers and percentages to enable accurate monitoring of HIV-infected adults and children. However, our observations suggest that the Auto40 mini flow cytometer is not suitable for all laboratories as it is oversized and ultimately very expensive. Conclusion The Chad experience with the Auto40 flow cytometer suggests that its usage in resource-limited settings should be mainly reserved to reference (level 1) or district (level 2) laboratories, rather than to laboratories of health care centres (level 3). PMID:24083615

  1. Two-photon flow cytometry

    NASA Astrophysics Data System (ADS)

    Zhong, Cheng F.; Ye, Jing Yong; Myc, Andrzej; Thomas, Thommey P.; Bielinska, Anna; Baker, James R., Jr.; Norris, Theodore B.

    2005-03-01

    Flow cytometry is a powerful technique for obtaining quantitative information from fluorescence in cells. Quantization is achieved by assuring a high degree of uniformity in the optical excitation and detection, generally by using a highly controlled flow such as is obtained via hydrodynamic focusing. In this work, we demonstrate a two-beam, two-channel detection and two-photon excitation flow cytometry (T3FC) system that enables multi-dye analysis to be performed very simply, with greatly relaxed requirements on the fluid flow. Two-photon excitation using a femtosecond near-infrared (NIR) laser has the advantages that it enables simultaneous excitation of multiple dyes and achieves very high signal-to-noise ratio through simplified filtering and fluorescence background reduction. By matching the excitation volume to the size of a cell, single-cell detection is ensured. Labeling of cells by targeted nanoparticles with multiple fluorophores enables normalization of the fluorescence signal and thus ratiometric measurements under nonuniform excitation. Quantitative size measurements can also be done even under conditions of nonuniform flow via a two-beam layout. This innovative detection scheme not only considerably simplifies the fluid flow system and the excitation and collection optics, it opens the way to quantitative cytometry in simple and compact microfluidics systems, or in vivo.

  2. Development of a Human Whole Blood Screening Platform to Monitor JAK/STAT Signaling Using High-Throughput Flow Cytometry.

    PubMed

    Fereshteh, Mark P; Li, Xin; Li, Sha; Fan, Yi; Zhang, Rosemary; Farr, Glen A; Kolodin, Garrett; Lippy, Jonathan; Naglich, Joseph G; Schieven, Gary; Schweizer, Liang; Zhang, Litao

    2016-09-01

    Oral agents targeting Janus-associated kinases (JAKs) are promising new agents in clinical development. To better understand the relationship between JAK inhibition and biological outcome, compounds targeting JAKs were evaluated in peripheral human whole blood. To date, these analyses are low throughput and costly. Here, we developed a robust 384-well, high-throughput flow-based assay approach to screen small molecules for JAK/STAT signaling inhibition in human whole blood. This assay platform provides a highly sensitive analysis of signaling events in blood and facilitates measurement of target engagement. Further, the automation technologies and process optimizations developed here overcame sample integrity, handling, and multiparametric data analysis bottlenecks without affecting assay performance. Together these efforts dramatically increased sample throughput compared to conventional manual flow cytometric approaches and enabled development of novel JAK/STAT inhibitors. PMID:27142718

  3. Nanotechnology-based molecular photoacoustic and photothermal flow cytometry platform for in vivo detection and killing of circulating cancer stem cells

    PubMed Central

    Kim, Jin-Woo; Zharov, Vladimir P.

    2010-01-01

    In vivo multicolor photoacoustic (PA) flow cytometry for ultra-sensitive molecular detection of the CD44+ circulating tumor cells (CTCs) is demonstrated on a mouse model of human breast cancer. Targeting of CTCs with stem-like phenotype, which are naturally shed from parent tumors, was performed with functionalized gold and magnetic nanoparticles. Results in vivo were verified in vitro with a multifunctional microscope, which integrates PA, photothermal (PT), fluorescent and transmission modules. Magnet-induced clustering of magnetic nanoparticles in individual cells significantly amplified PT and PA signals. The novel noninvasive platform, which integrates multispectral PA detection and PT therapy with a potential for multiplex targeting of many cancer biomarkers using multicolor nanoparticles, may prospectively solve grand challenges in cancer research for diagnosis and purging of undetectable yet tumor-initiating cells in circulation before they form metastasis. PMID:19957272

  4. Nanotechnology-based molecular photoacoustic and photothermal flow cytometry platform for in-vivo detection and killing of circulating cancer stem cells.

    PubMed

    Galanzha, Ekaterina I; Kim, Jin-Woo; Zharov, Vladimir P

    2009-12-01

    In-vivo multicolor photoacoustic (PA) flow cytometry for ultrasensitive molecular detection of the CD44+ circulating tumor cells (CTCs) is demonstrated on a mouse model of human breast cancer. Targeting of CTCs with stem-like phenotype, which are naturally shed from parent tumors, was performed with functionalized gold and magnetic nanoparticles. Results in vivo were verified in vitro with a multifunctional microscope, which integrates PA, photothermal (PT), fluorescent and transmission modules. Magnet-induced clustering of magnetic nanoparticles in individual cells significantly amplified PT and PA signals. The novel noninvasive platform, which integrates multispectral PA detection and PT therapy with a potential for multiplex targeting of many cancer biomarkers using multicolor nanoparticles, may prospectively solve grand challenges in cancer research for diagnosis and purging of undetectable yet tumor-initiating cells in circulation before they form metastasis. PMID:19957272

  5. 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

  6. Quantitative Functional Morphology by Imaging Flow Cytometry.

    PubMed

    Vorobjev, Ivan A; Barteneva, Natasha S

    2016-01-01

    This chapter describes advantages and limitations of imaging flow cytometry (IFC) based on Imagestream instrumentation using a hybrid approach of morphometric measurement and quantitation of multiparametric fluorescent intensities' distribution in cells and particles. Brief comparison is given of IFC with conventional flow cytometry and fluorescent microscopy. Some future directions of the IFC technology are described and discussed. PMID:27460234

  7. 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

  8. 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.

  9. Multiplex immunoassay for persistent organic pollutants in tilapia: Comparison of imaging- and flow cytometry-based platforms using spectrally encoded paramagnetic microspheres

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recent developments in spectrally encoded microspheres (SEMs)-based technologies provide high multiplexing possibilities. Most SEMs-based assays required a flow cytometer with sophisticated fluidics and optics. The new imaging superparamagnetic SEMs-based platform transports SEMs with considerably ...

  10. Flow Cytometry: Impact On Early Drug Discovery

    PubMed Central

    Edwards, Bruce S.; Sklar, Larry A.

    2015-01-01

    Summary Modern flow cytometers can make optical measurements of 10 or more parameters per cell at tens-of-thousands of cells per second and over five orders of magnitude dynamic range. Although flow cytometry is used in most drug discovery stages, “sip-and-spit” sampling technology has restricted it to low sample throughput applications. The advent of HyperCyt sampling technology has recently made possible primary screening applications in which tens-of-thousands of compounds are analyzed per day. Target-multiplexing methodologies in combination with extended multi-parameter analyses enable profiling of lead candidates early in the discovery process, when the greatest numbers of candidates are available for evaluation. The ability to sample small volumes with negligible waste reduces reagent costs, compound usage and consumption of cells. Improved compound library formatting strategies can further extend primary screening opportunities when samples are scarce. Dozens of targets have been screened in 384- and 1536-well assay formats, predominantly in academic screening lab settings. In concert with commercial platform evolution and trending drug discovery strategies, HyperCyt-based systems are now finding their way into mainstream screening labs. Recent advances in flow-based imaging, mass spectrometry and parallel sample processing promise dramatically expanded single cell profiling capabilities to bolster systems level approaches to drug discovery. PMID:25805180

  11. Flow Cytometry: Impact on Early Drug Discovery.

    PubMed

    Edwards, Bruce S; Sklar, Larry A

    2015-07-01

    Modern flow cytometers can make optical measurements of 10 or more parameters per cell at tens of thousands of cells per second and more than five orders of magnitude dynamic range. Although flow cytometry is used in most drug discovery stages, "sip-and-spit" sampling technology has restricted it to low-sample-throughput applications. The advent of HyperCyt sampling technology has recently made possible primary screening applications in which tens of thousands of compounds are analyzed per day. Target-multiplexing methodologies in combination with extended multiparameter analyses enable profiling of lead candidates early in the discovery process, when the greatest numbers of candidates are available for evaluation. The ability to sample small volumes with negligible waste reduces reagent costs, compound usage, and consumption of cells. Improved compound library formatting strategies can further extend primary screening opportunities when samples are scarce. Dozens of targets have been screened in 384- and 1536-well assay formats, predominantly in academic screening lab settings. In concert with commercial platform evolution and trending drug discovery strategies, HyperCyt-based systems are now finding their way into mainstream screening labs. Recent advances in flow-based imaging, mass spectrometry, and parallel sample processing promise dramatically expanded single-cell profiling capabilities to bolster systems-level approaches to drug discovery. PMID:25805180

  12. New flow cytometry approaches in equine andrology.

    PubMed

    Peña, Fernando J; Ortega Ferrusola, Cristina; Martín Muñoz, Patricia

    2016-07-01

    Flow cytometry is currently recognized as a robust tool for the evaluation of sperm quality and function. However, within equine reproduction, this technique has not reached the sophistication of other areas of biology and medicine. In recent years, more sophisticated flow cytometers have been introduced in andrology laboratories, and the number of tests that can be potentially used in the evaluation of sperm physiology has increased accordingly. In this review, recent advances in the evaluation of stallion spermatozoa will be discussed. These new techniques in flow cytometry are able to simultaneously measure damage to different sperm regions and/or changes in functionality. PMID:27160445

  13. 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.

  14. Microfluidic devices and methods for integrated flow cytometry

    DOEpatents

    Srivastava, Nimisha; Singh, Anup K.

    2011-08-16

    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.

  15. Flow cytometry-based apoptosis detection

    PubMed Central

    Wlodkowic, Donald; Skommer, Joanna; Darzynkiewicz, Zbigniew

    2013-01-01

    An apoptosing cell demonstrates multitude of characteristic morphological and biochemical features, which vary depending on the stimuli and cell type. The gross majority of classical apoptotic hallmarks can be rapidly examined by flow and image cytometry. Cytometry thus became a technology of choice in diverse studies of cellular demise. A large variety of cytometric methods designed to identify apoptotic cells and probe mechanisms associated with this mode of cell demise have been developed during the past two decades. In the present chapter we outline a handful of commonly used methods that are based on the assessment of: mitochondrial transmembrane potential, activation of caspases, plasma membrane alterations and DNA fragmentation. PMID:19609746

  16. A clinical flow cytometry data analysis assistant

    SciTech Connect

    Salzman, G.C. ); Stewart, C.C. ); Duque, R.E. ); Braylan, R.C. . Coll. of Medicine)

    1990-01-01

    A rule-based expert system is being developed to assist clinicians in the analysis of multivariate flow cytometry data for patients with leukemias or lymphomas. The cells are stained with fluorescently labeled monoclonal antibodies and the cell fluorescence is measured with a flow cytometer. Cluster analysis is used to isolate subpopulations in the data on which the clinical decisions are made. Symbolic facts for the expert system are instantiated using these numerical data and the knowledge of the clinicians and experts in flow cytometry. The first prototype used a decision tree and rigid rules. Is successfully classified only nine of eleven leukemia cases. A second prototype incorporating certainty factors into the rules is now being developed that should remove the need for a rigid decision tree. 9 refs.

  17. Assaying Cell Cycle Status Using Flow Cytometry.

    PubMed

    Kim, Kang Ho; Sederstrom, Joel M

    2015-01-01

    In this unit, two protocols are described for analyzing cell cycle status using flow cytometry. The first is based on the simultaneous analysis of proliferation-specific marker (Ki-67) and cellular DNA content, which discriminate resting/quiescent cell populations (G0 cell) and quantify cell cycle distribution (G1, S, or G2/M), respectively. The second is based on differential staining of DNA and RNA through co-staining of Hoechst 33342 and Pyronin Y, which is also useful to identify G0 cells from G1 cells. Along with these methods for analyzing cell cycle status, two additional methods for cell proliferation assays with recent updates of newly developed fluorophores, which allow multiplex analysis of cell cycle status, cell proliferation, and a gene of interest using flow cytometry, are outlined. PMID:26131851

  18. Flow: Statistics, visualization and informatics for flow cytometry

    PubMed Central

    Frelinger, Jacob; Kepler, Thomas B; Chan, Cliburn

    2008-01-01

    Flow is an open source software application for clinical and experimental researchers to perform exploratory data analysis, clustering and annotation of flow cytometric data. Flow is an extensible system that offers the ease of use commonly found in commercial flow cytometry software packages and the statistical power of academic packages like the R BioConductor project. PMID:18559108

  19. Applications of Imaging Flow Cytometry for Microalgae.

    PubMed

    Hildebrand, Mark; Davis, Aubrey; Abbriano, Raffaela; Pugsley, Haley R; Traller, Jesse C; Smith, Sarah R; Shrestha, Roshan P; Cook, Orna; Sánchez-Alvarez, Eva L; Manandhar-Shrestha, Kalpana; Alderete, Benjamin

    2016-01-01

    The ability to image large numbers of cells at high resolution enhances flow cytometric analysis of cells and cell populations. In particular, the ability to image intracellular features adds a unique aspect to analyses, and can enable correlation between molecular phenomena resulting in alterations in cellular phenotype. Unicellular microalgae are amenable to high-throughput analysis to capture the diversity of cell types in natural samples, or diverse cellular responses in clonal populations, especially using imaging cytometry. Using examples from our laboratory, we review applications of imaging cytometry, specifically using an Amnis(®) ImageStream(®)X instrument, to characterize photosynthetic microalgae. Some of these examples highlight advantages of imaging flow cytometry for certain research objectives, but we also include examples that would not necessarily require imaging and could be performed on a conventional cytometer to demonstrate other concepts in cytometric evaluation of microalgae. We demonstrate the value of these approaches for (1) analysis of populations, (2) documentation of cellular features, and (3) analysis of gene expression. PMID:27460237

  20. Coaxial-Flow System for Chemical Cytometry

    PubMed Central

    Marc, Paul J.; Sims, Christopher E.; Allbritton, Nancy L.

    2008-01-01

    Over the past decade, chemical cytometry performed by capillary electrophoresis (CE) has become increasingly valuable as a bio-analytical tool to quantify analytes from single cells. However, extensive use of CE-based chemical cytometry has been hindered by the relatively low throughput for the analysis of single adherent cells. In order to overcome the low throughput of CE-based analysis of adherent cells and increase its utility in evaluating cellular attributes, new higher throughput methods are needed. Integration of a coaxial buffer exchange system with CE-based chemical cytometry increased the rate of serial analyses of cells. In the designed system, fluid flow through a tube coaxial to the separation capillary was used to supply electrophoretic buffer to the capillary. This sheath or coaxial fluid was turned off between analysis of cells and on during cell sampling and electrophoresis. Thus, living cells were not exposed to the nonphysiologic electrophoretic buffer prior to lysis. Key parameters of the system such as the relative capillary-sheath positions, buffer flow velocities, and the cell chamber design were optimized. To demonstrate the utility of the system, rat basophilic leukemic cells loaded with Oregon Green and fluorescein were serially lysed and loaded into a capillary. Separation of the contents of 20 cells at a rate of 0.5 cells/min was demonstrated. PMID:17979298

  1. Flow cytometry for health monitoring in space

    SciTech Connect

    Jett, J.H.; Martin, J.C.; Saunders, G.C.; Stewart, C.C.

    1984-01-01

    Monitoring the health of space station or lunar base residents will be necessary to provide knowledge of the physiological status of astronauts. Flow cytometric techniques are uniquely capable of providing cellular, chromosome, hormone level and enzyme level information. The use of dyes provides the basis for fluorescently labeling specific cellular components. Laser induced fluorescence from stained cells is quantitated in a flow cytometer to measure cellular components such as DNA, RNA and protein. One major application of a flow cytometer will be to perform a complete blood count including hematocrit, hemoglobin content, and numbers of platelets, erythrocytes, granulocytes, lymphocytes and monocytes. A newly developed flow cytometry based fluoroimmunoassay will be able to measure levels of serum enzymes and hormones. It will also be possible to quantitate radiation exposure and some forms of chromosome damage with flow cytometric measurements. With relatively simple modifications to existing technology, it will be possible to construct a flight rated cytometer. 11 references, 6 figures, 2 tables.

  2. Resources for flow and image cytometry

    SciTech Connect

    Cassidy, M.

    1990-01-01

    This paper describes resources available to the flow and image cytometry community. I have been asked to limit the discussion to resources available in the United States, so reference to resources exclusively available in Japan, Europe, or Australia are not included. It is not the intention of this paper to include each and every resource available, rather, to describe the types available and give some examples. Included in this manuscript are listings of some of the examples of resources which readers may find useful. Addresses of commercial companies are not included in the interest of space. Most of the examples listed advertise on a regular basis in journals publishing in cytometry fields. The resources to be described are divided into five categories: instrument resources, computer and software resources, standards, physical or user'' resources, and instructional resources. Each of these resources will be discussed separately. 4 tabs.

  3. Optimized flow cytometry isolation of murine spermatocytes.

    PubMed

    Gaysinskaya, Valeriya; Soh, Ina Y; van der Heijden, Godfried W; Bortvin, Alex

    2014-06-01

    Meiotic prophase I (MPI), is an initial stage of meiosis characterized by intricate homologous chromosome interactions, synapsis, and DNA recombination. These processes depend on the complex, but poorly understood early MPI events of homologous chromosome search, alignment, and pairing. Detailed molecular investigation of these early events requires isolation of individual MPI substages. Enrichment for Pachytene (P) and Diplotene (D) substages of late MPI was previously accomplished using flow cytometry. However, separation of early MPI spermatocytes, specifically, of Leptotene (L) and Zygotene (Z) substages, has been a challenge due to these cells' similar characteristics. In this report, we describe an optimized Hoechst-33342 (Hoechst)-based flow cytometry approach for isolating individual MPI populations from adult mouse testis. We get significant enrichment for individual L and Z spermatocytes, previously inseparable from each other, and optimize the isolation of other MPI substages. Our flow cytometry approach is a combination of three optimized strategies. The first is optimization of testis dissociation protocol that yields more consistent and reproducible testicular single cell suspension. The second involves optimization of flow cytometric gating protocol where a critical addition to the standard protocol for cell discrimination based on Hoechst fluorescence, involves a back-gating technique based on light scattering parameters. This step specifies selection of individual MPI substages. The third, is an addition of DNA content restriction to the gating protocol to minimize contamination from non-meiotic cells. Finally, we confirm significant enrichment of high-purity Preleptotene (PreL), L, Z, P, and D MPI spermatocytes using stage-specific marker distribution. The technique will facilitate understanding of the molecular events underlying MPI. PMID:24664803

  4. Multiparameter Flow Cytometry For Clinical Applications

    NASA Astrophysics Data System (ADS)

    Stewart, Carleton C.

    1989-06-01

    Flow Cytometry facilities are well established and provide immunophenotyping and DNA content measurement services. The application of immunophenotyping has been primarily in monitoring therapy and in providing further information to aid in the definitive diagnosis of immunological and neoplastic disease such as: immunodeficiency disease, auto immune disease, organ transplantation, and leukemia and lymphoma. DNA content measurements have been particularly important in determining the fraction of cycling cells and presence of aneuploid cells in neoplasia. This information has been useful in the management of patients with solid tumors.

  5. Honey Bee Hemocyte Profiling by Flow Cytometry

    PubMed Central

    Marringa, William J.; Krueger, Michael J.; Burritt, Nancy L.; Burritt, James B.

    2014-01-01

    Multiple stress factors in honey bees are causing loss of bee colonies worldwide. Several infectious agents of bees are believed to contribute to this problem. The mechanisms of honey bee immunity are not completely understood, in part due to limited information about the types and abundances of hemocytes that help bees resist disease. Our study utilized flow cytometry and microscopy to examine populations of hemolymph particulates in honey bees. We found bee hemolymph includes permeabilized cells, plasmatocytes, and acellular objects that resemble microparticles, listed in order of increasing abundance. The permeabilized cells and plasmatocytes showed unexpected differences with respect to properties of the plasma membrane and labeling with annexin V. Both permeabilized cells and plasmatocytes failed to show measurable mitochondrial membrane potential by flow cytometry using the JC-1 probe. Our results suggest hemolymph particulate populations are dynamic, revealing significant differences when comparing individual hive members, and when comparing colonies exposed to diverse conditions. Shifts in hemocyte populations in bees likely represent changing conditions or metabolic differences of colony members. A better understanding of hemocyte profiles may provide insight into physiological responses of honey bees to stress factors, some of which may be related to colony failure. PMID:25285798

  6. Optical clearing in photoacoustic flow cytometry.

    PubMed

    Menyaev, Yulian A; Nedosekin, Dmitry A; Sarimollaoglu, Mustafa; Juratli, Mazen A; Galanzha, Ekaterina I; Tuchin, Valery V; Zharov, Vladimir P

    2013-01-01

    Clinical applications of photoacoustic (PA) flow cytometry (PAFC) for detection of circulating tumor cells in deep blood vessels are hindered by laser beam scattering, that result in loss of PAFC sensitivity and resolution. We demonstrate biocompatible and rapid optical clearing (OC) of skin to minimize light scattering and thus, increase optical resolution and sensitivity of PAFC. OC effect was achieved in 20 min by sequent skin cleaning, microdermabrasion, and glycerol application enhanced by massage and sonophoresis. Using 0.8 mm mouse skin layer over a blood vessel in vitro phantom we demonstrated 1.6-fold decrease in laser spot blurring accompanied by 1.6-fold increase in PA signal amplitude from blood background. As a result, peak rate for B16F10 melanoma cells in blood flow increased 1.7-fold. By using OC we also demonstrated the feasibility of PA contrast improvement for human hand veins. PMID:24409398

  7. Optical clearing in photoacoustic flow cytometry

    PubMed Central

    Menyaev, Yulian A.; Nedosekin, Dmitry A.; Sarimollaoglu, Mustafa; Juratli, Mazen A.; Galanzha, Ekaterina I.; Tuchin, Valery V.; Zharov, Vladimir P.

    2013-01-01

    Clinical applications of photoacoustic (PA) flow cytometry (PAFC) for detection of circulating tumor cells in deep blood vessels are hindered by laser beam scattering, that result in loss of PAFC sensitivity and resolution. We demonstrate biocompatible and rapid optical clearing (OC) of skin to minimize light scattering and thus, increase optical resolution and sensitivity of PAFC. OC effect was achieved in 20 min by sequent skin cleaning, microdermabrasion, and glycerol application enhanced by massage and sonophoresis. Using 0.8 mm mouse skin layer over a blood vessel in vitro phantom we demonstrated 1.6-fold decrease in laser spot blurring accompanied by 1.6-fold increase in PA signal amplitude from blood background. As a result, peak rate for B16F10 melanoma cells in blood flow increased 1.7-fold. By using OC we also demonstrated the feasibility of PA contrast improvement for human hand veins. PMID:24409398

  8. Deep profiling of multitube flow cytometry data

    PubMed Central

    O’Neill, Kieran; Aghaeepour, Nima; Parker, Jeremy; Hogge, Donna; Karsan, Aly; Dalal, Bakul; Brinkman, Ryan R.

    2015-01-01

    Motivation: Deep profiling the phenotypic landscape of tissues using high-throughput flow cytometry (FCM) can provide important new insights into the interplay of cells in both healthy and diseased tissue. But often, especially in clinical settings, the cytometer cannot measure all the desired markers in a single aliquot. In these cases, tissue is separated into independently analysed samples, leaving a need to electronically recombine these to increase dimensionality. Nearest-neighbour (NN) based imputation fulfils this need but can produce artificial subpopulations. Clustering-based NNs can reduce these, but requires prior domain knowledge to be able to parameterize the clustering, so is unsuited to discovery settings. Results: We present flowBin, a parameterization-free method for combining multitube FCM data into a higher-dimensional form suitable for deep profiling and discovery. FlowBin allocates cells to bins defined by the common markers across tubes in a multitube experiment, then computes aggregate expression for each bin within each tube, to create a matrix of expression of all markers assayed in each tube. We show, using simulated multitube data, that flowType analysis of flowBin output reproduces the results of that same analysis on the original data for cell types of >10% abundance. We used flowBin in conjunction with classifiers to distinguish normal from cancerous cells. We used flowBin together with flowType and RchyOptimyx to profile the immunophenotypic landscape of NPM1-mutated acute myeloid leukemia, and present a series of novel cell types associated with that mutation. Availability and implementation: FlowBin is available in Bioconductor under the Artistic 2.0 free open source license. All data used are available in FlowRepository under accessions: FR-FCM-ZZYA, FR-FCM-ZZZK and FR-FCM-ZZES. Contact: rbrinkman@bccrc.ca. Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25600947

  9. 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

  10. Cellometer image cytometry as a complementary tool to flow cytometry for verifying gated cell populations.

    PubMed

    Kuksin, Dmitry; Kuksin, Christina Arieta; Qiu, Jean; Chan, Leo Li-Ying

    2016-06-15

    Traditionally, many cell-based assays that analyze cell populations and functionalities have been performed using flow cytometry. However, flow cytometers remain relatively expensive and require highly trained operators for routine maintenance and data analysis. Recently, an image cytometry system has been developed by Nexcelom Bioscience (Lawrence, MA, USA) for automated cell concentration and viability measurement using bright-field and fluorescent imaging methods. Image cytometry is analogous to flow cytometry in that gating operations can be performed on the cell population based on size and fluorescent intensity. In addition, the image cytometer is capable of capturing bright-field and fluorescent images, allowing for the measurement of cellular size and fluorescence intensity data. In this study, we labeled a population of cells with an enzymatic vitality stain (calcein-AM) and a cell viability dye (propidium iodide) and compared the data generated by flow and image cytometry. We report that measuring vitality and viability using the image cytometer is as effective as flow cytometric assays and allows for visual confirmation of the sample to exclude cellular debris. Image cytometry offers a direct method for performing fluorescent cell-based assays but also may be used as a complementary tool to flow cytometers for aiding the analysis of more complex samples. PMID:27033005

  11. Flow cytometry: A powerful technology for measuring biomarkers

    SciTech Connect

    Jett, J.H.

    1994-09-01

    A broad definition of a biomarker is that it is a measurable characteristic of a biological system that changes upon exposure to a physical or chemical insult. While the definition can be further refined, it is sufficient for the purposes of demonstrating the advantages of flow cytometry for making quantitative measurements of biomarkers. Flow cytometry and cell sorting technologies have emerged during the past 25 years to take their place alongside other essential tools used in biology such as optical and electron microscopy. This paper describes the basics of flow cytometry technology, provides illustrative examples of applications of the technology in the field of biomarkers, describes recent developments in flow cytometry that have not yet been applied to biomarker measurements, and projects future developments of the technology. The examples of uses of flow cytometry for biomarker quantification cited in this paper are meant to be illustrative and not exhaustive in the sense of providing a review of the field.

  12. [Flow cytometry: applications in transfusion medicine].

    PubMed

    Boval, B

    2000-06-01

    In transfusion medicine, flow cytometry (FCM) is a methodology combining laser radiation, optics and a computerized treatment of numerous results. We can measure size, cellularity and fluorescence intensity of cells or particles in suspension after the binding of appropriate fluorescent antibodies or fluorescent dyes. The main utilisation of FCM in transfusion medicine is for quality control of the process of leukocyte reduction in red cell concentrates or in platelet units, using commercial kits. In addition, it is used for the enumeration of CD 34 positive cells before bone marrow transplantation and for control of platelet function in platelet units. For clinical investigations, FCM may be used for red cell phenotyping, essentially to detect minor populations (chimerism), for the estimation of red cell survival, or for the detection of fetal erythrocytes. In the field of platelet immunology, FCM is an essential tool for detecting platelet antibodies (auto or allo), for platelet phenotyping or for cross-matching. In the future perhaps, FCM will permit us to detect bacterial contamination or prion protein in transfused blood cells. PMID:10919227

  13. Bioaerosol characterization by flow cytometry with fluorochrome.

    PubMed

    Chen, Pei-Shih; Li, Chih-Shan

    2005-10-01

    Traditional culture and microscopy methods for evaluation of bioaerosols are slow, tedious, and rather imprecise. In this study, the application of flow cytometry that was combined with a fluorescent technique (FCM/FL) was evaluated as a technique to quickly and accurately determine and quantify the total concentration and viability of bioaerosols. The optimal conditions of five fluorescent dyes [acridine orange (AO), SYTO-13, propidium iodide (PI), YOPRO-1, and 5-cyano-2,3-ditolytetrazolium chloride (CTC)] used in FCM/FL were determined for laboratory samples of bacterial aerosols (Escherichia coli, and endospores of Bacillus subtilis) and fungal aerosols (Candida famata and Penicillium citrinum spores). Based on the measured cell concentration, fluorescence intensity, and staining efficiency as indicators for dye performance evaluation, SYTO-13 was found to be the most suitable fluorescent dye for determining the total concentration of the bioaerosols, as well as YOPRO-1 was the most suitable for determining viability. Moreover, the established optimal FCM/FL with dyes was validated for characterizing microorganism profiles from both air and water samples from the aeration tank of hospital wastewater treatment plant. In conclusion, the FCM/FL successfully assessed the total concentration and viability for bacterial and fungal microorganisms in environmental field samples. PMID:16193165

  14. Flow-cytometry techniques in radiation biology

    SciTech Connect

    McCarthy, K.F.; Hale, M.L.

    1988-01-01

    Considerable evidence exists that all blood cells are derived from HSC. These cells are of interest to radiobiologists because they are highly sensitive to low doses of ionizing radiation. Hematopoietic stem cells (HSC) are present in the marrow at a concentration of approximately 2-3 HSC per 1000 nucleated marrow cells. In the past, only clonogenic assays requiring 8-13 days and ten irradiated recipient rodents were available for assaying HSC. Because of the importance of HSC in the post-irradiation syndrome, the authors developed a new rapid method based on flow cytometry not only to assay but also to purify and characterize HSC. This new method makes extensive use of non-clonal antibodies conjugated to fluorescent phycobiliproteins through the sulfhydryls of the hinge region of the IgG molecule. An optical bench arrangement with a dye laser and an argon laser was used for dual excitation of the phycobiliprotein-monoclonal antibody conjugates and various cellular and DNA probes. Using 4', 6-diamidino 2-phenylindole dihydrochloride (DAP) exclusion to identify viable cells, it was possible to follow regeneration of post-irradiated rat marrow HSC.

  15. New Horizons in Platelets Flow Cytometry

    PubMed Central

    Saboor, Muhammad; Moinuddin, Moinuddin; Ilyas, Samina

    2013-01-01

    Platelet flow cytometry is an emerging tool in diagnostic and therapeutic hematology. It is eminently suited to study the expression of platelet surface receptors both qualitatively as well as quantitatively. It can serve as a useful marker for the documentation of in vivo platelet activation, and thus, fore-warn the risk of thromboembolism in patients with diabetes mellitus, coronary syndromes, peripheral vascular diseases, and pre-eclampsia. This technique can also be extended to study and compare the effect of various antiplatelet drugs on the level of activation of platelets and to establish any dose-effect relationship of these drugs. Topographical localization of platelet granules and study of platelet-platelet and platelet-leukocyte interaction is also possible by this procedure. All these parameters serve as pointers towards the presence of activated platelets in the circulation with its thromboembolic consequences. This is a simple reliable and cost effective technique which has a wide application in the diagnosis of various inherited and acquired platelet disorders. Study of platelet cluster of differentiation (CD) markers in various inherited disorders i.e. Bernard Soulier’s disease, von Willebrand disease, Glanzman’s disease, and Grey platelet syndrome may help categories the molecular lesions in these oft under-studied disorders. PMID:23983579

  16. 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. PMID:27078044

  17. 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. PMID:27068992

  18. The application of flow cytometry to histocompatibility testing.

    PubMed

    Horsburgh, T; Martin, S; Robson, A J

    2000-03-01

    Flow cytometry is a powerful technique that enables the sensitive and quantitative detection of both cellular antigens and bound biological moieties. This article reviews how flow cytometry is increasingly being used as histocompatibility laboratories for the analysis of antibody specificity and HLA antigen expression. A basic description of flow cytometry principles and standardisation is given, together with an outline of clinical application in the areas of pre-transplant cross-matching, antibody screening, post-transplant antibody monitoring and HLA-B27 detection. It is concluded that flow cytometry is a useful multi-parametric analytical tool, yielding clinical benefit especially in the identification of patients at risk of early transplant rejection. PMID:10834606

  19. Visible and Near Infrared Fluorescence Spectral Flow Cytometry

    PubMed Central

    Nolan, John P.; Condello, Danilo; Duggan, Erika; Naivar, Mark; Novo, David

    2013-01-01

    There is a long standing interest in measuring complete emission spectra from individual cells in flow cytometry. We have developed flow cytometry instruments and analysis approaches to enable this to be done routinely and robustly. Our spectral flow cytometers use a holographic grating to disperse light from single cells onto a CCD for high speed, wavelength-resolved detection. Customized software allows the single cell spectral data to be displayed and analyzed to produce new spectra-derived parameters. We show that familiar reference and calibration beads can be employed to quantitatively assess instrument performance. We use microspheres stained with six different quantum dots to compare a virtual bandpass filter approach with classic least squares (CLS) spectral unmixing, and then use antibody capture beads and CLS unmixing to demonstrate immunophenotyping of peripheral blood mononuclear cells using spectral flow cytometry. Finally, we characterize and evaluate several near infrared (NIR) emitting fluorophores for use in spectral flow cytometry. Spectral flow cytometry offers a number of attractive features for single cell analysis, including a simplified optical path, high spectral resolution, and streamlined approaches to quantitative multiparameter measurements. The availability of robust instrumentation, software, and analysis approaches will facilitate the development of spectral flow cytometry applications. PMID:23225549

  20. flowCore: a Bioconductor package for high throughput flow cytometry

    PubMed Central

    Hahne, Florian; LeMeur, Nolwenn; Brinkman, Ryan R; Ellis, Byron; Haaland, Perry; Sarkar, Deepayan; Spidlen, Josef; Strain, Errol; Gentleman, Robert

    2009-01-01

    Background Recent advances in automation technologies have enabled the use of flow cytometry for high throughput screening, generating large complex data sets often in clinical trials or drug discovery settings. However, data management and data analysis methods have not advanced sufficiently far from the initial small-scale studies to support modeling in the presence of multiple covariates. Results We developed a set of flexible open source computational tools in the R package flowCore to facilitate the analysis of these complex data. A key component of which is having suitable data structures that support the application of similar operations to a collection of samples or a clinical cohort. In addition, our software constitutes a shared and extensible research platform that enables collaboration between bioinformaticians, computer scientists, statisticians, biologists and clinicians. This platform will foster the development of novel analytic methods for flow cytometry. Conclusion The software has been applied in the analysis of various data sets and its data structures have proven to be highly efficient in capturing and organizing the analytic work flow. Finally, a number of additional Bioconductor packages successfully build on the infrastructure provided by flowCore, open new avenues for flow data analysis. PMID:19358741

  1. Computational analysis of high-throughput flow cytometry data

    PubMed Central

    Robinson, J Paul; Rajwa, Bartek; Patsekin, Valery; Davisson, Vincent Jo

    2015-01-01

    Introduction Flow cytometry has been around for over 40 years, but only recently has the opportunity arisen to move into the high-throughput domain. The technology is now available and is highly competitive with imaging tools under the right conditions. Flow cytometry has, however, been a technology that has focused on its unique ability to study single cells and appropriate analytical tools are readily available to handle this traditional role of the technology. Areas covered Expansion of flow cytometry to a high-throughput (HT) and high-content technology requires both advances in hardware and analytical tools. The historical perspective of flow cytometry operation as well as how the field has changed and what the key changes have been discussed. The authors provide a background and compelling arguments for moving toward HT flow, where there are many innovative opportunities. With alternative approaches now available for flow cytometry, there will be a considerable number of new applications. These opportunities show strong capability for drug screening and functional studies with cells in suspension. Expert opinion There is no doubt that HT flow is a rich technology awaiting acceptance by the pharmaceutical community. It can provide a powerful phenotypic analytical toolset that has the capacity to change many current approaches to HT screening. The previous restrictions on the technology, based on its reduced capacity for sample throughput, are no longer a major issue. Overcoming this barrier has transformed a mature technology into one that can focus on systems biology questions not previously considered possible. PMID:22708834

  2. 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…

  3. Application of Flow Cytometry in the Evaluation of Primary Immunodeficiencies.

    PubMed

    Fleisher, Thomas A; Madkaikar, Manisha; Rosenzweig, Sergio D

    2016-05-01

    Primary immunodeficiency disorders (PIDDs) are a heterogeneous group of inherited disorders of the immune system. Currently more than 250 different PIDDs with a known genetic defect have been recognized. The diagnosis of many of these disorders is supported strongly by a wide variety of flow cytometry applications. Flow cytometry offers a rapid and sensitive tool for diagnosis and classification of PIDDs. It is applicable in the initial workup and subsequent management of several primary immunodeficiency diseases. As our understanding of the pathogenesis and management of these diseases increases, the majority of these tests can be easily established in the diagnostic laboratory. Thus, the focus of this article is on the application of flow cytometry in the diagnosis and/or evaluation of PIDDs. PMID:26865168

  4. Flow cytometry measurements of human chromosome kinetochore labeling

    SciTech Connect

    Fantes, J.A.; Green, D.K.; Malloy, P.; Sumner, A.T.

    1989-03-01

    A method for the preparation and measurement of immunofluorescent human chromosome centromeres in suspension is described using CREST antibodies, which bind to the centromeric region of chromosomes. Fluorescein isothiocyanate (FITC)-conjugated antihuman antibodies provide the fluorescent label. Labeled chromosomes are examined on microscope slides and by flow cytometry. In both cases a dye which binds to DNA is added to provide identification of the chromosome groups. Sera from different CREST patients vary in their ability to bind to chromosome arms in addition to the centromeric region. Flow cytometry and microfluorimetry measurements have shown that with a given CREST serum the differences in kinetochore fluorescence between chromosomes are only minor. Flow cytometry experiments to relate the number of dicentric chromosomes, induced by in vitro radiation of peripheral blood cells to the slightly increased number of chromosomes with above-average kinetochore fluorescence did not produce decisive radiation dosimetry results.

  5. A Semiconductor Microlaser for Intracavity Flow Cytometry

    SciTech Connect

    Akhil, O.; Copeland, G.C.; Dunne, J.L.; Gourley, P.L.; Hendricks, J.K.; McDonald, A.E.

    1999-01-20

    Semiconductor microlasers are attractive components for micro-analysis systems because of their ability to emit coherent intense light from a small aperture. By using a surface-emitting semiconductor geometry, we were able to incorporate fluid flow inside a laser microcavity for the first time. This confers significant advantages for high throughput screening of cells, particulates and fluid analytes in a sensitive microdevice. In this paper we discuss the intracavity microfluidics and present preliminary results with flowing blood and brain cells.

  6. flowClust: a Bioconductor package for automated gating of flow cytometry data

    PubMed Central

    Lo, Kenneth; Hahne, Florian; Brinkman, Ryan R; Gottardo, Raphael

    2009-01-01

    Background As a high-throughput technology that offers rapid quantification of multidimensional characteristics for millions of cells, flow cytometry (FCM) is widely used in health research, medical diagnosis and treatment, and vaccine development. Nevertheless, there is an increasing concern about the lack of appropriate software tools to provide an automated analysis platform to parallelize the high-throughput data-generation platform. Currently, to a large extent, FCM data analysis relies on the manual selection of sequential regions in 2-D graphical projections to extract the cell populations of interest. This is a time-consuming task that ignores the high-dimensionality of FCM data. Results In view of the aforementioned issues, we have developed an R package called flowClust to automate FCM analysis. flowClust implements a robust model-based clustering approach based on multivariate t mixture models with the Box-Cox transformation. The package provides the functionality to identify cell populations whilst simultaneously handling the commonly encountered issues of outlier identification and data transformation. It offers various tools to summarize and visualize a wealth of features of the clustering results. In addition, to ensure its convenience of use, flowClust has been adapted for the current FCM data format, and integrated with existing Bioconductor packages dedicated to FCM analysis. Conclusion flowClust addresses the issue of a dearth of software that helps automate FCM analysis with a sound theoretical foundation. It tends to give reproducible results, and helps reduce the significant subjectivity and human time cost encountered in FCM analysis. The package contributes to the cytometry community by offering an efficient, automated analysis platform which facilitates the active, ongoing technological advancement. PMID:19442304

  7. Nanocrystal-based biomimetic system for quantitative flow cytometry

    NASA Astrophysics Data System (ADS)

    Yim, Peter; Dobrovolskaia, Marina; Kang, HyeongGon; Clarke, Matthew; Patri, Anil K.; Hwang, Jeeseong

    2007-02-01

    Flow cytometry has been instrumental in rapid analysis of single cells since the 1970s. One of the common approaches is the immunofluorescence study involving labeling of cells with antibodies conjugated to organic fluorophores. More recently, as the application of flow cytometry extended from simple cell detection to single-cell proteomic analysis, the need of determining the actual number of antigens in a single cell has driven the flow cytomery technique towards a quantitative methodology. However, organic fluorophores are challenging to use as probes for quantitative detection due to the lack of photostability and of quantitative fluorescence standards. National Institute of Standards and Technologies (NIST) provides a set of fluorescein isothiocyanate (FITC) labeled beads, RM 8640, which is the only nationally recognized fluorescent particle standard. On the other hand, optical characteristics of semiconductor nanocrystals or quantum dots or QDs are superior to traditional dye molecules for the use as tags for biological and chemical fluorescent sensors and detectors. Compelling advantages of QDs include long photostability, broad spectral coverage, easy excitation, and suitability for multiplexed sensing. Recently, novel surface coatings have been developed to render QDs water soluble and bio-conjugation ready, leading to their use as fluorescent tags and sensors for a variety of biological applications including immunolabeling of cells. Here, we describe our approach of using fluorescent semiconductor QDs as a novel tool for quantitative flow cytometry detection. Our strategy involves the development of immuno-labeled QD-conjugated silica beads as "biomimetic cells." In addition to flow cytometry, the QD-conjugated silica beads were characterized by fluorescence microscopy to quantitate the number of QDs attached to a single silica bead. Our approach enables flow cytometry analysis to be highly sensitive, quantitative, and encompass a wide dynamic range of

  8. Human CD4+ lymphocytes for antigen quantification: characterization using conventional flow cytometry and mass cytometry.

    PubMed

    Wang, Lili; Abbasi, Fatima; Ornatsky, Olga; Cole, Kenneth D; Misakian, Martin; Gaigalas, Adolfas K; He, Hua-Jun; Marti, Gerald E; Tanner, Scott; Stebbings, Richard

    2012-07-01

    To transform the linear fluorescence intensity scale obtained with fluorescent microspheres to an antibody bound per cell (ABC) scale, a biological cell reference material is needed. Optimally, this material should have a reproducible and tight ABC value for the expression of a known clinical reference biomarker. In this study, we characterized commercially available cryopreserved peripheral blood mononuclear cells (PBMCs) and two lyophilized PBMC preparations, Cyto-Trol and PBMC-National Institute for Biological Standard and Control (NIBSC) relative to freshly prepared PBMC and whole blood samples. It was found that the ABC values for CD4 expression on cryopreserved PBMC were consistent with those of freshly obtained PBMC and whole blood samples. By comparison, the ABC value for CD4 expression on Cyto-Trol is lower and the value on PBMC-NIBSC is much lower than those of freshly prepared cell samples using both conventional flow cytometry and CyTOF™ mass cytometry. By performing simultaneous surface and intracellular staining measurements on these two cell samples, we found that both cell membranes are mostly intact. Moreover, CD4(+) cell diameters from both lyophilized cell preparations are smaller than those of PBMC and whole blood. This could result in steric interference in antibody binding to the lyophilized cells. Further investigation of the fixation effect on the detected CD4 expression suggests that the very low ABC value obtained for CD4(+) cells from lyophilized PBMC-NIBSC is largely due to paraformaldehyde fixation; this significantly decreases available antibody binding sites. This study provides confirmation that the results obtained from the newly developed mass cytometry are directly comparable to the results from conventional flow cytometry when both methods are standardized using the same ABC approach. PMID:22539147

  9. Ultrafast quantitative time-stretch imaging flow cytometry of phytoplankton

    NASA Astrophysics Data System (ADS)

    Lai, Queenie T. K.; Lau, Andy K. S.; Tang, Anson H. L.; Wong, Kenneth K. Y.; Tsia, Kevin K.

    2016-03-01

    Comprehensive quantification of phytoplankton abundance, sizes and other parameters, e.g. biomasses, has been an important, yet daunting task in aquatic sciences and biofuel research. It is primarily because of the lack of effective tool to image and thus accurately profile individual microalgae in a large population. The phytoplankton species are highly diversified and heterogeneous in terms of their sizes and the richness in morphological complexity. This fact makes time-stretch imaging, a new ultrafast real-time optical imaging technology, particularly suitable for ultralarge-scale taxonomic classification of phytoplankton together with quantitative image recognition and analysis. We here demonstrate quantitative imaging flow cytometry of single phytoplankton based on quantitative asymmetric-detection time-stretch optical microscopy (Q-ATOM) - a new time-stretch imaging modality for label-free quantitative phase imaging without interferometric implementations. Sharing the similar concept of Schlieren imaging, Q-ATOM accesses multiple phase-gradient contrasts of each single phytoplankton, from which the quantitative phase profile is computed. We employ such system to capture, at an imaging line-scan rate of 11.6 MHz, high-resolution images of two phytoplankton populations (scenedesmus and chlamydomonas) in ultrafast microfluidic flow (3 m/s). We further perform quantitative taxonomic screening analysis enabled by this technique. More importantly, the system can also generate quantitative phase images of single phytoplankton. This is especially useful for label-free quantification of biomasses (e.g. lipid droplets) of the particular species of interest - an important task adopted in biofuel applications. Combining machine learning for automated classification, Q-ATOM could be an attractive platform for continuous and real-time ultralarge-scale single-phytoplankton analysis.

  10. The Intersection of Flow Cytometry with Microfluidics and Microfabrication

    PubMed Central

    Piyasena, Menake E.; Graves, Steven W.

    2014-01-01

    A modern flow cytometer can analyze and sort particles on a one by one basis at rates of 50,000 particles per second. Flow cytometers can also measure as many as 17 channels of fluorescence, several angles of scattered light, and other non-optical parameters such as particle impedance. More specialized flow cytometers can provide even greater analysis power, such as single molecule detection, imaging, and full spectral collection, at reduced rates. These capabilities have made flow cytometers an invaluable tool for numerous applications including cellular immunophenotyping, CD4+ T-cell counting, multiplex microsphere analysis, high-throughput screening, and rare cell analysis and sorting. Many bio-analytical techniques have been influenced by the advent of microfluidics as a component in analytical tools and flow cytometry is no exception. Here we detail the functions and uses of a modern flow cytometer, review the recent and historical contributions of microfluidics and microfabricated devices to field of flow cytometry, examine current application areas, and suggest opportunities for the synergistic application of microfabrication approaches to modern flow cytometry. PMID:24488050

  11. Per-channel basis normalization methods for flow cytometry data

    PubMed Central

    Hahne, Florian; Khodabakhshi, Alireza Hadj; Bashashati, Ali; Wong, Chao-Jen; Gascoyne, Randy D.; Weng, Andrew P.; Seifert-Margolis, Vicky; Bourcier, Katarzyna; Asare, Adam; Lumley, Thomas; Gentleman, Robert; Brinkman, Ryan R.

    2013-01-01

    Between-sample variation in high throughput flow cytometry data poses a significant challenge for analysis of large scale data sets, such as those derived from multi-center clinical trials. It is often hard to match biologically relevant cell populations across samples due to technical variation in sample acquisition and instrumentation differences. Thus normalization of data is a critical step prior to analysis, particularly in large-scale data sets from clinical trials, where group specific differences may be subtle and patient-to-patient variation common. We have developed two normalization methods that remove technical between-sample variation by aligning prominent features (landmarks) in the raw data on a per-channel basis. These algorithms were tested on two independent flow cytometry data sets by comparing manually gated data, either individually for each sample or using static gating templates, before and after normalization. Our results show a marked improvement in the overlap between manual and static gating when the data are normalized, thereby facilitating the use of automated analyses on large flow cytometry data sets. Such automated analyses are essential for high throughput flow cytometry. PMID:19899135

  12. Phasor plotting with frequency-domain flow cytometry.

    PubMed

    Cao, Ruofan; Jenkins, Patrick; Peria, William; Sands, Bryan; Naivar, Mark; Brent, Roger; Houston, Jessica P

    2016-06-27

    Interest in time resolved flow cytometry is growing. In this paper, we collect time-resolved flow cytometry data and use it to create polar plots showing distributions that are a function of measured fluorescence decay rates from individual fluorescently-labeled cells and fluorescent microspheres. Phasor, or polar, graphics are commonly used in fluorescence lifetime imaging microscopy (FLIM). In FLIM measurements, the plotted points on a phasor graph represent the phase-shift and demodulation of the frequency-domain fluorescence signal collected by the imaging system for each image pixel. Here, we take a flow cytometry cell counting system, introduce into it frequency-domain optoelectronics, and process the data so that each point on a phasor plot represents the phase shift and demodulation of an individual cell or particle. In order to demonstrate the value of this technique, we show that phasor graphs can be used to discriminate among populations of (i) fluorescent microspheres, which are labeled with one fluorophore type; (ii) Chinese hamster ovary (CHO) cells labeled with one and two different fluorophore types; and (iii) Saccharomyces cerevisiae cells that express combinations of fluorescent proteins with different fluorescence lifetimes. The resulting phasor plots reveal differences in the fluorescence lifetimes within each sample and provide a distribution from which we can infer the number of cells expressing unique single or dual fluorescence lifetimes. These methods should facilitate analysis time resolved flow cytometry data to reveal complex fluorescence decay kinetics. PMID:27410612

  13. Fluorescence lifetime measurements in flow cytometry

    NASA Astrophysics Data System (ADS)

    Beisker, Wolfgang; Klocke, Axel

    1997-05-01

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

  14. Critical assessment of automated flow cytometry data analysis techniques.

    PubMed

    Aghaeepour, Nima; Finak, Greg; Hoos, Holger; Mosmann, Tim R; Brinkman, Ryan; Gottardo, Raphael; Scheuermann, Richard H

    2013-03-01

    Traditional methods for flow cytometry (FCM) data processing rely on subjective manual gating. Recently, several groups have developed computational methods for identifying cell populations in multidimensional FCM data. The Flow Cytometry: Critical Assessment of Population Identification Methods (FlowCAP) challenges were established to compare the performance of these methods on two tasks: (i) mammalian cell population identification, to determine whether automated algorithms can reproduce expert manual gating and (ii) sample classification, to determine whether analysis pipelines can identify characteristics that correlate with external variables (such as clinical outcome). This analysis presents the results of the first FlowCAP challenges. Several methods performed well as compared to manual gating or external variables using statistical performance measures, which suggests that automated methods have reached a sufficient level of maturity and accuracy for reliable use in FCM data analysis. PMID:23396282

  15. Sizing of DNA fragments by flow cytometry

    NASA Astrophysics Data System (ADS)

    Johnson, Mitchell E.; Goodwin, Peter M.; Ambrose, W. Patrick; Martin, John C.; Marrone, Babetta L.; Jett, James H.; Keller, Richard A.

    1993-06-01

    Individual, stained DNA fragments were sized using a modified flow cytometer with high sensitivity fluorescence detection. The fluorescent intercalating dye ethidium homodimer was used to stain stoichiometrically lambda phage DNA and a Kpn I digest of lambda DNA. Stained, individual fragments of DNA were passed through a low average power, focused, mode-locked laser beam, and the fluorescence from each fragment was collected and quantified. Time-gated detection was used to discriminate against Raman scattering from the water solvent. The fluorescence burst from each fragment was related directly to its length, thus providing a means to size small quantities of kilobase lengths of DNA quickly. Improvements of several orders of magnitude in analysis time and sample size over current gel electrophoresis techniques were realized. Fragments of 17.1, 29.9, and 48.5 thousand base pairs were well resolved, and were sized in 164 seconds. Less than one pg of DNA was required for analysis.

  16. Sizing of DNA fragments by flow cytometry

    SciTech Connect

    Johnson, M.E.; Goodwin, P.M.; Ambrose, W.P.; Martin, J.C.; Marrone, B.L.; Jett, J.H.; Keller, R.A.

    1993-02-01

    Individual, stained DNA fragments were sized using a modified flow cytometer with high sensitivity fluorescence detection. The fluorescent intercalating dye ethidium homodimer was used to stain stoichiometrically lambda phage DNA and a Kpn I digest of lambda DNA. Stained, individual fragments of DNA were passed through a low average power, focused, mode-locked laser beam, and the fluorescence from each fragment was collected and quantified. Time-gated detection was used to discriminate against Raman scattering from the water solvent. The fluorescence burst from each fragment was related directly to its length, thus providing a means to size small quantities of kilobase lengths of DNA quickly. Improvements of several orders of magnitude in analysis time and sample size over current gel electrophoresis techniques were realized. Fragments of 17.1,29.9, and 48.5 thousand base pairs were well resolved, and were sized in 164 seconds. Less than one pg of DNA was required for analysis. We have demonstrated sizing of individual, stained DNA fragments with resolution approaching that of gel electrophoresis for moderately large fragments, but with significant reductions in the analysis time and the amount of sample required. Furthermore, system response is linear with DNA fragment length, in contrast to the logarithmic response in gel electrophoresis. There exists the potential to perform this sizing using relatively simple instrumentation, i.e. a continuous wave laser of low power and current mode detection.

  17. Sizing of DNA fragments by flow cytometry

    SciTech Connect

    Johnson, M.E.; Goodwin, P.M.; Ambrose, W.P.; Martin, J.C.; Marrone, B.L.; Jett, J.H.; Keller, R.A.

    1993-01-01

    Individual, stained DNA fragments were sized using a modified flow cytometer with high sensitivity fluorescence detection. The fluorescent intercalating dye ethidium homodimer was used to stain stoichiometrically lambda phage DNA and a Kpn I digest of lambda DNA. Stained, individual fragments of DNA were passed through a low average power, focused, mode-locked laser beam, and the fluorescence from each fragment was collected and quantified. Time-gated detection was used to discriminate against Raman scattering from the water solvent. The fluorescence burst from each fragment was related directly to its length, thus providing a means to size small quantities of kilobase lengths of DNA quickly. Improvements of several orders of magnitude in analysis time and sample size over current gel electrophoresis techniques were realized. Fragments of 17.1,29.9, and 48.5 thousand base pairs were well resolved, and were sized in 164 seconds. Less than one pg of DNA was required for analysis. We have demonstrated sizing of individual, stained DNA fragments with resolution approaching that of gel electrophoresis for moderately large fragments, but with significant reductions in the analysis time and the amount of sample required. Furthermore, system response is linear with DNA fragment length, in contrast to the logarithmic response in gel electrophoresis. There exists the potential to perform this sizing using relatively simple instrumentation, i.e. a continuous wave laser of low power and current mode detection.

  18. 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

  19. Microfluidic fluorescence in situ hybridization and flow cytometryFlowFISH)

    PubMed Central

    Liu, Peng; Meagher, Robert J.; Light, Yooli Kim; Yilmaz, Suzan; Chakraborty, Romy; Arkin, Adam P.; Hazen, Terry C.; Singh, Anup K.

    2011-01-01

    We describe an integrated microfluidic device (µFlowFISH) capable of performing 16S rRNA fluorescence in situ hybridization (FISH) followed by flow cytometric detection for identifying bacteria in natural microbial communities. The device was used for detection of species involved in bioremediation of Cr(VI) and other metals in groundwater samples from a highly-contaminated environmental site (Hanford, WA, USA). The µFlowFISH seamlessly integrates two components: a hybridization chamber formed between two photopolymerized membranes, where cells and probes are electrophoretically loaded, incubated and washed; and a downstream cross structure for electrokinetically focusing cells into a single-file flow for flow cytometry analysis. The device is capable of analyzing a wide variety of bacteria including aerobic, facultative and anaerobic bacteria and was initially tested and validated using cultured microbes, including Escherichia coli, as well as two strains isolated from Hanford site: Desulfovibrio vulgaris strain RCH1, and Pseudomonas sp. strain RCH2 that are involved in Cr(VI) reduction and immobilization. Combined labeling and detection efficiencies of 74–97% were observed in experiments with simple mixtures of cultured cells confirmed specific labeling. Results obtained were in excellent agreement with those obtained by conventional flow cytometry confirming the accuracy of µFlowFISH. Finally, the device was used for analyzing water samples collected on different dates from the Hanford Site. We were able to monitor the numbers of Pseudomonas sp. with only 100–200 cells loaded into the microchip. The µFlowFISH approach provides an automated platform for quantitative detection of microbial cells from complex samples, and is ideally suited for analysis of precious samples with low cell numbers such as those found at extreme environmental niches, bioremediation sites, and the human microbiome. PMID:21755095

  20. CRITICAL ASSESSMENT OF AUTOMATED FLOW CYTOMETRY DATA ANALYSIS TECHNIQUES

    PubMed Central

    Aghaeepour, Nima; Finak, Greg; Hoos, Holger; Mosmann, Tim R.; Gottardo, Raphael; Brinkman, Ryan; Scheuermann, Richard H.

    2013-01-01

    Traditional methods for flow cytometry (FCM) data processing rely on subjective manual gating. Recently, several groups have developed computational methods for identifying cell populations in multidimensional FCM data. The Flow Cytometry: Critical Assessment of Population Identification Methods (FlowCAP) challenges were established to compare the performance of these methods on two tasks – mammalian cell population identification to determine if automated algorithms can reproduce expert manual gating, and sample classification to determine if analysis pipelines can identify characteristics that correlate with external variables (e.g., clinical outcome). This analysis presents the results of the first of these challenges. Several methods performed well compared to manual gating or external variables using statistical performance measures, suggesting that automated methods have reached a sufficient level of maturity and accuracy for reliable use in FCM data analysis. PMID:23396282

  1. Managing Multi-center Flow Cytometry Data for Immune Monitoring.

    PubMed

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

    2014-01-01

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

  2. Managing Multi-center Flow Cytometry Data for Immune Monitoring

    PubMed Central

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

    2014-01-01

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

  3. Flow cytometry applications in the study of immunological lung disorders.

    PubMed

    Mortaz, Esmaeil; Gudarzi, Hoda; Tabarsi, Payam; M Adcock, Ian; Masjedi, Mohamad Reza; Jamaati, Hamid Reza; Garssen, Johan; Velayati, Ali Akbar; A Redegeld, Frank

    2015-02-01

    The use of flow cytometry in the clinical laboratory has grown substantially in the past decade. Flow cytometric analysis provides a rapid qualitative and quantitative description of multiple characteristics of individual cells. For example, it is possible to detect the cell size and granularity, aspects of DNA and RNA content and the presence of cell surface and nuclear markers which are used to characterize the phenotype of single cells. Flow cytometry has been used for the immunophenotyping of a variety of specimens including whole blood, bone marrow, serous cavity fluids, (cerebrospinal fluid) CSF, urine and all types of body fluids. The technique has also been applied to human bronchoalveolar lavage (BAL) fluid, peritoneal fluids and blood. In this review, we describe the current status of the application of flow cytometry as a diagnostic tool in various lung diseases. We focus on the analysis of BAL cell composition in chronic obstructive lung disease (COPD), asthma, lung cancer, sarcoidosis, tuberculosis and idiopathic eosinophilic pneumonia (IEP). PMID:25530134

  4. Laser rastering flow cytometry: fast cell counting and identification

    NASA Astrophysics Data System (ADS)

    Vacca, G.; Junnarkar, M. R.; Goldblatt, N. R.; Yee, M. W.; Van Slyke, B. M.; Briese, T. C.

    2009-02-01

    We describe the concept of laser rastering flow cytometry, where a rapidly scanning laser beam allows counting and classification of cells at much higher rates than currently possible. Modifications to existing flow cytometers to implement the concept include an acousto-optic deflector, fast analog-to-digital conversion, and a two-step digital-signal-processing scheme that handles the high data rates and provides key assay information. Results are shown that prove the concept, demonstrating the ability to resolve closely spaced cells and to measure cells at rates more than an order of magnitude faster than on conventional flow-cytometer-based hematology analyzers.

  5. Fluorescent In Situ Hybridization in Suspension by Imaging Flow Cytometry.

    PubMed

    Maguire, Orla; Wallace, Paul K; Minderman, Hans

    2016-01-01

    The emergence of imaging flow cytometry (IFC) has brought novel applications exploiting its advantages over conventional flow cytometry and microscopy. One of the new applications is fluorescence in situ hybridization in suspension (FISH-IS). Conventional FISH is a slide-based approach in which the spotlike imagery resulting from hybridization with fluorescently tagged probes is evaluated by fluorescence microscopy. The FISH-IS approach evaluated by IFC enables the evaluation of tens to hundreds of thousands of cells in suspension and the analysis can be automated and standardized diminishing operator bias from the analysis. The high cell number throughput of FISH-IS improves the detection of rare events compared to conventional FISH. The applicability of FISH-IS is currently limited to detection of abnormal quantitative differences of hybridization targets such as occur in numerical chromosome abnormalities, deletions and amplifications.Here, we describe a protocol for FISH-IS using chromosome enumeration probes as an example. PMID:27460240

  6. Analyzing Schizosaccharomyces pombe DNA Content by Flow Cytometry.

    PubMed

    Boye, Erik; Anda, Silje; Rothe, Christiane; Stokke, Trond; Grallert, Beáta

    2016-01-01

    Flow cytometry can be used to measure the DNA content of individual cells. The data are usually presented as DNA histograms that can be used to examine the cells' progression through the cell cycle. Under standard growth conditions, fission yeast cells do not complete cytokinesis until after G1 phase; therefore, DNA histograms show one major peak representing cells in G1 (2×1C DNA) and G2 phase (1×2C DNA). By analysis of the duration of the fluorescence signal as well as the intensity of the DNA-related signal, it is possible to discriminate between cells in M/G1, S, and G2 This protocol describes how to prepare cells for flow cytometry and analyze them. We also describe the application of barcoding for more accurate comparison of samples. PMID:27250946

  7. A CLIPS expert system for clinical flow cytometry data analysis

    NASA Technical Reports Server (NTRS)

    Salzman, G. C.; Duque, R. E.; Braylan, R. C.; Stewart, C. C.

    1990-01-01

    An expert system is being developed using CLIPS to assist clinicians in the analysis of multivariate flow cytometry data from cancer patients. Cluster analysis is used to find subpopulations representing various cell types in multiple datasets each consisting of four to five measurements on each of 5000 cells. CLIPS facts are derived from results of the clustering. CLIPS rules are based on the expertise of Drs. Stewart, Duque, and Braylan. The rules incorporate certainty factors based on case histories.

  8. 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.

  9. Sample handling for kinetics and molecular assembly in flow cytometry

    SciTech Connect

    Sklar, L.A. |; Seamer, L.C.; Kuckuck, F.; Prossnitz, E.; Edwards, B.; Posner, G.

    1998-07-01

    Flow cytometry discriminates particle associated fluorescence from the fluorescence of the surrounding medium. It permits assemblies of macromolecular complexes on beads or cells to be detected in real-time with precision and specificity. The authors have investigated two types of robust sample handling systems which provide sub-second resolution and high throughput: (1) mixers which use stepper-motor driven syringes to initiate chemical reactions in msec time frames; and (2) flow injection controllers with valves and automated syringes used in chemical process control. In the former system, the authors used fast valves to overcome the disparity between mixing 100 {micro}ls of sample in 100 msecs and delivering sample to a flow cytometer at 1 {micro}l/sec. Particles were detected within 100 msec after mixing, but turbulence was created which lasted for 1 sec after injection of the sample into the flow cytometer. They used optical criteria to discriminate particles which were out of alignment due to the turbulent flow. Complex sample handling protocols involving multiple mixing steps and sample dilution have also been achieved. With the latter system they were able to automate sample handling and delivery with intervals of a few seconds. The authors used a fluidic approach to defeat turbulence caused by sample introduction. By controlling both sheath and sample with individual syringes, the period of turbulence was reduced to {approximately} 200 msecs. Automated sample handling and sub-second resolution should permit broad analytical and diagnostic applications of flow cytometry.

  10. 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. PMID:27460250

  11. Rapid Cell Population Identification in Flow Cytometry Data*

    PubMed Central

    Aghaeepour, Nima; Nikolic, Radina; Hoos, Holger H.; Brinkman, Ryan R.

    2011-01-01

    We have developed flowMeans, a time-efficient and accurate method for automated identification of cell populations in flow cytometry (FCM) data based on K-means clustering. Unlike traditional K-means, flowMeans can identify concave cell populations by modelling a single population with multiple clusters. flowMeans uses a change point detection algorithm to determine the number of sub-populations, enabling the method to be used in high throughput FCM data analysis pipelines. Our approach compares favourably to manual analysis by human experts and current state-of-the-art automated gating algorithms. flowMeans is freely available as an open source R package through Bioconductor. PMID:21182178

  12. Stochastic Measurement Models for Quantifying Lymphocyte Responses Using Flow Cytometry

    PubMed Central

    Kan, Andrey; Pavlyshyn, Damian; Markham, John F.; Dowling, Mark R.; Heinzel, Susanne; Zhou, Jie H. S.; Marchingo, Julia M.; Hodgkin, Philip D.

    2016-01-01

    Adaptive immune responses are complex dynamic processes whereby B and T cells undergo division and differentiation triggered by pathogenic stimuli. Deregulation of the response can lead to severe consequences for the host organism ranging from immune deficiencies to autoimmunity. Tracking cell division and differentiation by flow cytometry using fluorescent probes is a major method for measuring progression of lymphocyte responses, both in vitro and in vivo. In turn, mathematical modeling of cell numbers derived from such measurements has led to significant biological discoveries, and plays an increasingly important role in lymphocyte research. Fitting an appropriate parameterized model to such data is the goal of these studies but significant challenges are presented by the variability in measurements. This variation results from the sum of experimental noise and intrinsic probabilistic differences in cells and is difficult to characterize analytically. Current model fitting methods adopt different simplifying assumptions to describe the distribution of such measurements and these assumptions have not been tested directly. To help inform the choice and application of appropriate methods of model fitting to such data we studied the errors associated with flow cytometry measurements from a wide variety of experiments. We found that the mean and variance of the noise were related by a power law with an exponent between 1.3 and 1.8 for different datasets. This violated the assumptions inherent to commonly used least squares, linear variance scaling and log-transformation based methods. As a result of these findings we propose a new measurement model that we justify both theoretically, from the maximum entropy standpoint, and empirically using collected data. Our evaluation suggests that the new model can be reliably used for model fitting across a variety of conditions. Our work provides a foundation for modeling measurements in flow cytometry experiments thus

  13. Identification of contact and respiratory sensitizers using flow cytometry

    SciTech Connect

    Goutet, Michele . E-mail: michele.goutet@inrs.fr; Pepin, Elsa; Langonne, Isabelle; Huguet, Nelly; Ban, Masarin

    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 analyses 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.

  14. Stochastic Measurement Models for Quantifying Lymphocyte Responses Using Flow Cytometry.

    PubMed

    Kan, Andrey; Pavlyshyn, Damian; Markham, John F; Dowling, Mark R; Heinzel, Susanne; Zhou, Jie H S; Marchingo, Julia M; Hodgkin, Philip D

    2016-01-01

    Adaptive immune responses are complex dynamic processes whereby B and T cells undergo division and differentiation triggered by pathogenic stimuli. Deregulation of the response can lead to severe consequences for the host organism ranging from immune deficiencies to autoimmunity. Tracking cell division and differentiation by flow cytometry using fluorescent probes is a major method for measuring progression of lymphocyte responses, both in vitro and in vivo. In turn, mathematical modeling of cell numbers derived from such measurements has led to significant biological discoveries, and plays an increasingly important role in lymphocyte research. Fitting an appropriate parameterized model to such data is the goal of these studies but significant challenges are presented by the variability in measurements. This variation results from the sum of experimental noise and intrinsic probabilistic differences in cells and is difficult to characterize analytically. Current model fitting methods adopt different simplifying assumptions to describe the distribution of such measurements and these assumptions have not been tested directly. To help inform the choice and application of appropriate methods of model fitting to such data we studied the errors associated with flow cytometry measurements from a wide variety of experiments. We found that the mean and variance of the noise were related by a power law with an exponent between 1.3 and 1.8 for different datasets. This violated the assumptions inherent to commonly used least squares, linear variance scaling and log-transformation based methods. As a result of these findings we propose a new measurement model that we justify both theoretically, from the maximum entropy standpoint, and empirically using collected data. Our evaluation suggests that the new model can be reliably used for model fitting across a variety of conditions. Our work provides a foundation for modeling measurements in flow cytometry experiments thus

  15. Expert systems for flow cytometry data analysis: A preliminary report

    SciTech Connect

    Salzman, G.C. ); Stewart, C.C. . Lab. of Flow Cytometry); Duque, R.E. )

    1990-01-01

    Flow Cytometry has become an accepted technique in the clinical laboratory for rapid immunophenotyping of patient blood samples. Multiple, fluorescent labeled monoclonal antibodies are used to tag the cells, which are then analyzed one at a time at rates of several thousand cells a second. Patient samples are processed through the flow cytometer at more than one a minute. Clinicians are being overwhelmed by the large amount of data that must be analyzed to provide the information needed to assist in disease diagnosis. An expert system is being developed to assist clinicians in analyzing this multivariate flow cytometry data. The data from each sample are processed by a clustering algorithm, which finds the means of the distinct cell subpopulations in a sample. These mean values of fluorescence are translated into words such as negative,'' dim'' and bright'' and the words are combined into patterns that are matched against the premises on the left hand side of the rules used to identify the disease categories. This is a report of work in progress. 13 refs., 4 figs.

  16. Measurement of microvesicle levels in human blood using flow cytometry.

    PubMed

    Chandler, Wayne L

    2016-07-01

    Microvesicles are fragments of cells released when the cells are activated, injured, or apoptotic. Analysis of microvesicle levels in blood has the potential to shed new light on the pathophysiology of many diseases. Flow cytometry is currently the only method that can simultaneously separate true lipid microvesicles from other microparticles in blood, determine the cell of origin and other microvesicle characteristics, and handle large numbers of clinical samples with a reasonable effort, but expanded use of flow cytometric measurement of microvesicle levels as a clinical and research tool requires improved, standardized assays. The goal of this review is to aid investigators in applying current best practices to microvesicle measurements. First pre-analytical factors are evaluated and data summarized for anticoagulant effects, sample transport and centrifugation. Next flow cytometer optimization is reviewed including interference from background in buffers and reagents, accurate microvesicle counting, swarm interference, and other types of coincidence errors, size calibration, and detection limits using light scattering, impedance and fluorescence. Finally current progress on method standardization is discussed and a summary of current best practices provided. © 2016 Clinical Cytometry Society. PMID:26606416

  17. Diagnostic Utility of Flow Cytometry in Myelodysplastic Syndromes

    PubMed Central

    Aanei, Carmen Mariana; Picot, Tiphanie; Tavernier, Emmanuelle; Guyotat, Denis; Campos Catafal, Lydia

    2016-01-01

    Myelodysplastic syndromes (MDSs) are clonal disorders of hematopoiesis that exhibit heterogeneous clinical presentation and morphological findings, which complicates diagnosis, especially in early stages. Recently, refined definitions and standards in the diagnosis and treatment of MDS were proposed, but numerous questions remain. Multiparameter flow cytometry (MFC) is a helpful tool for the diagnostic workup of patients with suspected MDS, and various scores using MFC data have been developed. However, none of these methods have achieved the sensitivity that is required for a reassuring diagnosis in the absence of morphological abnormalities. One reason may be that each score evaluates one or two lineages without offering a broad view of the dysplastic process. The combination of two scores (e.g., Ogata and Red Score) improved the sensitivity from 50–60 to 88%, but the positive (PPV) and negative predictive values (NPV) must be improved. There are prominent differences between study groups when these scores are tested. Further research is needed to maximize the sensitivity of flow cytometric analysis in MDS. This review focuses on the application of flow cytometry for MDS diagnosis and discusses the advantages and limitations of different approaches. PMID:27446807

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

    PubMed

    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 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. PMID:23603893

  19. 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

  20. Defining human dendritic cell progenitors by multiparametric flow cytometry.

    PubMed

    Breton, Gaëlle; Lee, Jaeyop; Liu, Kang; Nussenzweig, Michel C

    2015-09-01

    Human dendritic cells (DCs) develop from progressively restricted bone marrow (BM) progenitors: these progenitor cells include granulocyte, monocyte and DC progenitor (GMDP) cells; monocyte and DC progenitor (MDP) cells; and common DC progenitor (CDP) and DC precursor (pre-DC) cells. These four DC progenitors can be defined on the basis of the expression of surface markers such as CD34 and hematopoietin receptors. In this protocol, we describe five multiparametric flow cytometry panels that can be used as a tool (i) to simultaneously detect or phenotype the four DC progenitors, (ii) to isolate DC progenitors to enable in vitro differentiation or (iii) to assess the in vitro differentiation and proliferation of DC progenitors. The entire procedure from isolation of cells to flow cytometry can be completed in 3-7 h. This protocol provides optimized antibody panels, as well as gating strategies, for immunostaining of BM and cord blood specimens to study human DC hematopoiesis in health, disease and vaccine settings. PMID:26292072

  1. Defining human dendritic cell progenitors by multiparametric flow cytometry

    PubMed Central

    Breton, Gaëlle; Lee, Jaeyop; Liu, Kang; Nussenzweig, Michel C

    2015-01-01

    Human dendritic cells (DCs) develop from progressively restricted bone marrow (BM) progenitors: these progenitor cells include granulocyte, monocyte and DC progenitor (GMDP) cells; monocyte and DC progenitor (MDP) cells; and common DC progenitor (CDP) and DC precursor (pre-DC) cells. These four DC progenitors can be defined on the basis of the expression of surface markers such as CD34 and hematopoietin receptors. In this protocol, we describe five multiparametric flow cytometry panels that can be used as a tool (i) to simultaneously detect or phenotype the four DC progenitors, (ii) to isolate DC progenitors to enable in vitro differentiation or (iii) to assess the in vitro differentiation and proliferation of DC progenitors. The entire procedure from isolation of cells to flow cytometry can be completed in 3–7 h. This protocol provides optimized antibody panels, as well as gating strategies, for immunostaining of BM and cord blood specimens to study human DC hematopoiesis in health, disease and vaccine settings. PMID:26292072

  2. Technical issues: flow cytometry and rare event analysis.

    PubMed

    Hedley, B D; Keeney, M

    2013-06-01

    Flow cytometry has become an essential tool for identification and characterization of hematological cancers and now, due to technological improvements, allows the identification and rapid enumeration of small tumor populations that may be present after induction therapy (minimal residual disease, MRD). The quantitation of MRD has been shown to correlate with relapse and survival rates in numerous diseases and in certain cases, and evidence of MRD is used to alter treatment protocols. Recent improvements in hardware allow for high data rate collection. Improved fluorochromes take advantage of violet laser excitation and maximize signal-to-noise ratio allowing the population of interest to be isolated in multiparameter space. This isolation, together with a low background rate, permits for detection of residual tumor populations in a background of normal cells. When counting such rare events, the distribution is governed by Poisson statistics, with precision increasing with higher numbers of cells collected. In several hematological malignancies, identification of populations at frequencies of 0.01% and lower has been attained. The choice of antibodies used in MRD detection facilitates the definition of a fingerprint to identify abnormal populations throughout treatment. Tumor populations can change phenotype, and an approach that relies on 'different from normal' has proven useful, particularly in the acute leukemias. Flow cytometry can and is used for detection of MRD in many hematological diseases; however, standardized approaches for specific diseases must be developed to ensure precise identification and enumeration that may alter the course of patient treatment. PMID:23590661

  3. Measurement and Characterization of Apoptosis by Flow Cytometry.

    PubMed

    Telford, William; Tamul, Karen; Bradford, Jolene

    2016-01-01

    Apoptosis is an important mechanism in cell biology, playing a critical regulatory role in virtually every organ system. It has been particularly well characterized in the immune system, with roles ranging from immature immune cell development and selection to down-regulation of the mature immune response. Apoptosis is also the primary mechanism of action of anti-cancer drugs. Flow cytometry has been the method of choice for analyzing apoptosis in suspension cells for more than 25 years. Numerous assays have been devised to measure both the earliest and latest steps in the apoptotic process, from the earliest signal-transduction events to the late morphological changes in cell shape and granularity, proteolysis, and chromatin condensation. These assays are particularly powerful when combined into multicolor assays determining several apoptotic characteristics simultaneously. The multiparametric nature of flow cytometry makes this technology particularly suited to measuring apoptosis. In this unit, we will describe the four main techniques for analyzing caspase activity in apoptotic cells, combined with annexin V and cell permeability analysis. These relatively simple multiparametric assays are powerful techniques for assessing cell death. © 2016 by John Wiley & Sons, Inc. PMID:27367289

  4. Functional Analysis of Human NK cells by Flow Cytometry

    PubMed Central

    Bryceson, Yenan T.; Fauriat, Cyril; Nunes, João M.; Wood, Stephanie M.; Björkström, Niklas K.; Long, Eric O.; Ljunggren, Hans-Gustaf

    2016-01-01

    Natural killer (NK) cells are a subset of lymphocytes that contribute to innate immunity through cytokine secretion and target cell lysis. NK cell function is regulated by a multiplicity of activating and inhibitory receptors. The advance in instrumentation for multi-color flow cytometry and the generation of specific mAbs for different epitopes related to phenotypic and functional parameters have facilitated our understanding of NK cell responses. Here, we provide protocols for flow cytometric evaluation of degranulation and cytokine production by human NK cells from peripheral blood at the single cell level. In addition to offering insight into the regulation of human NK cell responses, these techniques are applicable to the assessment of various clinical conditions, including the diagnosis of immunodeficiency syndromes. PMID:20033652

  5. Digital Analysis and Sorting of Fluorescence Lifetime by Flow Cytometry

    PubMed Central

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

    2010-01-01

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

  6. 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

  7. How validated receptor occupancy flow cytometry assays can impact decisions and support drug development.

    PubMed

    Moulard, Maxime; Ozoux, Marie-Laure

    2016-03-01

    Because of the pressure of significant attrition in drug development, demonstration of target engagement after drug administration enables dose and regimen optimization, patient selection, and stratification from the earliest stages of drug development. The determination of receptor occupancy (RO) can support these efforts. Flow cytometry is one of the preferred technologies to be used based on the important advances in the technology over the last years enabling the simultaneous determination on target cells, of multi intra or surface cell parameters with adequate precision in a regulated environment. Nevertheless, compared to other platforms using the same antigen-antibody binding concept, the flow cytometry approach has faced several challenges, not only due to the technology per se and the diversity of receptor occupancy approaches, but also related to the nature of the matrix where the determination is performed. To illustrate these points, three case studies (antibody-drug conjugate and naked antibody) are provided here to highlight the importance of the choice of the right antibody pair to measure both receptor density (RD) and occupancy by the drug on cancer cells in blood and in bone marrow and the possibility to circumvent the lack of a critical reagent with an innovative approach. In addition, the use of RO data to determine the minimum anticipated biological effect level (MABEL) with translational data from preclinical to human studies, selection of starting dose for the first in man study will be discussed. © 2015 International Clinical Cytometry Society. PMID:26332593

  8. 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...

  9. Analysis of High-Throughput Flow Cytometry Data Using plateCore

    PubMed Central

    Strain, Errol; Hahne, Florian; Brinkman, Ryan R.; Haaland, Perry

    2009-01-01

    Flow cytometry (FCM) software packages from R/Bioconductor, such as flowCore and flowViz, serve as an open platform for development of new analysis tools and methods. We created plateCore, a new package that extends the functionality in these core packages to enable automated negative control-based gating and make the processing and analysis of plate-based data sets from high-throughput FCM screening experiments easier. plateCore was used to analyze data from a BD FACS CAP screening experiment where five Peripheral Blood Mononucleocyte Cell (PBMC) samples were assayed for 189 different human cell surface markers. This same data set was also manually analyzed by a cytometry expert using the FlowJo data analysis software package (TreeStar, USA). We show that the expression values for markers characterized using the automated approach in plateCore are in good agreement with those from FlowJo, and that using plateCore allows for more reproducible analyses of FCM screening data. PMID:19956418

  10. 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.

  11. Control of continuous polyhydroxybutyrate synthesis using calorimetry and flow cytometry.

    PubMed

    Maskow, Thomas; Müller, Susann; Lösche, Andreas; Harms, Hauke; Kemp, Richard

    2006-02-20

    The substrate-carbon flow can be controlled in continuous bioreactor cultures by the medium composition, for example, by the C/N ratio. The carbon distribution is optimal when a maximum fraction flows into the desired product and the residual is just sufficient to compensate for the dilution of the microbial catalyst. Undershooting of the latter condition is reflected immediately by changes in the Gibbs energy dissipation and cellular states. Two calorimetric measurement principles were applied to optimize the continuous synthesis of polyhydroxybutyrate (PHB) by Variovorax paradoxus DSM4065 during growth with constantly increasing supply rates of fructose or toxic phenol. Firstly, the changed slope of the heat production rate in a complete heat balanced bioreactor (CHB) indicated optimum carbon channeling into PHB. The extent of the alteration depended directly on the toxic properties of the substrate. Secondly, a flow through calorimeter was connected with the bioreactor as a "measurement loop." The optimum substrate carbon distribution was indicated by a sudden change in the heat production rate independent of substrate toxicity. The sudden change was explained mathematically and exploited for the long-term control of phenol conversion into PHB. LASER flow cytometry measurements distinguished between subpopulations with completely different PHB-content. Populations grown on fructose preserved a constant ratio of two subpopulations with double and quadruple sets of DNA. Cells grown on phenol comprised a third subpopulation with a single DNA set. Rising phenol concentrations caused this subpopulation to increase. It may thus be considered as an indicator of chemostress. PMID:16245347

  12. Detection of Apoptotic Versus Autophagic Cell Death by Flow Cytometry.

    PubMed

    Sica, Valentina; Maiuri, M Chiara; Kroemer, Guido; Galluzzi, Lorenzo

    2016-01-01

    Different modes of regulated cell death (RCD) can be initiated by distinct molecular machineries and their morphological manifestations can be difficult to discriminate. Moreover, cells responding to stress often activate an adaptive response centered around autophagy, and whether such a response is cytoprotective or cytotoxic cannot be predicted based on morphological parameters only. Molecular definitions are therefore important to understand various RCD subroutines from a mechanistic perspective. In vitro, various forms of RCD including apoptosis and autophagic cell death can be easily discriminated from each other with assays that involve chemical or pharmacological interventions targeting key components of either pathway. Here, we detail a straightforward method to discriminate apoptosis from autophagic cell death by flow cytometry, based on the broad-spectrum caspase inhibitor Z-VAD-fmk and the genetic inhibition of ATG5. PMID:27108427

  13. Recent applications of flow cytometry in aquatic microbial ecology.

    PubMed

    Troussellier, M; Courties, C; Vaquer, A

    1993-01-01

    Microorganisms (unicellular algae, bacteria) constitute fundamental compartments of aquatic ecosystems because of their high concentrations and activities. The evaluation and understanding of their behavior and role raise different problems for which traditional methodologies are often inadequate, whether they refer to global or classical microscopic analyses. Flow cytometry (FCM) has been recently used to study microorganisms in aquatic environments. Although this technology is still applied on a limited scale in our field, a large number of works has been done showing that FCM seems to be a promising tool for aquatic microbial ecology. This paper summarizes, from the literature produced during the last decade and with original data obtained in our laboratory, the main questions related to the cell identification, the evaluation of cell viability, biomasses and productions and the measurements of bacterial and phytoplanktonic activities. The representatives of sampling and observation scales is also discussed within the framework of the FCM measurements. PMID:8220221

  14. Extended cavity laser enhanced two-photon flow cytometry

    PubMed Central

    Tkaczyk, Eric R.; Tkaczyk, Alan H.; Katnik, Steve; Ye, Jing Yong; Luker, Kathryn E.; Luker, Gary D.; Myc, Andrzej; Baker, James R.; Norris, Theodore B.

    2015-01-01

    We demonstrate enhanced sensitivity in two-photon flow cytometry with an extended cavity laser excitation source. At low power, the home-built 20-MHz oscillator was able to detect a significantly larger fraction, in either phosphate buffered saline (PBS) or whole blood, of green fluorescent protein (GFP) –expressing MCA-207 cells cross-labeled with the membrane-binding lipophilic dye DiD. A geometrical model is used to explain unique features of the signals resulting from the different spatial distribution of DiD and GFP. These unique features include sub-square law scaling of unsaturated two-photon signal, a sigmoidal sensitivity curve for detection under varying powers for cell detection thresholds as low as a single photon, and uncorrelated signal strengths in two detection channels. PMID:19021327

  15. Analysis of receptor tyrosine kinase internalization using flow cytometry.

    PubMed

    Li, Ning; Hill, Kristen S; Elferink, Lisa A

    2008-01-01

    The internalization of activated receptor tyrosine kinases (RTKs) by endocytosis and their subsequent down regulation in lysosomes plays a critical role in regulating the duration and intensity of downstream signaling events. Uncoupling of the RTK cMet from ligand-induced degradation was recently shown to correlate with sustained receptor signaling and increased cell tumorigenicity, suggesting that the corruption of these endocytic mechanisms could contribute to increased cMet signaling in metastatic cancers. To understand how cMet signaling for normal cell growth is controlled by endocytosis and how these mechanisms are dysregulated in metastatic cancers, we developed flow cytometry-based assays to examine cMet internalization. PMID:19066037

  16. Applications of Flow Cytometry to Characterize Bacterial Physiological Responses

    PubMed Central

    Contreras-Garduño, Jorge A.; Pedraza-Reyes, Mario

    2014-01-01

    Although reports of flow cytometry (FCM) applied to bacterial analysis are increasing, studies of FCM related to human cells still vastly outnumber other reports. However, current advances in FCM combined with a new generation of cellular reporter probes have made this technique suitable for analyzing physiological responses in bacteria. We review how FCM has been applied to characterize distinct physiological conditions in bacteria including responses to antibiotics and other cytotoxic chemicals and physical factors, pathogen-host interactions, cell differentiation during biofilm formation, and the mechanisms governing development pathways such as sporulation. Since FCM is suitable for performing studies at the single-cell level, we describe how this powerful technique has yielded invaluable information about the heterogeneous distribution of differently and even specialized responding cells and how it may help to provide insights about how cell interaction takes place in complex structures, such as those that prevail in bacterial biofilms. PMID:25276788

  17. Bead-Based Assays for Biodetection: From Flow-Cytometry to Microfluidics

    SciTech Connect

    Ozanich, Richard M.; Antolick, Kathryn C.; Bruckner-Lea, Cindy J.; Bunch, Kyle J.; Dockendorff, Brian P.; Grate, Jay W.; Nash, Michael A.; Tyler, Abby J.

    2009-05-04

    ABSTRACT The potential for the use of biological agents by terrorists is a real threat. Two approaches for detection of biological species will be described: 1) The use of microbead arrays for multiplexed flow cytometry detection of cytokines and botulinum neurotoxin simulant, and 2) a microfluidic platform for capture and separation of different size superparamagnetic nanoparticles followed by on-chip fluorescence detection of the sandwich complex. The methods and automated fluidic systems used for trapping functionalized microbeads will be described. This approach allows sample, assay reagents, and wash solutions to be perfused over a micro-column of beads, resulting in faster and more sensitive assays. The automated fluidic approach resulted in up to five-fold improvements in assay sensitivity/speed as compared to identical assays performed in a typical manual batch mode. A second approach for implementing multiplexed bead-based assays without using flow cytometry detection is currently under development. The goal of the microfluidic-based approach is to achieve rapid (<20 minutes), multiplexed (> 3 bioagents) detection using a simple and low-cost, integrated microfluidic/optical detection platform. Using fiber-optic guided laser-induced fluorescence, assay detection limits were shown to be in the 100’s of picomolar range (10’s of micrograms per liter) for botulinum neurotoxin simulant without any optimization of the microfluidic device or optical detection approach. Video taping magnetic nanoparticle capture and release was used to improve understanding of the process and revealed interesting behavior.

  18. Bead-based assays for biodetection: from flow-cytometry to microfluidics

    NASA Astrophysics Data System (ADS)

    Ozanich, Richard M., Jr.; Antolick, Kathryn; Bruckner-Lea, Cynthia J.; Bunch, Kyle J.; Dockendorff, Brian P.; Grate, Jay W.; Nash, Michael A.; Tyler, Abby; Warner, Cynthia L.; Warner, Marvin G.

    2009-05-01

    The potential for the use of biological agents by terrorists is a real threat. Two approaches for antibody-based detection of biological species are described in this paper: 1) The use of microbead arrays for multiplexed flow cytometry detection of cytokines and botulinum neurotoxin simulant, and 2) a microfluidic platform for capture and separation of different size superparamagnetic nanoparticles followed by on-chip fluorescence detection of the sandwich complex. These approaches both involve the use of automated fluidic systems for trapping antibody-functionalized microbeads, which allows sample, assay reagents, and wash solutions to be perfused over a micro-column of beads, resulting in faster and more sensitive immunoassays. The automated fluidic approach resulted in up to five-fold improvements in immunoassay sensitivity/speed as compared to identical immunoassays performed in a typical manual batch mode. A second approach for implementing multiplexed bead-based immunoassays without using flow cytometry detection is currently under development. The goal of the microfluidic-based approach is to achieve rapid (<20 minutes), multiplexed (>= 3 bioagents) detection using a simple and low-cost, integrated microfluidic/optical detection platform. Using fiber-optic guided laser-induced fluorescence, assay detection limits were shown to be in the 100's of picomolar range (10's of micrograms per liter) for botulinum neurotoxin simulant without any optimization of the microfluidic device or optical detection approach.

  19. RchyOptimyx: Cellular Hierarchy Optimization for Flow Cytometry

    PubMed Central

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

    2013-01-01

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

  20. Use of CCD sensors in flow cytometry for nonimaging applications

    NASA Astrophysics Data System (ADS)

    Beisker, Wolfgang

    1997-05-01

    The use of charge coupled devices (CCDs) as non-imaging sensors in flow cytometric systems to replace the classical photomultplier tubes (PMTs) is very advantageous: the quantum efficiency of the CCDs is about 5 to 10 times higher as for PMTs, the charge storage capability of CCDs avoids analogue processing of the fluorescence signals, the dynamic range is up to 18 bits and the fluorescence intensity at different wavelengths can be recorded on the same chip. In this report a full frame CCD imager is used in a thermoelectrically cooled environment. The output signal for the CCD is digitized with a 12-bit ADC and the data are sorted as list-mode data typically used in flow cytometric work. The performance of the system is demonstrated with DNA staining of mammalian cells with acridine-orange, propidium iodide and ethidium bromide. DNA histograms comparable with standard flow cytometry are recorded. From the same data set pulse-widths histograms can be processed and used for doublet discrimination. The high quantum efficiency of the CCD sensors is of special interest for fluorescing dyes in the dark red or near IR wavelength range.

  1. Dielectrophoresis switching with vertical sidewall electrodes for microfluidic flow cytometry

    PubMed Central

    Flanagan, Lisa A.; Jeon, Noo Li; Monuki, Edwin; Lee, Abraham P.

    2012-01-01

    A novel dielectrophoresis switching with vertical electrodes in the sidewall of microchannels for multiplexed switching of objects has been designed, fabricated and tested. With appropriate electrode design, lateral DEP force can be generated so that one can dynamically position particulates along the width of the channel. A set of interdigitated electrodes in the sidewall of the microchannels is used for the generation of non-uniform electrical fields to generate negative DEP forces that repel beads/cells from the sidewalls. A countering DEP force is generated from another set of electrodes patterned on the opposing sidewall. These lateral negative DEP forces can be adjusted by the voltage and frequency applied. By manipulating the coupled DEP forces, the particles flowing through the microchannel can be positioned at different equilibrium points along the width direction and continue to flow into different outlet channels. Experimental results for switching biological cells and polystyrene microbeads to multiple outlets (up to 5) have been achieved. This novel particle switching technique can be integrated with other particle detection components to enable microfluidic flow cytometry systems. PMID:17713608

  2. Digital analysis and sorting of fluorescence lifetime by flow cytometry.

    PubMed

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

    2010-09-01

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

  3. Flow cytometry for the diagnosis of autoimmune thrombocytopenia.

    PubMed

    Tomer, Aaron

    2006-03-01

    Autoimmune thrombocytopenia is a disorder characterized by antibody-mediated accelerated platelet destruction. Despite its clinical importance, the diagnosis of autoimmune thrombocytopenia is one of exclusion, thus inevitably associated with potential difficulties. Current clinically applicable methods used to determine antigen-specific antibodies, primarily directed to GPIIb/IIIa (CD41a) and GPIb (CD42b), include the monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay and the radioactive immunobead assay. Neither of these assays is commonly used by clinical laboratories, however, because of methodologic and practical limitations. As a result, diagnoses are generally based on clinical impression despite patient presentations that are sometimes complex. To overcome some of these difficulties, flow cytometric techniques have been developed, employing standard methods and equipment suitable for testing a single sample or multiple samples, as may occur in cases of autoimmune thrombocytopenia. The availability of a feasible technique such as flow cytometry, with improved sensitivity and specificity, should facilitate the routine use of a diagnostic method in the evaluation of thrombo-cytopenic patients suspected of having an autoimmune disorder and permit follow-up to determine immune remission. PMID:16537048

  4. [Detection of anti-granulocyte antibodies using flow cytometry].

    PubMed

    Kumagai, T; Furihata, K; Misawa, K; Aoki, M; Kameko, M; Ichikawa, T; Okubo, Y; Ito, S; Ohto, H

    1994-03-01

    To detect anti-granulocyte antibodies (AGAs) in the sera of granulocytopenic patients is important to study the mechanism of the disease. In this report, we studied neutrophil associated immunoglobulin (NAIg) and neutrophil binding immunoglobulin (NBIg) in patients' sera using flow cytometry (FCM). We investigated the interference of circulating immune complexes (CIC) on measuring the NAIg and NBIg. No apparent effect of CIC was observed at concentrations up to 140 micrograms/ml. NAIg and NBIg were semiquantitated by determining the relative fluorescence intensity (RFI) on a flow cytometer and the normal ranges of NAIg and NBIg were less than 15 RFI and less than 10 RFI, respectively. Of 100 sera from patients with neutropenia, 5 were positive for NBIg and 3 of them were positive for granulocyte-specific antibodies. One serum of a patient with benign chronic neutropenia showed clear specificity for NA1 alloantigen but the other 4 AGAs were not specific for NA alloantigen system. Our NAIg, NBIg screening procedure, including NA specificity testing of NBIg and detection of reactivity with normal lymphocytes using FCM, is simple and useful for measuring and studying serological and immunological characteristics of AGAs. PMID:8152165

  5. Evaluation of a green laser pointer for flow cytometry.

    PubMed

    Habbersett, Robert C; Naivar, Mark A; Woods, Travis A; Goddard, Gregory R; Graves, Steven W

    2007-10-01

    Flow cytometers typically incorporate expensive lasers with high-quality (TEM00) output beam structure and very stable output power, significantly increasing system cost and power requirements. Red diode lasers minimize power consumption and cost, but limit fluorophore selection. Low-cost DPSS laser pointer modules could possibly offer increased wavelength selection but presumed emission instability has limited their use. A $160 DPSS 532 nm laser pointer module was first evaluated for noise characteristics and then used as the excitation light source in a custom-built flow cytometer for the analysis of fluorescent calibration and alignment microspheres. Eight of ten modules tested were very quiet (RMS noise < or = 0.6% between 0 and 5 MHz). With a quiet laser pointer module as the light source in a slow-flow system, fluorescence measurements from alignment microspheres produced CVs of about 3.3%. Furthermore, the use of extended transit times and < or =1 mW of laser power produced both baseline resolution of all 8 peaks in a set of Rainbow microspheres, and a detection limit of <20 phycoerythrin molecules per particle. Data collected with the transit time reduced to 25 micros (in the same instrument but at 2.4 mW laser output) demonstrated a detection limit of approximately 75 phycoerythrin molecules and CVs of about 2.7%. The performance, cost, size, and power consumption of the tested laser pointer module suggests that it may be suitable for use in conventional flow cytometry, particularly if it were coupled with cytometers that support extended transit times. PMID:17712796

  6. Characterization, detection, and counting of metal nanoparticles using flow cytometry.

    PubMed

    Zucker, Robert M; Ortenzio, Jayna N R; Boyes, William K

    2016-02-01

    There is a need to accurately detect, characterize, and quantify nanoparticles in suspensions. This study helps to understand the complex interactions between similar types of nanoparticles. Before initiating a study of metal nanoparticles, five submicron PS beads with sizes between 200 nm and 1 µm were used to derive a reference scale that was useful in evaluating the flow cytometer for functionality, sensitivity, resolution, and reproducibility. Side scatter intensity (SSC) from metal nanoparticles was obtained simultaneously from 405 nm and 488 nm lasers. The 405 nm laser generally yielded histogram distributions with smaller CVs, less side scatter intensity, better separation indices between beads and decreased scatter differences between different sized particles compared with the 488 nm laser. Submicron particles must be diluted to 10(6) and 10(7) particles/mL before flow cytometer analysis to avoid coincidence counting artifacts. When particles were too concentrated the following occurred: swarm, electronic overload, coincidence counting, activation of doublet discrimination and rejection circuitry, increase of mean SSC histogram distributions, alterations of SSC and pulse width histogram shape, decrease and fluctuations in counting rate and decrease or elimination of particulate water noise and 1 µm reference bead. To insure that the concentrations were in the proper counting range, the nanoparticle samples were mixed with a known concentration of 1µm counting beads. Sequential dilutions of metal nanoparticles in a 1 µm counting bead suspension helped determine the diluted concentration needed for flow cytometer analysis. It was found that the original concentrated nanoparticle samples had to be diluted, between 1:10,000 and 1:100,000, before characterization by flow cytometry. The concentration of silver or gold nanoparticles in the undiluted sample were determined by comparing them with a known concentration (1.9 × 10(6) beads/mL) of 1 µm

  7. Setting objective thresholds for rare event detection in flow cytometry.

    PubMed

    Richards, Adam J; Staats, Janet; Enzor, Jennifer; McKinnon, Katherine; Frelinger, Jacob; Denny, Thomas N; Weinhold, Kent J; Chan, Cliburn

    2014-07-01

    The accurate identification of rare antigen-specific cytokine positive cells from peripheral blood mononuclear cells (PBMC) after antigenic stimulation in an intracellular staining (ICS) flow cytometry assay is challenging, as cytokine positive events may be fairly diffusely distributed and lack an obvious separation from the negative population. Traditionally, the approach by flow operators has been to manually set a positivity threshold to partition events into cytokine-positive and cytokine-negative. This approach suffers from subjectivity and inconsistency across different flow operators. The use of statistical clustering methods does not remove the need to find an objective threshold between between positive and negative events since consistent identification of rare event subsets is highly challenging for automated algorithms, especially when there is distributional overlap between the positive and negative events ("smear"). We present a new approach, based on the Fβ measure, that is similar to manual thresholding in providing a hard cutoff, but has the advantage of being determined objectively. The performance of this algorithm is compared with results obtained by expert visual gating. Several ICS data sets from the External Quality Assurance Program Oversight Laboratory (EQAPOL) proficiency program were used to make the comparisons. We first show that visually determined thresholds are difficult to reproduce and pose a problem when comparing results across operators or laboratories, as well as problems that occur with the use of commonly employed clustering algorithms. In contrast, a single parameterization for the Fβ method performs consistently across different centers, samples, and instruments because it optimizes the precision/recall tradeoff by using both negative and positive controls. PMID:24727143

  8. Comparative analysis of whole blood lysis methods for flow cytometry.

    PubMed

    Bossuyt, X; Marti, G E; Fleisher, T A

    1997-06-15

    We performed a parallel evaluation of six whole blood lysis methods comparing light scatter and quantitative fluorescence intensity based on quantitative flow cytometry, of selected lymphocyte subsets and CD34+ cells. Leukocytes prepared with FACS Lysing Solution (BDIS), Immunolyse (Coulter) and Optilyse B (Immunotech) consistently gave lower forward scatter values than those prepared with ACK (BioWhitaker), Ortho-mune (Ortho) and ImmunoPrep (Coulter). Debris, defined as CD45 negative events with the threshold off, accounted approximately 80% of all events with ACK and Ortho-mune. The other lysing methods consistently yielded less debris (approximately 50%) with Immunolyse generating only approximately 16% debris. Optilyse and FACS lyse consistently displayed the lowest percentage of lymphoid cells (CD45+/CD14-) in the three part differential. The percentage of CD3+, CD20+, CD5+, and CD16/CD56+ cells was consistent with all methods but CD4 and CD8 determinants showed inconsistent variation with ACK and Ortho-mune. In addition, the fluorescence intensity of CD14 PE and CD8 PE staining was markedly decreased on cells prepared with ImmunoPrep. Finally, the clearest separation of CD34+ cells was observed with ACK and Ortho-mune. Our data demonstrate that the method used for red cell lysis can have definite impact on immunophenotyping and selected methods appear to be more suitable for specific applications. PMID:9222098

  9. Monitoring circulating apoptotic cells by in-vivo flow cytometry

    NASA Astrophysics Data System (ADS)

    Wei, Xunbin; Tan, Yuan; Chen, Yun; Zhang, Li; Li, Yan; Liu, Guangda; Wu, Bin; Wang, Chen

    2008-02-01

    Chemotherapies currently constitute one main venue of cancer treatment. For a large number of adult and elderly patients, however, treatment options are poor. These patients may suffer from disease that is resistant to conventional chemotherapy or may not be candidates for curative therapies because of advanced age or poor medical conditions. To control disease in these patients, new therapies must be developed that are selectively targeted to unique characteristics of tumor cell growth and metastasis. A reliable early evaluation and prediction of response to the chemotherapy is critical to its success. Chemotherapies induce apoptosis in tumor cells and a portion of such apoptotic cancer cells may be present in the circulation. However, the fate of circulating tumor cells is difficult to assess with conventional methods that require blood sampling. We report the in situ measurement of circulating apoptotic cells in live animals using in vivo flow cytometry, a novel method that enables real-time detection and quantification of circulating cells without blood extraction. Apoptotic cells are rapidly cleared from the circulation with a half-life of ~10 minutes. Real-time monitoring of circulating apoptotic cells can be useful for detecting early changes in disease processes, as well as for monitoring response to therapeutic intervention.

  10. 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.

  11. 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.

  12. Tracking Immune Cell Proliferation and Cytotoxic Potential Using Flow Cytometry

    PubMed Central

    Tario, Joseph D.; Muirhead, Katharine A.; Pan, Dalin; Munson, Mark E.; Wallace, Paul K.

    2015-01-01

    In the second edition of this series, we described the use of cell tracking dyes in combination with tetramer reagents and traditional phenotyping protocols to monitor levels of proliferation and cytokine production in antigen-specific CD8+ T cells. In particular, we illustrated how tracking dye fluorescence profiles could be used to ascertain the precursor frequencies of different subsets in the T-cell pool that are able to bind tetramer, synthesize cytokines, undergo antigen-driven proliferation, and/or carry out various combinations of these functional responses. Analysis of antigen-specific proliferative responses represents just one of many functions that can be monitored using cell tracking dyes and flow cytometry. In this third edition, we address issues to be considered when combining two different tracking dyes with other phenotypic and viability probes for the assessment of cytotoxic effector activity and regulatory T-cell functions. We summarize key characteristics of and differences between general protein- and membrane-labeling dyes, discuss determination of optimal staining concentrations, and provide detailed labeling protocols for both dye types. Examples of the advantages of two-color cell tracking are provided in the form of protocols for (a) independent enumeration of viable effector and target cells in a direct cytotoxicity assay and (b) simultaneous monitoring of proliferative responses in effector and regulatory T cells. PMID:21116982

  13. Flow cytometry reliability analysis and variations in sugarcane DNA content.

    PubMed

    Oliveira, A C L; Pasqual, M; Bruzi, A T; Pio, L A S; Mendonça, P M S; Soares, J D R

    2015-01-01

    The aim of this study was to evaluate the reliability of flow cytometry analysis and the use of this technique to differentiate species and varieties of sugarcane (Saccharum spp) according to their relative DNA content. We analyzed 16 varieties and three species belonging to this genus. To determine a reliable protocol, we evaluated three extraction buffers (LB01, Marie, and Tris·MgCl2), the presence and absence of RNase, six doses of propidium iodide (10, 15, 20, 25, and 30 μg), four periods of exposure to propidium iodide (0, 5, 10, and 20 min), and seven external reference standards (peas, beans, corn, radish, rye, soybean, and tomato) with reference to the coefficient of variation and the DNA content. For statistical analyses, we used the programs Sisvar(®) and Xlstat(®). We recommend using the Marie extraction buffer and at least 15 μg propidium iodide. The samples should not be analyzed immediately after the addition of propidium iodide. The use of RNase is optional, and tomato should be used as an external reference standard. The results show that sugarcane has a variable genome size (8.42 to 12.12 pg/2C) and the individuals analyzed could be separated into four groups according to their DNA content with relative equality in the genome sizes of the commercial varieties. PMID:26125928

  14. 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

  15. Homogeneous agglutination assay based on micro-chip sheathless flow cytometry.

    PubMed

    Ma, Zengshuai; Zhang, Pan; Cheng, Yinuo; Xie, Shuai; Zhang, Shuai; Ye, Xiongying

    2015-11-01

    Homogeneous assays possess important advantages that no washing or physical separation is required, contributing to robust protocols and easy implementation which ensures potential point-of-care applications. Optimizing the detection strategy to reduce the number of reagents used and simplify the detection device is desirable. A method of homogeneous bead-agglutination assay based on micro-chip sheathless flow cytometry has been developed. The detection processes include mixing the capture-probe conjugated beads with an analyte containing sample, followed by flowing the reaction mixtures through the micro-chip sheathless flow cytometric device. The analyte concentrations were detected by counting the proportion of monomers in the reaction mixtures. Streptavidin-coated magnetic beads and biotinylated bovine serum albumin (bBSA) were used as a model system to verify the method, and detection limits of 0.15 pM and 1.5 pM for bBSA were achieved, using commercial Calibur and the developed micro-chip sheathless flow cytometric device, respectively. The setup of the micro-chip sheathless flow cytometric device is significantly simple; meanwhile, the system maintains relatively high sensitivity, which mainly benefits from the application of forward scattering to distinguish aggregates from monomers. The micro-chip sheathless flow cytometric device for bead agglutination detection provides us with a promising method for versatile immunoassays on microfluidic platforms. PMID:26649133

  16. Aequorea green fluorescent protein analysis by flow cytometry

    SciTech Connect

    Ropp, J.D.; Cuthbertson, R.A.; Donahue, C.J.; Wolfgang-Kimball, D.

    1995-12-01

    The isolation and expression of the cDNA for the green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria has highlighted its potential use as a marker for gene expression in a variety of cell types. The longer wavelength peak (470 nm) of GFP`s bimodal absorption spectrum better matches standard fluorescein filter sets; however, it has a considerably lower amplitude than the major absorption peak at 395. In an effort to increase the sensitivity of GFP with routinely available instrumentation, Heim et al. have generated a GFP mutant (serine-65 to threonine; S65T-GFP) which possesses a single absorption peak centered at 490 nm. We have constructed this mutant in order to determine whether it or wild-type GFP (wt-GFP) afforded greater sensitivity when excited near their respective absorption maxima. Using the conventionally available 488 nm and ultraviolet (UV) laser lines from the argon ion laser as well as the 407 nm line from a krypton ion laser with enhanced violet emission, we were able to closely match the absorption maxima of both the S65T and wild-type forms of Aequorea GFP and analyze differences in fluorescence intensity of transiently transfected 293 cells with flow cytometry. The highest fluorescence signal was observed with 488 nm excitation of S65T-GFP relative to all other laser line/GFP pairs. The wt-GFP fluorescence intensity, in contrast, was significantly higher at 407 nm relative to either 488 nm or UV. These results were consistent with parallel spectrofluorometric analysis of the emission spectrum for wt-GFP and S65T- GFP. The relative contribution of cellular autofluorescence at each wavelength was also investigated and shown to be significantly reduced at 407 nm relative to either UV or 488 nm. 29 refs., 5 figs.

  17. Understanding microbial/DOM interactions using fluorescence and flow cytometry

    NASA Astrophysics Data System (ADS)

    Fox, Bethany; Rushworth, Cathy; Attridge, John; Anesio, Alexandre; Cox, Tim; Reynolds, Darren

    2015-04-01

    The transformation and movement of dissolved organic carbon (DOC) within freshwater aquatic systems is an important factor in the global cycling of carbon. DOC within aquatic systems is known to underpin the microbial food web and therefore plays an essential role in supporting and maintaining the aquatic ecosystem. Despite this the interactions between bacteria and dissolved organic matter (DOM) are not well understood, although the literature indicates that the microbial processing of bioavailable DOM is essential during the production of autochthonous, labile, DOM. DOM can be broadly characterised by its fluorescing properties and Coble et al. (2014) define terrestrially derived DOM as exhibiting "peak C" fluorescence, whilst labile microbially derived DOM is defined as showing "peak T" fluorescence. Our work explores the microbial/DOM interactions by analysing aquatic samples using fluorescence excitation and emission matrices (EEMs) in conjunction with microbial consumption of dissolved oxygen. Environmental and synthetic water samples were subjected to fluorescence characterisation using both fluorescence spectroscopy and in situ fluorescence sensors (Chelsea Technologies Group Ltd.). PARAFAC analysis and peak picking were performed on EEMs and compared with flow cytometry data, used to quantify bacterial numbers present within samples. Synthetic samples were created using glucose, glutamic acid, nutrient-rich water and a standard bacterial seed. Synthetic samples were provided with terrestrially derived DOM via the addition of an aliquot of environmental water. Using a closed system approach, samples were incubated over time (up to a maximum of 20 days) and analysed at pre-defined intervals. The main focus of our work is to improve our understanding of microbial/DOM interactions and how these interactions affect both the DOM characteristics and microbial food web in freshwater aquatic systems. The information gained, in relation to the origin, microbial

  18. Performance of calibration standards for antigen quantitation with flow cytometry.

    PubMed

    Lenkei, R; Gratama, J W; Rothe, G; Schmitz, G; D'hautcourt, J L; Arekrans, A; Mandy, F; Marti, G

    1998-10-01

    In the frame of the activities initiated by the Task Force for Antigen Quantitation of the European Working Group on Clinical Cell Analysis (EWGCCA), an experiment was conducted to evaluate microbead standards used for quantitative flow cytometry (QFCM). An unified window of analysis (UWA) was established on three different instruments (EPICS XL [Coulter Corporation, Miami, FL], FACScan and FACS Calibur [Becton Dickinson, San Jose, CA]) with QC3 microbeads (FCSC, PR). By using this defined fluorescence intensity scale, the performance of several monoclonal antibodies directed to CD3, CD4, and CD8 (conjugated and unconjugated), from three manufacturers (BDIS, Coulter [Immunotech], and DAKO) was tested. In addition, the QIFI system (DAKO) and QuantiBRITE (BDIS), and a method of relative fluorescence intensity (RFI, method of Giorgi), were compared. mAbs reacting with three more antigens, CD16, CD19, and CD38 were tested on the FACScan instrument. Quantitation was carried out using a single batch of cryopreserved peripheral blood leukocytes, and all tests were performed as single color analyses. Significant correlations were observed between the antibody-binding capacity (ABC) values of the same CD antigen measured with various calibrators and with antibodies differing in respect to vendor, labeling and possible epitope recognition. Despite the significant correlations, the ABC values of most monoclonal antibodies differed by 20-40% when determined by the different fluorochrome conjugates and different calibrators. The results of this study indicate that, at the present stage of QFCM consistent ABC values may be attained between laboratories provided that a specific calibration system is used including specific calibrators, reagents, and protocols. PMID:9773879

  19. Flow cytometry systems for drug discovery and development

    NASA Astrophysics Data System (ADS)

    Ransom, John T.; Edwards, Bruce S.; Kuckuck, Frederick W., III; Okun, Alex; Mattox, David K.; Prossnitz, Eric R.; Sklar, Larry A.

    2000-04-01

    HT-PS is a fluidics-based pharmacology platform that uses viable cells and test compounds to rapidly identify active compounds and immediately determine their potency and specificity. Axiom employs this proprietary flow-through fluidics system coupled to a flow cytometer (FCM) as a detection system. Integration of FCM was enabled through a Plug-Flow Coupler (PFC) device that allows mixtures of cells and test compounds to be delivered to the FCM as discrete plugs of samples under positive air pressure. An FCM detector provides the advantages of multi parametric measurements and multiplexed, single cell analyses. Assays that combine two or more compatible, fluorescent bioresponse indicators simultaneously, such as measurements of intracellular pH and Ca2+, are possible. Alternatively, measurements of one or more bioresponses can be performed on several distinct cell populations individually stained with uniquely addressable fluorescent chromophores. These formats enable multiple experiments on a single sample and provide high content information thereby greatly increasing decision-making power regarding the activity, potency and selectivity of a test compound. Development of significant data with several hundred cells enables reduction in all requisite sample volumes. The PFC enables FCM sample analysis rates of at least 10 samples/minute. The data will illustrate HT-PS/PFC/FCM utility in the drug discovery arena.

  20. Discovery of Regulators of Receptor Internalization with High-Throughput Flow Cytometry

    PubMed Central

    Tapia, Phillip H.; Fisher, Gregory W.; Simons, Peter C.; Strouse, J. Jacob; Foutz, Terry; Waggoner, Alan S.; Jarvik, Jonathan; Sklar, Larry A.

    2012-01-01

    We developed a platform combining 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 facilitates drug discovery for trafficking receptors such as G protein-coupled receptors and was validated with the β2-adrenergic receptor (β2AR) system. When a chemical library containing ∼1200 off-patent drugs was screened against cells expressing FAP-tagged β2ARs, all 33 known β2AR-active ligands in the library were successfully identified, together with a number of compounds that might regulate receptor internalization in a nontraditional manner. Results indicated that the platform identified ligands of target proteins regardless of the associated signaling pathway; therefore, this approach presents opportunities to search for biased receptor modulators and is suitable for screening of multiplexed targets for improved efficiency. The results revealed that ligands may be biased with respect to the rate or duration of receptor internalization and that receptor internalization may be independent of activation of the mitogen-activated protein kinase pathway. PMID:22767611

  1. Analysis of Individual Molecular Events of DNA Damage Response by Flow and Image Assisted Cytometry

    PubMed Central

    Darzynkiewicz, Zbigniew; Traganos, Frank; Zhao, Hong; Halicka, H. Dorota; Skommer, Joanna; Wlodkowic, Donald

    2010-01-01

    This chapter describes molecular mechanisms of DNA damage response (DDR) and presents flow- and image-assisted cytometric approaches to assess these mechanisms and measure the extent of DDR in individual cells. DNA damage was induced by cell treatment with oxidizing agents, UV light, DNA topoisomerase I or II inhibitors, cisplatin, tobacco smoke, and by exogenous and endogenous oxidants. Chromatin relaxation (decondensation) is an early event of DDR chromatin that involves modification of high mobility group proteins (HMGs) and histone H1 and was detected by cytometry by analysis of the susceptibility of DNA in situ to denaturation using the metachromatic fluorochrome acridine orange. Translocation of the MRN complex consisting of Meiotic Recombination 11 Homolog A (Mre11), Rad50 homolog and Nijmegen Breakage Syndrome 1 (NMR1) into DNA damage sites was assessed by laser scanning cytometry as the increase in the intensity of maximal pixel as well as integral value of Mre11 immunofluorescence. Examples of cytometric detection of activation of Ataxia telangiectasia mutated (ATM), and Check 2 (Chk2) protein kinases using phospho-specific Abs targeting Ser1981 and Thr68 of these proteins, respectively are also presented. We also discuss approaches to correlate activation of ATM and Chk2 with phosphorylation of p53 on Ser15 and histone H2AX on Ser139 as well as with cell cycle position and DNA replication. The capability of laser scanning cytometry to quantify individual foci of phosphorylated H2AX and/or ATM that provides more dependable assessment of the presence of DNA double-strand breaks is outlined. The new microfluidic Lab-on-a-Chip platforms for interrogation of individual cells offer a novel approach for DDR cytometric analysis. PMID:21722802

  2. Computational flow cytometry: helping to make sense of high-dimensional immunology data.

    PubMed

    Saeys, Yvan; Gassen, Sofie Van; Lambrecht, Bart N

    2016-07-01

    Recent advances in flow cytometry allow scientists to measure an increasing number of parameters per cell, generating huge and high-dimensional datasets. To analyse, visualize and interpret these data, newly available computational techniques should be adopted, evaluated and improved upon by the immunological community. Computational flow cytometry is emerging as an important new field at the intersection of immunology and computational biology; it allows new biological knowledge to be extracted from high-throughput single-cell data. This Review provides non-experts with a broad and practical overview of the many recent developments in computational flow cytometry. PMID:27320317

  3. Minimally Invasive Assessment of Tumor Angiogenesis by Fine Needle Aspiration and Flow Cytometry

    PubMed Central

    Sottnik, Joseph L.; Guth, Amanda M.; Mitchell, Leah A.; Dow, Steven W.

    2011-01-01

    The development of a new, less invasive, and more rapidly implemented method of quantifying endothelial cell density in tumors could facilitate experimental and clinical studies of angiogenesis. Therefore, we evaluated the utility of tumor fine needle aspiration (FNA) coupled with flow cytometry for assessment of tumor angiogenesis. Samples were obtained from cutaneous tumors of mice using FNA, then immunostained and assessed by flow cytometry to determine the number of CD31+ endothelial cells. Results of the FNA/flow cytometry technique were compared with quantification of tumor microvessel density using immunohistochemistry. The ability of the FNA/cytometry technique to quantify the effects of anti-angiogenic therapy and to monitor changes in tumor angiogenesis over time in individual tumors was also determined. We found that endothelial cell percentages determined in tumor tissue aspirates by flow cytometry correlated well with the percentages of endothelial cells determined in whole tumor digests by flow cytometry and with tumor microvessel density measurements by immunohistochemistry. Moreover, we found that repeated FNA sampling of tumors did not induce endothelial cell changes. Interestingly, by employing repeated FNA sampling of the same tumors we were able to observe a sudden and marked decline in tumor angiogenesis triggered when tumors reached a certain size. Thus, we conclude that the FNA/flow cytometry technique is an efficient, reproducible, and relatively non-invasive method of rapidly assessing tumor angiogenesis, which could be readily applied to evaluation of tumor angiogenesis in clinical settings in humans. PMID:20734228

  4. Examination of Thymic Positive and Negative Selection by Flow Cytometry

    PubMed Central

    Baldwin, Troy A.

    2012-01-01

    premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCRα is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10. Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice. PMID:23093039

  5. Flow cytometry using Brillouin imaging and sensing via time-resolved optical (BISTRO) measurements.

    PubMed

    Meng, Zhaokai; Petrov, Georgi I; Yakovlev, Vladislav V

    2015-11-01

    A novel concept of Brillouin imaging and sensing via time-resolved optical (BISTRO) measurements is introduced for flow cytometry applications. The system affords robust, maintenance-free and high-speed elasticity-sensitive measurements. PMID:26347908

  6. Rapid Flow Cytometry Test for Identification of Different Carbapenemases in Enterobacteriaceae.

    PubMed

    Silva, Ana P; Faria-Ramos, Isabel; Ricardo, Elisabete; Miranda, Isabel M; Espinar, Maria J; Costa-de-Oliveira, Sofia; Cantón, Rafael; Rodrigues, Acácio G; Pina-Vaz, Cidália

    2016-06-01

    A flow cytometry test was developed to identify carbapenemase production by Enterobacteriaceae and to discriminate between the different types of carbapenemases (classes A, B, and D). It is based on the detection of meropenem activity against bacteria, coupled with different carbapenemase inhibitors, which is assessed by flow cytometry. It represents a convenient, fast, and reliable approach (100% sensitivity and 100% specificity) for the detection and characterization of different carbapenemases. PMID:27021318

  7. The emergence of flow cytometry for sensitive, real-time measurements of molecular interactions

    SciTech Connect

    Nolan, J.P.; Sklar, L.A. |

    1998-07-01

    The analysis of macromolecular interactions is an essential element of biomedical research. Flow cytometry is uniquely capable of making sensitive and quantitative measurements of molecular interactions. These measurements can be made in real time with subsecond kinetic resolution using purified biomolecules or living cells. Combined with automated sample handling, these features make flow cytometry a versatile and robust technology for the analysis of molecular interactions.

  8. Rapid Patterning of 1-D Collagenous Topography as an ECM Protein Fibril Platform for Image Cytometry

    PubMed Central

    Xue, Niannan; Li, Xia; Bertulli, Cristina; Li, Zhaoying; Patharagulpong, Atipat; Sadok, Amine; Huang, Yan Yan Shery

    2014-01-01

    Cellular behavior is strongly influenced by the architecture and pattern of its interfacing extracellular matrix (ECM). For an artificial culture system which could eventually benefit the translation of scientific findings into therapeutic development, the system should capture the key characteristics of a physiological microenvironment. At the same time, it should also enable standardized, high throughput data acquisition. Since an ECM is composed of different fibrous proteins, studying cellular interaction with individual fibrils will be of physiological relevance. In this study, we employ near-field electrospinning to create ordered patterns of collagenous fibrils of gelatin, based on an acetic acid and ethyl acetate aqueous co-solvent system. Tunable conformations of micro-fibrils were directly deposited onto soft polymeric substrates in a single step. We observe that global topographical features of straight lines, beads-on-strings, and curls are dictated by solution conductivity; whereas the finer details such as the fiber cross-sectional profile are tuned by solution viscosity. Using these fibril constructs as cellular assays, we study EA.hy926 endothelial cells' response to ROCK inhibition, because of ROCK's key role in the regulation of cell shape. The fibril array was shown to modulate the cellular morphology towards a pre-capillary cord-like phenotype, which was otherwise not observed on a flat 2-D substrate. Further facilitated by quantitative analysis of morphological parameters, the fibril platform also provides better dissection in the cells' response to a H1152 ROCK inhibitor. In conclusion, the near-field electrospun fibril constructs provide a more physiologically-relevant platform compared to a featureless 2-D surface, and simultaneously permit statistical single-cell image cytometry using conventional microscopy systems. The patterning approach described here is also expected to form the basics for depositing other protein fibrils, seen among

  9. A Competitive Flow Cytometry Screening System for Directed Evolution of Therapeutic Enzyme.

    PubMed

    Cheng, Feng; Kardashliev, Tsvetan; Pitzler, Christian; Shehzad, Aamir; Lue, Hongqi; Bernhagen, Jürgen; Zhu, Leilei; Schwaneberg, Ulrich

    2015-07-17

    A ligand-mediated eGFP-expression system (LiMEx) was developed as a novel flow cytometry based screening platform that relies on a competitive conversion/binding of arginine between arginine deiminase and arginine repressor. Unlike product-driven detection systems, the competitive screening platform allows to evolve enzymes toward efficient operation at low substrate concentrations under physiological conditions. The principle of LiMEx was validated by evolving arginine deiminase (ADI, an anticancer therapeutic) for stronger inhibition of tumor growth. After screening of ∼8.2 × 10(6) clones in three iterative rounds of epPCR libraries, PpADI (ADI from Pseudomonas plecoglossicida) variant M31 with reduced S0.5 value (0.17 mM compared to 1.23 mM (WT)) and, importantly, increased activity at physiological arginine concentration (M31:6.14 s(-1); WT: not detectable) was identified. Moreover, M31 showed a significant inhibitory effect against SK-MEL-28 and G361 melanoma cell lines. (IC50 = 0.02 μg/mL for SK-MEL-28 and G361). PMID:25658761

  10. Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer

    PubMed Central

    Pospichalova, Vendula; Svoboda, Jan; Dave, Zankruti; Kotrbova, Anna; Kaiser, Karol; Klemova, Dobromila; Ilkovics, Ladislav; Hampl, Ales; Crha, Igor; Jandakova, Eva; Minar, Lubos; Weinberger, Vit; Bryja, Vitezslav

    2015-01-01

    Flow cytometry is a powerful method, which is widely used for high-throughput quantitative and qualitative analysis of cells. However, its straightforward applicability for extracellular vesicles (EVs) and mainly exosomes is hampered by several challenges, reflecting mostly the small size of these vesicles (exosomes: ~80–200 nm, microvesicles: ~200–1,000 nm), their polydispersity, and low refractive index. The current best and most widely used protocol for beads-free flow cytometry of exosomes uses ultracentrifugation (UC) coupled with floatation in sucrose gradient for their isolation, labeling with lipophilic dye PKH67 and antibodies, and an optimized version of commercial high-end cytometer for analysis. However, this approach requires an experienced flow cytometer operator capable of manual hardware adjustments and calibration of the cytometer. Here, we provide a novel and fast approach for quantification and characterization of both exosomes and microvesicles isolated from cell culture media as well as from more complex human samples (ascites of ovarian cancer patients) suitable for multiuser labs by using a flow cytometer especially designed for small particles, which can be used without adjustments prior to data acquisition. EVs can be fluorescently labeled with protein-(Carboxyfluoresceinsuccinimidyl ester, CFSE) and/or lipid- (FM) specific dyes, without the necessity of removing the unbound fluorescent dye by UC, which further facilitates and speeds up the characterization of microvesicles and exosomes using flow cytometry. In addition, double labeling with protein- and lipid-specific dyes enables separation of EVs from common contaminants of EV preparations, such as protein aggregates or micelles formed by unbound lipophilic styryl dyes, thus not leading to overestimation of EV numbers. Moreover, our protocol is compatible with antibody labeling using fluorescently conjugated primary antibodies. The presented methodology opens the possibility for

  11. 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...

  12. flowCL: ontology-based cell population labelling in flow cytometry

    PubMed Central

    Courtot, Mélanie; Meskas, Justin; Diehl, Alexander D.; Droumeva, Radina; Gottardo, Raphael; Jalali, Adrin; Taghiyar, Mohammad Jafar; Maecker, Holden T.; McCoy, J. Philip; Ruttenberg, Alan; Scheuermann, Richard H.; Brinkman, Ryan R.

    2015-01-01

    Motivation: Finding one or more cell populations of interest, such as those correlating to a specific disease, is critical when analysing flow cytometry data. However, labelling of cell populations is not well defined, making it difficult to integrate the output of algorithms to external knowledge sources. Results: We developed flowCL, a software package that performs semantic labelling of cell populations based on their surface markers and applied it to labelling of the Federation of Clinical Immunology Societies Human Immunology Project Consortium lyoplate populations as a use case. Conclusion: By providing automated labelling of cell populations based on their immunophenotype, flowCL allows for unambiguous and reproducible identification of standardized cell types. Availability and implementation: Code, R script and documentation are available under the Artistic 2.0 license through Bioconductor (http://www.bioconductor.org/packages/devel/bioc/html/flowCL.html). Contact: rbrinkman@bccrc.ca Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25481008

  13. 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

  14. Monitoring of population dynamics of Corynebacterium glutamicum by multiparameter flow cytometry

    PubMed Central

    Neumeyer, Andrea; Hübschmann, Thomas; Müller, Susann; Frunzke, Julia

    2013-01-01

    Summary Phenotypic variation of microbial populations is a well-known phenomenon and may have significant impact on the success of industrial bioprocesses. Flow cytometry (FC) and the large repertoire of fluorescent dyes bring the high-throughput analysis of multiple parameters in single bacterial cells into reach. In this study, we evaluated a set of different fluorescent dyes for suitability in FC single cell analysis of the biotechnological platform organism Corynebacterium glutamicum. Already simple scattering properties of C. glutamicum cells in the flow cytometer were shown to provide valuable information on the growth activity of analysed cells. Furthermore, we used DAPI staining for a FC-based determination of the DNA content of C. glutamicum cells grown on standard minimal or complex media. Characteristic DNA patterns were observed mirroring the typical uncoupled DNA synthesis in the logarithmic (log) growth phase and are in agreement with a symmetric type of cell division of C. glutamicum. Application of the fluorescent dyes Syto 9, propidium iodide, and DiOC2(3) allowed the identification of subpopulations with reduced viability and membrane potential within early log and stationary phase populations. The presented data highlight the potential of FC-based analyses for online monitoring of C. glutamicum bioprocesses and provide a first reference for future applications and protocols. PMID:23279937

  15. Detection of cyclin D1 in B cell lymphoproliferative disorders by flow cytometry

    PubMed Central

    Jain, P; Giustolisi, G M; Atkinson, S; Elnenaei, M O; Morilla, R; Owusu-Ankomah, K; Rafiq-Mohammed, F; Matutes, E; Wotherspoon, A; Catovsky, D

    2002-01-01

    Aims: To describe and revise a flow cytometric assay for evaluating cyclin D1 overexpression in B cell lymphoproliferative disorders (B-LPDs). Methods: Cyclin D1 expression was evaluated in 11 healthy controls and 51 patients with B-LPD by flow cytometry using the 5D4 monoclonal antibody. In 25 cases, experiments were repeated up to four times with mononuclear cells (MNC) fixed in ethanol for 1–120 days to evaluate the consistency of cyclin D1 expression. Flow cytometry results were compared with fluorescence in situ hybridisation (FISH) for the t(11;14) translocation in 19 patients and with immunohistochemistry (IHC) using the DCS-6 monoclonal antibody in nine patients. Results: A mean fluorescence intensity ratio (MFIR) of 4.8 was defined as the cut off point for positivity based on cyclin D1 expression in healthy controls (mean + 3 SD). Ten patients overexpressed cyclin D1 by flow cytometry. These included five of eight patients with mantle cell lymphoma, four of 19 with chronic lymphocytic leukaemia, and one with follicular lymphoma. MFIR in the repeat experiments differed less than 25% in 20 of 25 patients and in no cases did it cross the cut off point. There was a good correlation between cyclin D1 expression by flow cytometry and FISH for t(11;14) in 15 of 19 patients and six of nine had concordant results with flow cytometry, FISH, and IHC. Conclusion: Cyclin D1 expression remains fairly stable once MNC are fixed in ethanol and the flow cytometric assay can be used for the routine screening of B-LPD. Further comparisons between flow cytometry, IHC, and FISH may be needed to ascertain the diagnostic value of the flow cytometric assay. PMID:12461064

  16. Role of Flow Cytometry in the Diagnosis and Prognosis of Plasma Cell Myeloma.

    PubMed

    Olteanu, Horatiu

    2016-03-01

    This article provides an overview of the role of flow cytometry in the diagnosis and follow-up of plasma cell myeloma. A brief introduction to the general immunophenotypic features of normal and myeloma plasma cells is provided, followed by a discussion of technical issues as they relate to the application of flow cytometry in this entity. The prognostic and therapeutic utility of flow cytometric immunophenotyping in myeloma is also analyzed, with an emphasis on the growing role of minimal residual analysis as potential biomarker for evaluating treatment efficacy and for tailoring risk-adapted treatment, in prospective clinical trials. PMID:26940271

  17. Enumeration and Biomass Estimation of Bacteria in Aquifer Microcosm Studies by Flow Cytometry

    PubMed Central

    DeLeo, P. C.; Baveye, P.

    1996-01-01

    Flow cytometry was used to enumerate and characterize bacteria from a sand column microcosm simulating aquifer conditions. Pure cultures of a species of Bacillus isolated from subsurface sediments or Bacillus megaterium were first evaluated to identify these organisms' characteristic histograms. Counting was then carried out with samples from the aquifer microcosms. Enumeration by flow cytometry was compared with more-traditional acridine orange direct counting. These two techniques gave statistically similar results. However, counting by flow cytometry, in this case, surveyed a sample size 700 times greater than did acridine orange direct counting (25 (mu)l versus 0.034 (mu)l) and required 1/10 the time (2 h versus 20 h). Flow cytometry was able to distinguish the same species of bacteria grown under different nutrient conditions, and it could distinguish changes in cell growth patterns, specifically single cell growth versus chained cell growth in different regions of an aquifer microcosm. A biomass estimate was calculated by calibrating the total fluorescence of a sample from a pure culture with the dry weight of a freeze-dried volume from the original pure culture. Growth conditions significantly affected histograms and biomass estimates, so the calibration was carried out with cells grown under conditions similar to those in the aquifer microcosm. Costs associated with using flow cytometry were minimal compared with the amount of time saved in counting cells and estimating biomass. PMID:16535470

  18. MIFlowCyt: The Minimum Information about a Flow Cytometry Experiment

    PubMed Central

    Lee, Jamie A.; Spidlen, Josef; Boyce, Keith; Cai, Jennifer; Crosbie, Nicholas; Dalphin, Mark; Furlong, Jeff; Gasparetto, Maura; Goldberg, Michael; Goralczyk, Elizabeth M.; Hyun, Bill; Jansen, Kirstin; Kollmann, Tobias; Kong, Megan; Leif, Robert; McWeeney, Shannon; Moloshok, Thomas D.; Moore, Wayne; Nolan, Garry; Nolan, John; Nikolich-Zugich, Janko; Parrish, David; Purcell, Barclay; Qian, Yu; Selvaraj, Biruntha; Smith, Clayton; Tchuvatkina, Olga; Wertheimer, Anne; Wilkinson, Peter; Wilson, Christopher; Wood, James; Zigon, Robert; Scheuermann, Richard H.; Brinkman, Ryan R.

    2009-01-01

    Background A fundamental tenet of scientific research is that published results are open to independent validation and refutation. Minimum data standards aid data providers, users and publishers by providing a specification of what is required to unambiguously interpret experimental findings. Here, we present the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard, stating the minimum information required to report flow cytometry (FCM) experiments. Methods We brought together a cross-disciplinary international collaborative group of bioinformaticians, computational statisticians, software developers, instrument manufacturers, and clinical and basic research scientists to develop the standard. The standard was subsequently vetted by the International Society for the Advancement of Cytometry (ISAC) Data Standards Task Force, Standards Committee, membership and Council. Results The MIFlowCyt standard includes recommendations about descriptions of the specimens and reagents included in the FCM experiment, the configuration of the instrument used to perform the assays and the data processing approaches used to interpret the primary output data. MIFlowCyt has been adopted as a standard by ISAC, representing the flow cytometry scientific community including scientists as well as software and hardware manufactures. Conclusions MIFlowCyt’s adoption by the scientific and publishing communities will facilitate third-party understanding and reuse of flow cytometry data. PMID:18752282

  19. Amphiphilic mediated sample preparation for micro-flow cytometry

    DOEpatents

    Clague, David S.; Wheeler, Elizabeth K.; Lee, Abraham P.

    2009-03-17

    A flow cytometer includes a flow cell for detecting the sample, an oil phase in the flow cell, a water phase in the flow cell, an oil-water interface between the oil phase and the water phase, a detector for detecting the sample at the oil-water interface, and a hydrophobic unit operatively connected to the sample. The hydrophobic unit is attached to the sample. The sample and the hydrophobic unit are placed in an oil and water combination. The sample is detected at the interface between the oil phase and the water phase.

  20. Amphiphilic mediated sample preparation for micro-flow cytometry

    DOEpatents

    Clague, David S.; Wheeler, Elizabeth K.; Lee, Abraham P.

    2006-07-25

    A flow cytometer includes a flow cell for detecting the sample, an oil phase in the flow cell, a water phase in the flow cell, an oil-water interface between the oil phase and the water phase, a detector for detecting the sample at the oil-water interface, and a hydrophobic unit operatively connected to the sample. The hydrophobic unit is attached to the sample. The sample and the hydrophobic unit are placed in an oil and water combination. The sample is detected at the interface between the oil phase and the water phase.

  1. Beyond conventional cell analysis: the latest science and technology in flow cytometry.

    PubMed

    Wright, Sharlene

    2016-01-01

    Combining powerful performance and innovative design and technology, it is possible to deliver a compact, easy-to-use flow cytometry system. Pushing the 'norms' of conventional flow cytometry, today's--and tomorrow's--systems enable complex research into high-content applications in cell biology, as well as a deeper understanding of the advantages gained from the emerging nanoparticle frontier. Flow cytometry is a powerful tool for interrogating complex biological questions at the forefront of biomedical and life science research and increasingly for clinical laboratory applications. Today's investigators want to harness that power and are demanding smaller and more powerful instruments that are more affordable and easier to use. Using innovation, engineers are able to deliver solutions to meet the challenge. PMID:26887094

  2. Intermittent blood flow in the KHT sarcoma--flow cytometry studies using Hoechst 33342.

    PubMed Central

    Minchinton, A. I.; Durand, R. E.; Chaplin, D. J.

    1990-01-01

    The administration of the fluorescent DNA stain, Hoechst 33342, to mice bearing the KHT sarcoma, combined with flow cytometry, can be used to select cells according to their proximity to functional vasculature. Different protocols of administration of Hoechst 33342 were used in order to differentiate between the presence of temporary and chronically hypoxic cells. The results show a large difference in radiosensitivity between cells close to, and distant from, functional vasculature. However, this pattern of radiosensitivity is observed only when the staining period with Hoechst 33342 is short and coincides with the period of irradiation. When the radiation treatment is temporally divorced from the staining period then the radiosensitivity and staining intensity are not related. This result can be interpreted as indicating that hypoxic cells exist within this tumour as a result of fluctuations in tumour blood flow. PMID:2386734

  3. Evidence for P-Glycoprotein Involvement in Cell Volume Regulation Using Coulter Sizing in Flow Cytometry.

    PubMed

    Pasquier, Jennifer; Rioult, Damien; Abu-Kaoud, Nadine; Hoarau-Véchot, Jessica; Marin, Matthieu; Le Foll, Frank

    2015-01-01

    The regulation of cell volume is an essential function that is coupled to a variety of physiological processes such as receptor recycling, excitability and contraction, cell proliferation, migration, and programmed cell death. Under stress, cells undergo emergency swelling and respond to such a phenomenon with a regulatory volume decrease (RVD) where they release cellular ions, and other osmolytes as well as a concomitant loss of water. The link between P-glycoprotein, a transmembrane transporter, and cell volume regulation is controversial, and changes in cells volume are measured using microscopy or electrophysiology. For instance, by using the patch-clamp method, our team demonstrated that chloride currents activated in the RVD were more intense and rapid in a breast cancer cell line overexpressing the P-glycoprotein (P-gp). The Cell Lab Quanta SC is a flow cytometry system that simultaneously measures electronic volume, side scatter and three fluorescent colors; altogether this provides unsurpassed population resolution and accurate cell counting. Therefore, here we propose a novel method to follow cellular volume. By using the Coulter-type channel of the cytometer Cell Lab Quanta SC MPL (multi-platform loading), we demonstrated a role for the P-gp during different osmotic treatments, but also a differential activity of the P-gp through the cell cycle. Altogether, our data strongly suggests a role of P-gp in cell volume regulation. PMID:26114386

  4. flowClean: Automated identification and removal of fluorescence anomalies in flow cytometry data.

    PubMed

    Fletez-Brant, Kipper; Špidlen, Josef; Brinkman, Ryan R; Roederer, Mario; Chattopadhyay, Pratip K

    2016-05-01

    Modern flow cytometry systems can be coupled to plate readers for high-throughput acquisition. These systems allow hundreds of samples to be analyzed in a single day. Quality control of the data remains challenging, however, and is further complicated when a large number of parameters is measured in an experiment. Our examination of 29,228 publicly available FCS files from laboratories worldwide indicates 13.7% have a fluorescence anomaly. In particular, fluorescence measurements for a sample over the collection time may not remain stable due to fluctuations in fluid dynamics; the impact of instabilities may differ between samples and among parameters. Therefore, we hypothesized that tracking cell populations (which represent a summary of all parameters) in centered log ratio space would provide a sensitive and consistent method of quality control. Here, we present flowClean, an algorithm to track subset frequency changes within a sample during acquisition, and flag time periods with fluorescence perturbations leading to the emergence of false populations. Aberrant time periods are reported as a new parameter and added to a revised data file, allowing users to easily review and exclude those events from further analysis. We apply this method to proof-of-concept datasets and also to a subset of data from a recent vaccine trial. The algorithm flags events that are suspicious by visual inspection, as well as those showing more subtle effects that might not be consistently flagged by investigators reviewing the data manually, and out-performs the current state-of-the-art. flowClean is available as an R package on Bioconductor, as a module on the free-to-use GenePattern web server, and as a plugin for FlowJo X. © 2016 International Society for Advancement of Cytometry. PMID:26990501

  5. 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.

  6. 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. PMID:2194018

  7. Microbial flow cytometry: An ideal tool for prospective antimicrobial drug development.

    PubMed

    Muthirulan, Pushpanathan; Chandrasekaran, Arun Richard

    2016-09-15

    Flow cytometry has tremendous applications in qualitative and quantitative analysis of characteristics of single microbial cells. Its ability to efficiently discriminate and quantify multiple parameters of microbial cells has made it a powerful tool to catalog the mechanism of action of antimicrobial peptides (AMPs) on target cells. Here, we provide a comprehensive overview and strategic design on how multi-parametric analysis of flow cytometry is unsurpassed in studying the antimicrobial process of AMPs in an accurate and rapid way. This strategy provides a conceptual framework for understanding distinct classes of AMPs and getting insights into antimicrobial mechanisms of novel AMPs. PMID:27288557

  8. 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. PMID:26849001

  9. National flow cytometry and sorting research resource. Annual progress report, July, 1, 1994--June 30, 1995, Year 12

    SciTech Connect

    Jett, J.H.

    1995-04-27

    Research progress utilizing flow cytometry is described. Topics include: rapid kinetics flow cytometry; characterization of size determinations for small DNA fragments; statistical analysis; energy transfer measurements of molecular confirmation in micelles; and enrichment of Mus spretus chromosomes by dual parameter flow sorting and identification of sorted fractions by fluorescence in-situ hybridization onto G-banded mouse metaphase spreads.

  10. Matching of flow-cytometry histograms using information theory in feature space.

    PubMed Central

    Zeng, Qing; Wand, Matthew; Young, Alan J.; Rawn, James; Milford, Edgar L.; Mentzer, Steven J.; Greenes, Robert A.

    2002-01-01

    Flow cytometry is a widely available technique for analyzing cell-surface protein expression. Data obtained from flow cytometry is frequently used to produce fluorescence intensity histograms. Comparison of histograms can be useful in the identification of unknown molecules and in the analysis of protein expression. In this study, we examined the combination of a new smoothing technique called SiZer with information theory to measure the difference between cytometry histograms. SiZer provides cross-bandwidth smoothing and allowed analysis in feature space. The new methods were tested on a panel of monoclonal antibodies raised against proteins expressed on peripheral blood lymphocytes and compared with previous methods. The findings suggest that comparing information content of histograms in feature space is effective and efficient for identifying antibodies with similar cell-surface binding patterns. PMID:12463961

  11. Minimal residual disease analysis by eight-color flow cytometry in relapsed childhood acute lymphoblastic leukemia

    PubMed Central

    Karawajew, Leonid; Dworzak, Michael; Ratei, Richard; Rhein, Peter; Gaipa, Giuseppe; Buldini, Barbara; Basso, Giuseppe; Hrusak, Ondrej; Ludwig, Wolf-Dieter; Henze, Günter; Seeger, Karl; von Stackelberg, Arend; Mejstrikova, Ester; Eckert, Cornelia

    2015-01-01

    Multiparametric flow cytometry is an alternative approach to the polymerase chain reaction method for evaluating minimal residual disease in treatment protocols for primary acute lymphoblastic leukemia. Given considerable differences between primary and relapsed acute lymphoblastic leukemia treatment regimens, flow cytometric assessment of minimal residual disease in relapsed leukemia requires an independent comprehensive investigation. In the present study we addressed evaluation of minimal residual disease by flow cytometry in the clinical trial for childhood relapsed acute lymphoblastic leukemia using eight-color flow cytometry. The major challenge of the study was to reliably identify low amounts of residual leukemic cells against the complex background of regeneration, characteristic of follow-up samples during relapse treatment. In a prospective study of 263 follow-up bone marrow samples from 122 patients with B-cell precursor acute lymphoblastic leukemia, we tested various B-cell markers, adapted the antibody panel to the treatment protocol, and evaluated its performance by a blinded parallel comparison with the polymerase chain reaction data. The resulting eight-color single-tube panel showed a consistently high overall concordance (P<0.001) and, under optimal conditions, sensitivity similar to that of the reference polymerase chain reaction method. Overall, evaluation of minimal residual disease by flow cytometry can be successfully integrated into the clinical management of relapsed childhood acute lymphoblastic leukemia either as complementary to the polymerase chain reaction or as an independent risk stratification tool. ALL-REZ BFM 2002 clinical trial information: NCT00114348 PMID:26001791

  12. 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

  13. Determination of natural killer cell function by flow cytometry.

    PubMed Central

    Kane, K L; Ashton, F A; Schmitz, J L; Folds, J D

    1996-01-01

    Natural killer cells (NK cells) are a subset of peripheral blood lymphocytes that mediate non-major histocompatibility complex-restricted cytotoxicity of foreign target cells. The "gold standard" assay for NK cell activity has been the chromium release assay. This method is not easily performed in the clinical laboratory because of difficulties with disposal of radioactive and hazardous materials, short reagent half-lives, expense, and difficulties with assay standardization. We describe a flow cytometric assay for the clinical measurement of NK cell activity. This study compared the chromium release assay and the flow cytometric assay by using clinically relevant specimens. There were no significant differences between the two assays in the measurement of lytic activity for 17 peripheral blood specimens or in reproducibility in repeated samplings of healthy individuals. We also established a normal range of values for NK activity in healthy adults and identified a small cluster of individuals who have exceptionally high or low levels of NK activity. The flow cytometric assay was validated by testing specimens from subjects expected to have abnormally low levels of NK activity (pregnant women) and specimens from healthy individuals in whom the activity of NK cells was enhanced by exposure to interleukin-2 or alpha interferon. Treatment with these agents was associated with a significant increase in NK activity. These results confirm and extend those of others, showing that the flow cytometric assay is a viable alternative to the chromium release assay for measuring NK cell activity. PMID:8705672

  14. Assessment of Equine Autoimmune Thrombocytopenia (EAT) by flow cytometry

    PubMed Central

    Nunez, Rafael; Gomes-Keller, M Alice; Schwarzwald, Colin; Feige, Karsten

    2001-01-01

    Rationale Thrombocytopenia is a platelet associated process that occurs in human and animals as result of i) decreased production; ii) increased utilization; iii) increased destruction coupled to the presence of antibodies, within a process know as immune-mediated thrombocytopenia (IMT); or iv) platelet sequestration. Thus, the differentiation of the origin of IMT and the development of reliable diagnostic approaches and methodologies are important in the clarification of IMT pathogenesis. Therefore, there is a growing need in the field for easy to perform assays for assessing platelet morphological characteristics paired with detection of platelet-bound IgG. Objectives This study is aimed to develop and characterize a single color flow cytometric assay for detection of platelet-bound IgG in horses, in combination with flow cytometric assessment of platelet morphological characteristics. Findings The FSC and SSC evaluation of the platelets obtained from the thrombocytopenic animals shows several distinctive features in comparison to the flow cytometric profile of platelets from healthy animals. The thrombocytopenic animals displayed i) increased number of platelets with high FSC and high SSC, ii) a significant number of those gigantic platelets had strong fluorescent signal (IgG bound), iii) very small platelets or platelet derived microparticles were found significantly enhanced in one of the thrombocytopenic horses, iv) significant numbers of these microplatelet/microparticles/platelet-fragments still carry very high fluorescence. Conclusions This study describes the development and characterization of an easy to perform, inexpensive, and noninvasive single color flow cytometric assay for detection of platelet-bound IgG, in combination with flow cytometric assessment of platelet morphological characteristics in horses. PMID:11313001

  15. Statistical Mixture Modeling for Cell Subtype Identification in Flow Cytometry

    PubMed Central

    Chan, Cliburn; Feng, Feng; Ottinger, Janet; Foster, David; West, Mike; Kepler, Thomas B

    2010-01-01

    Background Statistical mixture modeling provides an opportunity for automated identification and resolution of cell subtypes in flow cytometric data. The configuration of cells as represented by multiple markers simultaneously can be modeled arbitrarily well as a mixture of Gaussian distributions in the dimension of the number of markers. Cellular subtypes may be related to one or multiple components of such mixtures, and fitted mixture models can be evaluated in the full set of markers as an alternative, or adjunct, to traditional subjective gating methods that rely on choosing one or two dimensions. Methods Four color flow data from human blood cells labeled with FITC-conjugated anti-CD3, PE-conjugated anti-CD8, PE-Cy5-conjugated anti-CD4 and APC-conjugated anti-CD19 Abs was acquired on a FACSCalibur. Cells from four murine cell lines, JAWS II, RAW 264.7, CTLL-2 and A20, were also stained with FITC-conjugated anti-CD11c, PE-conjugated anti-CD11b, PE-Cy5-conjugated anti-CD8a and PE-Cy7-conjugated-CD45R/B220 Abs respectively, and single color flow data were collected on an LSRII. The data was fitted with a mixture of multivariate Gaussians using standard Bayesian statistical approaches and Markov chain Monte Carlo computations. Results Statistical mixture models were able to identify and purify major cell subsets in human peripheral blood, using an automated process that can be generalized to an arbitrary number of markers. Validation against both traditional expert gating and synthetic mixtures of murine cell lines with known mixing proportions was also performed. Conclusions This paper describes studies of statistical mixture modeling of flow cytometric data, and demonstrates their utility in examples with four-color flow data from human peripheral blood samples and synthetic mixtures of murine cell lines. PMID:18496851

  16. iFlow: A Graphical User Interface for Flow Cytometry Tools in Bioconductor

    PubMed Central

    Lee, Kyongryun; Hahne, Florian; Sarkar, Deepayan; Gentleman, Robert

    2009-01-01

    Flow cytometry (FCM) has become an important analysis technology in health care and medical research, but the large volume of data produced by modern high-throughput experiments has presented significant new challenges for computational analysis tools. The development of an FCM software suite in Bioconductor represents one approach to overcome these challenges. In the spirit of the R programming language (Tree Star Inc., “FlowJo,” http://www.owjo.com), these tools are predominantly console-driven, allowing for programmatic access and rapid development of novel algorithms. Using this software requires a solid understanding of programming concepts and of the R language. However, some of these tools|in particular the statistical graphics and novel analytical methods|are also useful for nonprogrammers. To this end, we have developed an open source, extensible graphical user interface (GUI) iFlow, which sits on top of the Bioconductor backbone, enabling basic analyses by means of convenient graphical menus and wizards. We envision iFlow to be easily extensible in order to quickly integrate novel methodological developments. PMID:20049160

  17. Ultrasonic analyte concentration and application in flow cytometry

    DOEpatents

    Kaduchak, Gregory; Goddard, Greg; Salzman, Gary; Sinha, Dipen; Martin, John C.; Kwiatkowski, Christopher; Graves, Steven

    2008-03-11

    The present invention includes an apparatus and corresponding method for concentrating analytes within a fluid flowing through a tube using acoustic radiation pressure. The apparatus includes a function generator that outputs a radio frequency electrical signal to a transducer that transforms the radio frequency electric signal to an acoustic signal and couples the acoustic signal to the tube. The acoustic signal is converted within the tube to acoustic pressure that concentrates the analytes within the fluid.

  18. Ultrasonic analyte concentration and application in flow cytometry

    SciTech Connect

    Kaduchak, Gregory; Goddard, Greg; Salzman, Gary; Sinha, Dipen; Martin, John C.; Kwiatkowski, Christopher; Graves, Steven

    2015-07-07

    The present invention includes an apparatus and corresponding method for concentrating analytes within a fluid flowing through a tube using acoustic radiation pressure. The apparatus includes a function generator that outputs a radio frequency electrical signal to a transducer that transforms the radio frequency electric signal to an acoustic signal and couples the acoustic signal to the tube. The acoustic signal is converted within the tube to acoustic pressure that concentrates the analytes within the fluid.

  19. Ultrasonic analyte concentration and application in flow cytometry

    SciTech Connect

    Kaduchak, Gregory; Goddard, Greg; Salzman, Gary; Sinha, Dipen; Martin, John C.; Kwiatkowski, Christopher; Graves, Steven

    2014-07-22

    The present invention includes an apparatus and corresponding method for concentrating analytes within a fluid flowing through a tube using acoustic radiation pressure. The apparatus includes a function generator that outputs a radio frequency electrical signal to a transducer that transforms the radio frequency electric signal to an acoustic signal and couples the acoustic signal to the tube. The acoustic signal is converted within the tube to acoustic pressure that concentrates the analytes within the fluid.

  20. Circulation times of cancer cells by in vivo flow cytometry

    NASA Astrophysics Data System (ADS)

    Zhang, Li; Li, Yan; Gu, Zhengqin; Chen, Tong; Wang, Cheng; Wei, Xunbin

    2012-03-01

    Liver cancer is one of the most common malignancies in the world, with approximately 1,000,000 cases reported every year. Hepatocellular carcinoma may metastasize to lung, bones, kidney, and many other organs. Surgical resection, liver transplantation, chemotherapy and radiation therapy are the foundation of current HCC therapies. However the outcomes are poor: the survival rate is almost zero for metastatic HCC patients. Molecular mechanisms of HCC metastasis need to be understood better and new therapies must be developed. A recently developed "in vivo flow cytometer" combined with real-time confocal fluorescence imaging are used to assess spreading and the circulation kinetics of liver tumor cells. The in vivo flow cytometer has the capability to detect and quantify continuously the number and flow characteristics of fluorescently labeled cells in vivo in real time without extracting blood sample. We have measured the depletion kinetics of two related human HCC cell lines, high-metastatic HCCLM3 cells and low-metastatic HepG2 cells, which were from the same origin and obtained by repetitive screenings in mice. >60% HCCLM3 cells are depleted within the first hour. Interestingly, the low-metastatic HepG2 cells possess noticeably slower depletion kinetics. In comparison, <40% HepG2 cells are depleted within the first hour. The differences in depletion kinetics might provide insights into early metastasis processes.

  1. SETTLING AND COAGULATION CHARACTERISTICS OF FLUORESCENT PARTICLES DETERMINED BY FLOW CYTOMETRY AND FLUOROMETRY

    EPA Science Inventory

    A new technique for detecting particles in natural waters relies upon analysis of fluorescent emission by flow cytometry. luorescent pigment particles ranging in radius from 0.1 to 5 um are available in sufficient quantity to be useful as model particles. aboratory coagulation an...

  2. Flow Cytometry To Assess Cerebrospinal Fluid Fungal Burden in Cryptococcal Meningitis

    PubMed Central

    Graham, Lisa M.; Schutz, Charlotte; Scriba, Thomas J.; Wilkinson, Robert J.; Boulware, David R.; Meintjes, Graeme; Lalloo, David G.; Urban, Britta C.

    2015-01-01

    Fungal burden in the cerebrospinal fluid is an important determinant of mortality in cryptococcal meningitis, but its use in aiding clinical decision making is hampered by the time involved to perform quantitative cultures. Here, we demonstrate the potential of flow cytometry as a novel and rapid technique to address this issue. PMID:26719441

  3. Ploidy of USDA (United States Department of Agriculture) world pear germplasm collection determined by flow cytometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Living germplasm collections representing world diversity of pear (Pyrus L.) are maintained by the U.S. Department of Agriculture at the National Clonal Germplasm Repository (NCGR) in Corvallis, Oregon, USA. Flow cytometry was performed on young leaf tissue from 1,284 genebank accessions to assess p...

  4. 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 bacteria…

  5. Data File Standard for Flow Cytometry, Version FCS 3.1

    SciTech Connect

    Spidlen, Josef; Moore, Wayne; Parks, David; Goldberg, Michael; Bray, Chris; 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.; Brierre, Pierre

    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 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.

  6. Report of the European Myeloma Network on multiparametric flow cytometry in multiple myeloma and related disorders.

    PubMed

    Rawstron, Andy C; Orfao, Alberto; Beksac, Meral; Bezdickova, Ludmila; Brooimans, Rik A; Bumbea, Horia; Dalva, Klara; Fuhler, Gwenny; Gratama, Jan; Hose, Dirk; Kovarova, Lucie; Lioznov, Michael; Mateo, Gema; Morilla, Ricardo; Mylin, Anne K; Omedé, Paola; Pellat-Deceunynck, Catherine; Perez Andres, Martin; Petrucci, Maria; Ruggeri, Marina; Rymkiewicz, Grzegorz; Schmitz, Alexander; Schreder, Martin; Seynaeve, Carine; Spacek, Martin; de Tute, Ruth M; Van Valckenborgh, Els; Weston-Bell, Nicky; Owen, Roger G; San Miguel, Jesús F; Sonneveld, Pieter; Johnsen, Hans E

    2008-03-01

    The European Myeloma Network (EMN) organized two flow cytometry workshops. The first aimed to identify specific indications for flow cytometry in patients with monoclonal gammopathies, and consensus technical approaches through a questionnaire-based review of current practice in participating laboratories. The second aimed to resolve outstanding technical issues and develop a consensus approach to analysis of plasma cells. The primary clinical applications identified were: differential diagnosis of neoplastic plasma cell disorders from reactive plasmacytosis; identifying risk of progression in patients with MGUS and detecting minimal residual disease. A range of technical recommendations were identified, including: 1) CD38, CD138 and CD45 should all be included in at least one tube for plasma cell identification and enumeration. The primary gate should be based on CD38 vs. CD138 expression; 2) after treatment, clonality assessment is only likely to be informative when combined with immunophenotype to detect abnormal cells. Flow cytometry is suitable for demonstrating a stringent complete remission; 3) for detection of abnormal plasma cells, a minimal panel should include CD19 and CD56. A preferred panel would also include CD20, CD117, CD28 and CD27; 4) discrepancies between the percentage of plasma cells detected by flow cytometry and morphology are primarily related to sample quality and it is, therefore, important to determine that marrow elements are present in follow-up samples, particularly normal plasma cells in MRD negative cases. PMID:18268286

  7. High-resolution microbiota flow cytometry reveals dynamic colitis-associated changes in fecal bacterial composition.

    PubMed

    Zimmermann, Jakob; Hübschmann, Thomas; Schattenberg, Florian; Schumann, Joachim; Durek, Pawel; Riedel, René; Friedrich, Marie; Glauben, Rainer; Siegmund, Britta; Radbruch, Andreas; Müller, Susann; Chang, Hyun-Dong

    2016-05-01

    Using high-resolution flow cytometry of bacterial shape (forward scatter) and DNA content (DAPI staining), we detected dramatic differences in the fecal microbiota composition during murine colitis that were validated using 16S rDNA sequencing. This innovative method provides a fast and inexpensive tool to interrogate the microbiota on the single-cell level. PMID:26909672

  8. Application of flow cytometry and cell sorting to the bacterial analysis of environmental aerosol samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Flow cytometry (FCM) combined with viability staining is a useful tool in discerning viable bacteria in environmental samples where traditional culture methods may fail. Contamination of aerosol samples with dust and other non-biological particles can interfere with accurate sample analysis and ther...

  9. Improved Method for Bacterial Cell Capture after Flow Cytometry Cell Sorting ▿

    PubMed Central

    Guillebault, D.; Laghdass, M.; Catala, P.; Obernosterer, I.; Lebaron, P.

    2010-01-01

    Fixed cells with different nucleic acid contents and scatter properties (low nucleic acid [LNA], high nucleic acid 1 [HNA1], and HNA2) were sorted by flow cytometry (FCM). For each sort, 10,000 cells were efficiently captured on poly-l-lysine-coated microplates, resulting in efficient and reproducible PCR amplification. PMID:20817799

  10. Application of flow cytometry and fluorescent in situ hybridization for assessment of exposures to airborne bacteria.

    PubMed Central

    Lange, J L; Thorne, P S; Lynch, N

    1997-01-01

    Current limitations in the methodology for enumeration and identification of airborne bacteria compromise the precision and accuracy of bioaerosol exposure assessment. In this study, flow cytometry and fluorescent in situ hybridization (FISH) were evaluated for the assessment of exposures to airborne bacteria. Laboratory-generated two-component bioaerosols in exposures chambers and complex native bioaerosols in swine barns were sampled with two types of liquid impingers (all-glass impinger-30 and May 3-stage impinger). Aliquots of collection media were processed and enumerated by a standard culture technique, microscopy, or flow cytometry after nucleic acid staining with 4',6-diamidino-2-phenylindole (DAPI) and identified taxonomically by FISH. DAPI-labeled impinger samples yielded comparable estimates of bioaerosol concentrations when enumerated by microscopy or flow cytometry. The standard culture method underestimated bioaerosol concentrations by 2 orders of magnitude when compared to microscopy or flow cytometry. In the FISH method, aliquots of collection media were incubated with a probe universally complementary to eubacteria, a probe specific for several Pseudomonas species, and a probe complementary to eubacteria for detection of nonspecific binding. With these probes, FISH allowed quantitative identification of Pseudomonas aeruginosa and Escherichia coli bioaerosols in the exposure chamber without measurable nonspecific binding. Impinger samples from the swine barn demonstrated the efficacy of the FISH method for the identification of eubacteria in a complex organic dust. This work demonstrates the potential of emerging molecular techniques to complement traditional methods of bioaerosol exposure assessment. PMID:9097451

  11. A flow cytometry-based dopamine transporter binding assay using antagonist-conjugated quantum dots

    SciTech Connect

    Kovtun, Oleg; Ross, Emily; Tomlinson, Ian; Rosenthal, Sandra

    2012-01-01

    Here we present the development and validation of a flow cytometry-based dopamine transporter (DAT) binding assay that uses antagonist-conjugated quantum dots (QDs).We anticipate that our QD-based assay is of immediate value to the high throughput screening of novel DAT modulators.

  12. Measuring Cell Death by Propidium Iodide Uptake and Flow Cytometry.

    PubMed

    Crowley, Lisa C; Scott, Adrian P; Marfell, Brooke J; Boughaba, Jeanne A; Chojnowski, Grace; Waterhouse, Nigel J

    2016-01-01

    Propidium iodide (PI) is a small fluorescent molecule that binds to DNA but cannot passively traverse into cells that possess an intact plasma membrane. PI uptake versus exclusion can be used to discriminate dead cells, in which plasma membranes become permeable regardless of the mechanism of death, from live cells with intact membranes. PI is excited by wavelengths between 400 and 600 nm and emits light between 600 and 700 nm, and is therefore compatible with lasers and photodetectors commonly available in flow cytometers. This protocol for PI staining can be used to quantitate cell death in most modern research facilities and universities. PMID:27371595

  13. Flow Cytometry Analysis of Thymic Epithelial Cells and Their Subpopulations.

    PubMed

    Ohigashi, Izumi; Takahama, Yousuke

    2016-01-01

    The parenchyma of the thymus is compartmentalized into the cortex and the medulla, which are constructed by cortical thymic epithelial cells (cortical TECs, cTECs) and medullary thymic epithelial cells (mTECs), respectively. cTECs and mTECs essentially and differentially regulate the development and repertoire selection of T cells. Consequently, the biology of T cell development and selection includes the study of TECs in addition to the study of developing T cells and other hematopoietic cells including dendritic cells. In this chapter, we describe the methods for flow cytometric analysis and sorting of TECs and their subpopulations, including cTECs and mTECs. PMID:26294398

  14. Detection of single lambda DNA fragments by flow cytometry

    SciTech Connect

    Johnson, M.E.; Goodwin, P.M.; Ambrose, W.P.; Martin, J.C.; Marrone, B.L.; Keller, R.A. )

    1993-01-01

    The authors have demonstrated flow cytometric detection and sizing of single pieces of fluorescently stained lambda DNA (48.5 kb) and individual Kpn I restriction fragments of lambda DNA at 17.05 kb and 29.95 kb. DNA fragments were stained stoichiometrically with an intercalating dye such that the fluorescence from each fragment was directly proportional to fragment length. Laser powers range from 10 to 100 mW and transit times through the focused laser beam were several milliseconds. Measurements were made using time-resolved single photon counting of the detected fluorescence emission from individual stained DNA fragments. Samples were analyzed at rates of about 50 fragments per second. The measured fluorescence intensities are linearly correlated with DNA fragment length over the range measured. Detection sensitivity and resolution needed for analysis of small pieces of DNA are discussed and a comparison of single photon counting measurements of DNA fragments to measurements using more conventional flow cytometers is made. Applications of this methodology to DNA sizing and DNA fingerprinting are discussed.

  15. 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. PMID:25573116

  16. Flow-cytometry-based DNA hybidization and polymorphism analysis

    NASA Astrophysics Data System (ADS)

    Cai, Hong; Kommander, Kristina; White, P. S.; Nolan, John P.

    1998-05-01

    Functional analysis of the human genome, including the quantification of differential gene expression and the identification of polymorphic sites and disease genes, is an important element of the Human Genome Project. Current methods of analysis are mainly gel-based assays that are not well- suited to rapid genome-scale analyses. To analyze DNA sequence on a large scale, robust and high throughput assays are needed. We are developing a suite of microsphere-based approaches employing fluorescence detection to screen and analyze genomic sequence. Our approaches include competitive DNA hybridization to measure DNA or RNA targets in unknown samples, and oligo ligation or extension assays to analyze single-nucleotide polymorphisms. Apart from the advantages of sensitivity, simplicity, and low sample consumption, these flow cytometric approaches have the potential for high throughput multiplexed analysis using multicolored microspheres and automated sample handling.

  17. Hyperspectral cytometry.

    PubMed

    Grégori, Gérald; Rajwa, Bartek; Patsekin, Valery; Jones, James; Furuki, Motohiro; Yamamoto, Masanobu; Paul Robinson, J

    2014-01-01

    Hyperspectral cytometry is an emerging technology for single-cell analysis that combines ultrafast optical spectroscopy and flow cytometry. Spectral cytometry systems utilize diffraction gratings or prism-based monochromators to disperse fluorescence signals from multiple labels (organic dyes, nanoparticles, or fluorescent proteins) present in each analyzed bioparticle onto linear detector arrays such as multianode photomultipliers or charge-coupled device sensors. The resultant data, consisting of a series of characterizing every analyzed cell, are not compensated by employing the traditional cytometry approach, but rather are spectrally unmixed utilizing algorithms such as constrained Poisson regression or non-negative matrix factorization. Although implementations of spectral cytometry were envisioned as early as the 1980s, only recently has the development of highly sensitive photomultiplier tube arrays led to design and construction of functional prototypes and subsequently to introduction of commercially available systems. This chapter summarizes the historical efforts and work in the field of spectral cytometry performed at Purdue University Cytometry Laboratories and describes the technology developed by Sony Corporation that resulted in release of the first commercial spectral cytometry system-the Sony SP6800. A brief introduction to spectral data analysis is also provided, with emphasis on the differences between traditional polychromatic and spectral cytometry approaches. PMID:24271566

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

    PubMed Central

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

    2011-01-01

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

  19. Real-time on-line flow cytometry for bioprocess monitoring.

    PubMed

    Broger, Tobias; Odermatt, Res P; Huber, Pascal; Sonnleitner, Bernhard

    2011-07-20

    As the understanding of variation is the key to a good process and product quality one should pay attention to dynamics on the single-cell level. The basic idea of this approach was to qualify and quantify variations on the single-cell level during bioreactor cultivations by monitoring the expression of an eGFP tagged target protein (human membrane protein) using fully automated real-time, flow injection flow cytometry (FI-FCM). The FI-FCM system consists of a sampling- and defoaming- as well as of a dilution-section. It allows a very short monitoring interval (5 min) and is able to dilute the reactor sample by a factor ranging up to more than 10,000. In bioreactor cultivations of recombinant Pichia pastoris expressing the eGFP tagged target protein, high correlations (R(2)≥ 0.97) between the FI-FCM fluorescent signal and other, however, population-averaged fluorescence signals (off-line fluorescence, in situ fluorescence probe) were obtained. FI-FCM is the only method able to distinguish between few cells with high fluorescence and many cells with low fluorescence intensity and proved that cells differ significantly from each other within the population during bioreactor cultivations. Single-cell fluorescence was distributed over a broad range within the cell population. These distributions strongly suggest that (a) the AOX-I promoter is leaky and (b) a fraction of the population is able to express more protein of interest within shorter time and (c) a fraction of the population does not express the fusion protein at all. These findings can help in the selection of high producing, stable strains. To show the platform-independency of the system, it has successfully been tested during bioreactor cultivations of three different strains (P. pastoris, Saccharomyces cerevisiae, Escherichia coli). Along with its applications in PAT, the FI-FCM could be used as a platform-independent (prokaryotes and eukaryotes) method in various other applications; for example in the

  20. A flow injection flow cytometry system for on-line monitoring of bioreactors.

    PubMed

    Zhao, R; Natarajan, A; Srienc, F

    1999-03-01

    For direct and on-line study of the physiological states of cell cultures, a robust flow injection system has been designed and interfaced with flow cytometry (FI-FCM). The core of the flow injection system includes a microchamber designed for sample processing. The design of this microchamber allows not only an accurate on-line dilution but also on-line cell fixation, staining, and washing. The flow injection part of the system was tested by monitoring the optical density of a growing E.coli culture on-line using a spectrophotometer. The entire growth curve, from lag phase to stationary phase, was obtained with frequent sampling. The performance of the entire FI-FCM system is demonstrated in three applications. The first is the monitoring of green fluorescent protein fluorophore formation kinetics in E.coli by visualizing the fluorescence evolution after protein synthesis is inhibited. The data revealed a subpopulation of cells that do not become fluorescent. In addition, the data show that single-cell fluorescence is distributed over a wide range and that the fluorescent population contains cells that are capable of reaching significantly higher expression levels than that indicated by the population average. The second application is the detailed flow cytometric evaluation of the batch growth dynamics of E.coli expressing Gfp. The collected single-cell data visualize the batch growth phases and it is shown that a state of balanced growth is never reached by the culture. The third application is the determination of distribution of DNA content of a S. cerevisiae population by automatically staining cells using a DNA-specific stain. Reproducibility of the on-line staining reaction shows that the system is not restricted to measuring the native properties of cells; rather, a wider range of cellular components could be monitored after appropriate sample processing. The system is thus particularly useful because it operates automatically without direct operator

  1. Telomere analysis by fluorescence in situ hybridization and flow cytometry.

    PubMed Central

    Hultdin, M; Grönlund, E; Norrback, K; Eriksson-Lindström, E; Just, T; Roos, G

    1998-01-01

    Determination of telomere length is traditionally performed by Southern blotting and densitometry, giving a mean telomere restriction fragment (TRF) value for the total cell population studied. Fluorescence in situ hybridization (FISH) of telomere repeats has been used to calculate telomere length, a method called quantitative (Q)-FISH. We here present a quantitative flow cytometric approach, Q-FISHFCM, for evaluation of telomere length distribution in individual cells based on in situ hybridization using a fluorescein-labeled peptide nucleic acid (PNA) (CCCTAA)3probe and DNA staining with propidium iodide. A simple and rapid protocol with results within 30 h was developed giving high reproducibility. One important feature of the protocol was the use of an internal cell line control, giving an automatic compensation for potential differences in the hybridization steps. This protocol was tested successfully on cell lines and clinical samples from bone marrow, blood, lymph nodes and tonsils. A significant correlation was found between Southern blotting and Q-FISHFCMtelomere length values ( P = 0.002). The mean sub-telomeric DNA length of the tested cell lines and clinical samples was estimated to be 3.2 kbp. With the Q-FISHFCMmethod the fluorescence signal could be determined in different cell cycle phases, indicating that in human cells the vast majority of telomeric DNA is replicated early in S phase. PMID:9685479

  2. 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

  3. Non-Linear Optical Flow Cytometry Using a Scanned, Bessel Beam Light-Sheet

    PubMed Central

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

    2015-01-01

    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. PMID:26021750

  4. Label-free cell cycle analysis for high-throughput imaging flow cytometry

    PubMed Central

    Blasi, Thomas; Hennig, Holger; Summers, Huw D.; Theis, Fabian J.; Cerveira, Joana; Patterson, James O.; Davies, Derek; Filby, Andrew; Carpenter, Anne E.; Rees, Paul

    2016-01-01

    Imaging flow cytometry combines the high-throughput capabilities of conventional flow cytometry with single-cell imaging. Here we demonstrate label-free prediction of DNA content and quantification of the mitotic cell cycle phases by applying supervised machine learning to morphological features extracted from brightfield and the typically ignored darkfield images of cells from an imaging flow cytometer. This method facilitates non-destructive monitoring of cells avoiding potentially confounding effects of fluorescent stains while maximizing available fluorescence channels. The method is effective in cell cycle analysis for mammalian cells, both fixed and live, and accurately assesses the impact of a cell cycle mitotic phase blocking agent. As the same method is effective in predicting the DNA content of fission yeast, it is likely to have a broad application to other cell types. PMID:26739115

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

    PubMed

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

    2015-01-01

    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. PMID:26021750

  6. New fluorogenic dyes for analysis of cellular processes by flow cytometry and confocal microscopy.

    PubMed

    Nikolova, Kalina; Kaloyanova, Stefka; Mihaylova, Nikolina; Stoitsova, Stoyanka; Chausheva, Stela; Vasilev, Aleksey; Lesev, Nedyalko; Dimitrova, Petya; Deligeorgiev, Todor; Tchorbanov, Andrey

    2013-12-01

    Fluorescent microscopy and fluorescent imaging by flow cytometry are two of the fastest growing areas in the medical and biological research. Innovations in fluorescent chemistry and synthesis of new dye probes are closely related to the development of service equipment such as light sources, and detection techniques. Among compounds known as fluorescent labels, the cyanine-based dyes have become widely used since they have high excitation coefficients, narrow emission bands and high fluorescence upon binding to nucleic acids. The key methods for evaluation of apoptosis and cell cycle allow measuring DNA content by several flow cytometric techniques. We have synthesized new monomethine cyanine dyes and have characterized their applicability for staining of live and/or apoptotic cells. Imaging experiments by flow cytometry and confocal laser scanning microscopy (CLSM) have been also performed. Two of the dyes have shown high-affinity binding to the nuclei at high dilutions, up to 10(-9)M. Flow cytometry and CLSM have confirmed that these dyes labeled selectively non-living, e.g. ethanol-fixed cells that makes them appropriate for estimations of cell viability and apoptosis. The novel structures proved to be appropriate also for analysis of the cell cycle. PMID:24231377

  7. Application of flow cytometry to studies of pathogenic free-living amoebae.

    PubMed Central

    Muldrow, L L; Tyndall, R L; Fliermans, C B

    1982-01-01

    Species of small, free-living amoebae of the genera Naegleria and Acanthamoeba can cause fatal amoebic meningoencephalitis. Previous investigations have shown that pathogenic amoebae are associated with thermally altered water. Flow cytometric techniques for identifying species of pathogenic and nonpathogenic amoebae from such water have been developed, using immunofluorescence and fluorescein-bound concanavalin A. Flow cytometry is accomplished with a cytofluorograph, in which cells are dispersed in a suspended carrier liquid and passed in front of a focused argon ion laser beam. Cells are then distinguished by the degree of scattered light (size) or fluorescence. Flow cytometry techniques have proven efficient for environmental samples, as indicated by the identification of pathogenic Naegleria fowleri and nonpathogenic Naegleri gruberi and Acanthamoeba castellanii isolated from the Savannah River Plant in South Carolina. Cytofluorographic analysis of environmental samples has several advantages over the current methods of isolation and classification of free-living amoebae. With this system, it is possible to rapidly identify species and quantitate mixtures of pathogenic amoebae in environmental samples. Cytofluorographic analysis of amoebic isolates reduces the time presently required to screen environmental sites for pathogenic amoebae. The cytofluorograph permits detection and species identification of nonthermophilic Naegleria spp. and Acanthamoeba spp. that could not easily be isolated for species identification by conventional methods. Other advantages of flow cytometry over fluorescent microscopy include a high degree of statistical precision due to the large numbers measured, high immunofluorescent titers, and elimination of subjectivity and fluorescence fading. PMID:6186196

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

    PubMed

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

    2016-01-01

    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. PMID:27596736

  9. 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

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

    PubMed Central

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

    2015-01-01

    Recognition, binding, internalization, and elimination of pathogens and cell debris are important functions of professional as well as non-professional phagocytes. However, high-throughput methods for quantifying cell-associated particles and discriminating bound from internalized particles have been lacking. Here we describe a protocol for using imaging flow cytometry to quantify the attached and phagocytosed particles that are associated with a population of cells. Cells were exposed to fluorescent particles, fixed, and exposed to an antibody of a different fluorophore that recognizes the particles. The antibody is added without cell permeabilization, such that the antibody only binds extracellular particles. Cells with and without associated particles were identified by imaging flow cytometry. For each cell with associated particles, a spot count algorithm was employed to quantify the number of extracellular (double fluorescent) and intracellular (single fluorescent) particles per cell, from which the percent particle internalization was determined. The spot count algorithm was empirically validated by examining the fluorescence and phase contrast images acquired by the flow cytometer. We used this protocol to measure binding and internalization of the bacterium Neisseria gonorrhoeae by primary human neutrophils, using different bacterial variants and under different cellular conditions. The results acquired using imaging flow cytometry agreed with findings that were previously obtained using conventional immunofluorescence microscopy. This protocol provides a rapid, powerful method for measuring the association and internalization of any particle by any cell type. PMID:25967947

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

    PubMed Central

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

    2016-01-01

    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. PMID:27596736

  12. 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.

  13. Multi-parameter flow cytometry for discovery of disease mechanisms in rheumatic diseases

    PubMed Central

    Soloski, Mark J.; Chrest, Francis J.

    2013-01-01

    Flow cytometry has emerged as an essential tool for investigators in the study of the complexity of the immune system and the examination of its role in human health and disease. This technology has developed to the point where one can readily generate a large descriptive data set that details the levels of important immune cell subsets and defines an individual immune cell signature or “Immune-cellome”. This immune cell signature would clearly display individual variation but also would change in a manner reflective of disease state. Analysis of the “immune-cellome” may provide novel insight into disease pathophysiology, provide new biomarkers of disease activity and perhaps identify therapeutic targets. In this brief review we will cover current advances in complex flow cytometry and suggest ways this may be applied to the study of rheumatic diseases. PMID:23334929

  14. AutoGate: A Macintosh cluster analysis program for flow cytometry data

    SciTech Connect

    Salzman, G.C.; Parson, J.D.; Beckman, R.J. ); Stewart, S.J.; Stewart, C.C. )

    1993-01-01

    AutoGate, a cluster analysis program for Flow Cytometry Standard Data, has been developed for use on the Macintosh computer. AutoGate reads FCS format list mode files. It partitions the list mode events into a user-selected number of populations using K-means cluster analysis. One or more of the populations can be displayed as colored, bivariate dot plots. Eight variate data and up to twelve clusters can be analyzed. The dot plots can be saved as PICT format files. Data for individual clusters can be saved as FCS or ASCII format files. AutoGate is available from the authors through the National Flow Cytometry and Sorting Research Resource at Los Alamos.

  15. Evolution of flow cytometry standard, FCS3.0, into a DICOM-compatible format

    NASA Astrophysics Data System (ADS)

    Leif, Robert C.; Leif, Suzanne B.

    1997-05-01

    The International Society for Analytical Cytology, ISAC, has developed a flow cytometry standard (FCS) to permit data interchange, ISAC will soon replace FCS 2.0 with FCS 3.0. Unfortunately, the proposed FCS 3.0 is still fraught with problems, which are of sufficient magnitude as to warrant its early replacement. The most reasonable replacement is as a supplement to the digital imaging and communications in medicine, DICOM 3.0, standard. The recent digital microscopy extension of DICOM can be extended and modified to include flow cytometry data. DICOM includes: image graphics objects, specifications for describing: studies, reports, the acquisition of the data and the individuals involved, physician, patient, etc. Storing the present FCS data in a database, which has already been accomplished with the QC tracker software, will facilitate the transition of FCS to DICOM.

  16. Monitoring of microbial communities by flow cytometry and rRNA-targeted hybridization probes

    NASA Astrophysics Data System (ADS)

    Wallner, Guenter; Amann, Rudolf

    1995-10-01

    Flow cytometry in combination with ribosomal RNA (rRNA) based fluorescence in situ hybridization is a new technique for the analysis of microbial communities. Oligonucleotide probes directed against ribosomal RNA allow the identification of species or groups of micro- organisms. Combined with flow cytometry, up to several thousand cells per second can be classified. In addition to the identification and specific enumeration of micro-organisms, further information on the distribution of cell size, DNA and ribosome content -- and therefore an assessment of activity -- within the entire community or subpopulations can be obtained. This technique is much more accurate, informative, and rapid than classical culture-dependent methods. Data of activated sludge samples hybridized with fluorescein labeled oligonucleotides and counterstained with the DNA-specific dye Hoechst 33342 are presented as examples for its applicability to complex microbial communities.

  17. Antimicrobial properties of biosynthesized silver nanoparticles studied by flow cytometry and related techniques.

    PubMed

    Railean-Plugaru, Viorica; Pomastowski, Pawel; Rafinska, Katarzyna; Wypij, Magdalena; Kupczyk, Wojciech; Dahm, Hanna; Jackowski, Marek; Buszewski, Boguslaw

    2016-03-01

    This work reports the effect of silver bionanoparticles (Bio(AgNPs) synthesized by Actinobacteria CGG 11n on selected Gram (+) and Gram (-) bacteria. Flow cytometry, classical antibiogram method and fluorescent microscopy approach was used for evaluation of antimicrobial activity of Bio(AgNPs) and their combination with antibiotics. Furthermore, the performed research specified the capacity of flow cytometry method as an alternative to the standard ones and as a complementary method to electromigration techniques. The study showed antibacterial activity of both BioAgNPs and the combination of antibiotics/BioAgNPs against all the tested bacteria strains in comparison with a diffusion, dilution and bioautographic methods. The synergistic effect of antibiotics/BioAgNPs combination (e.g. kanamycin, ampicillin, neomycin and streptomycin) was found to be more notable against Pseudomonas aeruginosa representing a prototype of multi-drug resistant "superbugs" for which effective therapeutic options are very limited. PMID:26763104

  18. In Vivo Monitoring of Multiple Circulating Cell Populations Using Two-photon Flow Cytometry

    PubMed Central

    Tkaczyk, Eric R.; Zhong, Cheng Frank; Ye, Jing Yong; Myc, Andrzej; Thomas, Thommey; Cao, Zhengyi; Duran-Struuck, Raimon; Luker, Kathryn E.; Luker, Gary D.; Norris, Theodore B.; Baker, James R.

    2008-01-01

    To detect and quantify multiple distinct populations of cells circulating simultaneously in the blood of living animals, we developed a novel optical system for two-channel, two-photon flow cytometry in vivo. We used this system to investigate the circulation dynamics in live animals of breast cancer cells with low (MCF-7) and high (MDA-MB-435) metastatic potential, showing for the first time that two different populations of circulating cells can be quantified simultaneously in the vasculature of a single live mouse. We also non-invasively monitored a population of labeled, circulating red blood cells for more than two weeks, demonstrating that this technique can also quantify the dynamics of abundant cells in the vascular system for prolonged periods of time. These data are the first in vivo application of multichannel flow cytometry utilizing two-photon excitation, which will greatly enhance our capability to study circulating cells in cancer and other disease processes. PMID:19221581

  19. Application of flow cytometry in diagnosing lymphomas in dogs and cats.

    PubMed

    Aniołek, Olga; Gajewski, Zdzisław; Giziński, Sławomir

    2014-01-01

    Classification of types of lymphomas is done by interpreting cell morphology results obtained in cytological and/or histological examinations. In recent years, additional methods like immunocytochemistry (ICC), immunohistochemistry (IHC), immunophenotyping by flow cytometry and polymerase chain reaction (PCR) for antigen receptor rearrangements (PARR), have been used to diagnose and classify lymphomas. Unfortunately, none of these methods is completely specific and sensitive. Thus, a combination of several diagnostic methods or use of all available techniques allows for evaluation of morphological properties of cells like their maturity and diversification. Owing to the use of sets of antibodies it is possible to identify the phenotype of hyperplastic cells as well as their origin. Combination of results obtained through phenotypical analysis with flow cytometry examination with morphological, histological and genetic testing enables a detailed analysis of, in this case, lymphoproliferative diseases including reaction changes, primary and secondary immunological deficits as well as autoimmune diseases. PMID:26155143

  20. Use of Flow Cytometry to Measure Biogeochemical Rates and Processes in the Ocean

    NASA Astrophysics Data System (ADS)

    Lomas, Michael W.; Bronk, Deborah A.; van den Engh, Ger

    2011-01-01

    An important goal of marine biogeochemists is to quantify the rates at which elements cycle through the ocean's diverse microbial assemblage, as well as to determine how these rates vary in time and space. The traditional view that phytoplankton are producers and bacteria are consumers has been found to be overly simplistic, and environmental metagenomics is discovering new and important microbial metabolisms at an accelerating rate. Many nutritional strategies previously attributed to one microorganism or functional group are also or instead carried out by other groups. To tease apart which organism is doing what will require new analytical approaches. Flow cytometry, when combined with other techniques, has great potential for expanding our understanding of microbial interactions because groups can be distinguished optically, sorted, and then collected for subsequent analyses. Herein, we review the advances in our understanding of marine biogeochemistry that have arisen from the use of flow cytometry.

  1. A comparative study of frozen-section immunoperoxidase and flow cytometry for immunophenotypic analysis of lymph node biopsies.

    PubMed Central

    Biesemier, K W; Dent, G A; Pryzwansky, K B; Folds, J D

    1994-01-01

    Immunophenotyping by flow cytometry and frozen-section immunoperoxidase was compared on 21 consecutive lymph node biopsy specimens, of which a diagnosis of lymphoma was made for 11 specimens. Samples for flow cytometry were obtained by a fine-needle aspiration technique. Concordance between frozen-section immunoperoxidase and flow cytometry for all routine markers on all specimens ranged from 76 to 100%. In general, B-cell markers showed poorer concordance than T-cell markers, with kappa and lambda light chains having the poorest concordance, at 76% each. Flow cytometry was significantly more sensitive (90 versus 30%; P < 0.006) and had a significantly higher negative predictive value (100 versus 63%; P < 0.006) than frozen-section immunoperoxidase for demonstrating light-chain restriction. There was no significant difference in the specificities (100 versus 91%) or positive predictive values (100% each) between the two methods. Both methods demonstrated characteristic immunophenotypes for intermediate cell lymphomas, small lymphocytic lymphomas, and T-cell lymphoblastic lymphomas. Frozen-section immunoperoxidase and flow cytometry appear to be significantly concordant methods for immunophenotypic analysis of lymph node biopsies. Light-chain restriction is more readily demonstrated by flow cytometry than frozen-section immunoperoxidase. We believe that ex vivo fine-needle aspiration is a simple and reliable method of obtaining cell suspensions of lymph nodes for flow cytometry. PMID:7496966

  2. Assessment of the infectivity potential of Leishmania infantum, using flow cytometry.

    PubMed

    Kanellopoulos, Panagiotis; Dokianakis, Emmanouil; Tsirigotakis, Nikolaos; Koutala, Eleni; Antoniou, Maria

    2014-10-01

    The protozoan parasite Leishmania infantum causes leishmaniases, a sandfly-borne disease of humans and dogs, in all countries of the Mediterranean basin. The promastigote, infective stage of the parasite, once inoculated to the mammalian host by the vector, is ingested by macrophages. Leishmania lives within the lysosome of the phagocytic immune cells inactivating the enzymes contained. The ability of an isolate to survive within the macrophage and its rate of multiplication in this environment is an important factor determining the infectivity potential of the isolate and the manifestation of the disease. This capacity of the parasite is measured as the percentage of infected cells and the mean value of parasites per cell. The infectivity potential, of clinical isolates of L. infantum infecting THP-1 cells in vitro, was studied by flow cytometry and light microscopy. The percentages of cells in a sample containing a specific number of parasites, as recorded by light microscopy, were used in flow cytometry to manually gate the mean fluorescence intensity which corresponded to the percentage of cells with that number of parasites. The gating obtained, was then used as a "standard reference curve" to evaluate results by flow cytometry compared to those obtained by light microscopy. The results, of the overall percentage of infected cells and the number of parasites per cell in the culture, matched in the two methods. So, flow cytometry can be used as a rapid, cost effective, easy and reproducible method to study the infectivity potential of isolates, either in biological, epidemiological, or clinical tests, particularly for the assessment of drug efficiency trials. PMID:25045849

  3. Demonstration of cytoplasmic and nuclear antigens in acute leukaemia using flow cytometry.

    PubMed Central

    Farahat, N; van der Plas, D; Praxedes, M; Morilla, R; Matutes, E; Catovsky, D

    1994-01-01

    AIMS--To detect cytoplasmic and nuclear antigens using flow cytometry in acute leukaemia and to use this technique for double marker combinations. METHODS--Cytoplasmic staining was carried out in samples from 40 cases of acute leukaemia with monoclonal antibodies against the myeloid antigen CD13, the lymphoid antigens CD3, CD22, mu chain and the enzymes terminal deoxynucleotidyl transferase (TdT) and myeloperoxidase (MPO). The cells were fixed with paraformaldehyde and permeabilised with Tween 20 and Becton Dickinson's FACS lysing solution. Flow cytometry results were compared in the same cases with immunocytochemistry results using the alkaline phosphatase anti-alkaline phosphatase method. RESULTS--The gentle permeabilisation induced by this method permitted preservation of the membrane antigens and the size and morphology of the cells. The results using flow cytometry were comparable with those obtained using immunocytochemistry, with nearly complete concordance in most cases. CONCLUSIONS--This technique is simple, rapid, sensitive and reproducible and it is suitable for double staining procedures, such as nuclear and cytoplasmic, nuclear and membrane, or cytoplasmic and membrane. It therefore provides a powerful tool for extending the use of immunophenotyping for the diagnosis and follow up of acute leukaemia. It could also be used for the investigation of minimal residual disease. PMID:7962655

  4. 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).

  5. Flow cytometry community fingerprinting and amplicon sequencing for the assessment of landfill leachate cellulolytic bioaugmentation.

    PubMed

    Kinet, R; Dzaomuho, P; Baert, J; Taminiau, B; Daube, G; Nezer, C; Brostaux, Y; Nguyen, F; Dumont, G; Thonart, P; Delvigne, F

    2016-08-01

    Flow cytometry (FCM) is a high throughput single cell technology that is actually becoming widely used for studying phenotypic and genotypic diversity among microbial communities. This technology is considered in this work for the assessment of a bioaugmentation treatment in order to enhance cellulolytic potential of landfill leachate. The experimental results reveal the relevant increase of leachate cellulolytic potential due to bioaugmentation. Cytometric monitoring of microbial dynamics along these assays is then realized. The flow FP package is used to establish microbial samples fingerprint from initial 2D cytometry histograms. This procedure allows highlighting microbial communities' variation along the assays. Cytometric and 16S rRNA gene sequencing fingerprinting methods are then compared. The two approaches give same evidence about microbial dynamics throughout digestion assay. There are however a lack of significant correlation between cytometric and amplicon sequencing fingerprint at genus or species level. Same phenotypical profiles of microbiota during assays matched to several 16S rRNA gene sequencing ones. Flow cytometry fingerprinting can thus be considered as a promising routine on-site method suitable for the detection of stability/variation/disturbance of complex microbial communities involved in bioprocesses. PMID:27160955

  6. Quantification of mitochondrial toxicity in HIV-infected individuals by quantitative PCR compared to flow cytometry

    PubMed Central

    Wagner, Thor A.; Lin, Chen-Han; Tobin, Nicole H.; Côté, Hélène C.F.; Sloan, Derek D.; Jerome, Keith R.; Frenkel, Lisa M.

    2014-01-01

    Background Non-invasive diagnostic assays to evaluate mitochondrial toxicity could have significant clinical utility for HIV-infected individuals on antiretroviral therapy (ART). Methods This study compared the ratio of mitochondrial to nuclear DNA determined by quantitative PCR to the ratio of mitochondrial to nuclear-encoded proteins by flow cytometry, in peripheral blood mononuclear cells from 73 HIV-infected individuals with and without risk factors for mitochondrial toxicity. Results PCR detected similar mitochondrial/nuclear DNA in HIV-infected individuals without a history of ART, and those receiving ART with lipodystrophy, lipoatrophy or a history of suspected lactic acidosis. However, the ratio was significantly greater in ART-untreated compared to those receiving either stavudine or didanosine. In contrast, flow cytometry did not detect any differences in mitochondrial/nuclear protein (1). There was no correlation between the assays (rho = −0.05, p = 0.65). Conclusions Assessment of the mitochondrial/nuclear DNA ratio by quantitative PCR performed better than the mitochondrial/nuclear-encoded protein ratio by flow cytometry to detect adverse effects of nucleoside analogues on mitochondria. PMID:23044657

  7. Monitoring antibacterial permeabilization in real time using time-resolved flow cytometry.

    PubMed

    Freire, João Miguel; Gaspar, Diana; de la Torre, Beatriz Garcia; Veiga, Ana Salomé; Andreu, David; Castanho, Miguel A R B

    2015-02-01

    Despite the intensive study of antibiotic-induced bacterial permeabilization, its kinetics and molecular mechanism remain largely elusive. A new methodology that extends the concept of the live-dead assay in flow cytometry to real time-resolved detection was used to overcome these limitations. The antimicrobial activity of pepR was monitored in time-resolved flow cytometry for three bacterial strains: Escherichia coli (ATCC 25922), E. coli K-12 (CGSC Strain 4401) and E. coli JW3596-1 (CGSC Strain 11805). The latter strain has truncated lipopolysaccharides (LPS) in the outer membrane. This new methodology provided information on the efficacy of the antibiotics and sheds light on their mode of action at membrane-level. Kinetic data regarding antibiotic binding and lytic action were retrieved. Membrane interaction and permeabilization events differ significantly among strains. The truncation of LPS moieties does not hamper AMP binding but compromises membrane disruption and bacterial killing. We demonstrated the usefulness of time-resolved flow cytometry to study antimicrobial-induced permeabilization by collecting kinetic data that contribute to characterize the action of antibiotics directly on bacteria. PMID:25445678

  8. Discovering cell types in flow cytometry data with random matrix theory

    NASA Astrophysics Data System (ADS)

    Shen, Yang; Nussenblatt, Robert; Losert, Wolfgang

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

  9. Procoagulant and platelet-derived microvesicle absolute counts determined by flow cytometry correlates with a measurement of their functional capacity

    PubMed Central

    Ayers, Lisa; Harrison, Paul; Kohler, Malcolm; Ferry, Berne

    2014-01-01

    Background Flow cytometry is the most commonly used technology to measure microvesicles (MVs). Despite reported limitations of this technique, MV levels obtained using conventional flow cytometry have yielded many clinically relevant findings, such as associations with disease severity and ability to predict clinical outcomes. This study aims to determine if MV enumeration by flow cytometry correlates with a measurement of their functional capacity, as this may explain how flow cytometry generates clinically relevant results. Methods One hundred samples from healthy individuals and patients with obstructive sleep apnoea were analysed by conventional flow cytometry (FACSCalibur) and by three functional MV assays: Zymuphen MP-activity in which data were given as phosphatidylserine equivalent, STA® Phospholipid Procoag Assay expressed as clotting time and Endogenous Thrombin Potential (ETP) reflecting in vitro thrombin generation. Correlations were determined by Spearman correlation. Results Absolute counts of lactadherin+ procoagulant MVs generated by flow cytometry weakly correlated with the results obtained from the Zymuphen MP-activity (r=0.5370, p<0.0001); correlated with ETP (r=0.7444, p<0.0001); negatively correlated with STA® Phospholipid Procoag Assay clotting time (−0.7872, p<0.0001), reflecting a positive correlation between clotting activity and flow cytometry. Levels of Annexin V+ procoagulant and platelet-derived MVs were also associated with functional assays. Absolute counts of MVs derived from other cell types were not correlated with the functional results. Conclusions Quantitative results of procoagulant and platelet-derived MVs from conventional flow cytometry are associated with the functional capability of the MVs, as defined by three functional MV assays. Flow cytometry is a valuable technique for the quantification of MVs from different cellular origins; however, a combination of several analytical techniques may give the most comprehensive

  10. 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

  11. An improved protocol for flow cytometry analysis of phytoplankton cultures and natural samples.

    PubMed

    Marie, Dominique; Rigaut-Jalabert, Fabienne; Vaulot, Daniel

    2014-11-01

    Preservation of cells, choice of fixative, storage, and thawing conditions are recurrent issues for the analysis of phytoplankton by flow cytometry. We examined the effects of addition of the surfactant Pluronic F68 to glutaraldehyde-fixed photosynthetic organisms in cultures and natural samples. In particular, we examined cell losses and modifications of side scatter (a proxy of cell size) and fluorescence of natural pigments. We found that different marine phytoplankton species react differently to the action of Pluronic F68. In particular, photosynthetic prokaryotes are less sensitive than eukaryotes. Observed cell losses may result from cell lysis or from cell adhesion to the walls of plastic tubes that are commonly used for flow cytometry analysis. The addition of the surfactant, Pluronic F68, has a positive effect on cells for long-term storage. We recommend to modify current protocols for preservation of natural marine planktonic samples, by fixing them with glutaraldehyde 0.25% (final concentration) and adding Pluronic F68 at a final concentration of 0.01% in the samples before preservation. Pluronic F68 also appears effective for preserving samples without fixation for subsequent sorting, e.g. for molecular biology analyses. © 2014 International Society for Advancement of Cytometry. PMID:25155102

  12. Flow Cytometry Enables Multiplexed Measurements of Genetically Encoded Intramolecular FRET Sensors Suitable for Screening.

    PubMed

    Doucette, Jaimee; Zhao, Ziyan; Geyer, Rory J; Barra, Melanie M; Balunas, Marcy J; Zweifach, Adam

    2016-07-01

    Genetically encoded sensors based on intramolecular FRET between CFP and YFP are used extensively in cell biology research. Flow cytometry has been shown to offer a means to measure CFP-YFP FRET; we suspected it would provide a unique way to conduct multiplexed measurements from cells expressing different FRET sensors, which is difficult to do with microscopy, and that this could be used for screening. We confirmed that flow cytometry accurately measures FRET signals using cells transiently transfected with an ERK activity reporter, comparing responses measured with imaging and cytometry. We created polyclonal long-term transfectant lines, each expressing a different intramolecular FRET sensor, and devised a way to bar-code four distinct populations of cells. We demonstrated the feasibility of multiplexed measurements and determined that robust multiplexed measurements can be conducted in plate format. To validate the suitability of the method for screening, we measured responses from a plate of bacterial extracts that in unrelated experiments we had determined contained the protein kinase C (PKC)-activating compound teleocidin A-1. The multiplexed assay correctly identifying the teleocidin A-1-containing well. We propose that multiplexed cytometric FRET measurements will be useful for analyzing cellular function and for screening compound collections. PMID:26908592

  13. Flow cytometry and single nucleus sorting for Cre-based analysis of changes in transcriptional states.

    PubMed

    Samadder, Partha; Weng, Ning; Doetschman, Thomas; Heimark, Ronald L; Galbraith, David W

    2016-05-01

    The organs of eukaryotic organisms comprise complex interspersions of cell types, whose different molecular activities, and corresponding cellular states, cooperate during development to produce the final, functional organ. Dysfunction of organs in disease, particularly oncogenesis, initiates with changes of state of a minor subset of cells. It therefore is hard to detect early molecular indicators of disease within an overwhelming background of normal cells. Flow cytometry and sorting provides a convenient way to purify minority subpopulations, if a specific fluorophore can be unambiguously and exclusively associated with this subpopulation. We have generated a number of transgenic mouse lines expressing a nuclear-localized version of the Green Fluorescent Protein (GFP), within which the production of a chimeric histone 2B-GFP protein occurs under the control of a constitutively-active, actin-derived promoter, separated by a Floxed-STOP sequence. In the presence of Cre recombinase, within F1 progeny of these mouse lines, excision of the STOP sequence activates transcription which results in the emergence of cells containing green fluorescent nuclei. We describe the characterization of these lines using a combination of microscopic imaging, flow cytometry and sorting, and Reverse-Transcription polymerase chain reaction of transcripts within single sorted nuclei isolated from tissue homogenates. These lines should be particularly useful for analysis of transcriptional changes in oncogenesis. © 2016 International Society for Advancement of Cytometry. PMID:27003621

  14. Barrett's esophagus. Correlation between mucin histochemistry, flow cytometry, and histologic diagnosis for predicting increased cancer risk.

    PubMed Central

    Haggitt, R. C.; Reid, B. J.; Rabinovitch, P. S.; Rubin, C. E.

    1988-01-01

    A predominance of sulfated mucin in the nongoblet columnar cells of Barrett's specialized metaplastic epithelium has been postulated to be a form of mild dysplasia and to indicate an increased risk of adenocarcinoma. Flow cytometry for the analysis of nuclear DNA content and cell cycle parameters has also been postulated to be an objective aid in the diagnosis of dysplasia and carcinoma in Barrett's esophagus. The authors investigated the relationship among sulfated mucin, flow cytometric data, and histologic diagnosis in each of 152 biopsies from 42 patients who had Barrett's specialized metaplastic epithelium. Sulfated mucin, as detected by the high iron diamine-Alcian blue stain, was present in biopsies from 8 of 11 (73%) patients with the histologic diagnosis of dysplasia or carcinoma, in 7 of 9 (78%) patients whose biopsies were indefinite for dysplasia, and in 12 of 22 (55%) patients whose biopsies were negative for dysplasia (P = 0.37). Sulfated mucins predominated in 9%, 22%, and 9% of the patients, respectively (P = 0.56). Abnormal flow cytometry (aneuploidy or increased G2/tetraploid fraction) was found in all patients with the histologic diagnosis of dysplasia or carcinoma, in 3 of 9 (33%) indefinite for dysplasia, and in 1 of 22 (5%) negative for dysplasia (P = less than 0.0001). Neither the presence nor the predominance of sulfated mucin in the specialized metaplastic epithelium of Barrett's esophagus has sufficiently high sensitivity or specificity for dysplasia or carcinoma to be of value in managing patients. Abnormal flow cytometry shows excellent correlation with the histologic diagnosis of dysplasia and carcinoma; it detects a subset of patients whose biopsies are histologically indefinite or negative for dysplasia, but who have flow cytometric abnormalities similar to those otherwise seen only in dysplasia and carcinoma. Images Figure 1 Figure 2 Figure 3 PMID:3354644

  15. A high-throughput method for detection of DNA in chloroplasts using flow cytometry

    PubMed Central

    Rowan, Beth A; Oldenburg, Delene J; Bendich, Arnold J

    2007-01-01

    Background The amount of DNA in the chloroplasts of some plant species has been shown recently to decline dramatically during leaf development. A high-throughput method of DNA detection in chloroplasts is now needed in order to facilitate the further investigation of this process using large numbers of tissue samples. Results The DNA-binding fluorophores 4',6-diamidino-2-phenylindole (DAPI), SYBR Green I (SG), SYTO 42, and SYTO 45 were assessed for their utility in flow cytometric analysis of DNA in Arabidopsis chloroplasts. Fluorescence microscopy and real-time quantitative PCR (qPCR) were used to validate flow cytometry data. We found neither DAPI nor SYTO 45 suitable for flow cytometric analysis of chloroplast DNA (cpDNA) content, but did find changes in cpDNA content during development by flow cytometry using SG and SYTO 42. The latter dye provided more sensitive detection, and the results were similar to those from the fluorescence microscopic analysis. Differences in SYTO 42 fluorescence were found to correlate with differences in cpDNA content as determined by qPCR using three primer sets widely spaced across the chloroplast genome, suggesting that the whole genome undergoes copy number reduction during development, rather than selective reduction/degradation of subgenomic regions. Conclusion Flow cytometric analysis of chloroplasts stained with SYTO 42 is a high-throughput method suitable for determining changes in cpDNA content during development and for sorting chloroplasts on the basis of DNA content. PMID:17381841

  16. Deformation of double emulsions under conditions of flow cytometry hydrodynamic focusing.

    PubMed

    Ma, Shaohua; Huck, Wilhelm T S; Balabani, Stavroula

    2015-11-21

    Water-in-oil-in-water (w/o/w) microfluidics double emulsions offer a new route to compartmentalise reagents into isolated aqueous microenvironments while maintaining an aqueous carrier fluid phase; this enables compatibility with commercial flow cytometry systems such as fluorescence-activated cell sorting (FACS). Double emulsion (inner core) deformation under hydrodynamic focusing conditions that mimic the environment double emulsions experience in flow cytometry applications is of particular importance for droplet stability and cell viability. This paper reports on an experimental study of the dynamic deformation of aqueous cores of w/o/w double emulsions under hydrodynamic focusing, with the sheath flow directed at 45° to the sample flow. A number of factors affecting the inner core deformation and recovery were examined. Deformation was found to depend significantly on the core or shell viscosity, the droplet-to-sheath flow velocity ratio, and core and shell sizes. Core deformation was found to depend more on the type of surfactant rather concentration with high molecular weight surfactant exhibiting a negligible effect on deformation whereas low molecular weight surfactant enhancing deformation at low concentrations due to their lateral mobility at the interface. PMID:26394745

  17. Detection of an Abnormal Myeloid Clone by Flow Cytometry in Familial Platelet Disorder With Propensity to Myeloid Malignancy

    PubMed Central

    Ok, Chi Young; Leventaki, Vasiliki; Wang, Sa A.; Dinardo, Courtney; Medeiros, L. Jeffrey; Konoplev, Sergej

    2016-01-01

    Objectives To report aberrant myeloblasts detected by flow cytometry immunophenotypic studies in an asymptomatic patient with familial platelet disorder with propensity to myeloid malignancy, a rare autosomal dominant disease caused by germline heterozygous mutations in Runt-related transcription factor 1. Methods Morphologic evaluation, flow cytometry immunophenotypic studies, nanofluidics-based qualitative multiplex reverse transcriptase polymerase chain reaction, Sanger sequencing, and next-generation sequencing-based mutational hotspot analysis of 53 genes were performed on bone marrow biopsy and aspirate samples. Results Flow cytometry immunophenotypic analysis showed 0.6% CD34+ blasts with an abnormal immunophenotype: CD13 increased, CD33+, CD38 decreased, CD117 increased, and CD123 increased. Conclusions The acquisition of new phenotypic aberrancies in myeloblasts as detected by flow cytometry immunophenotypic studies might be a harbinger of impending myelodysplastic syndrome or acute myeloid leukemia in a patient with familial platelet disorder with propensity to myeloid malignancy. PMID:26800764

  18. Determination of cluster composition in heteroaggregation of binary particle systems by flow cytometry.

    PubMed

    Rollié, Sascha; Sundmacher, Kai

    2008-12-01

    Cluster composition in aggregation processes of multiple particle species can be dynamically determined by flow cytometry if particle populations are fluorescently labeled. By flow cytometric single particle analysis, aggregates can be characterized according to the exact amount of constituent particles, allowing the detailed and separate quantification of homo- and heteroaggregation. This contribution demonstrates the application of flow cytometry for the experimental detection of heteroaggregation in a binary particle mixture of oppositely charged polystyrene (PS) particles and Rhodamine-B labeled melamine-formaldehyde (MF-RhB) particles. Experiments with different particle concentration, temperature, mixing mode, ionic strength and particle mixing ratio are presented. Aggregation kinetics are enhanced with increasing particle concentration and temperature as well as by increased shear of mixing. These results represent well-known behavior published in previous investigations and validate the performance of flow cytometry for probing heteroaggregation processes. Physical insight with a novel level of detail is gained by the quantification of de- and restabilization phenomena. At low ionic strength, "raspberry"-type aggregates with PS cores are formed by primary heteroaggregation. At moderate particle number ratios, these aggregates are electrostatically destabilized and form more complex aggregates in a secondary heteroaggregation process. At high particle number ratios (> or =50:1), the raspberry-type aggregates are electrostatically restabilized and secondary heteroaggregation is prevented. The dynamic change of aggregate charge was verified by zeta-potential measurements. The elevation of salt concentration over several orders of magnitude retards aggregation dynamics, since attractive interparticle forces are diminished by an electrostatic double layer. This indicates that heteroaggregation induced by attractive interparticle forces is faster than aggregation

  19. Two New Nuclear Isolation Buffers for Plant DNA Flow Cytometry: A Test with 37 Species

    PubMed Central

    Loureiro, João; Rodriguez, Eleazar; Doležel, Jaroslav; Santos, Conceição

    2007-01-01

    Background and Aims After the initial boom in the application of flow cytometry in plant sciences in the late 1980s and early 1990s, which was accompanied by development of many nuclear isolation buffers, only a few efforts were made to develop new buffer formulas. In this work, recent data on the performance of nuclear isolation buffers are utilized in order to develop new buffers, general purpose buffer (GPB) and woody plant buffer (WPB), for plant DNA flow cytometry. Methods GPB and WPB were used to prepare samples for flow cytometric analysis of nuclear DNA content in a set of 37 plant species that included herbaceous and woody taxa with leaf tissues differing in structure and chemical composition. The following parameters of isolated nuclei were assessed: forward and side light scatter, propidium iodide fluorescence, coefficient of variation of DNA peaks, quantity of debris background, and the number of particles released from sample tissue. The nuclear genome size of 30 selected species was also estimated using the buffer that performed better for a given species. Key Results In unproblematic species, the use of both buffers resulted in high quality samples. The analysis of samples obtained with GPB usually resulted in histograms of DNA content with higher or similar resolution than those prepared with the WPB. In more recalcitrant tissues, such as those from woody plants, WPB performed better and GPB failed to provide acceptable results in some cases. Improved resolution of DNA content histograms in comparison with previously published buffers was achieved in most of the species analysed. Conclusions WPB is a reliable buffer which is also suitable for the analysis of problematic tissues/species. Although GPB failed with some plant species, it provided high-quality DNA histograms in species from which nuclear suspensions are easy to prepare. The results indicate that even with a broad range of species, either GPB or WPB is suitable for preparation of high

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

    PubMed Central

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

    2015-01-01

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

  1. Fluorogen Activating Proteins in Flow Cytometry for the Study of Surface Molecules and Receptors

    PubMed Central

    Saunders, Matthew J.; Szent-Gyorgyi, Christopher; Fisher, Gregory W.; Jarvik, Jonathan W.; Bruchez, Marcel P.; Waggoner, Alan S.

    2012-01-01

    The use of fluorescent proteins, particularly when genetically fused to proteins of biological interest, have greatly advanced many flow cytometry research applications. However, there remains a major limitation to this methodology in that only total cellular fluorescence is measured. Commonly used fluorescent proteins (e.g. EGFP and its variants) are fluorescent whether the fusion protein exists on the surface or in sub-cellular compartments. A flow cytometer cannot distinguish between these separate sources of fluorescence. This can be of great concern when using flow cytometry, plate readers or microscopy to quantify cell surface receptors or other surface proteins genetically fused to fluorescent proteins. Recently developed fluorogen activating proteins (FAPs) solve many of these issues by allowing the selective visualization of only those cell surface proteins that are exposed to the extra cellular milieu. FAPs are GFP-sized single chain antibodies that specifically bind to and generate fluorescence from otherwise non-fluorescent dyes (‘activate the fluorogen’). Like the fluorescent proteins, FAPs can be genetically fused to proteins of interest. When exogenously added fluorogens bind FAPs, fluorescence immediately increases by as much as 20,000 fold, rendering the FAP fusion proteins highly fluorescent. Moreover, since fluorogens can be made membrane impermeant, fluorescence can be limited to only those receptors expressed on the cell surface. Using cells expressing beta-2 adrenergic receptor (β2AR) fused at its N-terminus to a FAP, flow cytometry based receptor internalization assays have been developed and characterized. The fluorogen/FAP system is ideally suited to the study of cell surface proteins by fluorescence and avoids drawbacks of using receptor/fluorescent protein fusions, such as internal accumulation. We also briefly comment on extending FAP-based technologies to the study of events occurring inside of the cell as well. PMID:22366230

  2. Flow cytometry protocols for surface and intracellular antigen analyses of neural cell types.

    PubMed

    Menon, Vishal; Thomas, Ria; Ghale, Arun R; Reinhard, Christina; Pruszak, Jan

    2014-01-01

    Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of protocols for flow cytometric analysis of the cluster of differentiation (CD) surface antigens and intracellular antigens in neural cell types. Our step-by-step description of the methodological procedures include: the harvesting of neural in vitro cultures, an optional carboxyfluorescein succinimidyl ester (CFSE)-labeling step, followed by surface antigen staining with conjugated CD antibodies (e.g., CD24, CD54), and subsequent intracellar antigen detection via primary/secondary antibodies or fluorescently labeled Fab fragments (Zenon labeling). The video demonstrates the most critical steps. Moreover, principles of experimental planning, the inclusion of critical controls, and fundamentals of flow cytometric analysis (identification of target population and exclusion of debris; gating strategy; compensation for spectral overlap) are briefly explained in order to enable neurobiologists with limited prior knowledge or specific training in flow cytometry to assess its utility and to better exploit this powerful methodology. PMID:25549236

  3. Simultaneous evaluation of plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential in bovine spermatozoa by flow cytometry.

    PubMed

    Kanno, Chihiro; Kang, Sung-Sik; Kitade, Yasuyuki; Yanagawa, Yojiro; Takahashi, Yoshiyuki; Nagano, Masashi

    2016-08-01

    The present study aimed to develop an objective evaluation procedure to estimate the plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential of bull spermatozoa simultaneously by flow cytometry. Firstly, we used frozen-thawed semen mixed with 0, 25, 50, 75 or 100% dead spermatozoa. Semen was stained using three staining solutions: SYBR-14, propidium iodide (PI), and phycoerythrin-conjugated peanut agglutinin (PE-PNA), for the evaluation of plasma membrane integrity and acrosomal integrity. Then, characteristics evaluated by flow cytometry and by fluorescence microscopy were compared. Characteristics of spermatozoa (viability and acrosomal integrity) evaluated by flow cytometry and by fluorescence microscopy were found to be similar. Secondly, we attempted to evaluate the plasma membrane integrity, acrosomal integrity, and also mitochondrial membrane potential of spermatozoa by flow cytometry using conventional staining with three dyes (SYBR-14, PI, and PE-PNA) combined with MitoTracker Deep Red (MTDR) staining (quadruple staining). The spermatozoon characteristics evaluated by flow cytometry using quadruple staining were then compared with those of staining using SYBR-14, PI, and PE-PNA and staining using SYBR-14 and MTDR. There were no significant differences in all characteristics (viability, acrosomal integrity, and mitochondrial membrane potential) evaluated by quadruple staining and the other procedures. In conclusion, quadruple staining using SYBR-14, PI, PE-PNA, and MTDR for flow cytometry can be used to evaluate the plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential of bovine spermatozoa simultaneously. PMID:26369275

  4. A Flow Cytometry Method for Rapidly Assessing Mycobacterium tuberculosis Responses to Antibiotics with Different Modes of Action

    PubMed Central

    Hendon-Dunn, Charlotte Louise; Doris, Kathryn Sarah; Thomas, Stephen Richard; Allnutt, Jonathan Charles; Marriott, Alice Ann Neville; Hatch, Kim Alexandra; Watson, Robert James; Bottley, Graham; Marsh, Philip David; Taylor, Stephen Charles

    2016-01-01

    Current methods for assessing the drug susceptibility of Mycobacterium tuberculosis are lengthy and do not capture information about viable organisms that are not immediately culturable under standard laboratory conditions as a result of antibiotic exposure. We have developed a rapid dual-fluorescence flow cytometry method using markers for cell viability and death. We show that the fluorescent marker calcein violet with an acetoxy-methyl ester group (CV-AM) can differentiate between populations of M. tuberculosis growing at different rates, while Sytox green (SG) can differentiate between live and dead mycobacteria. M. tuberculosis was exposed to isoniazid or rifampin at different concentrations over time and either dual stained with CV-AM and SG and analyzed by flow cytometry or plated to determine the viability of the cells. Although similar trends in the loss of viability were observed when the results of flow cytometry and the plate counting methods were compared, there was a lack of correlation between these two approaches, as the flow cytometry analysis potentially captured information about cell populations that were unable to grow under standard conditions. The flow cytometry approach had an additional advantage in that it could provide insights into the mode of action of the drug: antibiotics targeting the cell wall gave a flow cytometry profile distinct from those inhibiting intracellular processes. This rapid drug susceptibility testing method could identify more effective antimycobacterials, provide information about their potential mode of action, and accelerate their progress to the clinic. PMID:26902767

  5. A cytometry microparticle platform approach for screening tobacco microRNA changes after agrobacterium delivery.

    PubMed

    Powell, Joshua D; Chen, Qiang; Mason, Hugh S

    2016-08-01

    MicroRNAs are a class of non-coding regulatory RNAs that can modulate development as well as alter innate antiviral defenses in plants. In this study we explored changes in Nicotiana benthamiana tobacco microRNA expression as it relates to expression of a recombinant anti-Ebola GP1 antibody. The antibody was delivered to tobacco leaves through a bacterial Agrobacterium tumefaciens "agroinfiltration" expression strategy. A multiplex microparticle-based cytometry assay tracked the expression changes of 53 host tobacco microRNAs. Our results revealed that the most abundant microRNAs in actively growing leaves corresponded to nanoparticle probes specific to nta-mir-6149 and nta-miR-168b. After agroinfiltration, probes specific for nta-mir-398, and nta-mir-482d were significantly altered in their respective expression levels, however changes were partially attributed to the infiltration broth medium used in the antibody delivery process. Confirmation of nta-mir-398 and nta-mir-482d expression changes was also verified through RT-qPCR. To our knowledge this study is the first to profile medium and Agrobacterium injection at the microRNA level through a multiplex microparticle approach. PMID:27343681

  6. 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

  7. 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

  8. Detection of Salmonella typhimurium in dairy products with flow cytometry and monoclonal antibodies.

    PubMed Central

    McClelland, R G; Pinder, A C

    1994-01-01

    Flow cytometry, combined with fluorescently labelled monoclonal antibodies, offers advantages of speed and sensitivity for the detection of specific pathogenic bacteria in foods. We investigated the detection of Salmonella typhimurium in eggs and milk. Using a sample clearing procedure, we determined that the detection limit was on the order of 10(3) cells per ml after a total analysis time of 40 min. After 6 h of nonselective enrichment, the detection limits were 10 cells per ml for milk and 1 cell per ml for eggs, even in the presence of a 10,000-fold excess of Escherichia coli cells. Images PMID:7811064

  9. Protocol for Apoptosis Assay by Flow Cytometry Using Annexin V Staining Method

    PubMed Central

    Lakshmanan, Imayavaramban; Batra, Surinder K

    2016-01-01

    This assay is used to count the number of cells that have undergone apoptosis. Apoptosis will be detected by initially staining the cells with Annexin V and propidium Iodide solution followed by flow cytometry analysis. It is based on the principle that normal cells are hydrophobic in nature as they express phosphatidyl serine in the inner membrane (side facing the cytoplasm) and when the cells undergo apoptosis, the inner membrane flips to become the outer membrane, thus exposing phosphatidyl serine. The exposed phosphatidyl serine is detected by Annexin V, and propidium iodide stains the necrotic cells, which have leaky DNA content that help to differentiate the apoptotic and necrotic cells.

  10. A comparison of avalanche photodiode and photomultiplier tube detectors for flow cytometry

    NASA Astrophysics Data System (ADS)

    Lawrence, William G.; Varadi, Gyula; Entine, Gerald; Podniesinski, Edward; Wallace, Paul K.

    2008-02-01

    Commercial flow cytometers use photomultiplier tubes (PMTs) for fluorescence detection. These detectors have high linear gain and broad dynamic range, but have limited sensitivity in the red and near infrared spectral regions. We present a comparison of avalanche photodiodes (APDs) and PMTs as detectors in flow cytometry instruments, and demonstrate improved sensitivity and resolution in the red and near infrared spectral regions using the APD. The relative performance of the PMT and APD were evaluated by simultaneously measuring the mean fluorescence intensity and coefficient of variation for emission from light emitting diode pulses, flow cytometry test beads, and fluorescently labeled cells. The relative signal to noise performance of the APD and PMT was evaluated over the 500 nm to 1050 nm wavelength range using pulsed light emitting diode light sources. While APDs have higher quantum efficiency but lower internal gain than PMTs, with appropriate external amplification the APD has signal to noise response that is comparable to PMTs in the 500 nm to 650 nm range and improved response in the 650 nm to 850 nm range The data demonstrates that the APD had performance comparable to the PMT in the spectral region between 500 to 650 nm and improved performance in the range of 650 to 1000 nm, where the PMT performance is quite poor. CD4 positive lymphocyte populations were easily identified in normal human blood both by APD and PMT using phycoerythrin labeled antibodies. In contrast, only the APD detector could resolve CD4 positive populations using 800 nm Quantum dot labeled antibodies.

  11. 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.

  12. Applications of flow cytometry in plant pathology for genome size determination, detection and physiological status.

    PubMed

    D'Hondt, Liesbet; Höfte, Monica; Van Bockstaele, Erik; Leus, Leen

    2011-10-01

    Flow cytometers are probably the most multipurpose laboratory devices available. They can analyse a vast and very diverse range of cell parameters. This technique has left its mark on cancer, human immunodeficiency virus and immunology research, and is indispensable in routine clinical diagnostics. Flow cytometry (FCM) is also a well-known tool for the detection and physiological status assessment of microorganisms in drinking water, marine environments, food and fermentation processes. However, flow cytometers are seldom used in plant pathology, despite FCM's major advantages as both a detection method and a research tool. Potential uses of FCM include the characterization of genome sizes of fungal and oomycete populations, multiplexed pathogen detection and the monitoring of the viability, culturability and gene expression of plant pathogens, and many others. This review provides an overview of the history, advantages and disadvantages of FCM, and focuses on the current applications and future possibilities of FCM in plant pathology. PMID:21726378

  13. An Improved Flow Cytometry Method For Precise Quantitation Of Natural-Killer Cell Activity

    NASA Technical Reports Server (NTRS)

    Crucian, Brian; Nehlsen-Cannarella, Sandra; Sams, Clarence

    2006-01-01

    The ability to assess NK cell cytotoxicity using flow cytometry has been previously described and can serve as a powerful tool to evaluate effector immune function in the clinical setting. Previous methods used membrane permeable dyes to identify target cells. The use of these dyes requires great care to achieve optimal staining and results in a broad spectral emission that can make multicolor cytometry difficult. Previous methods have also used negative staining (the elimination of target cells) to identify effector cells. This makes a precise quantitation of effector NK cells impossible due to the interfering presence of T and B lymphocytes, and the data highly subjective to the variable levels of NK cells normally found in human peripheral blood. In this study an improved version of the standard flow cytometry assay for NK activity is described that has several advantages of previous methods. Fluorescent antibody staining (CD45FITC) is used to positively identify target cells in place of membranepermeable dyes. Fluorescent antibody staining of target cells is less labor intensive and more easily reproducible than membrane dyes. NK cells (true effector lymphocytes) are also positively identified by fluorescent antibody staining (CD56PE) allowing a simultaneous absolute count assessment of both NK cells and target cells. Dead cells are identified by membrane disruption using the DNA intercalating dye PI. Using this method, an exact NK:target ratio may be determined for each assessment, including quantitation of NK target complexes. Backimmunoscatter gating may be used to track live vs. dead Target cells via scatter properties. If desired, NK activity may then be normalized to standardized ratios for clinical comparisons between patients, making the determination of PBMC counts or NK cell percentages prior to testing unnecessary. This method provides an exact cytometric determination of NK activity that highly reproducible and may be suitable for routine use in the

  14. Minimal residual disease monitoring by 8-color flow cytometry in mantle cell lymphoma: an EU-MCL and LYSA study.

    PubMed

    Cheminant, Morgane; Derrieux, Coralie; Touzart, Aurore; Schmit, Stéphanie; Grenier, Adrien; Trinquand, Amélie; Delfau-Larue, Marie-Hélène; Lhermitte, Ludovic; Thieblemont, Catherine; Ribrag, Vincent; Cheze, Stéphane; Sanhes, Laurence; Jardin, Fabrice; Lefrère, François; Delarue, Richard; Hoster, Eva; Dreyling, Martin; Asnafi, Vahid; Hermine, Olivier; Macintyre, Elizabeth

    2016-03-01

    Quantification of minimal residual disease may guide therapeutic strategies in mantle cell lymphoma. While multiparameter flow cytometry is used for diagnosis, the gold standard method for minimal residual disease analysis is real-time quantitative polymerase chain reaction (RQ-PCR). In this European Mantle Cell Lymphoma network (EU-MCL) pilot study, we compared flow cytometry with RQ-PCR for minimal residual disease detection. Of 113 patients with at least one minimal residual disease sample, RQ-PCR was applicable in 97 (86%). A total of 284 minimal residual disease samples from 61 patients were analyzed in parallel by flow cytometry and RQ-PCR. A single, 8-color, 10-antibody flow cytometry tube allowed specific minimal residual disease assessment in all patients, with a robust sensitivity of 0.01%. Using this cut-off level, the true-positive-rate of flow cytometry with respect to RQ-PCR was 80%, whereas the true-negative-rate was 92%. As expected, RQ-PCR frequently detected positivity below this 0.01% threshold, which is insufficiently sensitive for prognostic evaluation and would ideally be replaced with robust quantification down to a 0.001% (10-5) threshold. In 10 relapsing patients, the transition from negative to positive by RQ-PCR (median 22.5 months before relapse) nearly always preceded transition by flow cytometry (4.5 months), but transition to RQ-PCR positivity above 0.01% (5 months) was simultaneous. Pre-emptive rituximab treatment of 2 patients at minimal residual disease relapse allowed re-establishment of molecular and phenotypic complete remission. Flow cytometry minimal residual disease is a complementary approach to RQ-PCR and a promising tool in individual mantle cell lymphoma therapeutic management. (clinicaltrials identifiers: 00209209 and 00209222). PMID:26703963

  15. Minimal residual disease monitoring by 8-color flow cytometry in mantle cell lymphoma: an EU-MCL and LYSA study

    PubMed Central

    Cheminant, Morgane; Derrieux, Coralie; Touzart, Aurore; Schmit, Stéphanie; Grenier, Adrien; Trinquand, Amélie; Delfau-Larue, Marie-Hélène; Lhermitte, Ludovic; Thieblemont, Catherine; Ribrag, Vincent; Cheze, Stéphane; Sanhes, Laurence; Jardin, Fabrice; Lefrère, François; Delarue, Richard; Hoster, Eva; Dreyling, Martin; Asnafi, Vahid; Hermine, Olivier; Macintyre, Elizabeth

    2016-01-01

    Quantification of minimal residual disease may guide therapeutic strategies in mantle cell lymphoma. While multiparameter flow cytometry is used for diagnosis, the gold standard method for minimal residual disease analysis is real-time quantitative polymerase chain reaction (RQ-PCR). In this European Mantle Cell Lymphoma network (EU-MCL) pilot study, we compared flow cytometry with RQ-PCR for minimal residual disease detection. Of 113 patients with at least one minimal residual disease sample, RQ-PCR was applicable in 97 (86%). A total of 284 minimal residual disease samples from 61 patients were analyzed in parallel by flow cytometry and RQ-PCR. A single, 8-color, 10-antibody flow cytometry tube allowed specific minimal residual disease assessment in all patients, with a robust sensitivity of 0.01%. Using this cut-off level, the true-positive-rate of flow cytometry with respect to RQ-PCR was 80%, whereas the true-negative-rate was 92%. As expected, RQ-PCR frequently detected positivity below this 0.01% threshold, which is insufficiently sensitive for prognostic evaluation and would ideally be replaced with robust quantification down to a 0.001% (10-5) threshold. In 10 relapsing patients, the transition from negative to positive by RQ-PCR (median 22.5 months before relapse) nearly always preceded transition by flow cytometry (4.5 months), but transition to RQ-PCR positivity above 0.01% (5 months) was simultaneous. Pre-emptive rituximab treatment of 2 patients at minimal residual disease relapse allowed re-establishment of molecular and phenotypic complete remission. Flow cytometry minimal residual disease is a complementary approach to RQ-PCR and a promising tool in individual mantle cell lymphoma therapeutic management. PMID:26703963

  16. Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high-resolution flow cytometry.

    PubMed

    Groot Kormelink, Tom; Arkesteijn, Ger J A; Nauwelaers, Frans A; van den Engh, Ger; Nolte-'t Hoen, Esther N M; Wauben, Marca H M

    2016-02-01

    Submicron-sized vesicles released by cells are increasingly recognized for their role in intercellular communication and as biomarkers of disease. Methods for high-throughput, multi-parameter analysis of such extracellular vesicles (EVs) are crucial to further investigate their diversity and function. We recently developed a high-resolution flow cytometry-based method (using a modified BD Influx) for quantitative and qualitative analysis of EVs. The fact that the majority of EVs is <200 nm in size requires special attention with relation to specific conditions of the flow cytometer, as well as sample concentration and event rate. In this study, we investigated how (too) high particle concentrations affect high-resolution flow cytometry-based particle quantification and characterization. Increasing concentrations of submicron-sized particles (beads, liposomes, and EVs) were measured to identify coincidence and swarm effects, caused by the concurrent presence of multiple particles in the measuring spot. As a result, we demonstrate that analysis of highly concentrated samples resulted in an underestimation of the number of particles and an interdependent overestimation of light scattering and fluorescence signals. On the basis of this knowledge, and by varying nozzle size and sheath pressure, we developed a strategy for high-resolution flow cytometric sorting of submicron-sized particles. Using the adapted sort settings, subsets of EVs differentially labeled with two fluorescent antibodies could be sorted to high purity. Moreover, sufficient numbers of EVs could be sorted for subsequent analysis by western blotting. In conclusion, swarm effects that occur when measuring high particle concentrations severely hamper EV quantification and characterization. These effects can be easily overlooked without including proper controls (e.g., sample dilution series) or tools (e.g., oscilloscope). Providing that the event rate is well controlled, the sorting strategy we propose

  17. Diurnal Variations of Circulating Extracellular Vesicles Measured by Nano Flow Cytometry

    PubMed Central

    Tigges, John; Toxavidis, Vasilis; Camacho, Virginia; Felton, Edward J.; Khoory, Joseph; Kreimer, Simion; Ivanov, Alexander R.; Mantel, Pierre-Yves; Jones, Jennifer; Akuthota, Praveen; Das, Saumya; Ghiran, Ionita

    2016-01-01

    The identification of extracellular vesicles (EVs) as intercellular conveyors of biological information has recently emerged as a novel paradigm in signaling, leading to the exploitation of EVs and their contents as biomarkers of various diseases. However, whether there are diurnal variations in the size, number, and tissue of origin of blood EVs is currently not known, and could have significant implications when using EVs as biomarkers for disease progression. Currently available technologies for the measurement of EV size and number are either time consuming, require specialized equipment, or lack sufficient accuracy across a range of EV sizes. Flow cytometry represents an attractive alternative to these methods; however, traditional flow cytometers are only capable of measuring particles down to 500 nm, which is significantly larger than the average and median sizes of plasma EVs. Utilizing a Beckman Coulter MoFlo XDP flow cytometer with NanoView module, we employed nanoscale flow cytometry (termed nanoFCM) to examine the relative number and scatter distribution of plasma EVs at three different time points during the day in 6 healthy adults. Analysis of liposomes and plasma EVs proved that nanoFCM is capable of detecting biologically-relevant vesicles down to 100 nm in size. With this high resolution configuration, we observed variations in the relative size (FSC/SSC distributions) and concentration (proportions) of EVs in healthy adult plasma across the course of a day, suggesting that there are diurnal variations in the number and size distribution of circulating EV populations. The use of nanoFCM provides a valuable tool for the study of EVs in both health and disease; however, additional refinement of nanoscale flow cytometric methods is needed for use of these instruments for quantitative particle counting and sizing. Furthermore, larger scale studies are necessary to more clearly define the diurnal variations in circulating EVs, and thus further inform

  18. Discriminative variable subsets in Bayesian classification with mixture models, with application in flow cytometry studies.

    PubMed

    Lin, Lin; Chan, Cliburn; West, Mike

    2016-01-01

    We discuss the evaluation of subsets of variables for the discriminative evidence they provide in multivariate mixture modeling for classification. The novel development of Bayesian classification analysis presented is partly motivated by problems of design and selection of variables in biomolecular studies, particularly involving widely used assays of large-scale single-cell data generated using flow cytometry technology. For such studies and for mixture modeling generally, we define discriminative analysis that overlays fitted mixture models using a natural measure of concordance between mixture component densities, and define an effective and computationally feasible method for assessing and prioritizing subsets of variables according to their roles in discrimination of one or more mixture components. We relate the new discriminative information measures to Bayesian classification probabilities and error rates, and exemplify their use in Bayesian analysis of Dirichlet process mixture models fitted via Markov chain Monte Carlo methods as well as using a novel Bayesian expectation-maximization algorithm. We present a series of theoretical and simulated data examples to fix concepts and exhibit the utility of the approach, and compare with prior approaches. We demonstrate application in the context of automatic classification and discriminative variable selection in high-throughput systems biology using large flow cytometry datasets. PMID:26040910

  19. Synergy of photoacoustic and fluorescence flow cytometry of circulating cells with negative and positive contrasts.

    PubMed

    Nedosekin, Dmitry A; Sarimollaoglu, Mustafa; Galanzha, Ekaterina I; Sawant, Rupa; Torchilin, Vladimir P; Verkhusha, Vladislav V; Ma, Jie; Frank, Markus H; Biris, Alexandru S; Zharov, Vladimir P

    2013-05-01

    In vivo photoacoustic (PA) and fluorescence flow cytometry were previously applied separately using pulsed and continuous wave lasers respectively, and positive contrast detection mode only. This paper introduces a real-time integration of both techniques with positive and negative contrast modes using only pulsed lasers. Various applications of this new tool are summarized, including detection of liposomes loaded with Alexa-660 dye, red blood cells labeled with Indocyanine Green, B16F10 melanoma cells co-expressing melanin and green fluorescent protein (GFP), C8161-GFP melanoma cells targeted by magnetic nanoparticles, MTLn3 adenocarcinoma cells expressing novel near-infrared iRFP protein, and quantum dot-carbon nanotube conjugates. Negative contrast flow cytometry provided label-free detection of low absorbing or weakly fluorescent cells in blood absorption and autofluorescence background, respectively. The use of pulsed laser for time-resolved discrimination of objects with long fluorescence lifetime (e.g., quantum dots) from shorter autofluorescence background (e.g., blood plasma) is also highlighted in this paper. The supplementary nature of PA and fluorescence detection increased the versatility of the integrated method for simultaneous detection of probes and cells having various absorbing and fluorescent properties, and provided verification of PA data using a more established fluorescence based technique. PMID:22903924

  20. Using dual-laser flow cytometry for monitoring phytoplankton composition and integrity

    NASA Astrophysics Data System (ADS)

    Schaefer, Harald; Beisker, Wolfgang; Steinberg, Christian

    1995-10-01

    Dual laser flow cytometry can be used for determining phytoplankton populations in lakes and lowland rivers and streams. Apart from answering basic limnological questions such as the time course of algal blooms or the annual succession of phytoplankton composition further investigations can be made for estimating the integrity of a phytoplankton community using biomass distribution spectra. Thus anthropogenic influence such as eutrophication, acidification or effects of xenobiotica can be monitored. Dual laser flow cytometry with excitation wavelengths of 458 and 528 nm was used to measure photosynthesis pigment fluorescence (chlorophyll a (CHLa), Em greater than 665 nm) and phycoerythrin (PE, Em 575 nm) and cell density of phytoplankton organisms in water samples. CHLa is excited directly by 458 nm and by energy transfer from carotenoids (Ex 528 nm). The ratio of the two fluorescence parameters (CFR) allows users to identify pigment groups in the phytoplankton population (chlorophytes and euglenophytes from chrysophytes, diatoms and dinophytes). PE- containing cyanophytes and cryptophytes can be detected by their PE fluorescence (Ex 528 nm). As a result of preliminary studies for preparing biomass spectra of phytoplankton communities measurements of protein content by staining with fluorescein isothiocyanate (FITC, Ex 488 nm, Em 530 nm) are also shown.

  1. Evaluating the morphology of erythrocyte population: An approach based on atomic force microscopy and flow cytometry.

    PubMed

    Ghosh, Sayari; Chakraborty, Ishita; Chakraborty, Monojit; Mukhopadhyay, Ashis; Mishra, Raghwendra; Sarkar, Debasish

    2016-04-01

    Erythrocyte morphology is gaining importance as a powerful pathological index in identifying the severity of any blood related disease. However, the existing technique of quantitative microscopy is highly time consuming and prone to personalized bias. On the other hand, relatively unexplored, complementary technique based on flow cytometry has not been standardized till date, particularly due to the lack of a proper morphological scoring scale. In this article, we have presented a new approach to formulate a non-empirical scoring scale based on membrane roughness (R(rms)) data obtained from atomic force microscopy. Subsequently, the respective morphological quantifier of the whole erythrocyte population, commonly known as morphological index, was expressed as a function of highest correlated statistical parameters of scattered signal profiles generated by flow cytometry. Feed forward artificial neural network model with multilayer perceptron architecture was used to develop the intended functional form. High correlation coefficient (R(2) = 0.95), even for model-formulation exclusive samples, clearly indicates the universal validity of the proposed model. Moreover, a direct pathological application of the proposed model has been illustrated in relation to patients, diagnosed to be suffering from a wide variety of cancer. PMID:26824317

  2. Innovative Flow Cytometry Allows Accurate Identification of Rare Circulating Cells Involved in Endothelial Dysfunction

    PubMed Central

    Boraldi, Federica; Bartolomeo, Angelica; De Biasi, Sara; Orlando, Stefania; Costa, Sonia; Cossarizza, Andrea; Quaglino, Daniela

    2016-01-01

    Introduction Although rare, circulating endothelial and progenitor cells could be considered as markers of endothelial damage and repair potential, possibly predicting the severity of cardiovascular manifestations. A number of studies highlighted the role of these cells in age-related diseases, including those characterized by ectopic calcification. Nevertheless, their use in clinical practice is still controversial, mainly due to difficulties in finding reproducible and accurate methods for their determination. Methods Circulating mature cells (CMC, CD45-, CD34+, CD133-) and circulating progenitor cells (CPC, CD45dim, CD34bright, CD133+) were investigated by polychromatic high-speed flow cytometry to detect the expression of endothelial (CD309+) or osteogenic (BAP+) differentiation markers in healthy subjects and in patients affected by peripheral vascular manifestations associated with ectopic calcification. Results This study shows that: 1) polychromatic flow cytometry represents a valuable tool to accurately identify rare cells; 2) the balance of CD309+ on CMC/CD309+ on CPC is altered in patients affected by peripheral vascular manifestations, suggesting the occurrence of vascular damage and low repair potential; 3) the increase of circulating cells exhibiting a shift towards an osteoblast-like phenotype (BAP+) is observed in the presence of ectopic calcification. Conclusion Differences between healthy subjects and patients with ectopic calcification indicate that this approach may be useful to better evaluate endothelial dysfunction in a clinical context. PMID:27560136

  3. A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

    PubMed Central

    García-García, Erick; Uribe-Querol, Eileen; Rosales, Carlos

    2013-01-01

    Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-κB and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types. PMID:23603868

  4. Peripheral blood biomarkers of solid tumor angiogenesis in dogs: a polychromatic flow cytometry pilot study.

    PubMed

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

    2013-05-01

    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

  5. Flow Cytometry Analysis Reveals That Only a Subpopulation of Mouse Sperm Undergoes Hyperpolarization During Capacitation1

    PubMed Central

    Escoffier, Jessica; Navarrete, Felipe; Haddad, Doug; Santi, Celia M.; Darszon, Alberto; Visconti, Pablo E.

    2015-01-01

    To gain fertilizing capacity, mammalian sperm should reside in the female tract for a period of time. The physiological changes that render the sperm able to fertilize are known as capacitation. Capacitation is associated with an increase in intracellular pH, an increase in intracellular calcium, and phosphorylation of different proteins. This process is also accompanied by the hyperpolarization of the sperm plasma membrane potential (Em). In the present work, we used flow cytometry to analyze changes in sperm Em during capacitation in individual cells. Our results indicate that a subpopulation of hyperpolarized mouse sperm can be clearly distinguished by sperm flow cytometry analysis. Using sperm bearing green fluorescent protein in their acrosomes, we found that this hyperpolarized subpopulation is composed of sperm with intact acrosomes. In addition, we show that the capacitation-associated hyperpolarization is blocked by high extracellular K+, by PKA inhibitors, and by SLO3 inhibitors in CD1 mouse sperm, and undetectable in Slo3 knockout mouse sperm. On the other hand, in sperm incubated in conditions that do not support capacitation, sperm membrane hyperpolarization can be induced by amiloride, high extracellular NaHCO3, and cAMP agonists. Altogether, our observations are consistent with a model in which sperm Em hyperpolarization is downstream of a cAMP-dependent pathway and is mediated by the activation of SLO3 K+ channels. PMID:25855261

  6. Alternative flow cytometry strategies to analyze stem cells and cell death in planarians

    PubMed Central

    Peiris, Tanuja Harshani; García‐Ojeda, Marcos E.

    2016-01-01

    Abstract Planarians possess remarkable stem cell populations that continuously support cellular turnover and are instrumental in the regeneration of tissues upon injury. Cellular turnover and tissue regeneration in planarians rely on the proper integration of local and systemic signals that regulate cell proliferation and cell death. Thus, understanding the signals controlling cellular proliferation and cell death in planarians could provide valuable insights for maintenance of adult body homeostasis and the biology of regeneration. Flow cytometry techniques have been utilized widely to identify, isolate, and characterize planarian stem cell populations. We developed alternative flow cytometry strategies that reduce the number of reagents and the time of sample preparation to analyze stem cells and cell death in planarians. The sensitivity of these methods is validated with functional studies using RNA interference and treatment with  γ irradiation or stressful conditions that are known to trigger cell death. Altogether, we provide a community resource intended to minimize adverse effects during ex vivo studies of stem cells and cell death in planarians. PMID:27307993

  7. 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

  8. Improved flow cytometry based cytotoxicity and binding assay for clinical antibody HLA crossmatching.

    PubMed

    Alheim, Mats; Paul, Prashanta Kumer; Hauzenberger, Dan-Mikael; Wikström, Ann-Charlotte

    2015-11-01

    The presence of preformed donor-specific HLA antibodies leads to early antibody mediated kidney allograft rejection. Therefore, detection and avoidance of donor reactive HLA antibodies prior to transplantation is of outmost importance in order to minimize the risk of rejection. Detection of pre-formed HLA antibodies is currently performed using complement-dependent cytotoxicity (CDC) assay alone or together with a flow cytometry based crossmatch (FCXM). This study was initiated to further evaluate our recently developed flow cytometry based procedure for determination of both cytotoxicity of and IgG binding to donor-derived lymphocytes by HLA antibodies. Highly enriched immuno-magnetic bead purified T and B lymphocytes were used as target cells for patient sera using 96-well plates. Importantly, the assay shows high sensitivity and specificity as determined by HLA typed donor cells and serum with defined HLA antibody IgG and C1q. Based on this and additional data generated in this paper, such as evaluation of appropriate serum and complements incubation times and assay reproducibility and stability, will enable us to more rapidly implement this assay in our clinical laboratory routines. In addition, we demonstrate that FCtox crossmatching of deceased donor cells has superior specificity compared to conventional CDC assay especially regarding high frequencies of false-positive reactions. PMID:26429307

  9. Quantification of heterotypic granule fusion in human neutrophils by imaging flow cytometry

    PubMed Central

    Björnsdottir, Halla; Welin, Amanda; Dahlgren, Claes; Karlsson, Anna; Bylund, Johan

    2015-01-01

    Human neutrophils are filled with intracellular storage organelles, called granules and secretory vesicles, which differ in their content of soluble matrix proteins and membrane-bound molecules. To date, at least four distinct granule/vesicle subsets have been identified. These organelles may secrete their content extracellularly following mobilization to and fusion with the plasma membrane, but some of them may also fuse with internal membrane-enclosed organelles, typically a plasma membrane-derived phagosome. There are also instances where different granules appear to fuse with one another, a process that would enable mixing of their matrix and membrane components. Such granule fusion enables e.g., myeloperoxidase-processing of intragranular oxygen radicals, a key event in the formation of neutrophil extracellular traps (Björnsdottir et al., 2015) [1]. Described herein are data that show the quantification of such heterotypic granule–granule fusion by the use of imaging flow cytometry, a technique that combines flow cytometry with microscopy. The analysis described is based on immunofluorescent staining of established granule markers (lactoferrin and/or NGAL for one granule subset; the specific granules, and CD63 for another granule subset, the azurophil granules) and calculation of a colocalization score for resting and PMA-stimulated neutrophils. PMID:26862586

  10. The Analysis of Cell Cycle, Proliferation, and Asymmetric Cell Division by Imaging Flow Cytometry.

    PubMed

    Filby, Andrew; Day, William; Purewal, Sukhveer; Martinez-Martin, Nuria

    2016-01-01

    Measuring cellular DNA content by conventional flow cytometry (CFC) and fluorescent DNA-binding dyes is a highly robust method for analysing cell cycle distributions within heterogeneous populations. However, any conclusions drawn from single-parameter DNA analysis alone can often be confounded by the asynchronous nature of cell proliferation. We have shown that by combining fluorescent DNA stains with proliferation tracking dyes and antigenic staining for mitotic cells one can elucidate the division history and cell cycle position of any cell within an asynchronously dividing population. Furthermore if one applies this panel to an imaging flow cytometry (IFC) system then the spatial information allows resolution of the four main mitotic phases and the ability to study molecular distributions within these populations. We have employed such an approach to study the prevalence of asymmetric cell division (ACD) within activated immune cells by measuring the distribution of key fate determining molecules across the plane of cytokinesis in a high-throughput, objective, and internally controlled manner. Moreover the ability to perform high-resolution, temporal dissection of the cell division process lends itself perfectly to investigating the influence chemotherapeutic agents exert on the proliferative capacity of transformed cell lines. Here we describe the method in detail and its application to both ACD and general cell cycle analysis. PMID:27460238

  11. Recent Advances on Multi-Parameter Flow Cytometry to Characterize Antimicrobial Treatments.

    PubMed

    Léonard, Lucie; Bouarab Chibane, Lynda; Ouled Bouhedda, Balkis; Degraeve, Pascal; Oulahal, Nadia

    2016-01-01

    The investigation on antimicrobial mechanisms is a challenging and crucial issue in the fields of food or clinical microbiology, as it constitutes a prerequisite to the development of new antimicrobial processes or compounds, as well as to anticipate phenomenon of microbial resistance. Nowadays it is accepted that a cells population exposed to a stress can cause the appearance of different cell populations and in particular sub-lethally compromised cells which could be defined as viable but non-culturable (VBNC). Recent advances on flow cytometry (FCM) and especially on multi-parameter flow cytometry (MP-FCM) provide the opportunity to obtain high-speed information at real time on damage at single-cell level. This review gathers MP-FCM methodologies based on individual and simultaneous staining of microbial cells employed to investigate their physiological state following different physical and chemical antimicrobial treatments. Special attention will be paid to recent studies exploiting the possibility to corroborate MP-FCM results with additional techniques (plate counting, microscopy, spectroscopy, molecular biology techniques, membrane modeling) in order to elucidate the antimicrobial mechanism of action of a given antimicrobial treatment or compound. The combination of MP-FCM methodologies with these additional methods is namely a promising and increasingly used approach to give further insight in differences in microbial sub-population evolutions in response to antimicrobial treatments. PMID:27551279

  12. Alternative flow cytometry strategies to analyze stem cells and cell death in planarians.

    PubMed

    Peiris, Tanuja Harshani; García-Ojeda, Marcos E; Oviedo, Néstor J

    2016-04-01

    Planarians possess remarkable stem cell populations that continuously support cellular turnover and are instrumental in the regeneration of tissues upon injury. Cellular turnover and tissue regeneration in planarians rely on the proper integration of local and systemic signals that regulate cell proliferation and cell death. Thus, understanding the signals controlling cellular proliferation and cell death in planarians could provide valuable insights for maintenance of adult body homeostasis and the biology of regeneration. Flow cytometry techniques have been utilized widely to identify, isolate, and characterize planarian stem cell populations. We developed alternative flow cytometry strategies that reduce the number of reagents and the time of sample preparation to analyze stem cells and cell death in planarians. The sensitivity of these methods is validated with functional studies using RNA interference and treatment with  γ irradiation or stressful conditions that are known to trigger cell death. Altogether, we provide a community resource intended to minimize adverse effects during ex vivo studies of stem cells and cell death in planarians. PMID:27307993

  13. 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.

  14. Recent Advances on Multi-Parameter Flow Cytometry to Characterize Antimicrobial Treatments

    PubMed Central

    Léonard, Lucie; Bouarab Chibane, Lynda; Ouled Bouhedda, Balkis; Degraeve, Pascal; Oulahal, Nadia

    2016-01-01

    The investigation on antimicrobial mechanisms is a challenging and crucial issue in the fields of food or clinical microbiology, as it constitutes a prerequisite to the development of new antimicrobial processes or compounds, as well as to anticipate phenomenon of microbial resistance. Nowadays it is accepted that a cells population exposed to a stress can cause the appearance of different cell populations and in particular sub-lethally compromised cells which could be defined as viable but non-culturable (VBNC). Recent advances on flow cytometry (FCM) and especially on multi-parameter flow cytometry (MP-FCM) provide the opportunity to obtain high-speed information at real time on damage at single-cell level. This review gathers MP-FCM methodologies based on individual and simultaneous staining of microbial cells employed to investigate their physiological state following different physical and chemical antimicrobial treatments. Special attention will be paid to recent studies exploiting the possibility to corroborate MP-FCM results with additional techniques (plate counting, microscopy, spectroscopy, molecular biology techniques, membrane modeling) in order to elucidate the antimicrobial mechanism of action of a given antimicrobial treatment or compound. The combination of MP-FCM methodologies with these additional methods is namely a promising and increasingly used approach to give further insight in differences in microbial sub-population evolutions in response to antimicrobial treatments. PMID:27551279

  15. Using dual laser flow cytometry for monitoring phytoplankton composition and integrity

    SciTech Connect

    Schaefer, H.; Beisker, W.; Steinberg, C.

    1995-12-31

    Dual laser flow cytometry can be used for determining phytoplankton populations in lakes and lowland rivers and streams. Apart from answering basic limnological questions such as the time course of algal blooms or the annual succession of phytoplankton composition further investigations can be made for estimating the integrity of phytoplankton community using biomass distribution spectra. Thus anthropogenic influence such as eutrophication, acidification or effects of xenobiotica can be monitored. Dual laser flow cytometry with excitation wavelengths of 458 and 528 nm was used to measure photosynthesis pigment fluorescence (chlorophyll a (CHLa), Em>665 nm) and phycoerythrin (PE, Em 575 nm) and cell density of phytoplankton organisms in water samples. CHLa is excited directly by 458 nm and by energy transfer from carotenoids (Ex 528 nm). The ratio of the two fluorescence parameters (CFR) allows to identify pigment groups in the phytoplankton population (chlorophytes and euglenophytes from chrysophytes, diatoms and dinophytes). PE-containing cyanophytes and cryptophytes can be detected by their PE fluorescence (Ex 528 nm). As a result of preliminary studies for preparing biomass spectra of phytoplankton communities measurements of protein content by staining with fluorescein isothiocyanate (FITC, ex 488 nm, Em 530 nm) are also shown.

  16. 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.

  17. Measurement and purification of human chromosomes by flow cytometry and sorting.

    PubMed Central

    Carrano, A V; Gray, J W; Langlois, R G; Burkhart-Schultz, K J; Van Dilla, M A

    1979-01-01

    The 24 human chromosome types of normal diploid fibroblast cell strain were classified into 15 groups by high-resolution flow cytometry on the basis of 33258 Hoechst fluorescence. Chromosomes associated with each group were flow sorted onto microscope slides and identified by quinacrine banding analysis. DNA cytophotometry of metaphase chromosomes from the same cell strain supported and extended this identification. Four of the groups purified were due to chromosomes of a single type--namely, chromosomes 5, 6, 13, and 17. Eight additional groups were also separated and found to contain the following chromosomes: 1 and 2; 3 and 4; 7, 8, and X; 9--12; 14 and 15; 16 and 18; 20 and Y; and 19, 21, and 22. The average purity for the 12 sorted fractions was 78%. PMID:286320

  18. High-resolution analysis of human peripheral lymphocyte chromosomes by flow cytometry.

    PubMed Central

    Young, B D; Ferguson-Smith, M A; Sillar, R; Boyd, E

    1981-01-01

    A method for high-resolution analysis of the human karyotype by flow cytometry has been developed. Metaphase chromosomes are prepared from short-term peripheral blood cultures, stained with ethidium bromide, and analyzed on a standard fluorescence-activated cell sorter (FACS-II). Flow karyotypes with up to 20 peaks can be obtained with coefficients of variation in the range 1-2%. At this level of resolution the contribution of many of the human chromosomes can be evaluated separately. Significant and reproducible differences between normal individuals have been detected and have been correlated with differences in the centric heterochromatin of certain chromosomes as revealed in their C-banded karyotypes. Images PMID:6950411

  19. Advances in Complex Multiparameter Flow Cytometry Technology: Applications in Stem Cell Research

    PubMed Central

    Preffer, Frederic; Dombkowski, David

    2009-01-01

    Flow cytometry and cell sorting are critical tools in stem cell research. Recent advances in flow cytometric hardware, reagents and software have synergized to permit the stem cell biologist to more fully identify and isolate rare cells based on their immunofluorescent and light scatter characteristics. Some of these improvements include physically smaller air-cooled lasers, new designs in optics, new fluorescent conjugate-excitation pairs, and improved software to visualize data, all which combine to open up new horizons in the study of stem cells, by enhancing the resolution and specificity of inquiry. In this review, these recent improvements in technology will be outlined and important cell surface and functional antigenic markers useful for the study of stem cells described. PMID:19492350

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

    PubMed Central

    Davey, H M; Kell, D B

    1996-01-01

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

  1. 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 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. PMID:26267664

  2. The potential of flow cytometry in the study of Bacillus cereus.

    PubMed

    Cronin, U P; Wilkinson, M G

    2010-01-01

    Flow cytometry (FCM) is a rapid method allowing the acquisition of multiparametric data from thousands of individual cells within a sample. As well as measuring the intrinsic light scattering properties of cells, a plethora of fluorescent dyes may be employed to yield information on macromolecule content, surface antigens present or physiological status. Despite FCM's indispensability within other fields e.g. immunology, it is underutilized within microbiological research. In this review, a strong case is presented for the potential of FCM in the study of Gram-positive spore-former, Bacillus cereus. Previous reports where FCM was successfully used in the study of B. cereus are reviewed along with relevant studies involving other members of the genus. Under headings reflecting common research themes associated with B. cereus, specific instances where FCM has generated novel data, providing a unique insight into the organism, are discussed. Further applications are posited, based on the authors' own research with FCM and B. cereus and work extant in the broader field of microbial cytometry. The authors conclude that, while the expense of equipment and reagents is an undeniable disadvantage, FCM is a technique capable of generating significantly novel data and allows the design and execution of experiments that are not possible with any other technique. PMID:19486207

  3. Using multispectral imaging flow cytometry to assess an in vitro intracellular Burkholderia thailandensis infection model.

    PubMed

    Jenner, Dominic; Ducker, Catherine; Clark, Graeme; Prior, Jo; Rowland, Caroline A

    2016-04-01

    The use of in vitro models to understand the interaction of bacteria with host cells is well established. In vitro bacterial infection models are often used to quantify intracellular bacterial load by lysing cell populations and subsequently enumerating the bacteria. Modern established techniques employ the use of fluorescence technologies such as flow cytometry, fluorescent microscopy, and/or confocal microscopy. However, these techniques often lack either the quantification of large data sets (microscopy) or use of gross fluorescence signal which lacks the visual confirmation that can provide additional confidence in data sets. Multispectral imaging flow cytometry (MIFC) is a novel emerging field of technology. This technology captures a bright field and fluorescence image of cells in a flow using a charged coupled device camera. It allows the analysis of tens of thousands of single cell images, making it an extremely powerful technology. Here MIFC was used as an alternative method of analyzing intracellular bacterial infection using Burkholderia thailandensis E555 as a model organism. It has been demonstrated that the data produced using traditional enumeration is comparable to data analyzed using MIFC. It has also been shown that by using MIFC it is possible to generate other data on the dynamics of the infection model rather than viable counts alone. It has been demonstrated that it is possible to inhibit the uptake of bacteria into mammalian cells and identify differences between treated and untreated cell populations. The authors believe this to be the first use of MIFC to analyze a Burkholderia bacterial species during intracellular infection. © 2016 Crown copyright. Published by Wiley Periodicals Inc. on behalf of ISAC. PMID:26841315

  4. Optical analysis of nanomaterial-cell interactions: flow cytometry and digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Mues, Sarah; Antunovic, Jan; Ossig, Rainer; Kemper, Björn; Schnekenburger, Jürgen

    2015-05-01

    The in vitro cytotoxicity assessment of engineered nanoparticles commonly involves the measurement of different endpoints like the formation of reactive oxygen species, cell viability or cell death. Usually these parameters are determined by optical readouts of enzymatically converted substrates that often interfere with the tested nanomaterials. Using cell viability (WST-8) and cell death (LDH) as parameter we have initially investigated the toxic effects of spherical (NM 300) and rod shaped (NM 302) silver nanomaterials with a matrix of four cell lines representing different functions: lung and kidney epithelial cells, macrophages and fibroblasts. In addition, we have used a label-free flow cytometer configuration to investigate interactions of particles and macrophages by side scatter signal analysis. Finally, we explored digital holographic microscopy (DHM) for multimodal label-free analysis of nanomaterial toxicity. Quantitative DHM phase images were analyzed for cell thickness, volume, density, dry mass and refractive index. We could demonstrate that silver spheres lead to more cytotoxic effects than rods in all four examined cell lines and both assay. Exemplarily a dose dependent interaction increase of cells with NM 300 and NM 302 analyzed by flow cytometry is shown. Furthermore, we found that the refractive index of cells is influenced by incubation with NM 300 in a decreasing manner. A 24 hours time-lapse measurement revealed a dose dependent decrease of dry mass and surface area development indicating reduced cell viability and cell death. Our results demonstrate digital holographic microscopy and flow cytometry as valuable label-free tools for nanomaterial toxicity and cell interaction studies.

  5. 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. PMID:19670359

  6. Tomographic Diffuse Fluorescence Flow Cytometry for Enumeration of Rare Circulating Cells in Vitro and in Vivo

    NASA Astrophysics Data System (ADS)

    Zettergren, Eric William

    2011-12-01

    Accurate quantification of circulating cell populations is important in many areas of preclinical and clinical biomedical research including the study of metastasized cancers, T-Lymphotocyes and hematopoietic stem cells. Normally this is done either by extraction and analysis of small blood samples or more recently using microscopy-based in vivo fluorescence flow cytometry. In this thesis, a new technological approach to this problem is described using detection of diffuse fluorescent light from relatively large blood vessels in vivo. The 'tomographic diffuse fluorescence flow cytometer' (TDFFC) uses modulated lasers to illuminate a mouse limb and an array of optical fibers coupled to a high-sensitivity photomultiplier tube array operating in photon counting mode to detect weak fluorescence signals from cells. It is first demonstrated that the TDFFC instrument is capable of detecting fluorescent microspheres and Vybrant-DiD labeled cells with excellent accuracy in an optical flow phantom with similar size, optical properties, linear flow rates and autofluorescence as a mouse limb. Preliminary data demonstrating that the TDFFC is capable of detecting circulating cells in nude mice in vivo is also shown. Finally, a number of methods for performing coarse tomographic localization of fluorescent cells within the cross-section of a mouse limb using TDFFC data sets are described, and the feasibility of this approach is demonstrated using in vitro data sets. In principle, this device would allow interrogation of the whole blood volume of a mouse in minutes, with several orders of magnitude sensitivity improvement compared with current approaches.

  7. A microfluidic device for uniform-sized cell spheroids formation, culture, harvesting and flow cytometry analysis.

    PubMed

    Patra, Bishnubrata; Chen, Ying-Hua; Peng, Chien-Chung; Lin, Shiang-Chi; Lee, Chau-Hwang; Tung, Yi-Chung

    2013-01-01

    Culture of cells as three-dimensional (3D) aggregates, named spheroids, possesses great potential to improve in vitro cell models for basic biomedical research. However, such cell spheroid models are often complicated, cumbersome, and expensive compared to conventional Petri-dish cell cultures. In this work, we developed a simple microfluidic device for cell spheroid formation, culture, and harvesting. Using this device, cells could form uniformly sized spheroids due to strong cell-cell interactions and the spatial confinement of microfluidic culture chambers. We demonstrated cell spheroid formation and culture in the designed devices using embryonic stem cells, carcinoma cells, and fibroblasts. We further scaled up the device capable of simultaneously forming and culturing 5000 spheroids in a single chip. Finally, we demonstrated harvesting of the cultured spheroids from the device with a simple setup. The harvested spheroids possess great integrity, and the cells can be exploited for further flow cytometry assays due to the ample cell numbers. PMID:24396525

  8. Quantitating MHC class II trafficking in primary dendritic cells using imaging flow cytometry

    PubMed Central

    Hennies, Cassandra M.; Lehn, Maria A.; Janssen, Edith M.

    2015-01-01

    Presentation of antigenic peptides in MHC class II (MHCII) on dendritic cells (DCs) is the first step in the activation of antigen-specific CD4+T cells. The expression of surface MHCII-peptide complexes is tightly regulated as the frequency of MHCII-peptide complexes can affect the magnitude, as well as the phenotype of the ensuing CD4+T cell response. The surface MHCII-peptide levels are determined by the balance between expression of newly generated complexes, complex internalization, and their subsequent re-emergence or degradation. However, the molecular mechanisms that underpin these processes are still poorly understood. Here we describe a multispectral imaging flow cytometry assay to visualize MHCII trafficking that can be used as a tool to dissect the molecular mechanisms that regulate MHCII homeostasis in primary mouse and human DCs. PMID:25967952

  9. Application of alkaline elution, Fast Micromethod and flow cytometry in detection of marine contamination.

    PubMed

    Bihari, Nevenka; Hamer, Bojan; Jaksić, Zeljko; Fafandel, Maja; Micić, Milena; Batel, Renato

    2002-06-01

    DNA damage is an inescapable aspect of life in the biosphere. The presented investigations were an attempt to examine the response of a DNA damage as a biomarker of environmental quality in the mussels Mytilus galloprovincialis sampled at differently contaminated areas of Istrian coast, Northern Adriatic. The investigations were performed in order to get information about the genotoxic risk for marine organisms exposed to mixed environmental pollution, as well as the information about the presence of unknown mixture of genotoxic contaminants in the marine environment. Types of DNA damage detected are alkali-labile sites and single-strand breaks measured by Fast Micromethod, interstrand cross-links and DNA protein cross-links by alkaline filter elution and cell cycle disturbation by flow cytometry. The applicability of all three methods for marine quality control is discussed. PMID:12064444

  10. Monitoring of Dynamic Microbiological Processes Using Real-Time Flow Cytometry

    PubMed Central

    Arnoldini, Markus; Heck, Tobias; Blanco-Fernández, Alfonso; Hammes, Frederik

    2013-01-01

    We describe a straightforward approach to continuously monitor a variety of highly dynamic microbiological processes in millisecond resolution with flow cytometry, using standard bench-top instrumentation. Four main experimental examples are provided, namely: (1) green fluorescent protein expression by antibiotic-stressed Escherichia coli, (2) fluorescent labeling of heat-induced membrane damage in an autochthonous freshwater bacterial community, (3) the initial growth response of late stationary E. coli cells inoculated into fresh growth media, and (4) oxidative disinfection of a mixed culture of auto-fluorescent microorganisms. These examples demonstrate the broad applicability of the method to diverse biological experiments, showing that it allows the collection of detailed, time-resolved information on complex processes. PMID:24244624

  11. Overview of clinical flow cytometry data analysis: recent advances and future challenges.

    PubMed

    Pedreira, Carlos E; Costa, Elaine S; Lecrevisse, Quentin; van Dongen, Jacques J M; Orfao, Alberto

    2013-07-01

    Major technological advances in flow cytometry (FC), both for instrumentation and reagents, have emerged over the past few decades. These advances facilitate simultaneous evaluation of more parameters in single cells analyzed at higher speed. Consequently, larger and more complex data files that contain information about tens of parameters for millions of cells are generated. This increasing complexity has challenged pre-existing data analysis tools and promoted the development of new algorithms and tools for data analysis and visualization. Here, we review the currently available (conventional and newly developed) data analysis and visualization strategies that aim for easier, more objective, and robust interpretation of FC data both in biomedical research and clinical diagnostic laboratories. PMID:23746659

  12. Flow cytometry for bacteria: enabling metabolic engineering, synthetic biology and the elucidation of complex phenotypes.

    PubMed

    Tracy, Bryan P; Gaida, Stefan M; Papoutsakis, Eleftherios T

    2010-02-01

    Flow cytometry (FC) and FC-based cell sorting have been established as critical tools in modern cell and developmental biology. Yet, their applications in bacteria, especially in the multiparametric mode, remain limited. We argue that FC technologies have the potential to greatly accelerate the analysis and development of microbial complex phenotypes through applications of metabolic engineering, synthetic biology, and evolutionary engineering. We demonstrate the importance of FC for elucidating population heterogeneity because of developmental processes or epigenetic regulation. FC can be engaged for both synthetic and analytical applications of complex phenotypes within a single species, multispecies, and microbial-library populations. Examples include methods to identify developmental microbial stages associated with productive metabolic phenotypes, select desirable promoters from a single species or metagenomic libraries, and to screen designer riboswitches for synthetic-biology applications. PMID:20206495

  13. Characterization of aggregate load and pattern in living yeast cells by flow cytometry.

    PubMed

    Hidalgo, Itahisa Hernández; Fleming, Thomas; Eckstein, Volker; Herzig, Stephan; Nawroth, Peter P; Tyedmers, Jens

    2016-01-01

    Protein aggregation is both a hallmark of and a driving force for a number of diseases. It is therefore important to identify the nature of these aggregates and the mechanism(s) by which the cell counteracts their detrimental properties. Currently, the study of aggregation in vivo is performed primarily using fluorescently tagged versions of proteins and analyzing the aggregates by fluorescence microscopy. While this strategy is considered the gold standard, it has several limitations, particularly with respect to its suitability for high-throughput screening (HTS). Here, using a GFP fusion of the well-characterized yeast prion amyloid protein [PSI+], we demonstrate that flow cytometry, which utilizes the same physical principles as fluorescence microscopy, can be used to determine the aggregate load and pattern in live and fixed yeast cells. Furthermore, our approach can easily be applied to high-throughput analyses such as screenings with a yeast deletion library. PMID:27625208

  14. Flow cytometry for monitoring contaminant exposure in black-crowned night-herons

    USGS Publications Warehouse

    Custer, T.W.; Bickham, J.W.; Lyne, T.B.; Lewis, T.; Ruedas, L.A.; Custer, Christine M.; Melancon, M.J.

    1994-01-01

    The flow cytometry method (FCM) was employed to determine cellular DNA content of black-crowned night-heron (Nycticorax nycticorax) embryos and 10-day-old chicks collected at sites differing in types of chemical contamination. The coefficient of variation of DNA content (CV) in blood collected from embryos suggested cytogenetic damage at a site in Louisiana known to be contaminated with petroleum. Blood CV from chicks suggested genetic damage at a site in Texas also known to be contaminated with petroleum. Spleen CVs in chicks were significantly lower than respective means from the reference site. the CVs of chick blood and liver and spleen negatively correlated, suggesting recovery of spleen and liver cells after exposure to a clastogenic compound. Thus, the lower CVs may also have been indicative of genetic damage. Based on the findings of this study, FCM is a potential indicator of certain environmental contaminants in black-crowned night-herons.

  15. Flow cytometry for monitoring contaminant exposure in black-crowned night-herons

    USGS Publications Warehouse

    Custer, T.W.; Bickham, J.W.; Lyne, T.B.; Lewis, T.; Ruedas, L.A.; Custer, Christine M.; Melancon, M.J.

    1994-01-01

    The flow cytometry method (FCM) was employed to determine cellular DNA content of black-crowned night-heron (Nycticorax nycticorax) embryos and 10-day-old chicks collected at sites differing in types of chemical contamination. The coefficient of variation of DNA content (CV) in blood collected from embryos suggested cytogenetic damage at a site in Louisiana known to be contaminated with petroleum. Blood CV from chicks suggested genetic damage at a site in Texas also known to be contaminated with petroleum. Spleen CVs in chicks were significantly lower than respective means from the reference site. The CVs of chick blood and liver and spleen negatively correlated, suggesting recovery of spleen and liver cells after exposure to a clastogenic compound. Thus, the lower CVs may also have been indicative of genetic damage. Based on the findings of this study, FCM is a potential indicator of certain environmental contaminants in black-crowned night-herons.

  16. Evaluation of Giardia duodenalis viability after metronidazole treatment by flow cytometry

    PubMed Central

    Barbosa, Joana; Rodrigues, Acácio Gonçalves; Pérez, Maria José; Pina-Vaz, Cidália

    2014-01-01

    Giardia duodenalis (syn. lamblia; syn. intestinalis) susceptibility testing is not routinely performed because the classical culture methods are very time-consuming and laborious. We developed a novel flow cytometry (FC) assay to evaluate the susceptibility of G. duodenalis trophozoites to metronidazole (MTZ). Different concentrations of MTZ were added to cultures of trophozoites (10 5 /mL) and the cultures were incubated for different periods. The 50% inhibitory concentration was calculated and propidium iodide (PI) was used to quantify the number of dead cells. After treatment, PI-positive trophozoites increased with increasing drug concentration and exposure time. An excellent correlation was found between FC and the classical method. A novel, accurate and reliable method is now available to evaluate G. duodenalis viability. PMID:25424449

  17. Dynamic Characterization of Growth and Gene Expression Using High-throughput Automated Flow cytometry

    PubMed Central

    Zuleta, Ignacio A.; Aranda-Díaz, Andrés; Li, Hao; El-Samad, Hana

    2014-01-01

    Cells adjust to changes in environmental conditions using complex regulatory programs. These cellular programs are the result of an intricate interplay between gene expression, cellular growth rate, and protein degradation fluxes. New technologies that enable simultaneous and time-resolved measurements of these variables are necessary to dissect cellular homeostatic strategies. Here, we report the development of a novel automated flow-cytometry robotic setup that enables real-time measurement of precise and simultaneous relative growth and protein synthesis rates of multiplexed microbial populations across many conditions. These measurements generate quantitative profiles of dynamically-evolving protein synthesis and degradation rates. We demonstrate this setup in the context of gene regulation of the unfolded protein response (UPR) and uncover a dynamic and complex landscape of gene expression, growth dynamics, and proteolysis following perturbations. PMID:24608180

  18. Flow cytometry for monitoring contaminant exposure in black-crowned night-herons.

    PubMed

    Custer, T W; Bickham, J W; Lyne, T B; Lewis, T; Ruedas, L A; Custer, C M; Melancon, M J

    1994-08-01

    The flow cytometry methods (FCM) was employed to determine cellular DNA content of black-crowned night-heron (Nycticorax nycticorax) embryos and 10-day-old chicks collected at sites differing in types of chemical contamination. The coefficient of variation of DNA content (CV) in blood collected from embryos suggested cytogenetic damage at a side in Louisiana known to be contaminated with petroleum. Blood CV from chicks suggested genetic damage at a site in Texas also known to be contaminated with petroleum. Spleen CVs in chicks were significantly lower than respective means from the reference site. The CVs of chick blood and liver and spleen negatively correlated, suggesting recovery of spleen and liver cells after exposure to a clastogenic compound. Thus, the lower CVs may also have been indicative of genetic damage. Based on the findings of this study, FCM is a potential indicator of certain environmental contaminants in black-crowned night-herons. PMID:8060161

  19. A Rapid and Quantitative Flow Cytometry Method for the Analysis of Membrane Disruptive Antimicrobial Activity

    PubMed Central

    O’Brien-Simpson, Neil M.; Pantarat, Namfon; Attard, Troy J.; Walsh, Katrina A.; Reynolds, Eric C.

    2016-01-01

    We describe a microbial flow cytometry method that quantifies within 3 hours antimicrobial peptide (AMP) activity, termed Minimum Membrane Disruptive Concentration (MDC). Increasing peptide concentration positively correlates with the extent of bacterial membrane disruption and the calculated MDC is equivalent to its MBC. The activity of AMPs representing three different membranolytic modes of action could be determined for a range of Gram positive and negative bacteria, including the ESKAPE pathogens, E. coli and MRSA. By using the MDC50 concentration of the parent AMP, the method provides high-throughput, quantitative screening of AMP analogues. A unique feature of the MDC assay is that it directly measures peptide/bacteria interactions and lysed cell numbers rather than bacteria survival as with MIC and MBC assays. With the threat of multi-drug resistant bacteria, this high-throughput MDC assay has the potential to aid in the development of novel antimicrobials that target bacteria with improved efficacy. PMID:26986223

  20. Assessment of immune parameters of manila clam Ruditapes philippinarum in different physiological conditions using flow cytometry

    NASA Astrophysics Data System (ADS)

    Park, Kyung-Il; Donaghy, Ludovic; Kang, Hyun-Sil; Hong, Hyun-Ki; Kim, Young-Ok; Choi, Kwang-Sik

    2012-03-01

    Cellular and humoral immune parameters are often used as biomarkers to trace environmental and physiological stresses in marine bivalves. In this study, we compared various immune parameters of Manila clams ( Ruditapes philippinarum) under normal conditions and under a high level of desiccation, using flow cytometry. The immune parameters analyzed included, total hemocyte count, hemocyte mortality, hemocyte DNA damage, reactive oxygen species (ROS) production, and phagocytosis activity. Total hemocyte count, hemocyte DNA damage, and hemocyte mortality were significantly elevated among clams under high desiccation stress, while phagocytosis activity and spontaneous ROS production were significantly lower compared to those parameters of the control clams ( p<0.05). These data suggest that the immune parameters analyzed in this study well reflect the physiological status of clams.

  1. Immunophenotype Discovery, Hierarchical Organization, and Template-Based Classification of Flow Cytometry Samples

    PubMed Central

    Azad, Ariful; Rajwa, Bartek; Pothen, Alex

    2016-01-01

    We describe algorithms for discovering immunophenotypes from large collections of flow cytometry samples and using them to organize the samples into a hierarchy based on phenotypic similarity. The hierarchical organization is helpful for effective and robust cytometry data mining, including the creation of collections of cell populations’ characteristic of different classes of samples, robust classification, and anomaly detection. We summarize a set of samples belonging to a biological class or category with a statistically derived template for the class. Whereas individual samples are represented in terms of their cell populations (clusters), a template consists of generic meta-populations (a group of homogeneous cell populations obtained from the samples in a class) that describe key phenotypes shared among all those samples. We organize an FC data collection in a hierarchical data structure that supports the identification of immunophenotypes relevant to clinical diagnosis. A robust template-based classification scheme is also developed, but our primary focus is in the discovery of phenotypic signatures and inter-sample relationships in an FC data collection. This collective analysis approach is more efficient and robust since templates describe phenotypic signatures common to cell populations in several samples while ignoring noise and small sample-specific variations. We have applied the template-based scheme to analyze several datasets, including one representing a healthy immune system and one of acute myeloid leukemia (AML) samples. The last task is challenging due to the phenotypic heterogeneity of the several subtypes of AML. However, we identified thirteen immunophenotypes corresponding to subtypes of AML and were able to distinguish acute promyelocytic leukemia (APL) samples with the markers provided. Clinically, this is helpful since APL has a different treatment regimen from other subtypes of AML. Core algorithms used in our data analysis are

  2. Establishment of pancreatic cancer stem cells by flow cytometry and their biological characteristics.

    PubMed

    Li, Dandan; Su, Dongyue; Xue, Lei; Liu, Yang; Pang, Wuyan

    2015-01-01

    To investigate the method of separating human pancreatic cancer stem cells by Hoechst 33342 labeled flow cytometry and to analyze the biological properties of pancreatic cancer stem cells. The human pancreatic cancer cell line PC-3 was divided into SP and non-SP cells by flow cytometry. The number of two cell clone spheres and nude mice tumor formation rates were compared by cultivating in serum-free medium; The expression of CD133, Nestin mRNA and protein was analyzed by real-time fluorescence quantitative PCR and Western blot; The expression of two cell drug resistance genes (MDR1, ABCG2, ABCA2 and MRP1) was analyzed by real time fluorescent quantitative PCR. The number of the cloned spheres in SP cells in serum-free medium was significantly higher than that of non-SP cells (P<0.05). The incidence of SP cells in the tumor of immunodeficiency nude mice was significantly higher than that of non-SP cells, and the difference was statistically significant (P<0.05). Real-time fluorescence quantitative PCR analysis showed that the expression of CD133 and Nestin mRNA in SP cells was significantly higher than those of non-SP cells, and the expression of CD133 and Nestin protein in SP cells was also significantly higher than those of non-SP cells (P<0.05). In conclusion, SP side population pancreatic cancer cells by Hoechst 33342 separation have the stem cell characteristics, higher tumor formation rate and higher drug resistance, which may be related to chemotherapy resistance. PMID:26617845

  3. 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.

  4. Assessment of erythroid dysplasia by "difference from normal" in routine clinical flow cytometry work-up.

    PubMed

    Eidenschink Brodersen, Lisa; Menssen, Andrew J; Wangen, Jamie R; Stephenson, Christine F; de Baca, Monica E; Zehentner, Barbara K; Wells, Denise A; Loken, Michael R

    2014-10-21

    Introduction: While multidimensional flow cytometry (MDF) has great utility in diagnostic work-ups of patients with suspected myelodysplastic syndromes (MDS), only the myeloid lineage has demonstrated reproducible abnormalities from multiple laboratories. With the effects of ammonium chloride (NH4 Cl) lysis on erythroid progenitors previously described, we applied this protocol to a patient cohort with diagnosed MDS to investigate phenotypic abnormalities that indicate erythroid dysplasia. Method: Bone marrow specimens [39 MDS, 9 acute myeloid leukemia (AML), 7 JAK2(V617F) positive myeloproliferative neoplasms (MPN), 5 nutritional deficiencies] were processed by NH4 Cl lysis and Ficoll preparation and evaluated by MDF using a difference from normal algorithm. Results: For the MDS cohort, phenotypic abnormalities on the mature erythroid progenitors were frequent for CD71 and CD36 (36% for each antigen); abnormalities for CD235a (8%) were observed. Among immature erythroid progenitors, abnormal maturation patterns (≤5%) and increased CD105 intensity (9%) were seen. Increased frequency of CD105 bright cells was observed (18%). While antigenic abnormalities correlated between NH4 Cl lysis and Ficoll preparation, the lysis method demonstrated the most consistent quantitative antigen intensities. Mean erythroid phenotypic abnormalities and prognostic cytogenetic subgroups correlated strongly. Morphologic and erythroid phenotypic abnormalities correlated, as did increasing FCSS and number of erythroid abnormalities, albeit without further increase for AML patients. Discussion: These data expand the understanding of erythropoiesis and define immunophenotypic abnormalities that indicate dyserythropoiesis in MDS utilizing a lysis protocol practical for routine implementation in clinical flow cytometric work-up. Preliminary studies also indicate strong correlation between phenotypic erythroid dysplasia and poor prognosis, as classified cytogenetically. © 2014 Clinical

  5. 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. PMID:26490516

  6. Using flow cytometry to estimate pollen DNA content: improved methodology and applications

    PubMed Central

    Kron, Paul; Husband, Brian C.

    2012-01-01

    Background and Aims Flow cytometry has been used to measure nuclear DNA content in pollen, mostly to understand pollen development and detect unreduced gametes. Published data have not always met the high-quality standards required for some applications, in part due to difficulties inherent in the extraction of nuclei. Here we describe a simple and relatively novel method for extracting pollen nuclei, involving the bursting of pollen through a nylon mesh, compare it with other methods and demonstrate its broad applicability and utility. Methods The method was tested across 80 species, 64 genera and 33 families, and the data were evaluated using established criteria for estimating genome size and analysing cell cycle. Filter bursting was directly compared with chopping in five species, yields were compared with published values for sonicated samples, and the method was applied by comparing genome size estimates for leaf and pollen nuclei in six species. Key Results Data quality met generally applied standards for estimating genome size in 81 % of species and the higher best practice standards for cell cycle analysis in 51 %. In 41 % of species we met the most stringent criterion of screening 10 000 pollen grains per sample. In direct comparison with two chopping techniques, our method produced better quality histograms with consistently higher nuclei yields, and yields were higher than previously published results for sonication. In three binucleate and three trinucleate species we found that pollen-based genome size estimates differed from leaf tissue estimates by 1·5 % or less when 1C pollen nuclei were used, while estimates from 2C generative nuclei differed from leaf estimates by up to 2·5 %. Conclusions The high success rate, ease of use and wide applicability of the filter bursting method show that this method can facilitate the use of pollen for estimating genome size and dramatically improve unreduced pollen production estimation with flow cytometry. PMID

  7. Hierarchical modeling for rare event detection and cell subset alignment across flow cytometry samples.

    PubMed

    Cron, Andrew; Gouttefangeas, Cécile; Frelinger, Jacob; Lin, Lin; Singh, Satwinder K; Britten, Cedrik M; Welters, Marij J P; van der Burg, Sjoerd H; West, Mike; Chan, Cliburn

    2013-01-01

    Flow cytometry is the prototypical assay for multi-parameter single cell analysis, and is essential in vaccine and biomarker research for the enumeration of antigen-specific lymphocytes that are often found in extremely low frequencies (0.1% or less). Standard analysis of flow cytometry data relies on visual identification of cell subsets by experts, a process that is subjective and often difficult to reproduce. An alternative and more objective approach is the use of statistical models to identify cell subsets of interest in an automated fashion. Two specific challenges for automated analysis are to detect extremely low frequency event subsets without biasing the estimate by pre-processing enrichment, and the ability to align cell subsets across multiple data samples for comparative analysis. In this manuscript, we develop hierarchical modeling extensions to the Dirichlet Process Gaussian Mixture Model (DPGMM) approach we have previously described for cell subset identification, and show that the hierarchical DPGMM (HDPGMM) naturally generates an aligned data model that captures both commonalities and variations across multiple samples. HDPGMM also increases the sensitivity to extremely low frequency events by sharing information across multiple samples analyzed simultaneously. We validate the accuracy and reproducibility of HDPGMM estimates of antigen-specific T cells on clinically relevant reference peripheral blood mononuclear cell (PBMC) samples with known frequencies of antigen-specific T cells. These cell samples take advantage of retrovirally TCR-transduced T cells spiked into autologous PBMC samples to give a defined number of antigen-specific T cells detectable by HLA-peptide multimer binding. We provide open source software that can take advantage of both multiple processors and GPU-acceleration to perform the numerically-demanding computations. We show that hierarchical modeling is a useful probabilistic approach that can provide a consistent labeling

  8. Microsatellite and flow cytometry analysis to help understand the origin of Dioscorea alata polyploids

    PubMed Central

    Nemorin, A.; David, J.; Maledon, E.; Nudol, E.; Dalon, J.; Arnau, G.

    2013-01-01

    Background and Aims Dioscorea alata is a polyploid species with a ploidy level ranging from diploid (2n = 2x = 40) to tetraploid (2n = 4x = 80). Ploidy increase is correlated with better agronomic performance. The lack of knowledge about the origin of D. alata spontaneous polyploids (triploids and tetraploids) limits the efficiency of polyploid breeding. The objective of the present study was to use flow cytometry and microsatellite markers to understand the origin of D. alata polyploids. Methods Different progeny generated by intracytotype crosses (2x × 2x) and intercytotype crosses (2x × 4x and 3x × 2x) were analysed in order to understand endosperm incompatibility phenomena and gamete origins via the heterozygosity rate transmitted to progeny. Results This work shows that in a 2x × 2x cross, triploids with viable seeds are obtained only via a phenomenon of diploid female non-gametic reduction. The study of the transmission of heterozygosity made it possible to exclude polyspermy and polyembryony as the mechanisms at the origin of triploids. The fact that no seedlings were obtained by a 3x × 2x cross made it possible to confirm the sterility of triploid females. Flow cytometry analyses carried out on the endosperm of seeds resulting from 2x × 4x crosses revealed endosperm incompatibility phenomena. Conclusions The major conclusion is that the polyploids of D. alata would have appeared through the formation of unreduced gametes. The triploid pool would have been built and diversified through the formation of 2n gametes in diploid females as the result of the non-viability of seeds resulting from the formation of 2n sperm and of the non-viability of intercytotype crosses. The tetraploids would have appeared through bilateral sexual polyploidization via the union of two unreduced gametes due to the sterility of triploids. PMID:23912697

  9. Use of flow cytometry to assess chondrocyte viability after laser reshaping of cartilage

    NASA Astrophysics Data System (ADS)

    Rasouli, Alexandre; Kim, Charlton C.; Basu, Reshmi; Wong, Brian J.

    2000-05-01

    Lasers have been shown to cause permanent shape change in cartilage via photothermally induced mechanical stress relaxation. While the biophysical properties of cartilage during laser irradiation have been studied, tissue viability following laser irradiation has not been fully characterized. In this study, cell viability staining and flow cytometry were used to determine chondrocyte viability following photothermal stress relaxation. Porcine septal cartilage slabs (10 X 25 X 1.5 mm) were irradiated with light from a Nd:YAG laser ((lambda) equals 1.32 micrometer, 25 W/cm2) while surface temperature, stress relaxation, and diffuse reflectance were recorded. Each slab received one, two, or three laser exposures (respective exposure times of 6.7, 7.2, 10 s), determined from measurements of diffuse reflectance, which correlate with mechanical stress relaxation. Irradiated samples were then divided into two groups analyzed immediately and at five days following laser exposure (the latter group was maintained in culture). Chondrocytes were isolated following serial enzymatic digestion with hyaluronidase, protease, and collagenase II for a total of 17 hours. Chondrocytes were then stained using SYTOR/DEAD RedTM (Molecular Probes; Eugene, OR) wherein live cells stained green (530 nm) and dead cells stained red (630 nm) when excited at 488 nm. A flow cytometer (FACScan, Becton Dickinson, Franklin Lakes, NJ) was then used to detect differential cell fluorescence; size; granularity; and the number of live cells, dead cells, and post irradiation debris in each treatment population. Nearly 60% of chondrocytes from reshaped cartilage samples isolated shortly after irradiation, were viable as determined using flow cytometry while non- irradiated controls were 100 percent viable. Specimens irradiated two or three times with the laser demonstrated increasing amounts of cellular debris along with a reduction in chondrocyte viability: 31 percent following two laser exposures, and 16

  10. Enhanced Red and Near Infrared Detection in Flow Cytometry Using Avalanche Photodiodes

    PubMed Central

    Lawrence, William G.; Varadi, Gyula; Entine, Gerald; Podniesinski, Edward; Wallace, Paul K.

    2008-01-01

    Background Polychromatic flow cytometry enables detailed identification of cell phenotype using multiple fluorescent parameters. The photomultiplier tubes used to detect fluorescence in current instruments limit the sensitivity in the long wavelength spectral range. We demonstrate the flow cytometric applications of silicon avalanche photodiodes, which have improved red sensitivity and a working fluorescence detection range beyond 1000 nm. Methods A comparison of the wavelength dependent performance of the avalanche photodiode and photomultiplier tube was carried out using pulsed light emitting diode sources, calibrated test beads and biological samples. A breadboard flow cytometer test bench was constructed to compare the performance of photomultiplier tubes and avalanche photodiode detectors. The avalanche photodiode used an additional amplifier stage to match the internal gain of the photomultiplier tube. Results The resolution of the avalanche photodiode and photomultiplier tube was compared for flow cytometry applications using a pulsed light emitting diode source over the 500 nm to 1060 nm spectral range. These measurements showed the relative changes in the signal to noise performance of the APD and PMT over a broad spectral range. Both the avalanche photodiode and photomultiplier tubes were used to measure the signal to noise response for a set of 6 peak calibration beads over the 530 to 800 nm wavelength range. CD4 positive cells labeled with antibody conjugated phycoerythrin or 800 nm quantum dots were identified by simultaneous detection using the avalanche photodiode and the photomultiplier tube. The ratios of the intensities of the CD4− and CD4+ populations were found to be similar for both detectors in the visible wavelengths, but only the avalanche photodiode was able to separate these populations at wavelengths above 800 nm. Conclusions These measurements illustrate the differences in APD and PMT performance at different wavelengths and signal

  11. In vivo quantitation of rare circulating tumor cells by multiphoton intravital flow cytometry

    PubMed Central

    He, Wei; Wang, Haifeng; Hartmann, Lynn C.; Cheng, Ji-Xin; Low, Philip S.

    2007-01-01

    Quantitation of circulating tumor cells (CTCs) constitutes an emerging tool for the diagnosis and staging of cancer, assessment of response to therapy, and evaluation of residual disease after surgery. Unfortunately, no existing technology has the sensitivity to measure the low numbers of tumor cells (<1 CTC per ml of whole blood) that characterize minimal levels of disease. We present a method, intravital flow cytometry, that noninvasively counts rare CTCs in vivo as they flow through the peripheral vasculature. The method involves i.v. injection of a tumor-specific fluorescent ligand followed by multiphoton fluorescence imaging of superficial blood vessels to quantitate the flowing CTCs. Studies in mice with metastatic tumors demonstrate that CTCs can be quantitated weeks before metastatic disease is detected by other means. Analysis of whole blood samples from cancer patients further establishes that human CTCs can be selectively labeled and quantitated when present at ≈2 CTCs per ml, opening opportunities for earlier assessment of metastatic disease. PMID:17601776

  12. 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.

  13. Light-scattering polarization measurements as a new parameter in flow cytometry

    SciTech Connect

    de Grooth, B.G.; Terstappen, L.W.; Puppels, G.J.; Greve, J.

    1987-11-01

    Polarization measurement of orthogonal light scattering is introduced as a new optical parameter in flow cytometry. In the experimental setup, the electrical field of the incident laser beam is polarized in the direction of the sample flow. The intensity of the orthogonal light scattering polarized along the direction of the incoming laser beam is called depolarized orthogonal light scattering. Theoretical analysis shows that for small values of the detection aperture, the measured depolarization is caused by anisotropic cell structures and multiple scattering processes inside the cell. Measurements of the orthogonal depolarized light scattering in combination with the normal orthogonal light scattering of human leucocytes revealed two populations of granulocytes. By means of cell sorting it was shown that the granulocytes with a relatively high depolarization are eosinophilic granulocytes. Similar experiments with human lymphocytes revealed a minor subpopulation of yet-unidentified lymphocytes with a relative large orthogonal light-scattering depolarization. The results were obtained with an argon ion laser tuned at different wavelengths as well as with a 630-nm helium neon laser. These results show that measurement of depolarized orthogonal light scattering is a useful new parameter for flow-cytometric cell differentiation.

  14. Measurement of Low-Abundance Intracellular mRNA Using Amplified FISH Staining and Image-Based Flow Cytometry.

    PubMed

    Henning, Andrea L; Sampson, Jill N Best; McFarlin, Brian Keith

    2016-01-01

    Recent advances in instrument design and reagent development have enabled the rapid progression in available measurement techniques in the field of flow cytometry. In particular, image-based flow cytometry extends the analysis capacity found in traditional flow cytometry. Until recently, it was not possible to measure intracellular mRNA in specific phenotypes of cells by flow cytometry. In this protocol, a method of completing simultaneous intracellular measurement of mRNA and protein for PPAR-gamma in peripheral blood monocytes, which have been exposed in vitro to modified LDL, is described. The process of PPAR-gamma activation following uptake of modified LDL is believed to play a role in the development of atherogenesis. PPAR-gamma mRNA measurement was made possible using an amplified FISH technique (PrimeFlow RNA Assay) that allowed for detection of low-abundant intracellular mRNA expression. This protocol represents a continued effort by the authors' laboratory to establish and validate new techniques to assess the role of the immune system in chronic disease. © 2016 by John Wiley & Sons, Inc. PMID:27037579

  15. Using fluorescence-activated flow cytometry to determine reactive oxygen species formation and membrane lipid peroxidation in viable boar spermatozoa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fluorescence-activated flow cytometry analyses were developed for determination of reactive oxygen species (ROS) formation and membrane lipid peroxidation in live spermatozoa loaded with, respectively, hydroethidine (HE) or the lipophilic probe 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-d...

  16. Flow cytometry and K-mer analysis estimates of the genome sizes of Bemisia tabaci B and Q (Hemiptera: Aleyrodidae)

    PubMed Central

    Guo, Li T.; Wang, Shao L.; Wu, Qing J.; Zhou, Xu G.; Xie, Wen; Zhang, You J.

    2015-01-01

    The genome sizes of the B- and Q-types of the whitefly Bemisia tabaci (Gennnadius) were estimated using flow cytometry (Drosophila melanogaster as the DNA reference standard and propidium iodide (PI) as the fluorochrome) and k-mer analysis. For flow cytometry, the mean nuclear DNA content was 0.686 pg for B-type males, 1.392 pg for B-type females, 0.680 pg for Q-type males, and 1.306 pg for Q-type females. Based on the relationship between DNA content and genome size (1 pg DNA = 980 Mbp), the haploid genome size of B. tabaci ranged from 640 to 682 Mbp. For k-mer analysis, genome size of B-type by two methods were consistent highly, but the k-mer depth distribution graph of Q-type was not enough perfect and the genome size was estimated about 60 M larger than its flow cytometry result. These results corroborate previous reports of genome size based on karyotype analysis and chromosome counting. However, these estimates differ from previous flow cytometry estimates, probably because of differences in the DNA reference standard and dyeing time, which were superior in the current study. For Q-type genome size difference by two method, some discussion were also stated, and all these results represent a useful foundation for B. tabaci genomics research. PMID:26042041

  17. 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

  18. 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…

  19. Computationally efficient multidimensional analysis of complex flow cytometry data using second order polynomial histograms.

    PubMed

    Zaunders, John; Jing, Junmei; Leipold, Michael; Maecker, Holden; Kelleher, Anthony D; Koch, Inge

    2016-01-01

    Many methods have been described for automated clustering analysis of complex flow cytometry data, but so far the goal to efficiently estimate multivariate densities and their modes for a moderate number of dimensions and potentially millions of data points has not been attained. We have devised a novel approach to describing modes using second order polynomial histogram estimators (SOPHE). The method divides the data into multivariate bins and determines the shape of the data in each bin based on second order polynomials, which is an efficient computation. These calculations yield local maxima and allow joining of adjacent bins to identify clusters. The use of second order polynomials also optimally uses wide bins, such that in most cases each parameter (dimension) need only be divided into 4-8 bins, again reducing computational load. We have validated this method using defined mixtures of up to 17 fluorescent beads in 16 dimensions, correctly identifying all populations in data files of 100,000 beads in <10 s, on a standard laptop. The method also correctly clustered granulocytes, lymphocytes, including standard T, B, and NK cell subsets, and monocytes in 9-color stained peripheral blood, within seconds. SOPHE successfully clustered up to 36 subsets of memory CD4 T cells using differentiation and trafficking markers, in 14-color flow analysis, and up to 65 subpopulations of PBMC in 33-dimensional CyTOF data, showing its usefulness in discovery research. SOPHE has the potential to greatly increase efficiency of analysing complex mixtures of cells in higher dimensions. PMID:26097104

  20. An Introduction to Automated Flow Cytometry Gating Tools and Their Implementation

    PubMed Central

    Verschoor, Chris P.; Lelic, Alina; Bramson, Jonathan L.; Bowdish, Dawn M. E.

    2015-01-01

    Current flow cytometry (FCM) reagents and instrumentation allow for the measurement of an unprecedented number of parameters for any given cell within a homogenous or heterogeneous population. While this provides a great deal of power for hypothesis testing, it also generates a vast amount of data, which is typically analyzed manually through a processing called “gating.” For large experiments, such as high-content screens, in which many parameters are measured, the time required for manual analysis as well as the technical variability inherent to manual gating can increase dramatically, even becoming prohibitive depending on the clinical or research goal. In the following article, we aim to provide the reader an overview of automated FCM analysis as well as an example of the implementation of FLOw Clustering without K, a tool that we consider accessible to researchers of all levels of computational expertise. In most cases, computational assistance methods are more reproducible and much faster than manual gating, and for some, also allow for the discovery of cellular populations that might not be expected or evident to the researcher. We urge any researcher who is planning or has previously performed large FCM experiments to consider implementing computational assistance into their analysis pipeline. PMID:26284066

  1. Panel development for multicolor flow-cytometry testing of proliferation and immunophenotype in hMSCs.

    PubMed

    Bradford, Jolene A; Clarke, Scott T

    2011-01-01

    Adult human mesenchymal stem cells (hMSC) are rare fibroblast-like cells capable of differentiation into a variety of cell tissues which include bone, cartilage, muscle, ligament, tendon, and adipose. Normal adult bone marrow and adipose tissue are the most common sources of these cells. The International Society for Cellular Therapy (ISCT) has proposed a set of standards to define hMSC for laboratory investigations and preclinical studies: adherence to plastic in standard culture conditions; in vitro differentiation into osteoblasts, adipocytes, and chondroblasts; and specific surface antigen expression. Direct measurement of proliferation combined with simultaneous detection of the ISCT-consensus immunophenotypic profile provides data that is used to determine the differentiation status and health of the cells. Flow cytometry provides a powerful technology that is routinely used to simultaneously and rapidly measure multiple parameters in a single sample. This chapter describes a flow cytometric panel for the simultaneous detection of immunophenotypic profile, proliferative capacity, and DNA content measurement in hMSC. Because a relatively small number of cells are needed with this approach, measurements can be made with minimal impact on expansion potential. The ability to assess antigen expression and proliferative status enables the investigator to make informed decisions on expansion and harvesting. PMID:21431532

  2. Use of fluorescent sequence-specific polyamides to discriminate human chromosomes by microscopy and flow cytometry.

    PubMed

    Gygi, Melanie P; Ferguson, Mark D; Mefford, Heather C; Lund, Kevin P; O'Day, Christine; Zhou, Peiwen; Friedman, Cynthia; van den Engh, Ger; Stolowitz, Mark L; Trask, Barbara J

    2002-07-01

    In this paper, we demonstrate the use of synthetic polyamide probes to fluorescently label heterochromatic regions on human chromosomes for discrimination in cytogenetic preparations and by flow cytometry. Polyamides bind to the minor groove of DNA in a sequence-specific manner. Unlike conventional sequence-specific DNA or RNA probes, polyamides can recognize their target sequence without the need to subject chromosomes to harsh denaturing conditions. For this study, we designed and synthesized a polyamide to target the TTCCA-motif repeated in the heterochromatic regions of chromosome 9, Y and 1. We demonstrate that the fluorescently labeled polyamide binds to its target sequence in both conventional cytogenetic preparations of metaphase chromosomes and suspended chromosomes without denaturation. Chromosomes 9 and Y can be discriminated and purified by flow sorting on the basis of polyamide binding and Hoechst 33258 staining. We generate chromosome 9- and Y-specific 'paints' from the sorted fractions. We demonstrate the utility of this technology by characterizing the sequence of an olfactory receptor gene that is duplicated on multiple chromosomes. By separating chromosome 9 from chromosomes 10-12 on the basis of polyamide fluorescence, we determine and differentiate the haplotypes of the highly similar copies of this gene on chromosomes 9 and 11. PMID:12087162

  3. Petrochemical-related DNA damage in wild rodents detected by flow cytometry

    SciTech Connect

    McBee, K.; Bickham, J.W.

    1988-03-01

    The need for quick, reliable, in situ tests of environmental mutagenicity is evidenced by increasing public concern about potential health effects of pollutants. Conventional tests of clastogenicity usually involve treatment of laboratory test systems with pure samples of suspect compounds followed by scoring numerous metaphase cells for chromosome aberrations. There are at least two shortcomings of these test protocols. They are very time consumptive and are generally restricted to controlled laboratory situations which may not realistically indicate the effects of environmental pollution. The use of flow cytometry to study resident rodent species as bioindicators provides a system by which cytogenetic effects of environmental pollutants upon exposed organisms rapidly and accurately can be ascertained. The authors found that two species of wild rodents (Peromyscus leucopus and Sigmodon hispidus) living at a dump site polluted with a complex mixture of oil, grease, polychlorinated biphenols, hexachlorobenzene, zinc, manganese, cadmium, chromium, copper, and lead had significantly higher frequencies of chromosomal aberrations than did animals from two unpolluted control sites. These data suggest that resident small mammals may be useful as in situ monitors of the presence and action of mutagenic pollutants in the environment. This study was conducted to determine if changes in patterns of DNA content indicative of the action of mutagens could be detected by flow cytometric analysis of tissues from these same animals.

  4. Lectin staining and flow cytometry reveals female-induced sperm acrosome reaction and surface carbohydrate reorganization

    PubMed Central

    Kekäläinen, Jukka; Larma, Irma; Linden, Matthew; Evans, Jonathan P.

    2015-01-01

    All cells are covered by glycans, an individually unique layer of oligo- and polysaccharides that are critical moderators of self-recognition and other cellular-level interactions (e.g. fertilization). The functional similarity between these processes suggests that gamete surface glycans may also have an important, but currently overlooked, role in sexual selection. Here we develop a user-friendly methodological approach designed to facilitate future tests of this possibility. Our proposed method is based on flow cytometric quantification of female-induced sperm acrosome reaction and sperm surface glycan modifications in the Mediterranean mussel Mytilus galloprovincialis. In this species, as with many other taxa, eggs release water-soluble factors that attract conspecific sperm (chemoattraction) and promote potentially measurable changes in sperm behavior and physiology. We demonstrate that flow cytometry is able to identify sperm from other seawater particles as well as accurately measure both acrosome reaction and structural modifications in sperm glycans. This methodological approach can increase our understanding of chemically-moderated gamete-level interactions and individual-specific gamete recognition in Mytilus sp. and other taxa with similar, easily identifiable acrosome structure. Our approach is also likely to be applicable to several other species, since carbohydrate-mediated cellular-level interactions between gametes are universal among externally and internally fertilizing species. PMID:26470849

  5. Determination by flow cytometry polyploidy inducing-capacity of colchicine in Cajanus cajan (L.) Mill sp.

    PubMed

    Udensi, O U; Ontui, V

    2013-07-01

    The need to optimize flow cytometric analysis for the determination of ploidy level is a worthwhile venture to precisely know at what concentration of a mutagen and at what time of exposure polyploidy could be induced. Flow cytometry was used to determine the polyploidy inducing-capacity of colchicine in pigeon pea (Cajanus cajan (L.) Mill sp). Seeds of pigeon pea were soaked in three different concentrations of colchicine-5 mg, 10 and 15 mg L(-1) for 24, 48 and 72 h, respectively, while the control group was soaked in water. Treated seeds and those from the control were planted in a greenhouse using a Completely Randomized Design (CRD). Results show that colchicine induced tetraploids (4n) and mixoploids (2n+ 4n) as the concentration of colchicine increased and soaking duration. Days to seedling emergence increased as concentration of colchicine and duration of soaking increased while germination rate decreased proportionately with the increase in colchicine concentration and soaking duration but did not significantly affect percentage seedling survival. Explicitly, colchicine has the capacity of inducing polyploidy; especially tetraploids on the seeds of pigeon pea, which obviously could be harnessed for further breeding and improvement of the pigeon pea. PMID:24505986

  6. Release of FITC-BSA from poly(l-lactic acid) microspheres analysis using flow cytometry.

    PubMed

    Kuo, Chih-Feng; Tsao, Nina; Chou, Hsin-Hao; Liu, Yi-Ling; Hsieh, Wen-Chuan

    2012-01-01

    In this investigation, biodegradable polymer poly(L-lactic acid) (PLA) microspheres were prepared by the W(1)/O/W(2) solvent evaporation method. The inner phase was aqueous solution (W(1)) that contained bovine serum albumin that was labeled with fluorescein isothiocyanate (FITC-BSA). PLA was dissolved in chloroform with emulsifier sorbitan monooleate (span 80) as the dispersed phase (O). These two solutions (W(1)/O) were emulsified by a homogenizer to form a primary emulsion. Polyvinyl alcohol (PVA) used as surfactant, was applied in the formation of microspheres (W(2)). 0.5% (w/v) PLA was stirred at 3000 rpm using a homogenizer. Microspheres with sizes of up to around 10 μm were produced. These microspheres were separated by the glycerol gradient method, and take microspheres at part of 25% glycerol gradient concentration was analyzed by flow cytometry, indicating a more homogeneous particle size distribution than that not separated. The microspheres were degraded using several enzymes, and around 40% was degraded by 72 h. This result reveals the effectiveness of drug delivery by PLA microspheres, which was evaluated by performing a drug release test and flow cytometric analysis. The FITC-BSA concentration in the supernatant increased with the experimental time. At the phagocytosis experiments, encapsulated with FITC-BSA drug of microspheres can be used by the cell, as particle size approximately 1 μm. PMID:21992796

  7. A Bead-Based Method for Multiplexed Identification and Quantitation of DNA Sequences Using Flow Cytometry

    PubMed Central

    Spiro, Alexander; Lowe, Mary; Brown, Drew

    2000-01-01

    A new multiplexed, bead-based method which utilizes nucleic acid hybridizations on the surface of microscopic polystyrene spheres to identify specific sequences in heterogeneous mixtures of DNA sequences is described. The method consists of three elements: beads (5.6-μm diameter) with oligomer capture probes attached to the surface, three fluorophores for multiplexed detection, and flow cytometry instrumentation. Two fluorophores are impregnated within each bead in varying amounts to create different bead types, each associated with a unique probe. The third fluorophore is a reporter. Following capture of fluorescent cDNA sequences from environmental samples, the beads are analyzed by flow cytometric techniques which yield a signal intensity for each capture probe proportional to the amount of target sequences in the analyte. In this study, a direct hybrid capture assay was developed and evaluated with regard to sequence discrimination and quantitation of abundances. The target sequences (628 to 728 bp in length) were obtained from the 16S/23S intergenic spacer region of microorganisms collected from polluted groundwater at the nuclear waste site in Hanford, Wash. A fluorescence standard consisting of beads with a known number of fluorescent DNA molecules on the surface was developed, and the resolution, sensitivity, and lower detection limit for measuring abundances were determined. The results were compared with those of a DNA microarray using the same sequences. The bead method exhibited far superior sequence discrimination and possesses features which facilitate accurate quantitation. PMID:11010868

  8. Cutting-Edge Analysis of Extracellular Microparticles using ImageStreamX Imaging Flow Cytometry

    PubMed Central

    Headland, Sarah E.; Jones, Hefin R.; D'Sa, Adelina S. V.; Perretti, Mauro; Norling, Lucy V.

    2014-01-01

    Interest in extracellular vesicle biology has exploded in the past decade, since these microstructures seem endowed with multiple roles, from blood coagulation to inter-cellular communication in pathophysiology. In order for microparticle research to evolve as a preclinical and clinical tool, accurate quantification of microparticle levels is a fundamental requirement, but their size and the complexity of sample fluids present major technical challenges. Flow cytometry is commonly used, but suffers from low sensitivity and accuracy. Use of Amnis ImageStreamX Mk II imaging flow cytometer afforded accurate analysis of calibration beads ranging from 1 μm to 20 nm; and microparticles, which could be observed and quantified in whole blood, platelet-rich and platelet-free plasma and in leukocyte supernatants. Another advantage was the minimal sample preparation and volume required. Use of this high throughput analyzer allowed simultaneous phenotypic definition of the parent cells and offspring microparticles along with real time microparticle generation kinetics. With the current paucity of reliable techniques for the analysis of microparticles, we propose that the ImageStreamX could be used effectively to advance this scientific field. PMID:24913598

  9. Cutting-edge analysis of extracellular microparticles using ImageStream(X) imaging flow cytometry.

    PubMed

    Headland, Sarah E; Jones, Hefin R; D'Sa, Adelina S V; Perretti, Mauro; Norling, Lucy V

    2014-01-01

    Interest in extracellular vesicle biology has exploded in the past decade, since these microstructures seem endowed with multiple roles, from blood coagulation to inter-cellular communication in pathophysiology. In order for microparticle research to evolve as a preclinical and clinical tool, accurate quantification of microparticle levels is a fundamental requirement, but their size and the complexity of sample fluids present major technical challenges. Flow cytometry is commonly used, but suffers from low sensitivity and accuracy. Use of Amnis ImageStream(X) Mk II imaging flow cytometer afforded accurate analysis of calibration beads ranging from 1 μm to 20 nm; and microparticles, which could be observed and quantified in whole blood, platelet-rich and platelet-free plasma and in leukocyte supernatants. Another advantage was the minimal sample preparation and volume required. Use of this high throughput analyzer allowed simultaneous phenotypic definition of the parent cells and offspring microparticles along with real time microparticle generation kinetics. With the current paucity of reliable techniques for the analysis of microparticles, we propose that the ImageStream(X) could be used effectively to advance this scientific field. PMID:24913598

  10. Estimation of the frequency of malformed sperm by slit scan flow cytometry

    SciTech Connect

    Halamka, J.; Gray, J.W.; Gledhill, B.L.; Lake, S.; Wyrobek, A.J.

    1984-01-01

    An investigation was made of the utility of Slit Scan Flow Cytometry (SSFCM) for measuring the frequencies of malformed sperm heads in control and mutagen treated B6C3F1/CRL mice. In SSFCM, fluorescence profiles of sperm heads stained with the DNA-specific fluorescent dye acriflavine were recorded for sperm flowing lengthwise through a 2.5-..mu..m-thick laser beam. Malformed sperm were detected as having fluorescence profiles that differed substantially from an average fluorescence profile for sperm from untreated mice. Fluorescence profiles were measured for 500 sperm per mouse from five control mice, five mice injected intraperitoneally daily for 5 days with a total of 375 mg/kg of body weight methyl methane sulfonate (MMS), and for 30 mice injected intraperitoneally daily for 5 days with total doses of procarbazine ranging from 125 mg/kg to 1250 mg/kg. Sperm were collected from the caudae epididymides 35 days after the last injection. Frequencies of malformed sperm in these samples were also estimated by visual analysis. All samples were analyzed in double blind fashion. The visual and SSFCM malformed sperm frequencies for the samples from control, MMS-treated, and procarbazine-treated mice were correlated. A dose effect was seen with both the visual and SSFCM estimates for the sperm from the procarbazine-treated mice. 8 references 3 figures.

  11. Flow cytometry compared with indirect immunofluorescence for rapid detection of dengue virus type 1 after amplification in tissue culture.

    PubMed

    Kao, C L; Wu, M C; Chiu, Y H; Lin, J L; Wu, Y C; Yueh, Y Y; Chen, L K; Shaio, M F; King, C C

    2001-10-01

    Dengue virus (DV) was detected early in infected mosquito C6/36 cells by using indirect immunofluorescence (IF) in conjunction with flow cytometry. Three fixation-permeabilization methods and three DV serotype 1 (DEN-1)-specific monoclonal antibodies, 8-8 (anti-E), 16-4 (anti-NS1), and 15F3-1 (anti-NS1), were evaluated for the detection of DEN-1 in infected C6/36 cells. We found that these three monoclonal antibodies were capable of detecting DV in C6/36 cells as early as 24 h postinoculation by using a conventional indirect IF stain. Both 8-8 and 16-4 detected DV earlier and showed a greater number of DV-positive cells than 15F3-1. In flow cytometry, 3% paraformaldehyde plus 0.1% Triton X-100 with 16-4, the best fixation-permeabilization method for testing DV, showed higher sensitivity (up to 1 PFU) than indirect IF stain. The higher sensitivity of 16-4 in detecting DEN-1 was found with both IF and flow cytometry. Flow cytometry, which had a sensitivity similar to that of nested reverse transcription-PCR, was more sensitive in detecting DV in the infected mosquito cells 10 h earlier than the conventional IF stain. When clinical specimens were amplified in mosquito C6/36 cells and then assayed for DV using flow cytometry and conventional virus isolation at day 7 postinfection, both methods had 97.22% (35 out of 36) agreement. Moreover, among 12 positive samples which were detected by conventional culture method, the flow cytometry assay could detect DV in 58.33% (7 out of 12) of samples even at day 3 postinfection. In conclusion, both monoclonal antibodies 8-8 and 16-4 can be used for the early detection of DEN-1-infected C6/36 cells, with 16-4 (anti-NS1) being the best choice for the rapid diagnosis of DV by both the IF staining and flow cytometry methods. PMID:11574589

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

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

  13. Quantification methods for human and large animal leukocytes using DNA dyes by flow cytometry.

    PubMed

    Pieper, Ina Laura; Radley, Gemma; Chan, Chris H H; Friedmann, Yasmin; Foster, Graham; Thornton, Catherine A

    2016-06-01

    Ovine and bovine blood is used heavily within the development of blood-handling medical devices, such as heart pumps (left ventricular assist devices, LVADs), for which blood cell damage needs to be monitored during in vitro testing. Hematology analyzers provide cell counts but no information about cell viability. The anthraquinone DNA dyes CyTRAK Orange™ and DRAQ7™ have practical and spectral properties rendering them suitable for multicolor assays. Compared to other DNA dyes such as Vybrant Dyecycle, CyTRAK Orange enables a faster staining protocol and does not require incubation at +37°C. Compared to traditional viability dyes such as propidium iodide and 7AAD, DRAQ7's unique spectral profile of excitation in both blue and red lasers and far-red emission enables identification of dual positive dead cell events and frees up detectors for use with other reagents. CyTRAK Orange and DRAQ7 could be used in combination with absolute counting bead standards to provide cell counts and viability but the combination of these dyes has previously only been used for microscopy on rodent cells. The purpose of this study was to evaluate the use of these dyes in combination in large animal blood samples for flow cytometry. A viability and cell counting protocol for bovine, ovine, and human leukocytes using CyTRAK Orange and DRAQ7 was prepared. Four different counting bead standards were evaluated using the Navios and FACSAria cytometers and compared to counts obtained from hematology analyzers. CyTRAK Orange successfully detected CD45(+) leukocytes in all species. The DRAQ7 single-stained dead cell counts correlated well with the CyTRAK Orange/DRAQ7 double-stained dead cell counts in human and bovine blood, but not in ovine blood, which could be related to the blood source. In conclusion, for human and bovine blood, this method works well for viability counts using different flow cytometers and bead standards. © 2016 International Society for Advancement of Cytometry

  14. Development of a new fertility prediction model for stallion semen, including flow cytometry.

    PubMed

    Barrier Battut, I; Kempfer, A; Becker, J; Lebailly, L; Camugli, S; Chevrier, L

    2016-09-01

    Several laboratories routinely use flow cytometry to evaluate stallion semen quality. However, objective and practical tools for the on-field interpretation of data concerning fertilizing potential are scarce. A panel of nine tests, evaluating a large number of compartments or functions of the spermatozoa: motility, morphology, viability, mitochondrial activity, oxidation level, acrosome integrity, DNA integrity, "organization" of the plasma membrane, and hypoosmotic resistance, was applied to a population of 43 stallions, 33 of which showing widely differing fertilities (19%-84% pregnancy rate per cycle [PRC]). Analyses were performed either within 2 hours after semen collection or after 24-hour storage at 4 °C in INRA96 extender, on three to six ejaculates for each stallion. The aim was to provide data on the distribution of values among said population, showing within-stallion and between-stallion variability, and to determine whether appropriate combinations of tests could evaluate the fertilizing potential of each stallion. Within-stallion repeatability, defined as intrastallion correlation (r = between-stallion variance/total variance) ranged between 0.29 and 0.84 for "conventional" variables (viability, morphology, and motility), and between 0.15 and 0.81 for "cytometric" variables. Those data suggested that analyzing six ejaculates would be adequate to characterize a stallion. For most variables, except those related to DNA integrity and some motility variables, results differed significantly between immediately performed analyses and analyses performed after 24 hours at 4 °C. Two "best-fit" combinations of variables were determined. Factorial discriminant analysis using a first combination of seven variables, including the polarization of mitochondria, acrosome integrity, DNA integrity, and hypoosmotic resistance, permitted exact determination of the fertility group for each stallion: fertile, that is, PRC higher than 55%; intermediate, that is, 45

  15. Toward the measurement of multiple fluorescence lifetimes in flow cytometry: maximizing multi-harmonic content from cells and microspheres.

    PubMed

    Jenkins, Patrick; Naivar, Mark A; Houston, Jessica P

    2015-11-01

    Flow cytometry is a powerful means for in vitro cellular analyses where multi-fluorescence and multi-angle light scattering can indicate unique biochemical or morphological features of single cells. Yet, to date, flow cytometry systems have lacked the ability to capture complex fluorescence dynamics due to the transient nature of flowing cells. In this contribution we introduce a simple approach for measuring multiple fluorescence lifetimes from a single cytometric event. We leverage square wave modulation, Fourier analysis, and high frequency digitization and show the ability to resolve more than one fluorescence lifetime from fluorescently-labelled cells and microspheres. Illustration of a flow cytometer capable of capturing multiple fluorescence lifetime measurements; creating potential for multi-parametric, time-resolved signals to be captured for every color channel. PMID:25727072

  16. FIND: A new software tool and development platform for enhanced multicolor flow analysis

    PubMed Central

    2011-01-01

    Background Flow Cytometry is a process by which cells, and other microscopic particles, can be identified, counted, and sorted mechanically through the use of hydrodynamic pressure and laser-activated fluorescence labeling. As immunostained cells pass individually through the flow chamber of the instrument, laser pulses cause fluorescence emissions that are recorded digitally for later analysis as multidimensional vectors. Current, widely adopted analysis software limits users to manual separation of events based on viewing two or three simultaneous dimensions. While this may be adequate for experiments using four or fewer colors, advances have lead to laser flow cytometers capable of recording 20 different colors simultaneously. In addition, mass-spectrometry based machines capable of recording at least 100 separate channels are being developed. Analysis of such high-dimensional data by visual exploration alone can be error-prone and susceptible to unnecessary bias. Fortunately, the field of Data Mining provides many tools for automated group classification of multi-dimensional data, and many algorithms have been adapted or created for flow cytometry. However, the majority of this research has not been made available to users through analysis software packages and, as such, are not in wide use. Results We have developed a new software application for analysis of multi-color flow cytometry data. The main goals of this effort were to provide a user-friendly tool for automated gating (classification) of multi-color data as well as a platform for development and dissemination of new analysis tools. With this software, users can easily load single or multiple data sets, perform automated event classification, and graphically compare results within and between experiments. We also make available a simple plugin system that enables researchers to implement and share their data analysis and classification/population discovery algorithms. Conclusions The FIND (Flow

  17. Measurement of Separase Proteolytic Activity in Single Living Cells by a Fluorogenic Flow Cytometry Assay

    PubMed Central

    Haaß, Wiltrud; Kleiner, Helga; Müller, Martin C.; Hofmann, Wolf-Karsten; Fabarius, Alice; Seifarth, Wolfgang

    2015-01-01

    ESPL1/Separase, an endopeptidase, is required for centrosome duplication and separation of sister-chromatides in anaphase of mitosis. Overexpression and deregulated proteolytic activity of Separase as frequently observed in human cancers is associated with the occurrence of supernumerary centrosomes, chromosomal missegregation and aneuploidy. Recently, we have hypothesized that increased Separase proteolytic activity in a small subpopulation of tumor cells may serve as driver of tumor heterogeneity and clonal evolution in chronic myeloid leukemia (CML). Currently, there is no quantitative assay to measure Separase activity levels in single cells. Therefore, we have designed a flow cytometry-based assay that utilizes a Cy5- and rhodamine 110 (Rh110)-biconjugated Rad21 cleavage site peptide ([Cy5-D-R-E-I-M-R]2-Rh110) as smart probe and intracellular substrate for detection of Separase enzyme activity in living cells. As measured by Cy5 fluorescence the cellular uptake of the fluorogenic peptide was fast and reached saturation after 210 min of incubation in human histiocytic lymphoma U937 cells. Separase activity was recorded as the intensity of Rh110 fluorescence released after intracellular peptide cleavage providing a linear signal gain within a 90–180 min time slot. Compared to conventional cell extract-based methods the flow cytometric assay delivers equivalent results but is more reliable, bypasses the problem of vague loading controls and unspecific proteolysis associated with whole cell extracts. Especially suited for the investigaton of blood- and bone marrow-derived hematopoietic cells the flow cytometric Separase assay allows generation of Separase activity profiles that tell about the number of Separase positive cells within a sample i.e. cells that currently progress through mitosis and about the range of intercellular variation in Separase activity levels within a cell population. The assay was used to quantify Separase proteolytic activity in leukemic

  18. Flow cytometry analysis of platelet P-selectin expression in whole blood--methodological considerations.

    PubMed

    Ritchie, J L; Alexander, H D; Rea, I M

    2000-12-01

    P-selectin is an adhesion molecule found in the alpha granules of platelets. Activation occurs in response to a range of inflammatory and thrombotic agents resulting in rapid up-regulation. Flow cytometry methods have recently been described for the analysis of platelet P-selectin expression in whole blood. While introducing these methods into our laboratory it was noted that expression could be stimulated, in vitro, in a number of ways. This study shows that red cell lysis, the anticoagulant K3 EDTA and the time elapse between blood collection and antibody labelling had statistically significant effects on P-selectin expression. Post-labelling fixation, with CellFIX, caused no significant effect. We conclude that blood for P-selectin analysis should be collected in sodium citrate and that red cell lysis and centrifugation should be avoided. When comparing samples, the time between collection and labelling should be standardized. The relatively high CV for the assay indicates that all samples should be labelled and analysed in duplicate with the mean level reported. PMID:11318803

  19. A neutralizing scFv antibody against infectious bursal disease virus screened by flow cytometry.

    PubMed

    Zhou, Yao; Xie, Zhi-Gang

    2015-09-01

    Infectious bursal disease (IBD) is considered a vital viral disease that threatens the poultry industry worldwide. In this study, a recombinant single chain variable fragment (scFv) antibody library derived from chickens immunized with VP2 protein of infectious bursal disease virus (IBDV) was constructed. The library was subjected to three rounds of screening by flow cytometry (FCM) against VP2/IBDV through a bacteria display technology, resulting in the enrichment of scFvs. Three scFv clones with different fluorescence intensity were obtained by colony pick up at random. The obtained scFv antibodies were expressed and purified. Relative affinity assay showed the three clones had different sensitivity to VP2, in accordance with fluorescence activity cell sorting analysis (FACS). The potential use of the isolated IBDV-specific scFv antibodies was demonstrated by the successful application of these antibodies in Western blotting and ELISA assay. What's more, in vitro neutralization measurement showed that one of the three isolated antibodies possessed the neutralization function against IBDV. This study provides new strategies for screening of antibody library, and scFv antibodies isolated in this study may be utilized as lead candidates for further development of diagnostic or therapeutic antibodies for detection and treatment of IBDV infection. PMID:26003676

  20. Evaluation of DNA degradation using flow cytometry: promising tool for postmortem interval determination.

    PubMed

    Williams, Teddric; Soni, Shivani; White, Jason; Can, Gunay; Javan, Gulnaz T

    2015-06-01

    Over the years, there have been numerous formulas proposed for use in determining the postmortem interval (PMI); however, no method is all encompassing and absolute. Even so, very little research has been undertaken to determine if there is a viable correlation between the rate of DNA degradation and PMI, which can be calculated from analysis by flow cytometry. In this study, we analyzed the rate of DNA degradation of spleen and brain tissues from 15 cadavers over a 96-hour period of time at 2 temperature conditions, that is, 21°C (room temperature) and 4°C (refrigerator) to mimic summer and winter weather, respectively. The resulting data were used to form a pattern that correlates DNA degradation to cell death occurrence. Statistical analyses were performed to determine the significance of the relationship between PMI and DNA degradation. Moreover, in search of alternative reliable organs of interest for PMI estimation, the results demonstrate that the brain has lesser DNA degradation as compared with the spleen. Thus, the current study suggests that the brain can be an organ of choice for PMI studies, but more research is underway in this aspect. PMID:25893913

  1. Flow cytometry used to assess genetic damage in frogs from farm ponds

    USGS Publications Warehouse

    Bly, B.L.; Knutson, M.G.; Sandheinrich, M.B.; Gray, B.R.; Jobe, D.A.

    2004-01-01

    Flow cytometry (FC) is a laboratory method used to detect genetic damage induced by environmental contaminants and other stressors in animals, including amphibians. We tested FC methods on three species of ranid frogs collected from farm ponds and natural wetlands in southeastern Minnesota. We compared FC metrics for Rana clamitans between ponds with direct exposure to agricultural contaminants and reference (unexposed) ponds. Concentrations of atrazine in water from our farm ponds ranged from 0.04 to 0.55 ppb. We found that R. clamitans from exposed ponds had DNA content similar to frogs from unexposed ponds. Pond-averaged C-values (a measure of DNA content) ranged from 6.53 to 7.08 for R. pipiens (n . 13), 6.55 to 6.60 for R. clamitans (n . 40) and 6.74 for R. palustris (n . 5). Among all species, the mean sample CVs ranged from 1.91 (R. palustris) to 6.31 (R. pipiens). Deformities were observed in only 2 of 796 individuals among all species and occurred in both reference and exposed ponds. Although we did not detect evidence of DNA damage associated with agriculture in our study, we demonstrated the potential of FC for screening amphibian populations for genetic damage. Metrics from a variety of amphibian species and locations as well as laboratory studies are needed to further assess the value of FC for monitoring amphibian genetic integrity in contaminated sites.

  2. Evaluation of Bt (Bacillus thuringiensis) corn on mouse testicular development by dual parameter flow cytometry.

    PubMed

    Brake, Denise G; Thaler, Robert; Evenson, Donald P

    2004-04-01

    The health safety of Bt (Bacillus thuringiensis) corn (Zea mays L.) was studied using mouse testes as a sensitive biomonitor of potential toxic effects. Pregnant mice were fed a Bt corn or a nontransgenic (conventional) diet during gestation and lactation. After they were weaned, young male mice were maintained on the respective diets. At 8, 16, 26, 32, 63, and 87 days after birth, three male mice and an adult reference mouse were killed, the testes were surgically removed, and the percentage of germ cell populations was measured by flow cytometry. Multigenerational studies were conducted in the same manner. There were no apparent differences in percentages of testicular cell populations (haploid, diploid, and tetraploid) between the mice fed the Bt corn diet and those fed the conventional diet. Because of the high rate of cell proliferation and extensive differentiation that makes testicular germ cells highly susceptible to some toxic agents, it was concluded that the Bt corn diet had no measurable or observable effect on fetal, postnatal, pubertal, or adult testicular development. If data from this study were extrapolated to humans, Bt corn is not harmful to human reproductive development. PMID:15053558

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

    PubMed Central

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

    2014-01-01

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

  4. Evaluation of intranuclear BrdU detection procedures for use in multicolor flow cytometry*

    PubMed Central

    Rothaeusler, Kristina; Baumgarth, Nicole

    2010-01-01

    Background Measurement of cell proliferation via BrdU incorporation in combination with multicolor cell surface staining would facilitate studies on cell subsets that require multiple markers for their identification. However, the extent to which the often harsh cell preparation procedures required affect the staining quality of more recently developed fluorescent dyes has not been assessed. Methods Three cell preparation protocols for BrdU measurement were compared for their ability to maintain fluorescent surface staining and scatter parameters of in vivo BrdU-labeled cells by flow cytometry. A 10-color fluorescent panel was developed to test the quality of surface staining following cell treatment and the ability to perform BrdU measurements on even small B lymphocyte subsets. Results All cell preparation procedures affected the quality of fluorescent and/or scatter parameters to varying degrees. Paraformaldehyde / saponin-based procedures preserved sufficient fluorescent surface staining to determine BrdU incorporation rates among all splenic B cell subsets, including B-1a cells, which constitute roughly 0.5% of cells. Turnover rates of B-1a cells were similar to immature B cells and higher than those of the other mature B cell subsets. Conclusion Paraformaldehyde / saponin-based cell preparation procedures facilitate detailed cell turnover studies on small cell subsets in vivo, revealing new functional information on rare cell populations. PMID:16538653

  5. Assessing microbiological water quality in drinking water distribution systems with disinfectant residual using flow cytometry.

    PubMed

    Gillespie, Simon; Lipphaus, Patrick; Green, James; Parsons, Simon; Weir, Paul; Juskowiak, Kes; Jefferson, Bruce; Jarvis, Peter; Nocker, Andreas

    2014-11-15

    Flow cytometry (FCM) as a diagnostic tool for enumeration and characterization of microorganisms is rapidly gaining popularity and is increasingly applied in the water industry. In this study we applied the method to obtain a better understanding of total and intact cell concentrations in three different drinking water distribution systems (one using chlorine and two using chloramines as secondary disinfectants). Chloramine tended to result in lower proportions of intact cells than chlorine over a wider residual range, in agreement with existing knowledge that chloramine suppresses regrowth more efficiently. For chlorinated systems, free chlorine concentrations above 0.5 mg L(-1) were found to be associated with relatively low proportions of intact cells, whereas lower disinfectant levels could result in substantially higher percentages of intact cells. The threshold for chlorinated systems is in good agreement with guidelines from the World Health Organization. The fact that the vast majority of samples failing the regulatory coliform standard also showed elevated proportions of intact cells suggests that this parameter might be useful for evaluating risk of failure. Another interesting parameter for judging the microbiological status of water, the biological regrowth potential, greatly varied among different finished waters providing potential help for investment decisions. For its measurement, a simple method was introduced that can easily be performed by water utilities with FCM capability. PMID:25123436

  6. Autofluorescence as a Signal to Sort Developing Glandular Trichomes by Flow Cytometry.

    PubMed

    Bergau, Nick; Navarette Santos, Alexander; Henning, Anja; Balcke, Gerd U; Tissier, Alain

    2016-01-01

    The industrial relevance of a number of metabolites produced in plant glandular trichomes (GTs) has spurred research on these specialized organs for a number of years. Most of the research, however, has focused on the elucidation of secondary metabolite pathways and comparatively little has been undertaken on the development and differentiation of GTs. One way to gain insight into these developmental processes is to generate stage-specific transcriptome and metabolome data. The difficulty for this resides in the isolation of early stages of development of the GTs. Here we describe a method for the separation and isolation of intact young and mature type VI trichomes from the wild tomato species Solanum habrochaites. The final and key step of the method uses cell sorting based on distinct autofluorescence signals of the young and mature trichomes. We demonstrate that sorting by flow cytometry allows recovering pure fractions of young and mature trichomes. Furthermore, we show that the sorted trichomes can be used for transcript and metabolite analyses. Because many plant tissues or cells have distinct autofluorescence components, the principles of this method can be generally applicable for the isolation of specific cell types without prior labeling. PMID:27446176

  7. Utilization of flow cytometry for festulolium breeding (Lolium multiflorum (2x) × Festuca arundinacea (6x))

    PubMed Central

    Akiyama, Yukio; Ueyama, Yasufumi; Hamada, Seiya; Kubota, Akito; Kato, Daisuke; Yamada-Akiyama, Hitomi; Takahara, Yoshinori; Fujimori, Masahiro

    2016-01-01

    Festulolium is a hybrid between Festuca and Lolium species that has valuable agronomic traits from both grass species. The purpose of our breeding program is to produce hexaploid festulolium that introduces tolerance to summer depression into Italian ryegrass (Lolium multiflorum) by crossing it with tall fescue (Festuca arundinacea). However, we found the DNA ploidy of hexaploids was not stable and was reduced in successive generations. We aimed to find out how to obtain stable high-ploidy festulolium. F1 hybrids of L. multiflorum and F. arundinacea were produced. The F3 generation was produced from putative hexaploid F2 individuals by open pollination. The F4 to F6 generations were obtained by polycrossing. The DNA ploidy levels of F2 to F6 individuals were estimated by flow cytometry. Cytological characteristics of the F5 and F6 individuals were investigated by FISH and GISH. The DNA ploidy level of hexaploid festulolium was reduced and stabilized at almost the same level as a tetraploid. Seed fertility was inversely correlated with an increase in ploidy level. GISH revealed no preferential Lolium transmission. FISH with a telomere probe revealed that counting the exact number of chromosomes in festulolium was difficult. DNA ploidy level was strongly correlated with the number of chromosomes. PMID:27162495

  8. DNA flow cytometry of colorectal carcinoma: correlation of DNA stemlines with other prognostic indices.

    PubMed

    Bawani, M; Tibrewala, S; Copur, S; Harris, D; Gilman-Sachs, A

    1991-02-01

    DNA flow cytometry (FCM) was performed on paraffin-embedded tissue blocks of 38 surgically resected colorectal carcinomas (CRC). Forty-seven percent of tumors exhibited aneuploidy and 53% were diploid. Seventy-two percent of patients in the aneuploid but only 35% in the diploid group were alive after a mean follow-up of 30.7 and 28.8 months (p = 0.01), and 5-yr survival of 56.7% and 11.7%, respectively (p less than 0.05). The site of tumor location, Dukes' stage, and serum CEA level did not predict a certain DNA stemline. However, irrespective of the ploidy pattern, a serum CEA level greater than 5.0 was associated with a higher mortality and poor 5-yr survival (p less than 0.005). Similarly, advanced Dukes' stage was associated with higher mortality (p less than 0.05). Forty-six percent of the patients with lesions that were Dukes' B2 or advanced stage received adjuvant therapy. Eighty-five percent of this subgroup of patients died; 18% of these patients had aneuploid tumors. The role of FCM in the assessment of prognosis of CRC deserves further clinical evaluation in a randomized control trial. PMID:1992633

  9. Intracellular Cytokine Staining and Flow Cytometry: Considerations for Application in Clinical Trials of Novel Tuberculosis Vaccines

    PubMed Central

    Smith, Steven G.; Smits, Kaatje; Joosten, Simone A.; van Meijgaarden, Krista E.; Satti, Iman; Fletcher, Helen A.; Caccamo, Nadia; Dieli, Francesco; Mascart, Francoise; McShane, Helen; Dockrell, Hazel M.; Ottenhoff, Tom H. M.

    2015-01-01

    Intracellular cytokine staining combined with flow cytometry is one of a number of assays designed to assess T-cell immune responses. It has the specific advantage of enabling the simultaneous assessment of multiple phenotypic, differentiation and functional parameters pertaining to responding T-cells, most notably, the expression of multiple effector cytokines. These attributes make the technique particularly suitable for the assessment of T-cell immune responses induced by novel tuberculosis vaccines in clinical trials. However, depending upon the particular nature of a given vaccine and trial setting, there are approaches that may be taken at different stages of the assay that are more suitable than other alternatives. In this paper, the Tuberculosis Vaccine Initiative (TBVI) TB Biomarker Working group reports on efforts to assess the conditions that will determine when particular assay approaches should be employed. We have found that choices relating to the use of fresh whole blood or peripheral blood mononuclear cells (PBMC) and frozen PBMC; use of serum-containing or serum-free medium; length of stimulation period and use of co-stimulatory antibodies can all affect the sensitivity of intracellular cytokine assays. In the case of sample material, frozen PBMC, despite some loss of sensitivity, may be more advantageous for batch analysis. We also recommend that for multi-site studies, common antibody panels, gating strategies and analysis approaches should be employed for better comparability. PMID:26367374

  10. Autofluorescence as a Signal to Sort Developing Glandular Trichomes by Flow Cytometry

    PubMed Central

    Bergau, Nick; Navarette Santos, Alexander; Henning, Anja; Balcke, Gerd U.; Tissier, Alain

    2016-01-01

    The industrial relevance of a number of metabolites produced in plant glandular trichomes (GTs) has spurred research on these specialized organs for a number of years. Most of the research, however, has focused on the elucidation of secondary metabolite pathways and comparatively little has been undertaken on the development and differentiation of GTs. One way to gain insight into these developmental processes is to generate stage-specific transcriptome and metabolome data. The difficulty for this resides in the isolation of early stages of development of the GTs. Here we describe a method for the separation and isolation of intact young and mature type VI trichomes from the wild tomato species Solanum habrochaites. The final and key step of the method uses cell sorting based on distinct autofluorescence signals of the young and mature trichomes. We demonstrate that sorting by flow cytometry allows recovering pure fractions of young and mature trichomes. Furthermore, we show that the sorted trichomes can be used for transcript and metabolite analyses. Because many plant tissues or cells have distinct autofluorescence components, the principles of this method can be generally applicable for the isolation of specific cell types without prior labeling. PMID:27446176

  11. High throughput screening of scFv antibodies against viral hemorrhagic septicaemia virus by flow cytometry.

    PubMed

    Zhou, Yao; Xie, Zhi-Gang

    2015-07-01

    Viral hemorrhagic septicaemia (VHS) is an economically important disease that affects salmon and trout worldwide. In this study, a recombinant single chain variable fragment (scFv) antibody library derived from rainbow trout immunized with purified viral hemorrhagic septicaemia virus (VHSV) was constructed. The library was subjected to three rounds of screening by flow cytometry (FCM) against VHSV through a bacteria display technology, resulting in the enrichment of scFv. Four scFv clones with different fluorescence intensity were obtained by colony pick up at random following three rounds of screening. The isolated scFv antibodies were expressed and purified. Relative affinity assay showed the four clones had different sensitivity to VHSV, in accordance with FCM. The potential use of the selected VHSV-specific scFv antibodies was demonstrated by the successful application in Western blotting assay, ELISA and immunofluorescence antibody test (IFAT), and one of the isolated scFv molecular showed excellent in vitro and in vivo blocking activities against VHSV. scFv isolated in this study can be promising diagnostic and/or therapeutic reagents for VHS. This study provides powerful strategies for screening antibodies against new diseases. PMID:25813596

  12. Flow cytometry may allow microscope-independent detection of holocentric chromosomes in plants.

    PubMed

    Zedek, František; Veselý, Pavel; Horová, Lucie; Bureš, Petr

    2016-01-01

    Two chromosomal structures, known as monocentric and holocentric chromosomes, have evolved in eukaryotes. Acentric fragments of monocentric chromosomes are unequally distributed to daughter cells and/or lost, while holocentric fragments are inherited normally. In monocentric species, unequal distribution should generate chimeras of cells with different nuclear DNA content. We investigated whether such differences in monocentric species are detectable by flow cytometry (FCM) as (i) a decreased nuclear DNA content and (ii) an increased coefficient of variance (CV) of the G1 peak after gamma radiation-induced fragmentation. We compared 13 monocentric and 9 holocentric plant species. Unexpectedly, monocentrics and holocentrics did not differ with respect to parameters (i) and (ii) in their response to gamma irradiation. However, we found that the proportion of G2 nuclei was highly elevated in monocentrics after irradiation, while holocentrics were negligibly affected. Therefore, we hypothesize that DNA-damaging agents induce cell cycle arrest leading to endopolyploidy only in monocentric and not (or to much lesser extent) in holocentric plants. While current microscope-dependent methods for holocentrism detection are unreliable for small and numerous chromosomes, which are common in holocentrics, FCM can use somatic nuclei. Thus, FCM may be a rapid and reliable method of high-throughput screening for holocentric candidates across plant phylogeny. PMID:27255216

  13. Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry.

    PubMed

    Pohl, Marie O; Stertz, Silke

    2015-01-01

    Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 °C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV. PMID:26575457

  14. 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.

  15. Genome size of Alexandrium catenella and Gracilariopsis lemaneiformis estimated by flow cytometry

    NASA Astrophysics Data System (ADS)

    Du, Qingwei; Sui, Zhenghong; Chang, Lianpeng; Wei, Huihui; Liu, Yuan; Mi, Ping; Shang, Erlei; Zeeshan, Niaz; Que, Zhou

    2016-08-01

    Flow cytometry (FCM) technique has been widely applied to estimating the genome size of various higher plants. However, there is few report about its application in algae. In this study, an optimized procedure of FCM was exploited to estimate the genome size of two eukaryotic algae. For analyzing Alexandrium catenella, an important red tide species, the whole cell instead of isolated nucleus was studied, and chicken erythrocytes were used as an internal reference. The genome size of A. catenella was estimated to be 56.48 ± 4.14 Gb (1C), approximately nineteen times larger than that of human genome. For analyzing Gracilariopsis lemaneiformis, an important economical red alga, the purified nucleus was employed, and Arabidopsis thaliana and Chondrus crispus were used as internal references, respectively. The genome size of Gp. lemaneiformis was 97.35 ± 2.58 Mb (1C) and 112.73 ± 14.00 Mb (1C), respectively, depending on the different internal references. The results of this research will promote the related studies on the genomics and evolution of these two species.

  16. Antifungal Susceptibility Testing of Fluconazole by Flow Cytometry Correlates with Clinical Outcome

    PubMed Central

    Wenisch, Christoph; Moore, Caroline B.; Krause, Robert; Presterl, Elisabeth; Pichna, Peter; Denning, David W.

    2001-01-01

    Susceptibility testing of fungi by flow cytometry (also called fluorescence-activated cell sorting [FACS]) using vital staining with FUN-1 showed a good correlation with the standard M27-A procedure for assessing MICs. In this study we determined MICs for blood culture isolates from patients with candidemia by NCCLS M27-A and FACS methods and correlated the clinical outcome of these patients with in vitro antifungal resistance test results. A total of 24 patients with candidemia for whom one or more blood cultures were positive for a Candida sp. were included. Susceptibility testing was performed by NCCLS M27-A and FACS methods. The correlation of MICs (NCCLS M27-A and FACS) and clinical outcome was calculated. In 83% of the cases, the MICs of fluconazole determined by FACS were within 1 dilution of the MICs determined by the NCCLS M27-A method. For proposed susceptibility breakpoints, there was 100% agreement between the M27-A and FACS methods. In the FACS assay, a fluconazole MIC of <1 μg/ml was associated with cure (P < 0.001) whereas an MIC of ≥1 μg/ml was associated with death (P < 0.001). The M27-A-derived fluconazole MICs did not correlate with outcome (P = 1 and P = 0.133). PMID:11427554

  17. Antibiotic Susceptibility Testing of the Gram-Negative Bacteria Based on Flow Cytometry

    PubMed Central

    Saint-Ruf, Claude; Crussard, Steve; Franceschi, Christine; Orenga, Sylvain; Ouattara, Jasmine; Ramjeet, Mahendrasingh; Surre, Jérémy; Matic, Ivan

    2016-01-01

    Rapidly treating infections with adequate antibiotics is of major importance. This requires a fast and accurate determination of the antibiotic susceptibility of bacterial pathogens. The most frequently used methods are slow because they are based on the measurement of growth inhibition. Faster methods, such as PCR-based detection of determinants of antibiotic resistance, do not always provide relevant information on susceptibility, particularly that which is not genetically based. Consequently, new methods, such as the detection of changes in bacterial physiology caused by antibiotics using flow cytometry and fluorescent viability markers, are being explored. In this study, we assessed whether Alexa Fluor® 633 Hydrazide (AFH), which targets carbonyl groups, can be used for antibiotic susceptibility testing. Carbonylation of cellular macromolecules, which increases in antibiotic-treated cells, is a particularly appropriate to assess for this purpose because it is irreversible. We tested the susceptibility of clinical isolates of Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa, to antibiotics from the three classes: β-lactams, aminoglycosides, and fluoroquinolones. In addition to AFH, we used TO-PRO®-3, which enters cells with damaged membranes and binds to DNA, and DiBAC4 (3), which enters cells with depolarized membranes. We also monitored antibiotic-induced morphological alterations of bacterial cells by analyzing light scattering signals. Although all tested dyes and light scattering signals allowed for the detection of antibiotic-sensitive cells, AFH proved to be the most suitable for the fast and reliable detection of antibiotic susceptibility. PMID:27507962

  18. Flow cytometry evaluation of lead and cadmium effects on mouse spermatogenesis.

    PubMed

    Oliveira, Helena; Loureiro, João; Filipe, Luísa; Santos, Conceição; Ramalho-Santos, João; Sousa, Mário; Pereira, Maria de Lourdes

    2006-10-01

    Flow cytometry (FCM) is a powerful tool to evaluate cell DNA content and ploidy levels. We have assessed the accuracy of two protocols of nuclei isolation from paraffinized samples (P1 and P2) by comparing FCM results with those obtained using fresh material (F1-F3). After isolation, nuclei were stained with propidium iodide and quantitatively analysed by FCM for changes in germ cell ratios. Results obtained with Protocol P2 were similar to those obtained using the protocol that gave best results for fresh tissues (F2). Protocol P2 was then applied to paraffin embedded testicular samples from ICR-CD1 mice exposed to 1, 2 and 3 mg CdCl(2)/kg bw by single subcutaneous injection, and to 74 and 100 mg PbCl(2)/kg bw administered in four repeated doses. The highest doses of CdCl(2) decreased the number of haploid (1C) cells and increased the number of diploid (2C), S phase and tetraploid (4C) cells. Treatment with PbCl(2) did not induce significant changes in testicular cells subpopulations. These results support the usefulness of FCM in evaluating the effect of toxic substances on mouse spermatogenesis, using both fresh and paraffinized material. PMID:16650732

  19. Flow Cytometry Increases the Sensitivity of Detection of Leukemia and Lymphoma Cells in Bronchoalveolar Lavage Specimens

    PubMed Central

    Song, Joo Y.; Filie, Armando C.; Venzon, David; tevenson, Maryalice Stetler-S; Yuan, Constance M.

    2013-01-01

    Background Recent studies have definitively determined that Flow Cytometry (FC) is significantly more sensitive than cytomorphology (CM) in detection of hematolymphoid neoplasms (HLN). However, its utility in paucicellular bronchoalveolar lavage (BAL) specimens has not been established. Methods FC was performed on BAL specimens submitted from 44 patients with a prior diagnosis of HLN. Panels chosen were based upon cellularity of specimen and patient history. FC results were compared with concurrent CM evaluations. Results All 44 BALs were deemed satisfactory for FC and yielded informative results that assisted in diagnosis. Diagnoses included 22/44 B-cell neoplasms, 16/44 T-cell neoplasms, 4/44 myeloid neoplasms, and 2/44 plasma cell neoplasms. Overall concordance was demonstrated between FC and CM in 77% (34/44) of cases. In 9/44 cases (20%), one technique (FC or CM) clearly detected malignant cells when the other did not. FC was more sensitive than CM in detecting a HLN in 8/9 discordant cases. In only one case (1/44, 2%) were malignant HLN cells suspected by CM, but not identified by FC (1/44, 2%). Conclusion We demonstrate, in the largest series published to date, that FC can be performed on BAL specimens. FC is indicated in evaluation of BAL for HLN and improves sensitivity of detection of HLN over CM alone. An integrated FC and CM approach is superior to either technique alone in diagnostic evaluation of BAL. PMID:22837143

  20. Identification of Inhibitors of V-ATPase Pumps in Yeast by HTS Flow Cytometry

    PubMed Central

    Johnson, Rebecca M.; Allen, Chris; Melman, Sandra D.; Waller, Anna; Young, Susan M.; Sklar, Larry A.; Parra, Karlett J.

    2010-01-01

    Fluorescence intensity of the pH-sensitive carboxyfluorescein derivative BCECF was monitored by high throughput flow cytometry in living yeast cells. We measured fluorescence intensity of BCECF trapped in yeast vacuoles, acidic compartments equivalent to lysosomes where V-ATPases are abundant. Because V-ATPases maintain a low pH in the vacuolar lumen, V-ATPase inhibition by concanamycin A alkalinized the vacuole and increased BCECF fluorescence. Likewise, V-ATPase deficient mutant cells had greater fluorescence intensity than wild-type cells. Thus, we detected an increase of fluorescence intensity after short-term and long-term inhibition of V-ATPase function. We used yeast cells loaded with BCECF to screen a small chemical library of structurally diverse compounds in order to identify V-ATPase inhibitors. One compound, disulfiram, enhanced BCECF fluorescence intensity (although to a degree beyond anticipated for pH changes alone in the mutant cells). Once confirmed by dose response assays (EC50=26 μM), we verified V-ATPase inhibition by disulfiram in secondary assays which measured ATP hydrolysis in vacuolar membranes. The inhibitory action of disulfiram against V-ATPase pumps revealed a novel effect previously unknown for this compound. Because V-ATPases are highly conserved, new inhibitors identified could be used as research and therapeutic tools in cancer, viral infections, and other diseases where V-ATPases are involved. PMID:20018164

  1. Role of macrophages in circulating prostate cancer cells studied by in vivo flow cytometry

    NASA Astrophysics Data System (ADS)

    Liu, Rongrong; Guo, Jin; Gu, Zhengqin; Wei, Xunbin

    2013-02-01

    Macrophages appear to be directly involved in cancer progression and metastasis. However, the role of macrophages in influencing tumor metastasis has not been fully understood. Here, we have used an emerging technique, namely in vivo flow cytometry (IVFC) to study the depletion kinetics of circulating prostate cancer cells in mice and how depletion of macrophages by the liposome-encapsulated clodronate affects the depletion kinetics. Our results show different depletion kinetics of PC-3 prostate cancer cells between macrophage-deficient group and the control group. The number of circulating tumor cells (CTCs) in macrophage-deficient group decreases in a slower manner compared to the control mice group. The differences in depletion kinetics indicate that the absence of macrophages might facilitate the stay of prostate tumor cells in circulation. We speculate that macrophages might be able to arrest, phagocytose and digest PC-3 cancer cells. Therefore, the phagocytosis may mainly contribute to the differences in depletion kinetics. The developed methods here would be useful to study the relationship between macrophages and cancer metastasis in small animal tumor model.

  2. Studying the role of macrophages in circulating prostate cancer cells by in vivo flow cytometry

    NASA Astrophysics Data System (ADS)

    Cui, Xiaojun; Guo, Jin; Gu, Zhengqin; Wei, Xunbin

    2012-12-01

    Metastasis is a very complicated multi-step process and accounts for the low survival rate of the cancerous patients. To metastasize, the malignant cells must detach from the primary tumor and migrate to secondary sites in the body through either blood or lymph circulation. Macrophages appear to be directly involved in tumor progression and metastasis. However, the role of macrophages in affecting cancer metastasis has not been fully elucidated. Here, we have utilized an emerging technique, namely in vivo flow cytometry (IVFC) to study the depletion kinetics of circulating prostate cancer cells in mice and how depletion of macrophages by the liposome-encapsulated clodronate affects the depletion kinetics. Our results show different depletion kinetics of PC-3 cells between macrophage-deficient group and the control group. The number of circulating tumor cells (CTCs) in macrophage-deficient group decreases in a slower manner compared to the control mice group. The differences in depletion kinetics indicate that the absence of macrophages facilitates the stay of prostate cancer cells in circulation. We speculate that macrophages might be able to arrest, phagocytose and digest PC-3 cells. Therefore, the phagocytosis may mainly contribute to the depletion kinetic differences. The developed methods here would be useful to study the relationship between macrophages and tumor metastasis in small animal cancer model.

  3. New Method to Disaggregate and Analyze Single Isolated Helminthes Cells Using Flow Cytometry: Proof of Concept

    PubMed Central

    Nava-Castro, Karen; Hernández-Bello, Romel; Muñiz-Hernández, Saé; Escobedo, Galileo; Morales-Montor, Jorge

    2011-01-01

    In parasitology, particularly in helminthes studies, several methods have been used to look for the expression of specific molecules, such as RT-PCR, western blot, 2D-electrophoresis, and microscopy, among others. However, these methods require homogenization of the whole helminth parasite, preventing evaluation of individual cells or specific cell types in a given parasite tissue or organ. Also, the extremely high interaction between helminthes and host cells (particularly immune cells) is an important point to be considered. It is really hard to obtain fresh parasites without host cell contamination. Then, it becomes crucial to determine that the analyzed proteins are exclusively from parasitic origin, and not a consequence of host cell contamination. Flow cytometry is a fluorescence-based technique used to evaluate the expression of extra-and intracellular proteins in different type cells, including protozoan parasites. It also allows the isolation and recovery of single-cell populations. Here, we describe a method to isolate and obtain purified helminthes cells. PMID:22187522

  4. Antibiotic Susceptibility Testing of the Gram-Negative Bacteria Based on Flow Cytometry.

    PubMed

    Saint-Ruf, Claude; Crussard, Steve; Franceschi, Christine; Orenga, Sylvain; Ouattara, Jasmine; Ramjeet, Mahendrasingh; Surre, Jérémy; Matic, Ivan

    2016-01-01

    Rapidly treating infections with adequate antibiotics is of major importance. This requires a fast and accurate determination of the antibiotic susceptibility of bacterial pathogens. The most frequently used methods are slow because they are based on the measurement of growth inhibition. Faster methods, such as PCR-based detection of determinants of antibiotic resistance, do not always provide relevant information on susceptibility, particularly that which is not genetically based. Consequently, new methods, such as the detection of changes in bacterial physiology caused by antibiotics using flow cytometry and fluorescent viability markers, are being explored. In this study, we assessed whether Alexa Fluor® 633 Hydrazide (AFH), which targets carbonyl groups, can be used for antibiotic susceptibility testing. Carbonylation of cellular macromolecules, which increases in antibiotic-treated cells, is a particularly appropriate to assess for this purpose because it is irreversible. We tested the susceptibility of clinical isolates of Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa, to antibiotics from the three classes: β-lactams, aminoglycosides, and fluoroquinolones. In addition to AFH, we used TO-PRO®-3, which enters cells with damaged membranes and binds to DNA, and DiBAC4 (3), which enters cells with depolarized membranes. We also monitored antibiotic-induced morphological alterations of bacterial cells by analyzing light scattering signals. Although all tested dyes and light scattering signals allowed for the detection of antibiotic-sensitive cells, AFH proved to be the most suitable for the fast and reliable detection of antibiotic susceptibility. PMID:27507962

  5. Utilization of flow cytometry for festulolium breeding (Lolium multiflorum (2x) × Festuca arundinacea (6x)).

    PubMed

    Akiyama, Yukio; Ueyama, Yasufumi; Hamada, Seiya; Kubota, Akito; Kato, Daisuke; Yamada-Akiyama, Hitomi; Takahara, Yoshinori; Fujimori, Masahiro

    2016-03-01

    Festulolium is a hybrid between Festuca and Lolium species that has valuable agronomic traits from both grass species. The purpose of our breeding program is to produce hexaploid festulolium that introduces tolerance to summer depression into Italian ryegrass (Lolium multiflorum) by crossing it with tall fescue (Festuca arundinacea). However, we found the DNA ploidy of hexaploids was not stable and was reduced in successive generations. We aimed to find out how to obtain stable high-ploidy festulolium. F1 hybrids of L. multiflorum and F. arundinacea were produced. The F3 generation was produced from putative hexaploid F2 individuals by open pollination. The F4 to F6 generations were obtained by polycrossing. The DNA ploidy levels of F2 to F6 individuals were estimated by flow cytometry. Cytological characteristics of the F5 and F6 individuals were investigated by FISH and GISH. The DNA ploidy level of hexaploid festulolium was reduced and stabilized at almost the same level as a tetraploid. Seed fertility was inversely correlated with an increase in ploidy level. GISH revealed no preferential Lolium transmission. FISH with a telomere probe revealed that counting the exact number of chromosomes in festulolium was difficult. DNA ploidy level was strongly correlated with the number of chromosomes. PMID:27162495

  6. Analysis of bacterial-surface-specific antibodies in body fluids using bacterial flow cytometry.

    PubMed

    Moor, Kathrin; Fadlallah, Jehane; Toska, Albulena; Sterlin, Delphine; Balmer, Maria L; Macpherson, Andrew J; Gorochov, Guy; Larsen, Martin; Slack, Emma

    2016-08-01

    Antibacterial antibody responses that target surfaces of live bacteria or secreted toxins are likely to be relevant in controlling bacterial pathogenesis. The ability to specifically quantify bacterial-surface-binding antibodies is therefore highly attractive as a quantitative correlate of immune protection. Here, binding of antibodies from various body fluids to pure-cultured live bacteria is made visible with fluorophore-conjugated secondary antibodies and measured by flow cytometry. We indicate the necessary controls for excluding nonspecific binding and also demonstrate a cross-adsorption technique for determining the extent of cross-reactivity. This technique has numerous advantages over standard ELISA and western blotting techniques because of its independence from scaffold binding, exclusion of cross-reactive elements from lysed bacteria and ability to visualize bacterial subpopulations. In addition, less than 10(5) bacteria and less than 10 μg of antibody are required per sample. The technique requires 3-4 h of hands-on experimentation and analysis. Moreover, it can be combined with automation and mutliplexing for high-throughput applications. PMID:27466712

  7. Ploidy Levels among Species in the ‘Oxalis tuberosa Alliance’ as Inferred by Flow Cytometry

    PubMed Central

    EMSHWILLER, EVE

    2002-01-01

    The ‘Oxalis tuberosa alliance’ is a group of Andean Oxalis species allied to the Andean tuber crop O. tuberosa Molina (Oxalidaceae), commonly known as ‘oca’. As part of a larger project studying the origins of polyploidy and domestication of cultivated oca, flow cytometry was used to survey DNA ploidy levels among Bolivian and Peruvian accessions of alliance members. In addition, this study provided a first assessment of C‐values in the alliance by estimating nuclear DNA contents of these accessions using chicken erythrocytes as internal standard. Ten Bolivian accessions of cultivated O. tuberosa were confirmed to be octoploid, with a mean nuclear DNA content of approx. 3·6 pg/2C. Two Peruvian wild Oxalis species, O. phaeotricha and O. picchensis, were inferred to be tetraploid (both with approx. 1·67 pg/2C), the latter being one of the putative progenitors of O. tuberosa identified by chloroplast‐expressed glutamine synthetase data in prior work. The remaining accessions (from 78 populations provisionally identified as 35 species) were DNA diploid, with nuclear DNA contents varying from 0·79 to 1·34 pg/2C. PMID:12102530

  8. Minimal residual disease in myeloma by flow cytometry: independent prediction of survival benefit per log reduction

    PubMed Central

    Rawstron, Andy C.; Gregory, Walter M.; de Tute, Ruth M.; Davies, Faith E.; Bell, Sue E.; Drayson, Mark T.; Cook, Gordon; Jackson, Graham H.; Morgan, Gareth J.; Child, J. Anthony

    2015-01-01

    The detection of minimal residual disease (MRD) in myeloma using a 0.01% threshold (10−4) after treatment is an independent predictor of progression-free survival (PFS), but not always of overall survival (OS). However, MRD level is a continuous variable, and the predictive value of the depth of tumor depletion was evaluated in 397 patients treated intensively in the Medical Research Council Myeloma IX study. There was a significant improvement in OS for each log depletion in MRD level (median OS was 1 year for ≥10%, 4 years for 1% to <10%, 5.9 years for 0.1% to <1%, 6.8 years for 0.01% to <0.1%, and more than 7.5 years for <0.01% MRD). MRD level as a continuous variable determined by flow cytometry independently predicts both PFS and OS, with approximately 1 year median OS benefit per log depletion. The trial was registered at www.isrctn.com as #68454111. PMID:25645353

  9. Definition of the zebrafish genome using flow cytometry and cytogenetic mapping

    PubMed Central

    Freeman, Jennifer L; Adeniyi, Adeola; Banerjee, Ruby; Dallaire, Stephanie; Maguire, Sean F; Chi, Jianxiang; Ng, Bee Ling; Zepeda, Cinthya; Scott, Carol E; Humphray, Sean; Rogers, Jane; Zhou, Yi; Zon, Leonard I; Carter, Nigel P; Yang, Fengtang; Lee, Charles

    2007-01-01

    Background The zebrafish (Danio rerio) is an important vertebrate model organism system for biomedical research. The syntenic conservation between the zebrafish and human genome allows one to investigate the function of human genes using the zebrafish model. To facilitate analysis of the zebrafish genome, genetic maps have been constructed and sequence annotation of a reference zebrafish genome is ongoing. However, the duplicative nature of teleost genomes, including the zebrafish, complicates accurate assembly and annotation of a representative genome sequence. Cytogenetic approaches provide "anchors" that can be integrated with accumulating genomic data. Results Here, we cytogenetically define the zebrafish genome by first estimating the size of each linkage group (LG) chromosome using flow cytometry, followed by the cytogenetic mapping of 575 bacterial artificial chromosome (BAC) clones onto metaphase chromosomes. Of the 575 BAC clones, 544 clones localized to apparently unique chromosomal locations. 93.8% of these clones were assigned to a specific LG chromosome location using fluorescence in situ hybridization (FISH) and compared to the LG chromosome assignment reported in the zebrafish genome databases. Thirty-one BAC clones localized to multiple chromosomal locations in several different hybridization patterns. From these data, a refined second generation probe panel for each LG chromosome was also constructed. Conclusion The chromosomal mapping of the 575 large-insert DNA clones allows for these clones to be integrated into existing zebrafish mapping data. An accurately annotated zebrafish reference genome serves as a valuable resource for investigating the molecular basis of human diseases using zebrafish mutant models. PMID:17597531

  10. Optimization of the cryopreservation of biological resources, Toxoplasma gondii tachyzoites, using flow cytometry.

    PubMed

    Mzabi, Alexandre; Escotte-Binet, Sandie; Le Naour, Richard; Ortis, Naïma; Audonnet, Sandra; Dardé, Marie-Laure; Aubert, Dominique; Villena, Isabelle

    2015-12-01

    The conservation of Toxoplasma gondii strains isolated from humans and animals is essential for conducting studies on Toxoplasma. Conservation is the main function of the French Biological Toxoplasma Resource Centre (BRC Toxoplasma, France, http://www.toxocrb.com/). In this study, we have determined the suitability of a standard cryopreservation methodology for different Toxoplasma strains using the viability of tachyzoites assayed by flow cytometry with dual fluorescent labelling (calcein acetoxymethyl ester and propidium iodide) of tachyzoites. This method provides a comparative quantitative assessment of viability after thawing. The results helped to define and refine quality criteria before tachyzoite cryopreservation and optimization of the cryopreservation parameters. The optimized cryopreservation method uses a volume of 1.0 mL containing 8 × 10(6) tachyzoites, in Iscove's Modified Dulbecco's Medium (IMDM) containing 10% foetal calf serum (FCS). The cryoprotectant additive is 10% v/v Me2SO without incubation. A cooling rate of ∼1 °C/min to -80 °C followed, after 48 h, by storage in liquid nitrogen. Thawing was performed using a 37 °C water bath that produced a warming rate of ∼100 °C/min, and samples were then diluted 1:5 in IMDM with 5% FCS, and centrifuged and resuspended for viability assessment. PMID:26408852

  11. An Improved Method for High-throughput Discrimination and Enumeration of Sedimentary Cells Using Flow Cytometry

    NASA Astrophysics Data System (ADS)

    Morono, Y.; Kallmeyer, J.; Terada, T.; Inagaki, F.; IODP Expedition 329 Shipboard Science Party

    2011-12-01

    Detection and enumeration of microbial life in marine subsurface environments provides primary information on the extent and habitability of the Earth's biosphere. Flow cytometry (FCM) is a powerful tool for identifying and enumerating fluorescence-stained cells with high throughput, using fluorescent intensity, range of wavelength, and cell size. FCM is widely used in medical sciences and aquatic microbial ecology. However, mineral grains and difficulties in distinguishing between life cells and non-specific background fluorescence prevented FCM to be applied for counting microbial cells in sediment or rock samples. SYBR Green I-stained cells can be distinguished from non-biological background signals based on differences in their fluorescence spectra. Here we extended this technique to FCM analysis by modifying the cell detachment protocol using a density gradient method, and then standardized an FCM cell counting method for various types of marine subsurface sediments. Microbial cells in sediment samples could effectively be detached and analyzed discriminatively with FCM. The high capacity of FCM to count particles (up to 10,000 cells/sec) and its high sensitivity will provide information about microbial cell abundance at high spatial resolution and with unprecedented accuracy. This improved cell count method will be useful to evaluate samples with high depth resolution, including narrow geochemical and geological interfaces as potential specific microbial niches, and may even help to asses very low population densities at the fringe of the biosphere.

  12. Combination of immunomagnetic separation with flow cytometry for detection of Listeria monocytogenes.

    PubMed

    Hibi, Kyoko; Abe, Akihisa; Ohashi, Eiji; Mitsubayashi, Kohji; Ushio, Hideki; Hayashi, Tetsuhito; Ren, Huifeng; Endo, Hideaki

    2006-07-28

    Listeria monocytogenes can grow at the low temperature commonly used in the storage and transportation of food, and the number of cases of food poisoning caused by L. monocytogenes has increased recently in the US and Europe. Several methods of detecting L. monocytogenes cells have been proposed; however, all existing methods require approximately 48 h incubation. In this study, we attempted rapid detection of L. monocytogenes using flow cytometry (FCM). The method is based on measuring the number of L. monocytogenes cells by using a combination of FCM and immunomagnetic separation (IMS). First, polyclonal antibodies (anti-L. monocytogenes rabbit IgG-FITC) conjugated with fluorescein isothiocyanate (FITC) were reacted with L. monocytogenes cells, and then FCM was applied. The cell numbers were determined by FCM using a traditional colony-counting method in the range of 10(4)-10(8) cells ml(-1). Tetrameric antibody complexes (TAC) were used because they can recognize both magnetic and FITC molecules on the FITC-conjugated antibodies. FITC-labeled L. monocytogenes cells were reacted with a secondary antibody (TAC) bound to magnetic beads. Then, IMS was used. The method is suitable for detection in the range of 10(2)-10(8)cells ml(-1). The FCM assay enumerated the cells within 1 min and the total assay time, including sample preparation, was less than 2 h. PMID:17723519

  13. Use of flow cytometry to follow the physiological states of microorganisms in cider fermentation processes.

    PubMed

    Herrero, Mónica; Quirós, Covadonga; García, Luis A; Díaz, Mario

    2006-10-01

    The flow cytometry (FC) technique used with certain fluorescent dyes (ChemChrome V6 [CV6], DRAQ5, and PI) has proven useful to label and to detect different physiological states of yeast and malolactic bacterium starters conducting cider fermentation over time (by performing sequential inoculation of microorganisms). First, the technique was tested with pure cultures of both types of microorganisms grown in synthetic media under different induced stress conditions. Metabolically active cells detected by FC and by the standard plate-counting method for both types of microorganisms in fresh overnight pure cultures gave good correlations between the two techniques in samples taken at this stage. Otherwise, combining the results obtained by FC and plating during alcoholic and malolactic fermentation over time in the cider-making process, different subpopulations were detected, showing significant differences between the methods. A small number of studies have applied the FC technique to analyze fermentation processes and mixed cultures over time. The results were used to postulate equations explaining the different physiological states in cell populations taken from fresh, pure overnight cultures under nonstress conditions or cells subjected to stress conditions over time, either under a pure-culture fermentation process (in this work, corresponding to alcoholic fermentation) or under mixed-fermentation conditions (for the malolactic-fermentation phase), that could be useful to improve the control of the processes. PMID:17021224

  14. Techniques to improve detection and analysis of extracellular vesicles using flow cytometry.

    PubMed

    Inglis, Heather C; Danesh, Ali; Shah, Avani; Lacroix, Jacques; Spinella, Philip C; Norris, Philip J

    2015-11-01

    Extracellular vesicles (EVs) range in size from 50 nm to 1 µm. Flow cytometry (FCM) is the most commonly used method for analyzing EVs; however, accurate characterization of EVs remains challenging due to their small size and lack of discrete positive populations. Here we report the use of optimization techniques that are especially well-suited for analyzing EVs from a high volume of clinical samples. Utilizing a two pronged approach that included 1) pre-filtration of antibodies to remove aggregates, followed by 2) detergent lysis of a replicate sample to account for remaining false positive events, we were able to effectively limit false positive non-EV events. In addition, we show that lysed samples are a useful alternative to isotypes for setting gates to exclude background fluorescence. To reduce background, we developed an approach using filters to "wash" samples post-staining thus providing a faster alternative to ultracentrifugation and sucrose gradient fractionation. In conclusion, use of these optimized techniques enhances the accuracy and efficiency of EV detection using FCM. PMID:25847910

  15. Immune Monitoring in Cancer Vaccine Clinical Trials: Critical Issues of Functional Flow Cytometry-Based Assays

    PubMed Central

    Urbani, Francesca; Proietti, Enrico

    2013-01-01

    The development of immune monitoring assays is essential to determine the immune responses against tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs) and their possible correlation with clinical outcome in cancer patients receiving immunotherapies. Despite the wide range of techniques used, to date these assays have not shown consistent results among clinical trials and failed to define surrogate markers of clinical efficacy to antitumor vaccines. Multiparameter flow cytometry- (FCM-) based assays combining different phenotypic and functional markers have been developed in the past decade for informative and longitudinal analysis of polyfunctional T-cells. These technologies were designed to address the complexity and functional heterogeneity of cancer biology and cellular immunity and to define biomarkers predicting clinical response to anticancer treatment. So far, there is still a lack of standardization of some of these immunological tests. The aim of this review is to overview the latest technologies for immune monitoring and to highlight critical steps involved in some of the FCM-based cellular immune assays. In particular, our laboratory is focused on melanoma vaccine research and thus our main goal was the validation of a functional multiparameter test (FMT) combining different functional and lineage markers to be applied in clinical trials involving patients with melanoma. PMID:24195078

  16. Multicentre evaluation of stable reference whole blood for enumeration of lymphocyte subsets by flow cytometry.

    PubMed

    Edwards, Cherry; Belgrave, Danielle; Janossy, George; Bradley, Nicholas J; Stebbings, Richard; Gaines-Das, Rose; Thorpe, Robin; Sawle, Alex; Arroz, Maria Jorge; Brando, Bruno; Gratama, Jan Willem; Orfao de Matos, Alberto; Papa, Stephano; Papamichail, Michael; Lenkei, Rodica; Rothe, Gregor; Barnett, David

    2005-06-22

    BACKGROUND: Clinical indications for lymphocyte subset enumeration by flow cytometry include monitoring of disease progression and timing of therapeutic intervention in infection with human immunodeficiency virus. Until recently international standardisation has not been possible due to a lack of suitable stable reference material. METHODS: This study consisted of two trials of a stabilised whole blood preparation. Eleven participants were sent two standard protocols for staining plus gating strategy and asked to report absolute counts for lymphocyte subsets. RESULTS: No significant difference was detected between the two methods when results from the two assays and all partners were pooled. Significant differences in results from the different partners were observed. However, representative mean counts were obtained for geometric means, geometric coefficient of variation, and 95% confidence interval for CD3 910 cells/mul, 9%, and 888 to 933, respectively), CD4 (495 cells/mul, 12%, and 483 to 507), and CD8 (408 cells/mul, 13%, and 393 to 422). CONCLUSION: We have introduced a stabilised blood preparation and a well-characterized biological standard. The availability of this reference material greatly simplifies the validation of new techniques for CD4(+) T-cell enumeration and the expansion of external quality assurance programmes for clinical laboratories, including those that operate in resource-restricted environments. (c) 2005 Wiley-Liss, Inc. PMID:15973699

  17. Flow cytometry may allow microscope-independent detection of holocentric chromosomes in plants

    PubMed Central

    Zedek, František; Veselý, Pavel; Horová, Lucie; Bureš, Petr

    2016-01-01

    Two chromosomal structures, known as monocentric and holocentric chromosomes, have evolved in eukaryotes. Acentric fragments of monocentric chromosomes are unequally distributed to daughter cells and/or lost, while holocentric fragments are inherited normally. In monocentric species, unequal distribution should generate chimeras of cells with different nuclear DNA content. We investigated whether such differences in monocentric species are detectable by flow cytometry (FCM) as (i) a decreased nuclear DNA content and (ii) an increased coefficient of variance (CV) of the G1 peak after gamma radiation-induced fragmentation. We compared 13 monocentric and 9 holocentric plant species. Unexpectedly, monocentrics and holocentrics did not differ with respect to parameters (i) and (ii) in their response to gamma irradiation. However, we found that the proportion of G2 nuclei was highly elevated in monocentrics after irradiation, while holocentrics were negligibly affected. Therefore, we hypothesize that DNA-damaging agents induce cell cycle arrest leading to endopolyploidy only in monocentric and not (or to much lesser extent) in holocentric plants. While current microscope-dependent methods for holocentrism detection are unreliable for small and numerous chromosomes, which are common in holocentrics, FCM can use somatic nuclei. Thus, FCM may be a rapid and reliable method of high-throughput screening for holocentric candidates across plant phylogeny. PMID:27255216

  18. Quantification of mammalian sperm morphology by slit-scan flow cytometry

    SciTech Connect

    Benaron, D.A.; Gray, J.W.; Gledhill, B.L.; Lake, S.; Wyrobek, A.J.; Young, I.T.

    1982-03-01

    The head shapes of mammalian sperm have been measured by slit-scan flow cytometry (SSFCM). In this approach, the distribution of fluorescence along acriflavine stained mammalian sperm is recorded and used as a measure of head shape. Fluorescence profiles were measured for sperm from mice, rabbits, hamsters, and bulls, and for sperm from mice exposed to testicular x-irradiation from 0 to 900 rads. The profiles for sperm from nonirradiated animals were characteristic of each species and were reproducible from sperm to sperm. Some of the fluorescence profiles for sperm from the irradiated mice differed significantly from the profiles usually measured for sperm from exposed mice. An algorithm was developed to determine the frequency of these sperm. The estimated frequencies of atypical profiles correlated well (r . 0.99) with the frequencies of abnormally shaped sperm determined by microscopic scoring. The maximum SSFCM sensitivity (minimum detectable dose . 199 rad) was not as high as that for the visual assay (minimum detectable dose . 116 rad). However, only 100 profiles were measured by SSFCM at each dose while at least 500 sperm were scored visually at each dose. The sensitivity of the SSFCM assay should be increased substantially by measuring more profiles. The objective nature of SSFCM couple with the high correlation with results from the visually based assay of morphology suggests the use of SSFCM to measure frequencies of misshapen sperm when testing for mutagens or monitoring for effects of environmental contaminants.

  19. Quantification of mammalian sperm morphology by slit-scan flow cytometry

    SciTech Connect

    Benaron, D.A.; Gray, J.W.; Gledhill, B.L.; Lake, S.; Wyrobek, A.J.; Young, I.T.

    1982-01-01

    The head shapes of mammalian sperm were measured by slit-scan flow cytometry (SSFCM). Fluorescence profiles were measured for sperm from mice, rabbits, hamsters, and bulls, and for sperm from mice, rabbits, hamsters, and bulls, and for sperm from mice exposed to testicular x-irradiation from 0 to 900 rads. Some of the fluorescence profiles for sperm from the irradiated mice differed significantly from the profiles usually measured for sperm from unexposed mice. An algorithm was developed to determine the frequency of these sperm. The estimated frequencies of atypical profiles correlated well with the frequencies of abnormally shaped sperm determined by microscopic scoring. The maximum SSFCM sensitivity was not as high as that for the visual assay. However, only 100 profiles were measured by SSFCM at each dose while at least 500 sperm were scored visually at each dose. The sensitivity of the SSFCM assay should be increased substantially by measuring more profiles. The objective nature of SSFCM coupled with the high correlation with results from the visually based assay of morphology suggests the use of SSFCM to measure frequencies of misshapen sperm when testing for mutagens or monitoring for effects of environmental contaminants.

  20. Flow cytometry-based methods for assessing soluble scFv activities and detecting pathogen antigens in solution

    SciTech Connect

    Gray, Sean; Weigel, Kris M.; Miller, Keith D.; Ndung'u, Joseph; Buscher, Philippe; Tran, Thao N.; Baird, Cheryl L.; Cangelosi, Gerard A.

    2010-04-01

    Novel methods are reported for evaluating and utilizing single chain fragment variable (scFv) antibodies derived from yeast-display libraries. Yeast-display was used to select scFv specific to invariant surface glycoproteins (ISG) of Trypanosoma brucei. A limiting step in the isolation of scFv from nonimmune libraries is the conversion of highly active yeast-displayed scFv into soluble antibodies that can be used in standard immunoassays. Challenges include limited solubility or activity following secretion and purification of scFv. For this reason, few scFv derived from yeast-display platforms have moved into development and implementation as diagnostic reagents. To address this problem, assays were developed that employ both yeastdisplayed and secreted scFv as analytical reagents. The first is a competitive inhibition flow cytometry (CIFC) assay that detects secreted scFv by virtue of its ability to competitively inhibit the binding of biotinylated antigen to yeast-displayed scFv. The second is an epitope binning assay that uses secreted scFv toidentify additional yeast-displayed scFv that bind nonoverlapping or noncompeting epitopes on an antigen. The epitope binning assay was used not only to identify sandwich assay pairs with yeast-displayed scFv, but also to identify active soluble scFv present in low concentration in a crude expression extract. Finally, a CIFC assay was developed that bypasses entirely the need for soluble scFv expression, by using yeast displayed scFv to detect unlabeled antigen in samples. These methods will facilitate the continued development and practical implementation of scFv derived from yeast-display libraries.

  1. A dual-color flow cytometry protocol for the simultaneous detection of Vibrio parahaemolyticus and Salmonella typhimurium using aptamer conjugated quantum dots as labels.

    PubMed

    Duan, Nuo; Wu, Shijia; Yu, Ye; Ma, Xiaoyuan; Xia, Yu; Chen, Xiujuan; Huang, Yukun; Wang, Zhouping

    2013-12-01

    A sensitive, specific method for the collection and detection of pathogenic bacteria was demonstrated using quantum dots (QDs) as a fluorescence marker coupled with aptamers as the molecular recognition element by flow cytometry. The aptamer sequences were selected using a bacterium-based SELEX strategy in our laboratory for Vibrio parahaemolyticus and Salmonella typhimurium that, when applied in this method, allows for the specific recognition of the bacteria from complex mixtures including shrimp samples. Aptamer-modified QDs (QD-apt) were employed to selectively capture and simultaneously detect the target bacteria with high sensitivity using the fluorescence of the labeled QDs. The signal intensity is amplified due to the high photostability of QDs nanoparticles, resulting in improved sensitivity over methods using individual dye-labeled probes. This proposed method is promising for the sensitive detection of other pathogenic bacteria in food stuff if suitable aptamers are chosen. The method may also provide another potential platform for the application of aptamer-conjugated QDs in flow cytometry. PMID:24267076

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

    PubMed

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

    2009-04-01

    The Abbot Cell-Dyn Sapphire is a new generation haematology analyser. The system uses optical/fluorescence flow cytometry in combination with electronic impedance to produce a full blood count. Optical and impedance are the default methods for platelet counting while automated CD61-immunoplatelet analysis can be run as selectable test. The aim of this study was to determine the platelet count performance of the three counting methods available on the instrument and to compare the results with those provided by Becton Dickinson FACSCalibur flow cytometer used as reference method. A lipid interference experiment was also performed. Linearity, carryover and precision were good, and satisfactory agreement with reference method was found for the impedance, optical and CD61-immunoplatelet analysis, although this latter provided the closest results in comparison with flow cytometry. In the lipid interference experiment, a moderate inaccuracy of optical and immunoplatelet counts was observed starting from a very high lipid value. PMID:18177435

  3. Light scattering and morphology of the lymphocyte as applied to flow cytometry for distinguishing healthy and infected individuals.

    PubMed

    Ruban, Gennady I; Berdnik, Vladimir V; Marinitch, Dmitry V; Goncharova, Natalia V; Loiko, Valery A

    2010-01-01

    A simple optical model of single lymphocytes with smooth and nonsmooth surfaces has been developed for healthy and infected individuals. The model can be used for rapid (in the real-time scale) solution of the inverse light-scattering problem on the basis of optical data measured by label-free flow cytometry. Light scattering patterns have been calculated for the model developed. It has been shown that the smooth and nonsmooth cells can be resolved using the intensities of the sideward- and backward-scattered light. We have found by calculations and validated by the flow cytometer experiments that intensity distributions for the cells of lymphocyte populations can be used as a preliminary signatures of some virus infections. Potential biomedical applications of the findings for label-free flow cytometry detection of individuals infected with viruses of hepatitis B or C and some others viruses are presented. PMID:21054124

  4. A simple flow cytometry method improves the detection of phosphatidylserine-exposing extracellular vesicles

    PubMed Central

    Arraud, N; Gounou, C; Linares, R; Brisson, A R

    2015-01-01

    Background Plasma contains cell-derived extracellular vesicles (EVs), which participate in physiopathological processes and have potential applications as disease biomarker. However, the enumeration of EVs faces major problems, due to their sub-micrometer size and to intrinsic limitations in methods of characterization, mainly flow cytometry (FCM). Objectives Our objective is to enumerate EVs in plasma, by taking as the prototype the population of phosphatidylserine (PS)-exposing EVs, which constitute one of the major EV populations and are responsible for thrombotic disorders. Methods The concentration of PS-exposing EVs in platelet-free plasma (PFP) of healthy subjects was measured by FCM using either light scattering or fluorescence as the trigger and fluorescent Annexin-5 (Anx5) as the specific label. In addition, PS-exposing EVs were enumerated by electron microscopy (EM) after labeling with Anx5 gold nanoparticles and sedimentation on EM grids. Results We show that about 50× more Anx5-positive EVs are detected by FCM when detection is triggered on fluorescence as compared with light scattering. By fluorescence triggering, concentrations of 22 000–30 000 Anx5-positive EVs per μL PFP were determined, using two different flow cytometers. The limit of detection of the fluorescence triggering method was estimated at about 1000–2500 Anx5 molecules. Results from EM suggest that EVs down to 100–150 nm diameter are detected by fluorescence triggering. Conclusion This study presents a simple method for enumerating EVs. We believe that this method is applicable in a general context and will improve our understanding of the roles of EVs in pathophysiological situations, which will open avenues for the development of EV-based diagnosis assays. PMID:25348269

  5. A microfluidics-based technique for automated and rapid labeling of cells for flow cytometry

    NASA Astrophysics Data System (ADS)

    Patibandla, Phani K.; Estrada, Rosendo; Kannan, Manasaa; Sethu, Palaniappan

    2014-03-01

    Flow cytometry is a powerful technique capable of simultaneous multi-parametric analysis of heterogeneous cell populations for research and clinical applications. In recent years, the flow cytometer has been miniaturized and made portable for application in clinical- and resource-limited settings. The sample preparation procedure, i.e. labeling of cells with antibodies conjugated to fluorescent labels, is a time consuming (˜45 min) and labor-intensive procedure. Microfluidics provides enabling technologies to accomplish rapid and automated sample preparation. Using an integrated microfluidic device consisting of a labeling and washing module, we demonstrate a new protocol that can eliminate sample handling and accomplish sample and reagent metering, high-efficiency mixing, labeling and washing in rapid automated fashion. The labeling module consists of a long microfluidic channel with an integrated chaotic mixer. Samples and reagents are precisely metered into this device to accomplish rapid and high-efficiency mixing. The mixed sample and reagents are collected in a holding syringe and held for up to 8 min following which the mixture is introduced into an inertial washing module to obtain ‘analysis-ready’ samples. The washing module consists of a high aspect ratio channel capable of focusing cells to equilibrium positions close to the channel walls. By introducing the cells and labeling reagents in a narrow stream at the center of the channel flanked on both sides by a wash buffer, the elution of cells into the wash buffer away from the free unbound antibodies is accomplished. After initial calibration experiments to determine appropriate ‘holding time’ to allow antibody binding, both modules were used in conjunction to label MOLT-3 cells (T lymphoblast cell line) with three different antibodies simultaneously. Results confirm no significant difference in mean fluorescence intensity values for all three antibodies labels (p < 0.01) between the

  6. Green fiber lasers: An alternative to traditional DPSS green lasers for flow cytometry

    PubMed Central

    Telford, William G.; Babin, Sergey A.; Khorev, Serge V.; Rowe, Stephen H.

    2009-01-01

    Green and yellow diode-pumped solid state (DPSS) lasers (532 and 561 nm) have become common fixtures on flow cytometers, due to their efficient excitation of phycoerythrin (PE) and its tandems, and their ability to excite an expanding array of expressible red fluorescent proteins. Nevertheless, they have some disadvantages. DPSS 532 nm lasers emit very close to the fluorescein bandwidth, necessitating optical modifications to permit detection of fluorescein and GFP. DPSS 561 nm lasers likewise emit very close to the PE detection bandwidth, and also cause unwanted excitation of APC and its tandems, requiring high levels of crossbeam compensation to reduce spectral overlap into the PE tandems. In this paper, we report the development of a new generation of green fiber lasers that can be engineered to emit in the range between 532 and 561 nm. A 550 nm green fiber laser was integrated into both a BD LSR II™ cuvette and FACSVantage DiVa™ jet-in-air cell sorter. This laser wavelength avoided both the fluorescein and PE bandwidths, and provided better excitation of PE and the red fluorescent proteins DsRed and dTomato than a power-matched 532 nm source. Excitation at 550 nm also caused less incidental excitation of APC and its tandems, reducing the need for crossbeam compensation. Excitation in the 550 nm range therefore proved to be a good compromise between 532 and 561 nm sources. Fiber laser technology is therefore providing the flexibility necessary for precisely matching laser wavelengths to our flow cytometry applications. PMID:19777600

  7. Blood group antigen studies using CdTe quantum dots and flow cytometry

    PubMed Central

    Cabral Filho, Paulo E; Pereira, Maria IA; Fernandes, Heloise P; de Thomaz, Andre A; Cesar, Carlos L; Santos, Beate S; Barjas-Castro, Maria L; Fontes, Adriana

    2015-01-01

    New methods of analysis involving semiconductor nanocrystals (quantum dots [QDs]) as fluorescent probes have been highlighted in life science. QDs present some advantages when compared to organic dyes, such as size-tunable emission spectra, broad absorption bands, and principally exceptional resistance to photobleaching. Methods applying QDs can be simple, not laborious, and can present high sensibility, allowing biomolecule identification and quantification with high specificity. In this context, the aim of this work was to apply dual-color CdTe QDs to quantify red blood cell (RBC) antigen expression on cell surface by flow cytometric analysis. QDs were conjugated to anti-A or anti-B monoclonal antibodies, as well as to the anti-H (Ulex europaeus I) lectin, to investigate RBCs of A1, B, A1B, O, A2, and Aweak donors. Bioconjugates were capable of distinguishing the different expressions of RBC antigens, both by labeling efficiency and by flow cytometry histogram profile. Furthermore, results showed that RBCs from Aweak donors present fewer amounts of A antigens and higher amounts of H, when compared to A1 RBCs. In the A group, the amount of A antigens decreased as A1 > A3 > AX = Ael, while H antigens were AX = Ael > A1. Bioconjugates presented stability and remained active for at least 6 months. In conclusion, this methodology with high sensibility and specificity can be applied to study a variety of RBC antigens, and, as a quantitative tool, can help in achieving a better comprehension of the antigen expression patterns on RBC membranes. PMID:26185442

  8. 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. PMID:22843284

  9. Blood group antigen studies using CdTe quantum dots and flow cytometry.

    PubMed

    Cabral Filho, Paulo E; Pereira, Maria I A; Fernandes, Heloise P; de Thomaz, Andre A; Cesar, Carlos L; Santos, Beate S; Barjas-Castro, Maria L; Fontes, Adriana

    2015-01-01

    New methods of analysis involving semiconductor nanocrystals (quantum dots [QDs]) as fluorescent probes have been highlighted in life science. QDs present some advantages when compared to organic dyes, such as size-tunable emission spectra, broad absorption bands, and principally exceptional resistance to photobleaching. Methods applying QDs can be simple, not laborious, and can present high sensibility, allowing biomolecule identification and quantification with high specificity. In this context, the aim of this work was to apply dual-color CdTe QDs to quantify red blood cell (RBC) antigen expression on cell surface by flow cytometric analysis. QDs were conjugated to anti-A or anti-B monoclonal antibodies, as well as to the anti-H (Ulex europaeus I) lectin, to investigate RBCs of A1, B, A1B, O, A2, and Aweak donors. Bioconjugates were capable of distinguishing the different expressions of RBC antigens, both by labeling efficiency and by flow cytometry histogram profile. Furthermore, results showed that RBCs from Aweak donors present fewer amounts of A antigens and higher amounts of H, when compared to A1 RBCs. In the A group, the amount of A antigens decreased as A1 > A3 > AX = Ael, while H antigens were AX = Ael > A1. Bioconjugates presented stability and remained active for at least 6 months. In conclusion, this methodology with high sensibility and specificity can be applied to study a variety of RBC antigens, and, as a quantitative tool, can help in achieving a better comprehension of the antigen expression patterns on RBC membranes. PMID:26185442

  10. Whole Blood Activation Results in Altered T Cell and Monocyte Cytokine Production Profiles by Flow Cytometry

    NASA Technical Reports Server (NTRS)

    Crucian, Brian E.; Sams, Clarence F.

    2001-01-01

    An excellent monitor of the immune balance of peripheral circulating cells is to determine their cytokine production patterns in response to stimuli. Using flow cytometry, a positive identification of cytokine producing cells in a mixed culture may be achieved. Recently, the ability to assess cytokine production following a whole-blood activation culture has been described. In this study, whole blood activation was compared to traditional PBMC activation and the individual cytokine secretion patterns for both T cells, T cell subsets and monocytes was determined by flow cytometry. RESULTS: For T cell cytokine assessment (IFNg/IL-10 and IL-21/L-4) following PMA +ionomycin activation: (1) a Significantly greater percentages of T cells producing IFNgamma and IL-2 were observed following whole-blood culture and (2) altered T cell cytokine production kinetics were observed by varying whole blood culture times. Four-color analysiS was used to allow assessment of cytokine production by specific T cell subsets. It was found that IFNgamma production was significantly elevated in the CD3+/CD8+ T cell population as compared to the CD3+/CD8- population following five hours of whole blood activation. Conversely, IL-2 and IL-10 production were Significantly elevated in the CD3+/CD8- T cell population as compared to the CD3+/CD8+ population. Monocyte cytokine production was assessed in both culture systems following LPS activation for 24 hours. A three-color flow cytometric was used to assess two cytokines (IL-1a/IL-12 and TNFa/IL-10) in conjunction with CD14. Nearly all monocytes were stimulated to produce IL-1a, IL-12 and TNFa. equally well in both culture systems, however monocyte production of IL-10 was significantly elevated in whole blood culture as compared to PBMC culture. IL-12 producing monocytes appeared to be a distinct subpopulation of the IL-1a producing set, whereas IL-10 and TNFa producing monocytes were largely mutually exclusive. IL-10 and TNFa producing

  11. 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

  12. 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

  13. Generalized Unmixing Model for Multispectral Flow Cytometry Utilizing Nonsquare Compensation Matrices

    PubMed Central

    Novo, David; Grégori, Gérald; Rajwa, Bartek

    2014-01-01

    Multispectral and hyperspectral flow cytometry (FC) instruments allow measurement of fluorescence or Raman spectra from single cells in flow. As with conventional FC, spectral overlap results in the measured signal in any given detector being a mixture of signals from multiple labels present in the analyzed cells. In contrast to traditional polychromatic FC, these devices utilize a number of detectors (or channels in multispectral detector arrays) that is larger than the number of labels, and no particular detector is a priori dedicated to the measurement of any particular label. This data-acquisition modality requires a rigorous study and understanding of signal formation as well as unmixing procedures that are employed to estimate labels abundance. The simplest extension of the traditional compensation procedure to multispectral data sets is equivalent to an ordinary least-square (LS) solution for estimating abundance of labels in individual cells. This process is identical to the technique employed for unmixing spectral data in various imaging fields. The present study shows that multispectral FC data violate key assumptions of the LS process, and use of the LS method may lead to unmixing artifacts, such as population distortion (spreading) and the presence of negative values in biomarker abundances. Various alternative unmixing techniques were investigated, including relative-error minimization and variance-stabilization transformations. The most promising results were obtained by performing unmixing using Poisson regression with an identity-link function within a generalized linear model framework. This formulation accounts for the presence of Poisson noise in the model of signal formation and subsequently leads to superior unmixing results, particularly for dim fluorescent populations. The proposed Poisson unmixing technique is demonstrated using simulated 8-channel, 2-fluorochrome data and real 32-channel, 6-fluorochrome data. The quality of unmixing is

  14. Measurement of receptor cross-linking at the cell surface via multiparameter flow cytometry

    NASA Astrophysics Data System (ADS)

    Posner, Richard G.; Bold, Jennifer; Bernstein, Yael; Rasor, Joe; Braslow, Joshua; Hlavacek, William S.; Perelson, Alan S.

    1998-05-01

    Many cellular responses, particularly in the immune system, are triggered by ligand binding to a cell-surface receptor. However, as indicated by bell-shaped dose-response curves, ligand binding alone is sometimes insufficient to trigger a response. Often, ligand binding must also induce the aggregation of cell-surface receptors through crosslinking, which occurs when a ligand binds simultaneously to two or more receptors. Thus, an important goal in cell biology has been to establish quantitative relationships between the amount of ligand present on a cell surface and the number of crosslinked ligand-specific cell-surface receptors. To better understand ligand-induced receptor aggregation, we have been investigating the binding of a model multivalent antigen (DNP25PE) to cell-surface anti-DNP FITC-labeled IgE (FITC- IgE). To determine the kinetic and equilibrium parameters that characterize crosslinking in this system, we have developed a combined theoretical and experimental approach that is based on multiparameter flow cytometry. With this approach, we can measure both the average number of ligand molecules that are bound per cell and the average number of receptor binding sites that are bound per cell. The average number of DNP25PE per cell is determined by measuring the fluorescence of phycoerythrin. The average number of occupied IgE sites per cell is determined by measuring the fluorescence of FITC, which is quenched upon ligand binding. This novel approach, together with conventional methods for changes in intracellular calcium, allows us to correlate for the first time the dynamics of IgE crosslinking with cell activation.

  15. Seminal plasma applied post-thawing affects boar sperm physiology: a flow cytometry study.

    PubMed

    Fernández-Gago, Rocío; Domínguez, Juan Carlos; Martínez-Pastor, Felipe

    2013-09-01

    Cryopreservation induces extensive biophysical and biochemical changes in the sperm. In the present study, we used flow cytometry to assess the capacitation-like status of frozen-thawed boar spermatozoa and its relationship with intracellular calcium, assessment of membrane fluidity, modification of thiol groups in plasma membrane proteins, reactive oxygen species (ROS) levels, viability, acrosomal status, and mitochondrial activity. This experiment was performed to verify the effect of adding seminal plasma on post-thaw sperm functions. To determine these effects after cryopreservation, frozen-thawed semen from seven boars was examined after supplementation with different concentrations of pooled seminal plasma (0%, 10%, and 50%) at various times of incubation from 0 to 4 hours. Incubation caused a decrease in membrane integrity and an increase in acrosomal damage, with small changes in other parameters (P > 0.05). Although 10% seminal plasma showed few differences with 0% (ROS increase at 4 hours, P < 0.05), 50% seminal plasma caused important changes. Membrane fluidity increased considerably from the beginning of the experiment, and ROS and free thiols in the cell surface increased by 2 hours of incubation. By the end of the experiment, viability decreased and acrosomal damage increased in the 50% seminal plasma samples. The addition of 50% of seminal plasma seems to modify the physiology of thawed boar spermatozoa, possibly through membrane changes and ROS increase. Although some effects were detrimental, the stimulatory effect of 50% seminal plasma could favor the performance of post-thawed boar semen, as showed in the field (García JC, Domínguez JC, Peña FJ, Alegre B, Gonzalez R, Castro MJ, Habing GG, Kirkwood RN. Thawing boar semen in the presence of seminal plasma: effects on sperm quality and fertility. Anim Reprod Sci 2010;119:160-5). PMID:23756043

  16. Fibrocytes in the fibrotic lung: altered phenotype detected by flow cytometry.

    PubMed

    Reese, Charles; Lee, Rebecca; Bonner, Michael; Perry, Beth; Heywood, Jonathan; Silver, Richard M; Tourkina, Elena; Visconti, Richard P; Hoffman, Stanley

    2014-01-01

    Fibrocytes are bone marrow hematopoietic-derived cells that also express a mesenchymal cell marker (commonly collagen I) and participate in fibrotic diseases of multiple organs. Given their origin, they or their precursors must be circulating cells before recruitment into target tissues. While most previous studies focused on circulating fibrocytes, here we focus on the fibrocyte phenotype in fibrotic tissue. The study's relevance to human disease is heightened by use of a model in which bleomycin is delivered systemically, recapitulating several features of human scleroderma including multi-organ fibrosis not observed when bleomycin is delivered directly into the lungs. Using flow cytometry, we find in the fibrotic lung a large population of CD45(high) fibrocytes (called Region I) rarely found in vehicle-treated control mice. A second population of CD45+ fibrocytes (called Region II) is observed in both control and fibrotic lung. The level of CD45 in circulating fibrocytes is far lower than in either Region I or II lung fibrocytes. The chemokine receptors CXCR4 and CCR5 are expressed at higher levels in Region I than in Region II and are present at very low levels in all other lung cells including CD45+/collagen I- leucocytes. The collagen chaperone HSP47 is present at similar high levels in both Regions I and II, but at a higher level in fibrotic lung than in control lung. There is also a major population of HSP47(high)/CD45- cells in fibrotic lung not present in control lung. CD44 is present at higher levels in Region I than in Region II and at much lower levels in all other cells including CD45+/collagen I- leucocytes. When lung fibrosis is inhibited by restoring caveolin-1 activity using a caveolin-1 scaffolding domain peptide (CSD), a strong correlation is observed between fibrocyte number and fibrosis score. In summary, the distinctive phenotype of fibrotic lung fibrocytes suggests that fibrocyte differentiation occurs primarily within the target organ. PMID

  17. Characterization of subsets of bone marrow-derived macrophages by flow cytometry analysis

    SciTech Connect

    Walker, E.B.; Akporiaye, E.T.; Warner, N.L.; Stewart, C.C.

    1985-01-01

    Normal C3H bone marrow cells were grown 7 days in medium containing L cell-derived colony stimulating factor-1 (CSF-1). During the first 4 days of culture, erythroid and granulocytic cells decreased while macrophages increased exponentially with a doubling time of about 31 hr. Only 0.3% of all cells in the initial bone marrrow suspension formed discrete colonies of mononuclear phagocytes, but by day 6 60% of the nonadherent cells were capable of forming macrophage colonies, representing a 200-fold enrichment of the original progenitor population. Using flow cytometry, mononuclear phagocytes obtained after 4 days of culture were separated into two distinct phenotypes based on their autofluorescence. Nonadherent cells were a discrete population of small cells exhibiting low autofluorescence, and the adherent cells were a broad heterogenous population of large cells exhibiting high autofluorescence. A panel of currently available rat monoclonal antibodies (MABs) against murine hematopoietic cells were used to determine whether unique subsets of macrophages could be resolved. The MABs RA 31B6 and H-11 stained virtually all the nonadherent cells but not adherent cells. The MABs E-2 and 11-4.1 (anti-H-2K/sup k/) stained almost all the adherent cells and demonstrated no significant staining of nonadherent cells. Nearly all the nonadherent and adherent cells were stained by the MABs DNL 4.4 and MAC-1. Additionally, the data suggest that the epitopes for MAC-2 and MAC-3 and ..gamma..2a Fc receptors develop late in nonadherent progenitor cells as they mature into adherent macrophages. 37 references, 5 figures, 5 tables.

  18. Development of a novel cell sorting method that samples population diversity in flow cytometry.

    PubMed

    Osborne, Geoffrey W; Andersen, Stacey B; Battye, Francis L

    2015-11-01

    Flow cytometry based electrostatic cell sorting is an important tool in the separation of cell populations. Existing instruments can sort single cells into multi-well collection plates, and keep track of cell of origin and sorted well location. However currently single sorted cell results reflect the population distribution and fail to capture the population diversity. Software was designed that implements a novel sorting approach, "Slice and Dice Sorting," that links a graphical representation of a multi-well plate to logic that ensures that single cells are sampled and sorted from all areas defined by the sort region/s. Therefore the diversity of the total population is captured, and the more frequently occurring or rarer cell types are all sampled. The sorting approach was tested computationally, and using functional cell based assays. Computationally we demonstrate that conventional single cell sorting can sample as little as 50% of the population diversity dependant on the population distribution, and that Slice and Dice sorting samples much more of the variety present within a cell population. We then show by sorting single cells into wells using the Slice and Dice sorting method that there are cells sorted using this method that would be either rarely sorted, or not sorted at all using conventional single cell sorting approaches. The present study demonstrates a novel single cell sorting method that samples much more of the population diversity than current methods. It has implications in clonal selection, stem cell sorting, single cell sequencing and any areas where population heterogeneity is of importance. PMID:25944021

  19. Coconut genome size determined by flow cytometry: Tall versus Dwarf types.

    PubMed

    Freitas Neto, M; Pereira, T N S; Geronimo, I G C; Azevedo, A O N; Ramos, S R R; Pereira, M G

    2016-01-01

    Coconuts (Cocos nucifera L.) are tropical palm trees that are classified into Tall and Dwarf types based on height, and both types are diploid (2n = 2x = 32 chromosomes). The reproduction mode is autogamous for Dwarf types and allogamous for Tall types. One hypothesis for the origin of the Dwarf coconut suggests that it is a Tall variant that resulted from either mutation or inbreeding, and differences in genome size between the two types would support this hypothesis. In this study, we estimated the genome sizes of 14 coconut accessions (eight Tall and six Dwarf types) using flow cytometry. Nuclei were extracted from leaf discs and stained with propidium iodide, and Pisum sativum (2C = 9.07 pg DNA) was used as an internal standard. Histograms with good resolution and low coefficients of variation (2.5 to 3.2%) were obtained. The 2C DNA content ranged from 5.72 to 5.48 pg for Tall accessions and from 5.58 to 5.52 pg for Dwarf accessions. The mean genome sizes for Tall and Dwarf specimens were 5.59 and 5.55 pg, respectively. Among all accessions, Rennel Island Tall had the highest mean DNA content (5.72 pg), whereas West African Tall had the lowest (5.48 pg). The mean coconut genome size (2C = 5.57 pg, corresponding to 2723.73 Mbp/haploid set) was classified as small. Only small differences in genome size existed among the coconut accessions, suggesting that the Dwarf type did not evolve from the Tall type. PMID:26909966

  20. [THE TECHNQUE OF FLOW CYTOMETRY IN EVALUATION OF NK-CELLS AND THEIR ACTIVITY].

    PubMed

    Abakushina, E V

    2015-11-01

    The review presents main characteristics of human natural killer cells (NK-cells), their phenotype and methods of detection of functional activities using flow cytometry. The NK-cells play important role in inherent and adaptive immunity against infections and tumors. Initially these cells were characterized on the basis of capacity to lyse malignant and infected cells without preliminary sensibilization or immunization. The human NK-cells have phenotype CD3-CD56+CD16+ and can be separated in two subpopulations depending on level of expression CD56. The CD56 bright NK-cells predominantly participates immuno-regulation producing cytokines. The CD56dim NK-cells predominantly have cytolytic activity. The NK-cells mediate destruction of infected and neoplastic cells through several effector mechanisms: by means of perforins/granzyme containing granules, receptors of apoptosis, antibody-depended cellular mediated cytotoxicity. The cytotoxicicty of NK-cells and production of cytokines provide regulatory role of NK-cells as important participants of adaptive immune system. The NK-cells are fitted with various receptors stimulating and inhibiting their activity. The activation of NK-cells depends on balance between inhibiting and activating receptors. The activation of NK-cells depends on balance between inhibiting and activating receptors. "The golden standard" of detection offunctional activity of human NK-cells is a test of releasing of radioactive chrome from target cells. To apply this technique in clinical practice is not an easy task because of difficulties of utilization of radioactive waste products, short half lifetime, expensiveness and complicated standardization. The article presents cyto-fluorimetric techniques for clinical detection of activity of NK-cells. These techniques permit to avoid a number of problems related to application of radioactivity. They also are fast and can be standardized. PMID:26999864

  1. 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. PMID:25857288

  2. Flow cytometry confirms reticulate evolution and reveals triploidy in Central European Diphasiastrum taxa (Lycopodiaceae, Lycophyta)

    PubMed Central

    Bennert, H. Wilfried; Horn, Karsten; Kauth, Marion; Fuchs, Jörg; Bisgaard Jakobsen, Iben Sophie; Øllgaard, Benjamin; Schnittler, Martin; Steinberg, Matthias; Viane, Ronnie

    2011-01-01

    Background and Aims Interspecific Diphasiastrum hybrids have been assumed to be homoploid and to produce well-formed spores serving sexual reproduction. If this were the case, forms intermediate between hybrids and parents or hybrid swarms should be expected. The purpose of this study was: (1) to check whether homoploidy consistently applies to the three hybrids throughout their Central European range; (2) to examine whether their genome sizes confirm their parentage as assumed by morphology; and (3) to perform a screening for detection of ploidy levels other than diploid and variation in DNA content due to backcrossing. Methods Flow cytometry was used first to measure the relative DNA values [with 4′,6-diamidino-2-phenylindole (DAPI) staining] and ploidy level as a general screening, and secondly to determine the absolute DNA 2C values [with propidium iodide (PI) staining] in a number of selected samples with the main focus on the hybrids. Key Results A considerable variation of DNA 2C values (5·26–7·52 pg) was detected between the three European Diphasiastrum species. The values of the diploid hybrids are highly constant without significant variation between regions. They are also intermediate between their assumed parents and agree closely with those calculated from their putative parents. This confirms their hybrid origin, assumed parentage and homoploid status. Considerably higher DNA amounts (9·48–10·30 pg) were obtained for three populations, suggesting that these represent triploid hybrids, an interpretation that is strongly supported by their morphology. Conclusions Diploid hybrids have retained their genetic and morphological identites throughout their Central European range, and thus no indications for diploid backcrossing were found. The triploid hybrids have probably originated from backcrossing between a diploid gametophyte of a hybrid (derived from a diplospore) and a haploid gametophyte of a diploid parental species. By repeated crossing

  3. Circulating Tumor Cell Detection and Capture by Photoacoustic Flow Cytometry in Vivo and ex Vivo.

    PubMed

    Galanzha, Ekaterina I; Zharov, Vladimir P

    2013-01-01

    Despite progress in detecting circulating tumor cells (CTCs), existing assays still have low sensitivity (1-10 CTC/mL) due to the small volume of blood samples (5-10 mL). Consequently, they can miss up to 103-104 CTCs, resulting in the development of barely treatable metastasis. Here we analyze a new concept of in vivo CTC detection with enhanced sensitivity (up to 102-103 times) by the examination of the entire blood volume in vivo (5 L in adults). We focus on in vivo photoacoustic (PA) flow cytometry (PAFC) of CTCs using label-free or targeted detection, photoswitchable nanoparticles with ultrasharp PA resonances, magnetic trapping with fiber-magnetic-PA probes, optical clearance, real-time spectral identification, nonlinear signal amplification, and the integration with PAFC in vitro. We demonstrate PAFC's capability to detect rare leukemia, squamous carcinoma, melanoma, and bulk and stem breast CTCs and its clusters in preclinical animal models in blood, lymph, bone, and cerebrospinal fluid, as well as the release of CTCs from primary tumors triggered by palpation, biopsy or surgery, increasing the risk of metastasis. CTC lifetime as a balance between intravasation and extravasation rates was in the range of 0.5-4 h depending on a CTC metastatic potential. We introduced theranostics of CTCs as an integration of nanobubble-enhanced PA diagnosis, photothermal therapy, and feedback through CTC counting. In vivo data were verified with in vitro PAFC demonstrating a higher sensitivity (1 CTC/40 mL) and throughput (up to 10 mL/min) than conventional assays. Further developments include detection of circulating cancer-associated microparticles, and super-rsesolution PAFC beyond the diffraction and spectral limits. PMID:24335964

  4. Circulating Tumor Cell Detection and Capture by Photoacoustic Flow Cytometry in Vivo and ex Vivo

    PubMed Central

    Galanzha, Ekaterina I.; Zharov, Vladimir P.

    2013-01-01

    Despite progress in detecting circulating tumor cells (CTCs), existing assays still have low sensitivity (1–10 CTC/mL) due to the small volume of blood samples (5–10 mL). Consequently, they can miss up to 103–104 CTCs, resulting in the development of barely treatable metastasis. Here we analyze a new concept of in vivo CTC detection with enhanced sensitivity (up to 102–103 times) by the examination of the entire blood volume in vivo (5 L in adults). We focus on in vivo photoacoustic (PA) flow cytometry (PAFC) of CTCs using label-free or targeted detection, photoswitchable nanoparticles with ultrasharp PA resonances, magnetic trapping with fiber-magnetic-PA probes, optical clearance, real-time spectral identification, nonlinear signal amplification, and the integration with PAFC in vitro. We demonstrate PAFC’s capability to detect rare leukemia, squamous carcinoma, melanoma, and bulk and stem breast CTCs and its clusters in preclinical animal models in blood, lymph, bone, and cerebrospinal fluid, as well as the release of CTCs from primary tumors triggered by palpation, biopsy or surgery, increasing the risk of metastasis. CTC lifetime as a balance between intravasation and extravasation rates was in the range of 0.5–4 h depending on a CTC metastatic potential. We introduced theranostics of CTCs as an integration of nanobubble-enhanced PA diagnosis, photothermal therapy, and feedback through CTC counting. In vivo data were verified with in vitro PAFC demonstrating a higher sensitivity (1 CTC/40 mL) and throughput (up to 10 mL/min) than conventional assays. Further developments include detection of circulating cancer-associated microparticles, and super-resolution PAFC beyond the diffraction and spectral limits. PMID:24335964

  5. Flow Cytometry for the Analysis of α-Dystroglycan Glycosylation in Fibroblasts from Patients with Dystroglycanopathies

    PubMed Central

    Stevens, Elizabeth; Torelli, Silvia; Feng, Lucy; Phadke, Rahul; Walter, Maggie C.; Schneiderat, Peter; Eddaoudi, Ayad; Sewry, Caroline A.; Muntoni, Francesco

    2013-01-01

    α-dystroglycan (α-DG) is a peripheral membrane protein that is an integral component of the dystrophin-glycoprotein complex. In an inherited subset of muscular dystrophies known as dystroglycanopathies, α-DG has reduced glycosylation which results in lower affinity binding to several extracellular matrix proteins including laminins. The glycosylation status of α-DG is normally assessed by the binding of the α-DG antibody IIH6 to a specific glycan epitope on α-DG involved in laminin binding. Immunocytochemistry and immunoblotting are two of the most widely used methods to detect the amount of α-DG glycosylation in muscle. While the interpretation of the presence or absence of the epitope on muscle using these techniques is straightforward, the assessment of a mild defect can be challenging. In this study, flow cytometry was used to compare the amount of IIH6-reactive glycans in fibroblasts from dystroglycanopathy patients with defects in genes known to cause α-DG hypoglycosylation to the amount in fibroblasts from healthy and pathological control subjects. A total of twenty one dystroglycanopathy patient fibroblasts were assessed, as well as fibroblasts from three healthy controls and seven pathological controls. Control fibroblasts have clearly detectable amounts of IIH6-reactive glycans, and there is a significant difference in the amount of this glycosylation, as measured by the mean fluorescence intensity of an antibody recognising the epitope and the percentage of cells positive for the epitope, between these controls and dystroglycanopathy patient fibroblasts (p<0.0001 for both). Our results indicate that the amount of α-DG glycosylation in patient fibroblasts is comparable to that in patient skeletal muscle. This method could complement existing immunohistochemical assays in skeletal muscle as it is quantitative and simple to perform, and could be used when a muscle biopsy is not available. This test could also be used to assess the pathogenicity of

  6. Some early differentiation markers detected in cytoplasm of pre-B cells by flow cytometry.

    PubMed

    Koníková, E; Glasová, M; Kusenda, J; Babusíková, O

    1996-01-01

    Immunofluorescent staining of cytoplasmic IgM (heavy chains) and CD24 as well as their simultaneous staining with surface B cell markers was used to study immunophenotype changes in B cell differentiation. Human hematopoietic B cell lines P3HR1 and RAJI were used. We found that IgM and CD24 cell markers while absent on cell membrane could be detected in their cytoplasm (c). The presence of cIgM in cell lines RAJI, P3HRI indicates their early pre-B differentiation stage. The presence of cCD24 simultaneously with mCD22 and cIgM is the evidence that hematopoietic cell lines or leukemias may not accurately reflect normal differentiation pathway. Combinations of cIgM, cCD24 with surface B cell markers CD10, CD19 on these cell lines can be considered as leukemia associated phenotypes. Some of them were shown in bone marrow and peripheral blood of pre-B ALL and B-CLL patients and can be used for the detection of minimal residual disease. Different fixation/permeabilization methods were tested in order to choose the optimal one for simple detection of cytoplasmic markers or their simultaneous detection with surface markers by flow cytometry. They included "one-component-methods" (methanol-M, saponin-S), methods combining these components with paraformaldehyde (P+M, P+S) or buffered formaldehyde acetone (BFA). The choice depended on individual marker detected. General parameters like the proportion of debris, cell aggregation, cell loss and the changes of scatter parameters FSC and SSC were taken into consideration. The priorities of combined methods P+S, P+M1 and BFA over one-component methods are demonstrated. PMID:8996561

  7. Reconfigurable laser arrays with capillary fill microfluidics for chip-based flow cytometry (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Thomas, Robert

    2016-03-01

    Low cost, portable chip based flow cytometry has great potential for applications in resource poor and point of care settings. Typical approaches utilise low cost silicon or glass substrates with light emission and detection performed either off-chip using external equipment or incorporated on-chip using `pick and place' diode lasers and photo-detectors. The former approach adds cost and limits portability while the sub-micron alignment tolerances imposed by the application make the latter impractical for all but the simplest of systems. Use of an optically active semiconductor substrate, on the other hand, overcomes these limitations by allowing multiple laser/detector arrays to be formed in the substrate itself using high resolution lithographic techniques. The capacity for multiple emitters and detectors on a single chip not only enables parallel measurement for increased throughput but also allows multiple measurements to be performed on each cell as it passes through the system. Several different experiments can be performed simultaneously and throughput demand can be reduced with the facility for error checking. Furthermore, the fast switching times inherent with semiconductor lasers allows the active sections of the device to be reconfigured on a sub-microsecond time scale providing additional functionality. This is demonstrated here in a capillary fill system using pairs of laser/detectors that are operated in pulsed mode and alternated between lasing and detecting in an interleaved manner. Passing cells are alternately interrogated from opposing directions providing information that can be used to correct for differences in lateral cell position and ultimately differentiate blood cell type.

  8. Evaluation of a Prototype Flow Cytometry Test for Serodiagnosis of Canine Visceral Leishmaniasis

    PubMed Central

    Ker, Henrique Gama; Coura-Vital, Wendel; Aguiar-Soares, Rodrigo Dian de Oliveira; Roatt, Bruno Mendes; das Dores Moreira, Nádia; Carneiro, Cláudia Martins; Machado, Evandro Marques de Menezes; Teixeira-Carvalho, Andréa; Martins-Filho, Olindo Assis; Giunchetti, Rodolfo Cordeiro; Araújo, Márcio Sobreira Silva; Coelho, Eduardo Antonio Ferraz; da Silveira-Lemos, Denise

    2013-01-01

    Diagnosing canine visceral leishmaniasis (CVL) is a critical challenge since conventional immunoserological tests still present some deficiencies. The current study evaluated a prototype flow cytometry serology test, using antigens and fluorescent antibodies that had been stored for 1 year at 4°C, on a broad range of serum samples. Noninfected control dogs and Leishmania infantum-infected dogs were tested, and the prototype test showed excellent performance in differentiating these groups with high sensitivity, specificity, positive and negative predictive values, and accuracy (100% in all analyses). When the CVL group was evaluated according to the dogs' clinical status, the prototype test showed outstanding accuracy in all groups with positive serology (asymptomatic II, oligosymptomatic, and symptomatic). However, in dogs which had positive results by PCR-restriction fragment length polymorphism (RFLP) but negative results by conventional serology (asymptomatic I), serological reactivity was not observed. Additionally, sera from 40 dogs immunized with different vaccines (Leishmune, Leish-Tec, or LBSap) did not present serological reactivity in the prototype test. Eighty-eight dogs infected with other pathogens (Trypanosoma cruzi, Leishmania braziliensis, Ehrlichia canis, and Babesia canis) were used to determine cross-reactivity and specificity, and the prototype test performed well, particularly in dogs infected with B. canis and E. canis (100% and 93.3% specificities, respectively). In conclusion, our data reinforce the potential of the prototype test for use as a commercial kit and highlight its outstanding performance even after storage for 1 year at 4°C. Moreover, the prototype test efficiently provided accurate CVL serodiagnosis with an absence of false-positive results in vaccinated dogs and minor cross-reactivity against other canine pathogens. PMID:24108778

  9. [APPLICATION OF FLOW CYTOMETRY FOR THE ANALYSIS OF CIRCULATING HEMOCYTE POPULATIONS IN THE ASCIDIAN HALOCYNTHIA AURANTIUM (PALLAS, 1787)].

    PubMed

    Sukhachev, A N; Dyachkov, I S; Kudryavtsev, I V; Kumeiko, V V; Tsybulskiy, A V; Polevshchikov, A V

    2015-01-01

    This study addresses the potentialities of flow cytometry in analyzing the composition of circulating hemocyte populations in the ascidian Halocynthia aurantium (Pallas, 1787) both using monoclonal antibodies (mAbs) against some human leukocyte conservative adhesion molecules and without mAbs. Flow cytometry, based on the assessment of forward and side scattering revealed five hemocyte populations. From the wide panel of antibodies against human leukocyte adhesion molecules (CD15, CD29, CD34, CD54, CD62L, CD62P, CD90, CD94, CD117, CD 166), only two mAbs (against CD54, CD90) displayed cross-reactivity with the H. aurantium hemocyte surface antigens. Distribution patterns of these antigens across the hemocyte populations have been analyzed. PMID:26281224

  10. Online flow cytometry for monitoring apoptosis in mammalian cell cultures as an application for process analytical technology.

    PubMed

    Kuystermans, Darrin; Avesh, Mohd; Al-Rubeai, Mohamed

    2016-05-01

    Apoptosis is the main driver of cell death in bioreactor suspension cell cultures during the production of biopharmaceuticals from animal cell lines. It is known that apoptosis also has an effect on the quality and quantity of the expressed recombinant protein. This has raised the importance of studying apoptosis for implementing culture optimization strategies. The work here describes a novel approach to obtain near real time data on proportion of viable, early apoptotic, late apoptotic and necrotic cell populations in a suspension CHO culture using automated sample preparation in conjunction with flow cytometry. The resultant online flow cytometry data can track the progression of apoptotic events in culture, aligning with analogous manual methodologies and giving similar results. The obtained near-real time apoptosis data are a significant improvement in monitoring capabilities and can lead to improved control strategies and research data on complex biological systems in bioreactor cultures in both academic and industrial settings focused on process analytical technology applications. PMID:25352493

  11. Fluorescein Isothiocyanate-Labeled Lectin Analysis of the Surface of the Nitrogen-Fixing Bacterium Azospirillum brasilense by Flow Cytometry

    PubMed Central

    Yagoda-Shagam, Janet; Barton, Larry L.; Reed, William P.; Chiovetti, Robert

    1988-01-01

    The cell surface of Azospirillum brasilense was probed by using fluorescein isothiocyanate (FITC)-labeled lectins, with binding determined by fluorescence-activated flow cytometry. Cells from nitrogen-fixing or ammonium-assimilating cultures reacted similarly to FITC-labeled lectins, with lectin binding in the following order: Griffonia simplicifolia II agglutinin > Griffonia simplicifolia I agglutinin > Triticum vulgaris agglutinin > Glycine max agglutinin > Canavalia ensiformis agglutinin > Limax flavus agglutinin > Lotus tetragonolobus agglutinin. The fluorescence intensity of cells labeled with FITC-labeled G. simplicifolia I, C. ensiformis, T. vulgaris, and G. max agglutinins was influenced by lectin concentration. Flow cytometry measurements of lectin binding to cells was consistent with measurements of agglutination resulting from lectin-cell interaction. Capsules surrounding nitrogen-fixing and ammonium-assimilating cells were readily demonstrated by light and transmission electron microscopies. Images PMID:16347693

  12. Quantitative Flow Cytometry Measurements in Antibodies Bound per Cell Based on a CD4 Reference.

    PubMed

    Wang, Lili; Degheidy, Heba; Abbasi, Fatima; Mostowski, Howard; Marti, Gerald; Bauer, Steven; Hoffman, Robert A; Gaigalas, Adolfas K

    2016-01-01

    Multicolor flow cytometer assays with fluorescently labeled antibodies are routinely used in clinical laboratories to measure the cell number of specific immunophenotypes and to estimate expression levels of specific receptors/antigens either on the cell surface or intracellularly. The cell number and specific receptors/antigens serve as biomarkers for pathological conditions at various stages of a disease. Existing methods and cell reference materials for quantitative expression measurements have not yet produced results that are of wide clinical interest or are instrument-independent across all fluorescence channels. This unit details a procedure for quantifying surface and intracellular biomarkers by calibrating the output of a multicolor flow cytometer in units of antibody bound per cell (ABC). The procedure includes (1) quality control of the flow cytometer, (2) fluorescence intensity calibration using hard dyed microspheres assigned with fluorescence intensity values, (3) compensation for fluorescence spillover between adjacent fluorescence channels, and (4) application of a biological reference calibrator to establish an ABC scale. The unit also points out current efforts for quantifying biomarkers in a manner that is independent of instrument platforms and reagent differences. PMID:26742654

  13. Enumerating Virus-Like Particles and Bacterial Populations in the Sinuses of Chronic Rhinosinusitis Patients Using Flow Cytometry.

    PubMed

    Carlson-Jones, Jessica A P; Paterson, James S; Newton, Kelly; Smith, Renee J; Dann, Lisa M; Speck, Peter; Mitchell, James G; Wormald, Peter-John

    2016-01-01

    There is increasing evidence to suggest that the sinus microbiome plays a role in the pathogenesis of chronic rhinosinusitis (CRS). However, the concentration of these microorganisms within the sinuses is still unknown. We show that flow cytometry can be used to enumerate bacteria and virus-like particles (VLPs) in sinus flush samples of CRS patients. This was achieved through trialling 5 sample preparation techniques for flow cytometry. We found high concentrations of bacteria and VLPs in these samples. Untreated samples produced the highest average bacterial and VLP counts with 3.3 ± 0.74 x 107 bacteria ml-1 and 2.4 ± 1.23 x 109 VLP ml-1 of sinus flush (n = 9). These counts were significantly higher than most of the treated samples (p < 0.05). Results showed 103 and 104 times inter-patient variation for bacteria and VLP concentrations. This wide variation suggests that diagnosis and treatment need to be personalised and that utilising flow cytometry is useful and efficient for this. This study is the first to enumerate bacterial and VLP populations in the maxillary sinus of CRS patients. The relevance of enumeration is that with increasing antimicrobial resistance, antibiotics are becoming less effective at treating bacterial infections of the sinuses, so alternative therapies are needed. Phage therapy has been proposed as one such alternative, but for dosing, the abundance of bacteria is required. Knowledge of whether phages are normally present in the sinuses will assist in gauging the safety of applying phage therapy to sinuses. Our finding, that large numbers of VLP are frequently present in sinuses, indicates that phage therapy may represent a minimally disruptive intervention towards the nasal microbiome. We propose that flow cytometry can be used as a tool to assess microbial biomass dynamics in sinuses and other anatomical locations where infection can cause disease. PMID:27171169

  14. Erratum to: Automated Sample Preparation Method for Suspension Arrays using Renewable Surface Separations with Multiplexed Flow Cytometry Fluorescence Detection

    SciTech Connect

    Grate, Jay W.; Bruckner-Lea, Cindy J.; Jarrell, Ann E.; Chandler, Darrell P.

    2003-04-10

    In this paper we describe a new method of automated sample preparation for multiplexed biological analysis systems that use flow cytometry fluorescence detection. In this approach, color-encoded microspheres derivatized to capture particular biomolecules are temporarily trapped in a renewable surface separation column to enable perfusion with sample and reagents prior to delivery to the detector. This method provides for separation of the biomolecules of interest from other sample matrix components as well as from labeling solutions.

  15. Microfabrication and Test of a Three-Dimensional Polymer Hydro-focusing Unit for Flow Cytometry Applications

    NASA Technical Reports Server (NTRS)

    Yang, Ren; Feeback, Daniel L.; Wang, Wanjun

    2004-01-01

    This paper details a novel three-dimensional (3D) hydro-focusing micro cell sorter for micro flow cytometry applications. The unit was microfabricated by means of SU-8 3D lithography. The 3D microstructure for coaxial sheathing was designed, microfabricated, and tested. Three-dimensional hydro-focusing capability was demonstrated with an experiment to sort labeled tanned sheep erythrocytes (red blood cells). This polymer hydro-focusing microstructure is easily microfabricated and integrated with other polymer microfluidic structures.

  16. Multicolor flow cytometry analysis of blood cell subsets in patients given total body irradiation before bone marrow transplantation

    SciTech Connect

    Clave, E.; Socie, G.; Carosella, E.

    1995-11-01

    Bone marrow transplantation has often been closely linked with accidental or intentional therapeutical irradiation. In both situations, study of the radiosensitivity of human blood cell subsets is of interest. Using one-color flow cytometry analysis of B lymphocytes, T cell subsets, and natural killer cells, we previously reported that lymphocyte subsets exhibit equal radiosensitivity. Taking advantage of recent developments in the knowledge of leukocyte differentiation antigens and flow cytometry technology we undertook a study of blood cell subsets to search for rare populations exhibiting different radiosensitivity. Thirty patients, who were delivered a 12 Gy fractionated total body irradiation as part of their conditioning regimen before transplantation for malignant disorders, were studied using multicolor flow cytometry. T and B lymphocytes showed a sharp, radiation-induced decrease, with the B lymphocytes (cluster of differentiation (CD) 19+) being the most sensitive. When analyzed by multicolor flow cytometry all major lymphocyte subsets appeared equally sensitive to the in vivo irradiation. Therefore, all major lymphocyte subsets sharing the helper phenotype (naive or memory) and the cytotoxic phenotype appeared equally sensitive to in vivo whole body irradiation. In parallel, the CD34+ cell subset remained basically unchanged after whole body irradiation. Finally, the CD3{minus}, 56+, 16+ natural killer cell subset was relatively radioresistant (91 and 74% of its initial value, after 2 and 4 Gy, respectively) as compared to other lymphocyte subsets. Our study provides evidence that T and B cell subsets seem to be highly radiosensitive in vivo. The CD34+ progenitor/stem cells and NK cells seem to be more radioresistant. This latter result might provide clues to the understanding of the pathophysiogeny of radiation-induced aplasia and of the engrafment/rejection process following bone marrow transplantation. 20 refs., 3 figs., 1 tab.

  17. Enumerating Virus-Like Particles and Bacterial Populations in the Sinuses of Chronic Rhinosinusitis Patients Using Flow Cytometry

    PubMed Central

    Carlson-Jones, Jessica A. P.; Paterson, James S.; Newton, Kelly; Smith, Renee J.; Dann, Lisa M.; Speck, Peter; Mitchell, James G.; Wormald, Peter-John

    2016-01-01

    There is increasing evidence to suggest that the sinus microbiome plays a role in the pathogenesis of chronic rhinosinusitis (CRS). However, the concentration of these microorganisms within the sinuses is still unknown. We show that flow cytometry can be used to enumerate bacteria and virus-like particles (VLPs) in sinus flush samples of CRS patients. This was achieved through trialling 5 sample preparation techniques for flow cytometry. We found high concentrations of bacteria and VLPs in these samples. Untreated samples produced the highest average bacterial and VLP counts with 3.3 ± 0.74 x 107 bacteria ml-1 and 2.4 ± 1.23 x 109 VLP ml-1 of sinus flush (n = 9). These counts were significantly higher than most of the treated samples (p < 0.05). Results showed 103 and 104 times inter-patient variation for bacteria and VLP concentrations. This wide variation suggests that diagnosis and treatment need to be personalised and that utilising flow cytometry is useful and efficient for this. This study is the first to enumerate bacterial and VLP populations in the maxillary sinus of CRS patients. The relevance of enumeration is that with increasing antimicrobial resistance, antibiotics are becoming less effective at treating bacterial infections of the sinuses, so alternative therapies are needed. Phage therapy has been proposed as one such alternative, but for dosing, the abundance of bacteria is required. Knowledge of whether phages are normally present in the sinuses will assist in gauging the safety of applying phage therapy to sinuses. Our finding, that large numbers of VLP are frequently present in sinuses, indicates that phage therapy may represent a minimally disruptive intervention towards the nasal microbiome. We propose that flow cytometry can be used as a tool to assess microbial biomass dynamics in sinuses and other anatomical locations where infection can cause disease. PMID:27171169

  18. Proposal for the standardization of flow cytometry protocols to detect minimal residual disease in acute lymphoblastic leukemia.

    PubMed

    Ikoma, Maura Rosane Valério; Beltrame, Miriam Perlingeiro; Ferreira, Silvia Inês Alejandra Cordoba Pires; Souto, Elizabeth Xisto; Malvezzi, Mariester; Yamamoto, Mihoko

    2015-01-01

    Minimal residual disease is the most powerful predictor of outcome in acute leukemia and is useful in therapeutic stratification for acute lymphoblastic leukemia protocols. Nowadays, the most reliable methods for studying minimal residual disease in acute lymphoblastic leukemia are multiparametric flow cytometry and polymerase chain reaction. Both provide similar results at a minimal residual disease level of 0.01% of normal cells, that is, detection of one leukemic cell in up to 10,000 normal nucleated cells. Currently, therapeutic protocols establish the minimal residual disease threshold value at the most informative time points according to the appropriate methodology employed. The expertise of the laboratory in a cancer center or a cooperative group could be the most important factor in determining which method should be used. In Brazil, multiparametric flow cytometry laboratories are available in most leukemia treatment centers, but multiparametric flow cytometry processes must be standardized for minimal residual disease investigations in order to offer reliable and reproducible results that ensure quality in the clinical application of the method. The Minimal Residual Disease Working Group of the Brazilian Society of Bone Marrow Transplantation (SBTMO) was created with that aim. This paper presents recommendations for the detection of minimal residual disease in acute lymphoblastic leukemia based on the literature and expertise of the laboratories who participated in this consensus, including pre-analytical and analytical methods. This paper also recommends that both multiparametric flow cytometry and polymerase chain reaction are complementary methods, and so more laboratories with expertise in immunoglobulin/T cell receptor (Ig/TCR) gene assays are necessary in Brazil. PMID:26670404

  19. Proliferation and differentiation of oligodendrocyte progenitor cells induced from rat embryonic neural precursor cells followed by flow cytometry.

    PubMed

    Lü, He-Zuo; Wang, Yan-Xia; Li, Ying; Fu, Sai-Li; Hang, Qin; Lu, Pei-Hua

    2008-08-01

    Previous studies have shown that a cell-intrinsic timer might determine when oligodendrocyte progenitor cells (OPCs) isolated from the central nervous system (CNS) stop dividing and initiate differentiation in a defined environment. In this report, the proliferation and differentiation of OPCs induced from neural precursor cells (NPCs) were analyzed by flow cytometry combined with carboxyfluorescein diacetate succinimidyl ester labeling and propidium iodide staining, respectively. When OPCs were cultured in OPC-medium, more than 30% of cells were in S- and G2/M-phases, and continuously self-renewed without differentiation. After exposure to thyroid hormone, there was an obvious decrease in the fraction of cells in both S- and G2/M-phases (<10%). Furthermore, the OPCs no longer proliferated, but differentiated into oligodendrocytes. The dynamic proliferation and differentiation characteristics of OPCs induced from NPCs and analyzed by flow cytometry were similar to those of OPCs isolated from the CNS and analyzed by other methods. These studies indicated that the proliferation and differentiation of OPCs can be followed simply and rapidly by flow cytometry. PMID:18473382

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

    NASA Astrophysics Data System (ADS)

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

    2016-02-01

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

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

    PubMed Central

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

    2016-01-01

    Three-dimensional (3D) tumor spheroid possesses great potential as an in vitro model to improve predictive capacity for pre-clinical drug testing. In this paper, we combine advantages of flow cytometry and microfluidics to perform drug testing and analysis on a large number (5000) of uniform sized tumor spheroids. The spheroids are formed, cultured, and treated with drugs inside a microfluidic device. The spheroids can then be harvested from the device without tedious operation. Due to the ample cell numbers, the spheroids can be dissociated into single cells for flow cytometry analysis. Flow cytometry provides statistical information in single cell resolution that makes it feasible to better investigate drug functions on the cells in more in vivo-like 3D formation. In the experiments, human hepatocellular carcinoma cells (HepG2) are exploited to form tumor spheroids within the microfluidic device, and three anti-cancer drugs: Cisplatin, Resveratrol, and Tirapazamine (TPZ), and their combinations are tested on the tumor spheroids with two different sizes. The experimental results suggest the cell culture format (2D monolayer vs. 3D spheroid) and spheroid size play critical roles in drug responses, and also demonstrate the advantages of bridging the two techniques in pharmaceutical drug screening applications. PMID:26877244

  2. Detection of IgG Anti-Leishmania Antigen by Flow Cytometry as a Diagnostic Test for Cutaneous Leishmaniasis.

    PubMed

    Pedral-Sampaio, Geraldo; Alves, Jessé S; Schriefer, Albert; Magalhães, Andréa; Meyer, Roberto; Glesby, Marshall J; Carvalho, Edgar M; Carvalho, Lucas P

    2016-01-01

    Diagnosis of cutaneous leishmaniasis (CL) relies on clinical presentation, parasite isolation, histopathologic evaluation and positive Montenegro skin test. However, the low amounts of parasites in the lesion of these individuals make parasite isolation and histopatologic diagnosis unreliable, often leading to false-negative results. Also, 15% of people living in endemic areas have sub-clinical infection characterized by positive Montenegro skin test, which may contribute to misdiagnosis. Although the main Leishmania killing mechanism is through cell-mediated immune response, antibodies against Leishmania antigens are found in infected individuals. Here our goal was to develop a new serological technique using polystyrene microspheres sensitized with soluble Leishmania antigens as a tool for the detection of IgG in serum from CL patients by flow cytometry. To validate the assay we carried out a comparative test (ELISA) commonly used as a diagnostic test for parasitic diseases. To determine cross-reactivity we used serum from patients with Chagas disease, caused by a trypanosome that has several proteins with high homology to those of the Leishmania genus. We observed that the flow cytometry technique was more sensitive than the ELISA, but, less specific. Our results show that the flow cytometry serologic test can be used to confirm CL cases in L. braziliensis transmission areas, however, presence of Chagas disease has to be ruled out in these individuals. PMID:27622535

  3. Flow cytometry of p53 protein expression in some hematological malignancies.

    PubMed

    Koníková, E; Kusenda, J; Babusíková, O

    1999-01-01

    p53 is a tumor suppressor gene encoding a nuclear phosphoprotein that plays an important role in the control of normal cell proliferation. We have tried to establish the value of the p53 protein expression in peripheral blood (PB) and/or bone marrow (BM) cells of patients with some hematological malignancies. A recently developed fixation/permeabilization method was modified for flow cytometric assessment of p53 protein expression using two anti-p53 monoclonal antibodies. p53 quantitation expressed as molecules of equivalent soluble fluorochrome per cell (MESF) providing valuable data contributing to a more precise definition of leukemic cells, was also applied. Our findings showed higher percentage of p53 expression in cells of AML patients at the time of diagnosis opposite to the controls. These data, in association with immunophenotype of cells, accompanied diagnosis of relapse or definition of remission after allogeneic BM transplantation. We observed also elevated levels of p53 protein at initial diagnosis of early B-ALL. According to our results quantitation of p53 protein allows better characterization of selected population of BM cells and should be used for the monitoring of blast persistence during and after therapy and might also be one of the methods to indicate early relapse. Percentage of p53 protein positivity varied in our group of B-CLL patients tested in connection with progression of disease. We documented also one case of Burkitt's lymphoma with high percentage of p53 positivity. Measurement of p53 protein expression by flow cytometry may be of clinical importance by indicating levels of positivity. Our results suggest, that p53 alteration is frequently involved at initial diagnosis of AML, in some T-cell disorders and on the contrary more frequently during early B-ALL relapse, in advanced stages of B-CLL and in Burkitt's lymphoma. p53 protein quantitation is of value to ascertain malignancy and provides additional parameter suitable for the

  4. Determination of sperm concentration using flow cytometry with simultaneous analysis of sperm plasma membrane integrity in zebrafish Danio rerio.

    PubMed

    Yang, Huiping; Daly, Jonathan; Tiersch, Terrence R

    2016-04-01

    Control of sperm concentration is required to ensure consistent and reproducible results for cryopreservation and in vitro fertilization protocols. Determination of sperm concentration is traditionally performed with a counting chamber (e.g., hemocytometer), or more recently with a spectrophotometer. For small-sized biomedical model fishes, the availability of sperm sample is limited to microliters, so it is desirable to develop fast and accurate approaches for concentration determination that also minimize sample use. In this study, a new approach was developed for sperm concentration determination using a flow cytometer (Accuri C6, BD Biosciences, San Jose, CA) with simultaneous measurement of sperm membrane integrity after fluorescent staining with SYBR(®) -14 and propidium iodide (PI) in sperm from Zebrafish Danio rerio. The goal was to develop a protocol for simultaneous determination of sperm quality and quantity by flow cytometry. The objectives were to (1) determine the effects of sample volume (250 and 500 µl) and analysis volume (10 and 50 µl) on the accuracy of particle counting using standard volumetric validation beads; (2) identify the effective range of sperm concentrations that flow cytometry can measure; (3) test the precision and reproducibility of the sperm concentration measurements; and (4) verify the flow cytometry approach by comparison with measurement with a hemocytometer and a microspectrophotometer. Sample volumes of 250 and 500 µl and analysis volumes of 10 and 50 µl did not affect bead count with the factory-set flow rates of "medium" or "fast," and the precision and accuracy was retained across a concentration range of 1 × 10(3) -1 × 10(7) cells/ml. The approach developed in this study was comparable to traditional methodologies such as hemocytometer or microspectrophotometer. This study provides an efficient, accurate, and rapid method for determination of sperm concentration using flow cytometry while providing

  5. Mass cytometry as a platform for the discovery of cellular biomarkers to guide effective rheumatic disease therapy.

    PubMed

    Nair, Nitya; Mei, Henrik E; Chen, Shih-Yu; Hale, Matthew; Nolan, Garry P; Maecker, Holden T; Genovese, Mark; Fathman, C Garrison; Whiting, Chan C

    2015-01-01

    The development of biomarkers for autoimmune diseases has been hampered by a lack of understanding of disease etiopathogenesis and of the mechanisms underlying the induction and maintenance of inflammation, which involves complex activation dynamics of diverse cell types. The heterogeneous nature and suboptimal clinical response to treatment observed in many autoimmune syndromes highlight the need to develop improved strategies to predict patient outcome to therapy and personalize patient care. Mass cytometry, using CyTOF®, is an advanced technology that facilitates multiparametric, phenotypic analysis of immune cells at single-cell resolution. In this review, we outline the capabilities of mass cytometry and illustrate the potential of this technology to enhance the discovery of cellular biomarkers for rheumatoid arthritis, a prototypical autoimmune disease. PMID:25981462

  6. Phase-sensitive flow cytometry: New technology for analyzing biochemical, functional, and structural features in fluorochrome- labeled cells/particles

    SciTech Connect

    Steinkamp, J.A.

    1993-12-01

    Flow cytometry (FCM) instruments rapidly measure biochemical, functional, and cytological properties of individual cells and macromolecular components, e.g., chromosomes, for clinical diagnostic medicine and biomedical and envirorunental research applications. These measurements are based on labeling cells with multiple fluorochromes for correlated analysis of macromolecules, such as DNA RNA, protein, and cell-surface receptors. This report describes the development of a phase-sensitive flow cytometer that provides unique capabilities for making laser-excited, phase-resolved measurements on fluorochrome-labelled cells and particles.

  7. Real-Time Label-Free Embolus Detection Using In Vivo Photoacoustic Flow Cytometry

    PubMed Central

    Juratli, Mazen A.; Menyaev, Yulian A.; Sarimollaoglu, Mustafa; Siegel, Eric R.; Nedosekin, Dmitry A.; Suen, James Y.; Melerzanov, Alexander V.; Juratli, Tareq A.; Galanzha, Ekaterina I.; Zharov, Vladimir P.

    2016-01-01

    Thromboembolic events are one of the world’s leading causes of death among patients. Embolus or clot formations have several etiologies including paraneoplastic, post-surgery, cauterization, transplantation, or extracorporeal circuits. Despite its medical significance, little progress has been made in early embolus detection, screening and control. The aim of our study is to test the utility of the in vivo photoacoustic (PA) flow cytometry (PAFC) technique for non-invasive embolus detection in real-time. Using in vivo PAFC, emboli were non-invasively monitored in the bloodstream of two different mouse models. The tumor-free mouse model consisted of two groups, one in which the limbs were clamped to produce vessel stasis (7 procedures), and one where the mice underwent surgery (7 procedures). The melanoma-bearing mouse model also consisted of two groups, one in which the implanted tumor underwent compression (8 procedures), and one where a surgical excision of the implanted tumor was performed (8 procedures). We demonstrated that the PAFC can detect a single embolus, and has the ability to distinguish between erythrocyte–rich (red) and leukocyte/platelet-rich (white) emboli in small vessels. We show that, in tumor-bearing mice, the level of circulating emboli was increased compared to tumor-free mice (p = 0.0013). The number of circulating emboli temporarily increased in the tumor-free control mice during vessel stasis (p = 0.033) and after surgical excisions (signed-rank p = 0.031). Similar observations were noted during tumor compression (p = 0.013) and after tumor excisions (p = 0.012). For the first time, it was possible to detect unlabeled emboli in vivo non-invasively, and to confirm the presence of pigmented tumor cells within circulating emboli. The insight on embolus dynamics during cancer progression and medical procedures highlight the clinical potential of PAFC for early detection of cancer and surgery-induced emboli to prevent the fatal

  8. Microfluidic CARS cytometry

    PubMed Central

    Wang, Han-Wei; Bao, Ning; Le, Thuc T.; Lu, Chang; Cheng, Ji-Xin

    2009-01-01

    Coherent anti-stokes Raman scattering (CARS) flow cytometry was demonstrated by combining a laser-scanning CARS microscope with a polydimethylsiloxane (PDMS) based microfluidic device. Line-scanning across the hydrodynamically focused core stream was performed for detection of flowing objects. Parameters were optimized by utilizing polystyrene beads as flowing particles. Population measurements of adipocytes isolated from mouse fat tissues demonstrated the viability of microfluidic CARS cytometry for quantitation of adipocyte size distribution. CARS cytometry could be a new modality for quantitative analysis with vibrational selectivity. PMID:18542688

  9. Identification of a Murine Erythroblast Subpopulation Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

    PubMed Central

    Konstantinidis, Diamantis G.; Pushkaran, Suvarnamala; Giger, Katie; Manganaris, Stefanos; Zheng, Yi; Kalfa, Theodosia A.

    2014-01-01

    Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation. We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis. We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation. Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry. Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters “aspect ratio” of a cell in the bright-field channel that aids in the recognition of elongated cells and “delta centroid XY Ter119/Draq5” that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate. The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells. PMID:24962543

  10. Diagnosis of leptomeningeal disease in diffuse large B-cell lymphomas of the central nervous system by flow cytometry and cytopathology.

    PubMed

    Schroers, Roland; Baraniskin, Alexander; Heute, Christoph; Vorgerd, Matthias; Brunn, Anna; Kuhnhenn, Jan; Kowoll, Annika; Alekseyev, Andriy; Schmiegel, Wolff; Schlegel, Uwe; Deckert, Martina; Pels, Hendrik

    2010-12-01

    Reliable detection of leptomeningeal disease has the potential of facilitating the diagnosis of central nervous system (CNS) lymphoma and is important for therapeutic considerations. Currently, the standard diagnostic procedure for the detection of lymphoma in the cerebrospinal fluid is cytopathology. To improve the limited specificity and sensitivity of cytopathology, flow cytometry has been suggested as an alternative. Here, we evaluated multi-parameter flow cytometry in combination with conventional cytopathology in cerebrospinal fluid (CSF) samples from 30 patients with primary CNS lymphoma and seven patients with secondary CNS lymphoma. Overall, in 11 of 37 (29.7%) patients with CNS lymphoma, lymphoma cells were detected in CSF by flow cytometry, while cytopathology was less sensitive displaying unequivocally malignant CSF cells in only seven of all 37 (18.9%) patients. Six (16.2%) patients showed cytopathological results suspicious of lymphoma; however, in only one of these patients, the diagnosis of CSF lymphoma cells could be confirmed by flow cytometry. In primary CNS lymphomas (PCNSL), seven of 30 (23.3%) patients were positive for CSF lymphoma cells in flow cytometry, in contrast to four (13.3%) patients with PCNSL with definitely positive cytopathology. In summary, our results suggest that multi-parameter flow cytometry increases the sensitivity and specificity of leptomeningeal disease detection in CNS lymphomas. Both methods should be applied concurrently for complementary diagnostic assessment in patients with CNS lymphoma. PMID:20727005

  11. FlowCal: A User-Friendly, Open Source Software Tool for Automatically Converting Flow Cytometry Data from Arbitrary to Calibrated Units.

    PubMed

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

    2016-07-15

    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, nonproprietary 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 Venus 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

  12. Platelet antibody detection by flow cytometry: an effective method to evaluate and give transfusional support in platelet refractoriness

    PubMed Central

    Bub, Carolina Bonet; Martinelli, Beatriz Moraes; Avelino, Thayná Mendonça; Gonçalez, Ana Cláudia; Barjas-Castro, Maria de Lourdes; Castro, Vagner

    2013-01-01

    Background Immune platelet refractoriness is mainly caused by human leukocyte antigen antibodies (80-90% of cases) and, to a lesser extent, by human platelet antigen antibodies. Refractoriness can be diagnosed by laboratory tests and patients should receive compatible platelet transfusions. A fast, effective and low cost antibody-screening method which detects platelet human leukocyte/platelet antigen antibodies is essential in the management of immune platelet refractoriness. Objective The aim of this study was to evaluate the efficiency of the flow cytometry platelet immunofluorescence test to screen for immune platelet refractoriness. Methods A group of prospective hematologic patients with clinically suspected platelet refractoriness treated in a referral center in Campinas, SP during July 2006 and July 2011 was enrolled in this study. Platelet antibodies were screened using the flow cytometry platelet immunofluorescence test. Anti-human leukocyte antigen antibodies were detected by commercially available methods. The sensitivity, specificity and predictive values of the immunofluorescence test were determined taking into account that the majority of antiplatelet antibodies presented human leukocyte antigen specificity. Results Seventy-six samples from 32 female and 38 male patients with a median age of 43.5 years (range: 5-84 years) were analyzed. The sensitivity of the test was 86.11% and specificity 75.00% with a positive predictive value of 75.61% and a negative predictive value of 85.71%. The accuracy of the method was 80.26%. Conclusion This study shows that the flow cytometry platelet immunofluorescence test has a high correlation with the anti-human leukocyte antigen antibodies. Despite a few limitations, the method seems to be efficient, fast and feasible as the initial screening for platelet antibody detection and a useful tool to crossmatch platelets for the transfusional support of patients with immune platelet refractoriness. PMID:24106442

  13. Differential expression of T cell antigens in normal peripheral blood lymphocytes: a quantitative analysis by flow cytometry.

    PubMed Central

    Ginaldi, L; Farahat, N; Matutes, E; De Martinis, M; Morilla, R; Catovsky, D

    1996-01-01

    AIMS: To obtain reference values of the level of expression of T cell antigens on normal lymphocyte subsets in order to disclose differences which could reflect their function or maturation stages, or both. METHODS: Peripheral blood from 15 healthy donors was processed by flow cytometry with triple colour analysis. For each sample phycoerythrin (PE) conjugated CD2, CD4, CD5, CD8, and CD56 monoclonal antibodies were combined with Cy5-R-phycoerythrin (TC) conjugated CD3 and fluorescein isothiocyanate (FITC) conjugated CD7; CD2- and CD7-PE were also combined with CD3-TC and CD4-FITC. Standard microbeads with different capacities to bind mouse immunoglobulins were used to convert the mean fluorescence intensity (MFI) values of the lymphocyte subsets identified by multiparametric flow cytometry into the number of antigen molecules per cell, measured as antibody binding capacity (ABC). RESULTS: CD4+ (helper/inducer) T cells exhibit a higher CD3 antigen expression compared with CD8+ (suppressor/ cytotoxic) T lymphocytes. Within the CD4+ T cells, the CD4+CD7- subset expressed a lower level of CD3 compared with CD4+CD7+ and CD8+CD7+ cells, and higher CD2 and CD5 expression than the main CD3+CD7+ subset. Major differences in antigen expression were also detected between CD3+ T cells and CD3-CD56+ natural killer (NK) cells: NK cells exhibited higher levels of CD7 and CD56 and lower levels of CD2 and CD5 than T cells. Significantly lower CD5 expression was also detected in the small CD5+ B lymphocyte subset compared with T cells. CONCLUSIONS: Quantitative flow cytometry with triple colour analysis may be used to detect antigen modulations in disease states and to increase the accuracy of diagnosis by comparison with findings in normal counterparts. Images PMID:8813949

  14. Exhaustive expansion: A novel technique for analyzing complex data generated by higher-order polychromatic flow cytometry experiments

    PubMed Central

    2010-01-01

    Background The complex data sets generated by higher-order polychromatic flow cytometry experiments are a challenge to analyze. Here we describe Exhaustive Expansion, a data analysis approach for deriving hundreds to thousands of cell phenotypes from raw data, and for interrogating these phenotypes to identify populations of biological interest given the experimental context. Methods We apply this approach to two studies, illustrating its broad applicability. The first examines the longitudinal changes in circulating human memory T cell populations within individual patients in response to a melanoma peptide (gp100209-2M) cancer vaccine, using 5 monoclonal antibodies (mAbs) to delineate subpopulations of viable, gp100-specific, CD8+ T cells. The second study measures the mobilization of stem cells in porcine bone marrow that may be associated with wound healing, and uses 5 different staining panels consisting of 8 mAbs each. Results In the first study, our analysis suggests that the cell surface markers CD45RA, CD27 and CD28, commonly used in historical lower order (2-4 color) flow cytometry analysis to distinguish memory from naïve and effector T cells, may not be obligate parameters in defining central memory T cells (TCM). In the second study, we identify novel phenotypes such as CD29+CD31+CD56+CXCR4+CD90+Sca1-CD44+, which may characterize progenitor cells that are significantly increased in wounded animals as compared to controls. Conclusions Taken together, these results demonstrate that Exhaustive Expansion supports thorough interrogation of complex higher-order flow cytometry data sets and aids in the identification of potentially clinically relevant findings. PMID:21034498

  15. Microfabrication and Test of a Three-Dimensional Polymer Hydro-focusing Unit for Flow Cytometry Applications

    NASA Technical Reports Server (NTRS)

    Yang, Ren; Feeback, Daniel L.; Wang, Wan-Jun

    2005-01-01

    This paper details a novel three-dimensional (3D) hydro-focusing micro cell sorter for micro flow cytometry applications. The unit was microfabricated by means of SU-8 3D lithography. The 3D microstructure for coaxial sheathing was designed, microfabricated, and tested. Three-dimensional hydrofocusing capability was demonstrated with an experiment to sort labeled tanned sheep erythrocytes (red blood cells). This polymer hydro-focusing microstructure is easily microfabricated and integrated with other polymer microfluidic structures. Keywords: SU-8, three-dimensional hydro-focusing, microfluidic, microchannel, cytometer

  16. Microfabrication and Test of a Three-Dimensional Polymer Hydro-Focusing Unit for Flow Cytometry Applications

    NASA Technical Reports Server (NTRS)

    Yang, Ren; Feedback, Daniel L.; Wang, Wanjun

    2004-01-01

    This paper details a novel three-dimensional (3D) hydro-focusing micro cell sorter for micro flow cytometry applications. The unit was micro-fabricated by means of SU-8 3D lithography. The 3D microstructure for coaxial sheathing was designed, micro-fabricated, and tested. Three-dimensional hydrofocusing capability was demonstrated with an experiment to sort labeled tanned sheep erythrocytes (red blood cells). This polymer hydro-focusing microstructure is easily micro-fabricated and integrated with other polymer microfluidic structures.

  17. Standardization of a method to detect bovine sperm-bound anti-sperm antibodies by flow cytometry

    PubMed Central

    Sardoy, MC; Anderson, DE; George, A; Wilkerson, ME; Skinner, S; Ferrer, MS

    2012-01-01

    The objectives were to standardize some methodological and analytical aspects of a direct technique to detect sperm-bound anti-sperm antibodies (ASAs) in bovine semen using flow cytometry, including the effects of pre-fixation of sperm membranes with formalin buffer solution and inclusion of dead cells in the analysis. Fourteen Angus bulls, including ASA-positive (experimentally induced ASAs) and 10 reproductively normal ASA-negative bulls, were used. Fixation of sperm membranes had no significant effect on the percentage of IgG- or IgA-bound spermatozoa detected by flow cytometry. However, including dead cells in the analysis increased the percentage of IgG-bound spermatozoa in fixed (live and dead 18.6 ± 9.7% and live 1.3 ± 0.5%; median ± SEM) and non-fixed samples (live and dead 18.8 ± 9.2%, live 1.5 ± 0.6%; P = 0.0029), as well as IgA-bound spermatozoa in fixed (live and dead 16.3 ± 6.4%, live 0.3 ± 0.5%) and non-fixed samples (live and dead 21.4 ± 4.6%, live 1.0 ± 0.5%; P = 0.0041) in semen from ASA-negative bulls. Intra-sample, intra-assay and inter-assay coefficients of variation (CV) were 0.8, 4.6 and 5.3%, respectively, for determination of sperm-bound IgG, and were 2.8, 8.4 and 40.3% for determination of sperm-bound IgA. Despite the high inter-assay CV for IgA determination, all ASA-positive bulls consistently had high percentages of IgA-bound spermatozoa. Flow cytometry correctly identified ASA-positive bulls. Confocal laser microscopy confirmed binding of ASAs to sperm heads and cytoplasmic droplets, and less frequently to midpieces and principal piece. In conclusion, although fixation was not necessary, dead cells should be excluded from the analysis, since ejaculates with a large proportion of dead cells can yield false-positive results. Flow cytometry was accurate and reliable for detection of sperm-bound IgG and IgA and discrimination between ASA-positive and ASA-negative bulls. PMID:22925638

  18. HLA-B27 detection – comparison of genetic sequence-based method and flow cytometry assay

    PubMed Central

    Kozakiewicz, Anna; Maśliński, Włodzimierz; Jurkowska, Monika

    2015-01-01

    Objectives The presence of human leukocyte antigen B27 (HLA-B27) is strongly associated with ankylosing spondylitis. HLA-B27 testing is routinely applied in the diagnosis of this disease. The aim of the present study was to compare two methods of HLA-B27 detection – a genetic sequence-based method and a flow cytometry assay. Material and methods Peripheral blood was obtained from 300 individuals with suspected spondyloarthropathy. Expression of HLA-B27 on the T cell surface was analysed by flow cytometry assay using GS145.2 monoclonal antibody specific for HLA-B27. DNA was isolated from the whole blood. Genes coding for HLA-B27, -B40 and -B47:01 were detected by polymerase chain reaction using the MW02/MW09 primer pair. Then, positive samples were sequenced in order to discriminate allelic variations of the HLA-B27 gene. Results of sequencing were analysed using Chromas LITE 2.1.1 software, BLAST software and the IMGT/HLA database. Ambiguous samples were additionally analysed by polymerase chain reaction using E91 and E136 primers amplifying a 135-bp fragment of the human HLA-B27 gene. Results Among 300 samples, 76 were HLA-B27-positive on the basis of flow cytometry analysis. Genetic sequence analysis confirmed positivity of 73 from among 76 samples. Two hundred twenty six samples were HLA-B27-negative, whereas the result of one sample analysis was ambiguous. Fifty-three samples were identified as allelic variation 27:05, 19 samples as allelic variation 27:02, and one sample as allelic variation 27:07. Conclusions This study shows that the genetic sequence-based method and the flow cytometry assay give consistent results in 99% of cases. The performed genetic analysis proves that the majority of HLA-B27-positive samples belong to the 27:05 allelic variation, which is strongly associated with high risk of ankylosing spondylitis.

  19. Flow Cytometry-Assisted Basophil Activation Test as a Safe Diagnostic Tool for Aspirin/NSAID Hypersenstivity

    PubMed Central

    Kim, Myung Shin

    2012-01-01

    Purpose Aspirin and non-steroidal anti-inflammatory drugs (ASA/NSAIDs) are common causes of drug hypersensitivity. An oral provocation test is the only definitive diagnostic test. This study assessed the reliability of a flow cytometry-assisted basophil activation test (FAST) as a safe diagnostic method for ASA/NSAID-induced hypersensitivity, as its high sensitivity and specificity have been demonstrated for many other drugs. Methods Eighteen patients and 11 controls were enrolled. Using a Flow-CAST kit® (Bühlmann Laboratories AG, Schönenbuch, Switzerland), 29 analyses with aspirin, ibuprofen, and diclofenac were performed by flow cytometry to detect double-positive staining of anti-IgE and anti-CD63. The stimulation index was defined as the activated basophil percentage after drug stimulation/basally active basophil percentage. A stimulation index≥2 and an absolute activated basophil percentage≥5 were considered positive. Results Patients with hypersensitivity to ASA/NSAIDs were predominantly female, and the prevalence of atopy was higher in patients than in controls. A sensitivity of 61%, specificity of 91%, positive predictive value of 92%, and negative predictive value of 59% were achieved. Conclusions FAST is a useful additional method for diagnosis of hypersensitivity reactions to ASA/NSAIDs. Further development is required to increase the sensitivity of the test. PMID:22548206

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

    PubMed Central

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

    2012-01-01

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

  1. In vivo imaging flow cytometry based on laser scanning two-photon microscopy at kHz cross-sectional frame rate

    NASA Astrophysics Data System (ADS)

    Kong, Lingjie; Tang, Jianyong; Cui, Meng

    2016-03-01

    In vivo flow cytometry has found numerous applications in biology and pharmacology. However, conventional cytometry does not provide the detailed morphological information that is needed to fully determine the phenotype of individual circulating cells. Imaging cytometry, capable of visualizing the morphology and dynamics of the circulating cells at high spatiotemporal resolution, is highly desired. Current wide-field based image cytometers are limited in the imaging depth and provide only two-dimensional resolution. For deep tissue imaging, laser scanning two-photon fluorescence microscopy (TPM) is widely adopted. However, for applications in flow cytometry, the axial scanning speed of current TPMs is inadequate to provide high-speed cross-sectional imaging of vasculature. We have integrated an optical phase-locked ultrasound lens into a standard TPM and achieved microsecond-scale axial scanning. With a galvo scanner for transverse scanning, we achieved kHz cross-sectional frame rate. Here we report its applications for in vivo deformability cytometry and in vivo imaging flow cytometry, and demonstrate the capability of imaging dynamical morphologies of flowing cells, distinguishing cells and cellular clusters, and simultaneously quantifying different cell populations based on their fluorescent labels.

  2. 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.

  3. Comparative of three methods (ELIZA, MAIPA and flow cytometry) to determine anti-platelet antibody in children with ITP.

    PubMed

    Hamidpour, Mohsen; Khalili, Ghader; Tajic, Nader; Shamsian, Bi Bi Shahin; Hamidpour, Rafie

    2014-01-01

    Immune (idiopathic) thrombocytopenic purpurea (ITP) is an autoimmune disease characterized by the increased anti-platelet antibodies in the patient's sera and decreased platelets in the blood circulation. This study has determined and characterized the antiplatelet glycoproteins in children with ITP. Thirty eight children, who were hospitalized with clinical signs of ITP in Mofid Children Hospital (Tehran, Iran) during 18 months, went under our clinical studies in a research project. ELISA, Flow cytometry and MAIPA (Monoclonal Antibody Immobilization of Platelet Antigens) methods were employed to determine serum anti-platelet antibodies level. The anti-platelet antibodies level above mean + 3SD of control group was assumed as positive. The platelet counts ranged between 2 × 10(9)/L and 100 × 10(9)/L. Among the patients 63.5% of them were anti-platelet antibodies positive with ELISA method. Results of platelet lysate method showed that 51.7% of patients had antibodies against platelet antigens. Antibody against platelet GPIIb/IIIa, GPIb/IX and GPIa/IIa using MAIPA method were 48%, 54% and 25% respectively. In flow cytometry 62% of patients showed anti-platelet antibodies. The comparison of three methods shows that since MAIPA is the specific method for the detection of very small amount of antibody against glycoprotein antigens, it has the advantage of differentiating between immune and non-immune thrombocytopenia. PMID:25755908

  4. Comparative of three methods (ELIZA, MAIPA and flow cytometry) to determine anti-platelet antibody in children with ITP

    PubMed Central

    Hamidpour, Mohsen; Khalili, Ghader; Tajic, Nader; Shamsian, Bi Bi Shahin; Hamidpour, Rafie

    2014-01-01

    Immune (idiopathic) thrombocytopenic purpurea (ITP) is an autoimmune disease characterized by the increased anti-platelet antibodies in the patient’s sera and decreased platelets in the blood circulation. This study has determined and characterized the antiplatelet glycoproteins in children with ITP. Thirty eight children, who were hospitalized with clinical signs of ITP in Mofid Children Hospital (Tehran, Iran) during 18 months, went under our clinical studies in a research project. ELISA, Flow cytometry and MAIPA (Monoclonal Antibody Immobilization of Platelet Antigens) methods were employed to determine serum anti-platelet antibodies level. The anti-platelet antibodies level above mean + 3SD of control group was assumed as positive. The platelet counts ranged between 2 × 109/L and 100 × 109/L. Among the patients 63.5% of them were anti-platelet antibodies positive with ELISA method. Results of platelet lysate method showed that 51.7% of patients had antibodies against platelet antigens. Antibody against platelet GPIIb/IIIa, GPIb/IX and GPIa/IIa using MAIPA method were 48%, 54% and 25% respectively. In flow cytometry 62% of patients showed anti-platelet antibodies. The comparison of three methods shows that since MAIPA is the specific method for the detection of very small amount of antibody against glycoprotein antigens, it has the advantage of differentiating between immune and non-immune thrombocytopenia. PMID:25755908

  5. Evaluation of two new fluorochromes, TOTO and YOYO, for DNA content analysis in cells and chromosomes by flow cytometry

    SciTech Connect

    Hirons, G.T.; Crissman, H.A. )

    1993-01-01

    The fluorochromes TOTO and YOYO were evaluated for their effectiveness in staining for DNA content analysis by flow cytometry (FCM). The dyes are dimers of thiazole orange (TO) and yellow oxazole (YO), respectively (Molecular Probes, Eugene, OR), and both have a very high quantum efficiency. Spectrofluorometric analysis showed that TOTO and YOYO had little fluorescence until bound to DNA or RNA. YOYO, the brighter of the two dyes, had an emission peak at [approximately]510 nm and TOTO at [approximately]530 nm. Analysis by flow cytometry indicated that cells stained with either dye at a concentration of [approximately]4.0 [mu]M could be preferentially excited at either 457 or 488 nm. Unfixed nuclei and fixed cells both treated with RNase, stained with either TOTO or YOYO, and analyzed by FCM yielded coefficients of variation (CV) comparable to CVs obtained for the same samples stained with mithramycin (MI) when excited at 457 nm and propidium iodide (PI) when excited at 488 nm. Both TOTO and YOYO are also being evaluated for their effectiveness in staining Chinese hamster embryo chromosomes; these results are being compared with results obtained with PI stained chromosomes.

  6. Vesicles tethered to microbubbles by hybridized DNA oligonucleotides: Flow cytometry analysis of this new drug delivery vehicle design

    PubMed Central

    Lozano, Monica M.; Starkel, Cambrie D.; Longo, Marjorie L.

    2010-01-01

    Hybridization of complementary lipid-linked DNA oligonucleotides was used to tether small unilamellar vesicles (SUVs) to the lipid monolayer shells of air-microbubbles, a new attachment design for a drug delivery vehicle to be used in tandem with ultrasound imaging. Flow cytometry was used and a novel analysis was developed, based upon light scattering and fluorescence intensity, to quantify the fraction of microbubbles of chosen size-ranges with oligonucleotide-tethered fluorescently labeled SUVs. Fluorescence microscopy was used to verify that our methodology results in successful high-density SUV tethering to a similar fraction of the microbubbles when compared to the flow cytometry statistics. The fraction of successful tetherings increased with the concentration of the complementary lipid-linked oligonucleotide as expected and decreased with the time that microbubbles were incubated with SUVs, which was not expected. Also unexpected, a large fraction of microbubbles had only background fluorescence levels while a much smaller fraction (at most one-eighth, for the shortest incubation and highest concentration of lipid-linked oligonucleotide) had oligonucleotide-tethered fluorescently labeled SUVs and, according to our fluorescence microscopy, that small fraction was densely covered with SUVs. Ejection of the lipid-linked oligonucleotide during high surface pressure compression of the monolayer shells of actively shrinking microbubbles subjected to the Laplace overpressure is speculated as a qualitatively explanation for the statistics. PMID:20229988

  7. A novel flow cytometry-based tool for determining the efficiency of human cytomegalovirus infection in THP-1 derived macrophages.

    PubMed

    Li, Huifen; Mao, Genxiang; Carlson, Joshua; Leng, Sean X

    2015-09-01

    Human cytomegalovirus (hCMV) is a ubiquitous pathogen that causes congenital infection and severe infections in immunocompromised patients. Chronic hCMV infection may also play an important role in immunosenescence and adverse health outcomes in older adults. THP-1, a human monocytic cell line and its derived macrophages serve as a useful cell culture model for mechanistic studies of hCMV infection and its underlying biology. A major methodological challenge is the lack of a quick and reliable tool to accurately determine the efficiency of hCMV infection in THP-1 derived macrophages. In this study, we developed a flow cytometry based method using commercially available monoclonal antibody (MAb) against hCMV immediate early (IE) antigen that can accurately determine infection efficiency. We used 0.5% formaldehyde for fixation, 90% methanol for permeabilization, and incubation with FITC conjugated MAb at 37°C. The method was tested by hCMV infection with laboratory Towne strain in the presence or absence of hydrocortisone. It was also compared with the routine flow cytometry protocol using Cytofix/Cytoperm solution and with immunofluorescence. The results indicate that this new method is reliable and time saving for accurate determination of infection efficiency. It may facilitate further investigations into the underlying biological mechanisms of hCMV infection. PMID:25958130

  8. Flow cytometry analysis of hormone receptors on human peripheral blood mononuclear cells to identify stress-induced neuroendocrine effects

    NASA Technical Reports Server (NTRS)

    Meehan, R. T.

    1986-01-01

    Understanding the role of circulating peptide hormones in the pathogenesis of space-flight induced disorders would be greatly facilitated by a method which monitors chronic levels of hormones and their effects upon in vivo cell physiology. Single and simultaneous multiparameter flow cytometry analysis was employed to identify subpopulations of mononuclear cells bearing receptors for ACTH, Endorphin, and Somatomedin-C using monoclonal antibodies and monospecific antisera with indirect immunofluorescence. Blood samples were obtained from normal donors and subjects participating in decompression chamber studies (acute stress), medical student academic examination (chronic stress), and a drug study (Dexamethasone). Preliminary results indicate most ACTH and Endorphin receptor positive cells are monocytes and B-cells, exhibit little diurnal variation but the relative percentages of receptor positive cells are influenced by exposure to various stressors and ACTH inhibition. This study demonstrates the capability of flow cytometry analysis to study cell surface hormone receptor regulation which should allow insight into neuroendocrine modulation of the immune and other cellular systems during exposure to stress or microgravity.

  9. Comparative study of three analysis methods (TTGE, flow cytometry and HPLC) for xenobiotic impact assessment on phytoplankton communities.

    PubMed

    Stachowski-Haberkorn, Sabine; Quiniou, Louis; Beker, Beatriz; Haberkorn, Hansy; Marie, Dominique; de la Broise, Denis

    2009-04-01

    The impacts of the fungicide Opus (epoxiconazole) on marine phytoplankton communities were assessed in a 12-day field experiment using in situ microcosms maintained underwater at 6 m depth. Three community analysis methods were compared for their sensitivity threshold in fungicide impact detection. When phytoplankton communities were exposed to 1 microg l(-1) of epoxiconazole, no effects could be demonstrated using TTGE (Temporal Temperature Gradient gel Electrophoresis), flow cytometry or HPLC. When exposed to 10 microg l(-1), TTGE fingerprints from PCR amplified 18S rDNA of communities exhibited significant differences compared with controls (ANOSIM, P = 0.028). Neither flow cytometry counts, nor HPLC pigment profiles allowed to show significant differences in microcosms exposed to 10 microg l(-1) of epoxiconazole. When exposed to 100 microg l(-1), all three methods allowed to detect significant differences in treated microcosms, as compared to controls. The TTGE analysis appears in this study as the most sensitive method for fungicide impact assessment on eukaryote microbial communities. PMID:19096934

  10. Evaluation of Ultrasound-Induced Damage to Escherichia coli and Staphylococcus aureus by Flow Cytometry and Transmission Electron Microscopy.

    PubMed

    Li, Jiao; Ahn, Juhee; Liu, Donghong; Chen, Shiguo; Ye, Xingqian; Ding, Tian

    2016-03-01

    As a nonthermal sterilization technique, ultrasound has attracted great interest in the field of food preservation. In this study, flow cytometry and transmission electron microscopy were employed to investigate ultrasound-induced damage to Escherichia coli and Staphylococcus aureus. For flow cytometry studies, single staining with propidium iodide (PI) or carboxyfluorescein diacetate (cFDA) revealed that ultrasound treatment caused cell death by compromising membrane integrity, inactivating intracellular esterases, and inhibiting metabolic performance. The results showed that ultrasound damage was independent of initial bacterial concentrations, while the mechanism of cellular damage differed according to the bacterial species. For the Gram-negative bacterium E. coli, ultrasound worked first on the outer membrane rather than the cytoplasmic membrane. Based on the double-staining results, we inferred that ultrasound treatment might be an all-or-nothing process: cells ruptured and disintegrated by ultrasound cannot be revived, which can be considered an advantage of ultrasound over other nonthermal techniques. Transmission electron microscopy studies revealed that the mechanism of ultrasound-induced damage was multitarget inactivation, involving the cell wall, cytoplasmic membrane, and inner structure. Understanding of the irreversible antibacterial action of ultrasound has great significance for its further utilization in the food industry. PMID:26746712

  11. Detection of paralytic shellfish toxins by a solid-phase inhibition immunoassay using a microsphere-flow cytometry system.

    PubMed

    Fraga, María; Vilariño, Natalia; Louzao, M Carmen; Campbell, Katrina; Elliott, Christopher T; Kawatsu, Kentaro; Vieytes, Mercedes R; Botana, Luis M

    2012-05-15

    Paralytic shellfish poisoning is a toxic syndrome described in humans following the ingestion of seafood contaminated with saxitoxin and/or its derivatives. The presence of these toxins in shellfish is considered an important health threat and their levels in seafood destined to human consumption are regulated in many countries, as well as the levels of other chemically unrelated toxins. We studied the feasibility of immunodetection of saxitoxin and its analogs using a solid-phase microsphere assay coupled to flow cytometry detection in a Luminex 200 system. The technique consists of a competition assay where the toxins in solution compete with bead-bound saxitoxin for binding to an antigonyautoxin 2/3 monoclonal antibody (GT-13A). The assay allowed the detection of saxitoxin both in buffer and mussel extracts in the range of 2.2-19.7 ng/mL (IC(20)-IC(80)). Moreover, the assay cross-reactivity with other toxins of the group is similar to previously published immunoassays, with adequate detection of most analogs except N-1 hydroxy analogs. The recovery rate of the assay for saxitoxin was close to 100%. This microsphere-based immunoassay is suitable to be used as a screening method, detecting saxitoxin from 260 to 2360 μg/kg. This microsphere/flow cytometry system provided similar sensitivities to previously published immunoassays and provides a solid background for the development of easy, flexible multiplexing of toxin detection in one sample. PMID:22500610

  12. Evaluating the effects of allelochemical ferulic acid on Microcystis aeruginosa by pulse-amplitude-modulated (PAM) fluorometry and flow cytometry.

    PubMed

    Wang, Rui; Hua, Ming; Yu, Yang; Zhang, Min; Xian, Qi-Ming; Yin, Da-Qiang

    2016-03-01

    We investigated the effects of allelochemical ferulic acid (FA) on a series of physiological and biochemical processes of blue-green algae Microcystis aeruginosa, in order to find sensitive diagnostic variables for allelopathic effects. Algal cell density was significantly suppressed by FA (0.31-5.17 mM) only after 48 h exposure. Inhibitions of photosynthetic parameters (F(v)/F(m) and F(v)'/F(m)') occurred more rapidly than cell growth, and the stimulation of non-photochemical quenching was observed as a feed-back mechanisms induced by photosystem II blockage, determining by PAM fluorometry. Inhibitions on esterase activity, membrane potential and integrity, as well as disturbance on cell size, were all detected by flow cytometry with specific fluorescent markers, although exhibiting varied sensitivities. Membrane potential and esterase activity were identified as the most sensitive parameters (with relatively lower EC50 values), and responded more rapidly (significantly inhibited only after 8 h exposure) than photosynthetic parameters and cell growth, thus may be the primary responses of cyanobacteria to FA exposure. The use of PAM fluorometry and flow cytometry for rapid assessment of those sensitive variables may contribute to future mechanistic studies of allolepathic effects on phytoplankton. PMID:26766364

  13. Imaging flow cytometry for automated detection of hypoxia-induced erythrocyte shape change in sickle cell disease.

    PubMed

    van Beers, Eduard J; Samsel, Leigh; Mendelsohn, Laurel; Saiyed, Rehan; Fertrin, Kleber Y; Brantner, Christine A; Daniels, Mathew P; Nichols, James; McCoy, J Philip; Kato, Gregory J

    2014-06-01

    In preclinical and early phase pharmacologic trials in sickle cell disease, the percentage of sickled erythrocytes after deoxygenation, an ex vivo functional sickling assay, has been used as a measure of a patient's disease outcome. We developed a new sickle imaging flow cytometry assay (SIFCA) and investigated its application. To perform the SIFCA, peripheral blood was diluted, deoxygenated (2% oxygen) for 2 hr, fixed, and analyzed using imaging flow cytometry. We developed a software algorithm that correctly classified investigator tagged "sickled" and "normal" erythrocyte morphology with a sensitivity of 100% and a specificity of 99.1%. The percentage of sickled cells as measured by SIFCA correlated strongly with the percentage of sickle cell anemia blood in experimentally admixed samples (R = 0.98, P ≤ 0.001), negatively with fetal hemoglobin (HbF) levels (R = -0.558, P = 0.027), negatively with pH (R = -0.688, P = 0.026), negatively with pretreatment with the antisickling agent, Aes-103 (5-hydroxymethyl-2-furfural) (R = -0.766, P = 0.002), and positively with the presence of long intracellular fibers as visualized by transmission electron microscopy (R = 0.799, P = 0.002). This study shows proof of principle that the automated, operator-independent SIFCA is associated with predictable physiologic and clinical parameters and is altered by the putative antisickling agent, Aes-103. SIFCA is a new method that may be useful in sickle cell drug development. PMID:24585634

  14. Hierarchical Bayesian mixture modelling for antigen-specific T-cell subtyping in combinatorially encoded flow cytometry studies

    PubMed Central

    Lin, Lin; Chan, Cliburn; Hadrup, Sine R.; Froesig, Thomas M.; Wang, Quanli; West, Mike

    2014-01-01

    Novel uses of automated flow cytometry technology for measuring levels of protein markers on thousands to millions of cells are promoting increasing need for relevant, customized Bayesian mixture modelling approaches in many areas of biomedical research and application. In studies of immune profiling in many biological areas, traditional flow cytometry measures relative levels of abundance of marker proteins using fluorescently labeled tags that identify specific markers by a single-color. One specific and important recent development in this area is the use of combinatorial marker assays in which each marker is targeted with a probe that is labeled with two or more fluorescent tags. The use of several colors enables the identification of, in principle, combinatorially increasingly numbers of subtypes of cells, each identified by a subset of colors. This represents a major advance in the ability to characterize variation in immune responses involving larger numbers of functionally differentiated cell subtypes. We describe novel classes of Markov chain Monte Carlo methods for model fitting that exploit distributed GPU (graphics processing unit) implementation. We discuss issues of cellular subtype identification in this novel, general model framework, and provide a detailed example using simulated data. We then describe application to a data set from an experimental study of antigen-specific T-cell subtyping using combinatorially encoded assays in human blood samples. Summary comments discuss broader questions in applications in immunology, and aspects of statistical computation. PMID:23629459

  15. Multiple physiological states of a Pseudomonas fluorescens DR54 biocontrol inoculant monitored by a new flow cytometry protocol.

    PubMed

    Nielsen, Tommy Harder; Sjøholm, Ole Rüdiger; Sørensen, Jan

    2009-03-01

    A new fluorescence staining and flow cytometry protocol was developed to monitor several physiological states in biocontrol strain Pseudomonas fluorescens DR54 during storage survival in a stationary-phase culture, preparation of clay carrier for seed formulation, and establishment in a sugar beet spermosphere. The high load of impurities in the environmental samples was dealt with by adding a density-gradient purification step to the staining protocol. Staining by SYBR Green, combined with either propidium iodide or ethidium bromide (EB)+DiBAC((4))3, was used to quantify the total cell population and further divide this population into: (1) intact cells with an unaffected membrane and energy metabolism. (2) De-energized cells unable to maintain membrane export (EB exclusion). (3) Depolarized cells unable to maintain membrane potential. (4) Permeabilized cells with a damaged membrane. During both stationary-phase storage and steps for preparation of formulation carrier, loss of intact P. fluorescens DR54 cells was quantitatively accounted for by depolarized and permeabilized states. Surviving inoculum cells subsequently proliferated on the germinating seeds, but with a surprisingly high abundance of de-energized cells. The new protocol is the first for flow cytometry to include a recording of both intact and several subpopulations of physiologically affected bacteria in complex, environmental samples with high impurity loads. PMID:19159420

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

    PubMed

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

    2016-07-01

    The effect of the culture medium pH (3.5-6.0) on the carotenoid and lipid (as fatty acids) production by the yeast Rhodosporidium toruloides NCYC 921 was studied. Flow cytometry was used to evaluate the yeast's physiological response to different culture medium pH values. The yeast biomass concentration and lipid content were maxima at pH 4.0 (5.90 g/L and 21.85 % w/w, respectively), while the maximum carotenoid content (63.37 μg/g) was obtained at pH 5.0. At the exponential phase, the yeast cell size and internal complexity were similar, at different medium pH. At the stationary phase, the yeast cell size and internal complexity decreased as the medium pH increased. At the exponential phase, the proportion of cells with polarized membranes was always high (>80 %) but at the stationary phase, the proportion of yeast cells with depolarized membranes was dominant (>65 %) and increased with the medium pH increase. The results here reported may contribute for yeast bioprocesses optimization. For the first time, multiparameter flow cytometry was used to evaluate the impact of medium pH changes on the yeast cell physiological status, specifically on the yeast membrane potential, membrane integrity, cell size and internal complexity. PMID:26945576

  17. Acridine Orange and Flow Cytometry: Which Is Better to Measure the Effect of Varicocele on Sperm DNA Integrity?

    PubMed Central

    Mohammed, Essam-Elden M.; Mosad, Eman; Zahran, Asmaa M.; Hameed, Diaa A.; Taha, Emad A.; Mohamed, Mohamed A.

    2015-01-01

    We evaluated the effect of varicocelectomy on semen parameters and levels of sperm DNA damage in infertile men. A total of 75 infertile men with varicocele and 40 fertile men (controls) were included in this study. Semen analysis and sperm DNA damage expressed as the DNA fragmentation index using acridine orange staining and chromatin condensation test by flow cytometry were assessed before and 6 months after varicocelectomy. The patients were also followed up for 1 year for pregnancy outcome. Semen parameters were significantly lower in varicocele patients compared to controls (P < 0.05). Mean percentages of sperm DNA fragmentation and sperm DNA chromatin condensation in patients were significantly higher than those in controls (P < 0.05). After varicocelectomy, sperm DNA fragmentation improved significantly, whereas sperm chromatin condensation was not significantly changed. In 15 out of 75 varicocele patients, clinical pregnancy was diagnosed; those with positive pregnancy outcome had significant improvement in sperm count, progressive sperm motility, and sperm DNA fragmentation, but there was no significant difference in sperm DNA condensation compared to negative pregnancy outcome patients. We concluded from this study that acridine orange stain is more reliable method than flow cytometry in the evaluation of sperm DNA integrity after varicocelectomy. PMID:26681938

  18. Tumour growth rates in squamous carcinoma of the head and neck measured by in vivo bromodeoxyuridine incorporation and flow cytometry.

    PubMed Central

    Forster, G.; Cooke, T. G.; Cooke, L. D.; Stanton, P. D.; Bowie, G.; Stell, P. M.

    1992-01-01

    The cell kinetics of 82 squamous cell carcinomas of the head and neck were studied by in vivo administration of the thymidine analogue, bromodeoxyuridine (BrdUrd). Ploidy, BrdUrd labelling index (LI), duration of S-phase (Ts), potential doubling time (Tpot) and S-phase fraction (SPF) were measured by flow cytometry on 50 microns paraffin embedded sections. The range of values obtained compared well with other in vivo cell kinetic studies of head and neck cancer. Aneuploid tumours had a significantly higher BrdUrd labelling index and SPF, and a short Tpot than diploid tumours. To validate the use of 50 microns sections for measuring cell kinetic parameters by flow cytometry a comparison of values obtained by 50 microns sections and small blocks of tissue was made. No significant difference was found between the two methods. Reproducibility of values between two consecutive thick sections was also good. We conclude that reproducible cell kinetic measurements can be made in tumour samples using 50 microns sections of BrdUrd labelled tissue. PMID:1586597

  19. Long-term effects of triethylenemelamine exposure on mouse testis cells and sperm chromatin structure assayed by flow cytometry

    SciTech Connect

    Evenson, D.P.; Baer, R.K.; Jost, L.K. )

    1989-01-01

    The toxic and potentially mutagenic actions of triethylenemelamine (TEM) on mouse body and testis weights, testicular cell kinetics, sperm production, sperm head morphology, and sperm chromatin structure were assessed in two experiments. The first experiment examined effects of four dose levels of TEM, assayed 1, 4, or 10 wk after toxic exposure. In the second study, effects from five dosage levels were measured at 1, 4, and 10 wk, and the highest dosage level was evaluated over 44 wk. TEM produced an expected dose related loss of spermatogenic activity and subsequent recovery as determined by dual-parameter (DNA, RNA) flow cytometry (FCM) measurements of testicular cells. Both testicular weights and caudal sperm reserves remained generally below controls after 44 wk recovery following exposure to the highest dosage. Chromatin structure alterations, defined as increased susceptibility to DNA denaturation in situ, and sperm head morphology were highly correlated with dose and with each other. Sperm head morphology and sperm chromatic structure remained abnormal at 44 wk for the 1.0 mg/kg TEM dosage, suggesting that the abnormalities, present long after the initial toxic response, may be a result of mutation. This study demonstrates that flow cytometry provides a unique, rapid, and efficient means to measure effects of reproductive toxins and potential mutagens.

  20. 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. PMID:23062200

  1. 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

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

    PubMed

    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

  3. 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

  4. Quantum dots thermal stability improves simultaneous phenotype-specific telomere length measurement by FISH-flow cytometry.

    PubMed

    Kapoor, Veena; Hakim, Fran T; Rehman, Najibah; Gress, Ronald E; Telford, William G

    2009-05-15

    Telomere length analysis has been greatly simplified by the quantitative flow cytometry technique FISH-flow. In this method, a fluorescein-labeled synthetic oligonucleotide complementary to the telomere terminal repeat sequence is hybridized to the telomere sequence and the resulting fluorescence measured by flow cytometry. This technique has supplanted the traditional laborious Southern blot telomere length measurement techniques in many laboratories, and allows single cell analysis of telomere length in high-throughput sample formats. Nevertheless, the harsh conditions required for telomere probe annealing (82 degrees C) has made it difficult to successfully combine this technique with simultaneous immunolabeling. Most traditional organic fluorescent probes (i.e. fluorescein, phycoerythrin, etc.) have limited thermal stability and do not survive the high temperature annealing process, despite efforts to covalently crosslink the antigen-antibody-fluorophore complex. This loss of probe fluorescence has made it difficult to measure FISH-flow in complex lymphocyte populations, and has generally forced investigators to use fluorescent-activated cell sorting to pre-separate their populations, a laborious technique that requires prohibitively large numbers of cells. In this study, we have substituted quantum dots (nanoparticles) for traditional fluorophores in FISH-flow. Quantum dots were demonstrated to possess much greater thermal stability than traditional low molecular weight and phycobiliprotein fluorophores. Quantum dot antibody conjugates directed against monocyte and T cell antigens were found to retain most of their fluorescence following the high temperature annealing step, allowing simultaneous fluorescent immunophenotyping and telomere length measurement. Since quantum dots have very narrow emission bandwidths, we were able to analyze multiple quantum dot antibody conjugates (Qdot 605, 655 and 705) simultaneously with FISH-flow measurement to assess the age

  5. 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. PMID:27251036

  6. Rapid in vitro biocompatibility assay of endovascular stents by flow cytometry using platelet activation and platelet-leukocyte aggregation.

    PubMed

    Tárnok, A; Mahnke, A; Müller, M; Zotz, R J

    1999-02-15

    Clinical studies suggest that stent design and surface texture are responsible for differences in biocompatibility of metallic endovascular stents. A simple in vitro experimental setup was established to test stent-induced degree of platelet and leukocyte activation and platelet-leukocyte aggregation by flow cytometry. Heparin-coated tantalum stents and gold-coated and uncoated stainless steel stents were tested. Stents were implanted into silicone tubes and exposed to blood from healthy volunteers. Platelet and leukocyte activation and percentage of leukocyte-platelet aggregates were determined in a whole-blood assay by subsequent staining for activation-associated antigens (CD41a, CD42b, CD62p, and fibrinogen binding) and leukocyte antigens (CD14 and CD45) and flow cytometric analysis. Blood taken directly after venous puncture or exposed to the silicone tube alone was used as negative controls. Positive control was in vitro stimulation with thrombin receptor activating peptide (TRAP-6). Low degree of platelet activation and significant increase in monocyte- and neutrophil-platelet aggregation were observed in blood exposed to stents (P < 0.05). In addition, leukocyte activation was induced as measured by increased CD45 and CD14 expression. Heparin coated stents continuously induced less platelet activation and leukocyte-platelet aggregation than uncoated stainless steel stents of the same length and shorter stents of the same structure. Stent surface coating and texture plays a role in platelet and leukocyte activation and leukocyte-platelet aggregation. Using this simple in vitro assay and whole blood and flow cytometry, it seems possible to differentiate stents by their potency to activate platelets and/or leukocytes. This assay could be applied for improving the biocompatibility of coronary stents. PMID:10088974

  7. Phytoplankton distribution during two contrasted summers in a Mediterranean harbour: combining automated submersible flow cytometry with conventional techniques.

    PubMed

    Thyssen, Melilotus; Beker, Beatriz; Ediger, Dilek; Yilmaz, Doruk; Garcia, Nicole; Denis, Michel

    2011-02-01

    Automated in situ flow cytometry, high-pressure liquid chromatography (HPLC), optical microscopy and fluorometry were combined to monitor phytoplankton over two summer periods (2005 and 2006). In 2006, temperature was higher and nutrients lower than in 2005, generating differences in the phytoplankton assemblages (i.e., abundance and structure). Pigment-size classes based on daily HPLC analysis provided evidence for higher proportions of picoplankton and nanoplankton with higher biomass in 2005 and a dominance of microplankton with lower biomass in 2006, the latter with lower specific diversity, as evidenced by weekly microscopy analyses. Total chlorophyll a estimations from fluorometry measurements recorded every 30 min were higher in 2005 than in 2006, as for the HPLC chlorophyll a concentrations. An automated in situ flow cytometer (Thyssen et al., J Plankton Res 30(9):1027-1040, 2008a) sampled seawater every 30 min. Data analysis yielded the resolution of seven clusters based on light scatter and fluorescence. In 2006, an increase in abundance of the largest cells was observed, confirming pigment and microscopy data. The results suggest that the ecosystem was on a constant renewing process in summer 2005 due to a strong wind event and on a highly productive and recycling way in summer 2006 due to stratification of the upper water layer. Automated submersible flow cytometry confirms to be a powerful tool providing high-resolution data by monitoring phytoplankton at the single cell level. This technology gives access to the shape of the light scatter and fluorescence signals generated by each cell passing through a laser beam and that are linked to size, structure and pigment content of the target cell. When combined with conventional techniques, it further improves our understanding of phytoplankton assemblages. PMID:20221799

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

    SciTech Connect

    Universities Space Research Association; Chappell, Lori J.; Whalen, Mary K.; Gurai, Sheena; Ponomarev, Artem; Cucinotta, Francis A.; Pluth, Janice M.

    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 dose 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.

  9. In Vivo Ultra-Fast Photoacoustic Flow Cytometry of Circulating Human Melanoma Cells Using Near-Ingrared High-Pulse Rate Lasers

    PubMed Central

    Nedosekin, Dmitry A.; Sarimollaoglu, Mustafa; Ye, John; Galanzha, Ekaterina I.; Zharov, Vladimir P.

    2011-01-01

    The circulating tumor cells (CTCs) appear to be a marker of metastasis development, especially, for highly aggressive and epidemically growing melanoma malignancy that is often metastatic at early stages. Recently, we introduced in vivo photoacoustic (PA) flow cytometry (PAFC) for label-free detection of mouse B16F10 CTCs in melanoma-bearing mice using melanin as an intrinsic marker. Here, we significantly improve the speed of PAFC by using a high pulse repetition rate laser operating at 820 and 1064 nm wavelengths. This platform was used in preclinical studies for label-free PA detection of low pigmented human CTCs. Demonstrated label-free PAFC detection, low level of background signals, and favorable safety standards for near infrared irradiation suggest that a fiber laser operating at 1064 nm at pulse repetition rates up to 0.5 MHz could be a promising source for portable clinical PAFC devices. The possible applications can include early diagnosis of melanoma at the parallel progression of primary tumor and CTCs, detection of cancer recurrence, residual disease, and real-time monitoring of therapy efficiency by counting CTCs before, during and after therapeutic intervention. Herewith, we also address sensitivity of label-free PAFC melanoma CTCs detection and introduce in vivo CTCs targeting by magnetic nanoparticles conjugated with specific antibody and magnetic cells enrichment. PMID:21786417

  10. In vivo ultra-fast photoacoustic flow cytometry of circulating human melanoma cells using near-infrared high-pulse rate lasers.

    PubMed

    Nedosekin, Dmitry A; Sarimollaoglu, Mustafa; Ye, Jian-Hui; Galanzha, Ekaterina I; Zharov, Vladimir P

    2011-10-01

    The circulating tumor cells (CTCs) appear to be a marker of metastasis development, especially, for highly aggressive and epidemically growing melanoma malignancy that is often metastatic at early stages. Recently, we introduced in vivo photoacoustic (PA) flow cytometry (PAFC) for label-free detection of mouse B16F10 CTCs in melanoma-bearing mice using melanin as an intrinsic marker. Here, we significantly improve the speed of PAFC by using a high-pulse repetition rate laser operating at 820 and 1064 nm wavelengths. This platform was used in preclinical studies for label-free PA detection of low-pigmented human CTCs. Demonstrated label-free PAFC detection, low level of background signals, and favorable safety standards for near-infrared irradiation suggest that a fiber laser operating at 1064 nm at pulse repetition rates up to 0.5 MHz could be a promising source for portable clinical PAFC devices. The possible applications can include early diagnosis of melanoma at the parallel progression of primary tumor and CTCs, detection of cancer recurrence, residual disease and real-time monitoring of therapy efficiency by counting CTCs before, during, and after therapeutic intervention. Herewith, we also address sensitivity of label-free detection of melanoma CTCs and introduce in vivo CTC targeting by magnetic nanoparticles conjugated with specific antibody and magnetic cells enrichment. PMID:21786417

  11. Nucleic acid fluorochromes and flow cytometry prove useful in assessing the effect of chlorination on drinking water bacteria.

    PubMed

    Phe, Meng-Huot; Dossot, Manuel; Guilloteau, Hélène; Block, Jean-Claude

    2005-09-01

    Flow cytometry (FCM), combined with staining using two fluorochromes (propidium iodide, PI, or SYBR Green II RNA gel stain, SYBR-II), was used to asses