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Sample records for flow cytometry mutation

  1. Hypoxia/Reoxygenation-Induced Mutations in Mammalian Cells Detected by the Flow Cytometry Mutation Assay and Characterized by Mutant Spectrum

    PubMed Central

    Keysar, Stephen B.; Trncic, Nadira; LaRue, Susan M.; Fox, Michael H.

    2010-01-01

    Under hypoxic conditions, cells are more resistant to cell killing by ionizing radiation by a factor of 2.5 to 3, potentially compromising the efficacy of radiotherapy. It has been shown recently that hypoxic conditions alone are sufficient to generate mutations in vitro and in vivo, likely due to the creation of reactive oxygen species (ROS) and a decrease in mismatch and homologous recombination DNA repair activity. These factors are known precursors to the onset of genetic instability and poor prognosis. We have previously characterized the flow cytometry mutation assay and its sensitivity to detect significant mutant fractions induced by genotoxic agents that are not detected by other mammalian assays. Here we measure the mutant fraction induced by hypoxia. CHO AL cells cultured at <0.1% O2 for 24 h generated a significant mutant fraction of 120 × 10−5 and had growth kinetics and survival characteristics similar to those obtained with other mutagens. We investigated the role of ROS by treating cells with the radical scavenger DMSO, which significantly reduced hypoxia toxicity and mutagenesis. Single cells were sorted from the mutant population, and the resulting clonal populations were stained for five antigens encoded by genes found along chromosome 11 to generate mutant spectra. The mutations were primarily large deletions, similar to those in background mutants, but the frequency was higher. We have demonstrated that hypoxic conditions alone are sufficient to generate mutations in mammalian cells in culture and that the spectrum of mutations is similar to background mutations. PMID:20041756

  2. Lasers in flow cytometry.

    PubMed

    Telford, William G

    2011-01-01

    Laser technology has advanced tremendously since the first gas lasers were incorporated into early flow cytometers. Gas lasers have been largely replaced by solid-state laser technology, making virtually any desirable visible light wavelength available for flow cytometry. Multiwavelength, white light, and wavelength tunable lasers are poised to enhance our analytical capabilities even further. In this chapter, I summarize the role that lasers play in cytometry, and the practical characteristics that make a laser appropriate for flow cytometry. I then review the latest single wavelength lasers available for flow cytometry, and how they can be used to excite the ever-expanding array of available fluorochromes. Finally, I review the contribution and potential of the latest tunable laser technology to flow cytometry, and show several examples of these novel sources integrated into production instruments. Technical details and critical parameters for successful application of these lasers for biomedical analysis are covered in depth.

  3. 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.

  4. 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.

  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. Early and rapid detection of X-linked lymphoproliferative syndrome with SH2D1A mutations by flow cytometry.

    PubMed

    Zhao, Meina; Kanegane, Hirokazu; Kobayashi, Chie; Nakazawa, Yozo; Ishii, Eizaburo; Kasai, Mikio; Terui, Kiminori; Gocho, Yoshihiro; Imai, Kohsuke; Kiyasu, Junichi; Nonoyama, Shigeaki; Miyawaki, Toshio

    2011-01-01

    X-linked lymphoproliferative syndrome (XLP) is a rare immunodeficiency with extreme vulnerability to Epstein-Barr virus (EBV) infection. It presents with fatal infectious mononucleosis, lymphoproliferative disorder, or dysgammaglobulinemia. The majority of affected males have mutations in the SH2D1A/SLAM-associated protein (SAP) gene. We previously generated an antihuman SAP monoclonal antibody (KST-3) for a flow cytometric assay and described the activation of T cells to be necessary for the flow cytometric assessment of the SAP expression using an FITC-conjugated secondary antibody. Between 2005 and 2008, we recruited 23 male patients with suspected XLP, including mainly EBV-associated hemophagocytic lymphohistiocytosis (HLH), and attempted to evaluate SAP expression in fresh lymphoid cells using Alexa Fluor 488-conjugated secondary antibody instead of an FITC-conjugated one. The method demonstrated that SAP was intensely expressed in CD8(+) T cells and NK cells in normal fresh blood samples, thus suggesting the possible rapid identification of individuals with SAP deficiency. SH2D1A mutations were identified in six patients with SAP deficiency, but not in patients with normal SAP expression. The outcomes from this trial were verified by a flow cytometric assay using KST-3 and Alexa Fluor 488 secondary antibody. Based on the demonstration SAP deficiency in patients with suspected XLP, including mainly EBV-associated HLH, this approach could serve as a method for the early and rapid detection of patients with XLP-1. Copyright © 2010 International Clinical Cytometry Society.

  7. Basics of flow cytometry.

    PubMed

    Radcliff, G; Jaroszeski, M J

    1998-01-01

    In summary, a beginner requires fundamental knowledge about flow cytometric instrumentation in order to effectively use this technology. It is important to remember that flow cytometers are very complex instruments that are composed of four closely related systems. The fluidic system transports particles from a suspension through the cytometer for interrogation by an illumination system. The resulting light scattering and fluorescence is collected, filtered, and converted into electrical signals by the optical and electronics system. The data storage and computer control system saves acquired data and is also the user interface for controlling most instrument functions. These four systems provide a very unique and powerful analytical tool for researchers and clinicians. This is because they analyze the properties of individual particles, and thousands of particles can be analyzed in a matter of seconds. Thus, data for a flow cytometric sample are a collection of many measurements instead of a single bulk measurement. Basic knowledge of instrumentation is a tremendous aid to designing experiments that can be successfully analyzed using flow cytometry. For example, it is important to know the emission wavelength of the laser in the instrument that will be used for analysis. This wavelength is critical knowledge for selecting probes. It is also important to understand that a different range of wavelengths is detected for each fluorescent channel. This will aid selection of probes that are compatible with the flow cytometer. Understanding the complication that emission spectra overlap contributes to detection can be used to guide fluorochrome selections for multicolor analysis. All of these experiment design considerations that rely on knowledge of how flow cytometers work are a very practical and effective means of avoiding wasted time, energy, and costly reagents. Data analysis is a paramount issue in flow cytometry. Analysis includes interpreting as well as

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. Teaching phagocytosis using flow cytometry.

    PubMed

    Boothby, John T; Kibler, Ruthann; Rech, Sabine; Hicks, Robert

    2004-05-01

    Investigative microbiology on protists in a basic teaching laboratory environment is limited by student skill level, ease of microbial culture and manipulation, instrumentation, and time. The flow cytometer is gaining use as a mainstream instrument in research and clinical laboratories, but has had minimal application in teaching laboratories. Although the cost of a flow cytometer is currently prohibitive for many microbiology teaching environments and the number of trained instructors and teaching materials is limited, in many ways the flow cytometer is an ideal instrument for teaching basic microbiology. We report here on a laboratory module to study phagocytosis in Tetrahymena sp. using flow cytometry in a basic microbiology teaching laboratory. Students and instructors found the flow cytometry data analysis program, Paint-AGate(PRO-TM), to be very intuitive and easy to learn within a short period of time. Assessment of student learning about Tetrahymena sp., phagocytosis, flow cytometry, and investigative microbiology using an inquiry-based format demonstrated an overall positive response from students.

  14. Masks in imaging flow cytometry.

    PubMed

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

    2017-01-01

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

  15. A comparative study of detection of p53 mutations in human breast cancer by flow cytometry, single-strand conformation polymorphism and genomic sequencing.

    PubMed Central

    Chakravarty, G.; Redkar, A.; Mittra, I.

    1996-01-01

    The accuracy of immunodetection by dual parameter flow cytometry (FCM), polymerase chain reaction-mediated single strand conformation polymorphism (PCR-SSCP) and genomic sequencing to detect p53 mutations were compared. Analysis by the last two techniques was restricted to exons 5-8. Initially, 110 breast tumours were screened for p53 expression by FCM. Seventy (64%) of tumours were immunopositive. Fifteen highly immunopositive and 15 completely immunonegative tumours were selected for further analysis by PCR-SSCP and genomic sequencing. Eleven out of 15 immunopositive tumours were found to have mutation by PCR-SSCP. Genomic sequencing confirmed the presence of mutation in 10 of these 11 immunopositive tumours. Therefore, four immunopositive tumours failed to show mutation by SSCP and five by genomic sequencing. Of the 15 immunonegative tumours, one showed mutation by both PCR-SSCP and genomic sequencing and one tumour has undergone deletion of the p53 gene. Overall, immunoreactivity correlated with both PCR-SSCP and genomic sequencing in 80% of cases (24/30), and there was 96.5% (28/29) concordance between PCR-SSCP and genomic sequencing. We conclude that there is good concordance between mutations detected by PCR-SSCP and genomic sequencing, but immunochemical detection of p53 overexpression is not an absolute indicator of p53 gene mutation. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8883402

  16. Bacteria detection by flow cytometry.

    PubMed

    Karo, Oliver; Wahl, Alexandra; Nicol, Sven-Boris; Brachert, Julia; Lambrecht, Bernd; Spengler, Hans-Peter; Nauwelaers, Frans; Schmidt, Michael; Schneider, Christian K; Müller, Thomas H; Montag, Thomas

    2008-01-01

    Since bacterial infection of the recipient has become the most frequent infection risk in transfusion medicine, suitable methods for bacteria detection in blood components are of great interest. Platelet concentrates are currently the focus of attention, as they are stored under temperature conditions, which enable the multiplication of most bacteria species contaminating blood donations. Rapid methods for bacteria detection allow testing immediately before transfusion in a bed-side like manner. This approach would overcome the sampling error observed in early sampling combined with culturing of bacteria and would, at least, prevent the transfusion of highly contaminated blood components leading to acute septic shock or even death of the patient. Flow cytometry has been demonstrated to be a rapid and feasible approach for detection of bacteria in platelet concentrates. The general aim of the current study was to develop protocols for the application of this technique under routine conditions. The effect of improved test reagents on practicability and sensitivity of the method is evaluated. Furthermore, the implementation of fluorescent absolute count beads as an internal standard is demonstrated. A simplified pre-incubation procedure has been undertaken to diminish the detection limit in a pragmatic manner. Additionally, the application of bacteria detection by flow cytometry as a culture method is shown, i.e., transfer of samples from platelet concentrates into a satellite bag, incubation of the latter at 37 degrees C, and measuring the contaminating bacteria in a flow cytometer.

  17. Flow cytometry and laser scanning cytometry, a comparison of techniques.

    PubMed

    Mach, William J; Thimmesch, Amanda R; Orr, James A; Slusser, Joyce G; Pierce, Janet D

    2010-08-01

    Flow and laser scanning cytometry are used extensively in research and clinical settings. These techniques provide clinicians and scientists information about cell functioning in a variety of health and disease states. An in-depth knowledge and understanding of cytometry techniques can enhance interpretation of current research findings. Our goal with this review is to reacquaint clinicians and scientists with information concerning differences between flow and laser scanning cytometry by comparing their capabilities and applications. A Pubmed abstract search was conducted for articles on research, reviews and current texts relating to origins and use of flow and laser scanning cytometry. Attention was given to studies describing application of these techniques in the clinical setting. Both techniques exploit interactions between the physical properties of light. Data are immediately and automatically acquired; they are distinctly different. Flow cytometry provides valuable rapid information about a wide variety of cellular or particle characteristics. This technique does not provide the scanned high resolution image analysis needed for investigators to localize areas of interest within the cell for quantification. Flow cytometry requires that the sample contain a large amount disaggregated, single, suspended cells. Laser scanning cytometry is slide-based and does not require as large of a sample. The tissue sample is affixed to a slide allowing repeated sample analyses. These cytometry techniques are used in the clinical setting to understand pathophysiological derangements associated with many diseases; cardiovascular disease, diabetes, acute lung injury, hemorrhagic shock, surgery, cancer and Alzheimer's disease. Understanding the differences between FCM and LSCM can assist investigators in planning and design of their research or clinical testing. Researchers and clinicians optimize these technique capabilities with the cellular characteristics they wish to

  18. Inertial microfluidics for flow cytometry

    NASA Astrophysics Data System (ADS)

    Di Carlo, Dino

    2010-08-01

    Inertial components of the Navier-Stokes equations are usually not considered in microfluidic flows but have recently been shown to be of great practical use for continuous manipulation of particles and cells. After introducing the physical basis of the counter-intuitive self focusing of particles in a single inlet flow, I will discuss our current best focusing systems, and I will present results on using inertial focusing to create an extreme throughput flow cytometer for blood analysis. This system is an imaging cytometer implementation that can image 1 million focused blood cells per second, with the capability to increase to 20 million cells per second with appropriate wide-field of view imaging systems. The microfluidic device consists of 256 parallel high-aspect ratio microchannels in each of which two streams of focused cells assemble. These cells also form regular trains in the direction of flow such that cell coincidence is a rare occurrence, far below Poisson statistics suggest. Controlled inertially focused streams of particles are poised to provide next-generation filter-less filters and simplified flow cytometry instruments which ultimately may aid in water treatment environmental cleanup and cost-effective medical diagnostics.

  19. Flow cytometry: retrospective, fundamentals and recent instrumentation.

    PubMed

    Picot, Julien; Guerin, Coralie L; Le Van Kim, Caroline; Boulanger, Chantal M

    2012-03-01

    Flow cytometry is a complete technology given to biologists to study cellular populations with high precision. This technology elegantly combines sample dimension, data acquisition speed, precision and measurement multiplicity. Beyond the statistical aspect, flow cytometry offers the possibility to physically separate sub-populations. These performances come from the common endeavor of physicists, biophysicists, biologists and computer engineers, who succeeded, by providing new concepts, to bring flow cytometry to current maturity. The aim of this paper is to present a complete retrospective of the technique and remind flow cytometry fundamentals before focusing on recent commercial instrumentation.

  20. Flow cytometry: basic principles and applications.

    PubMed

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

    2017-03-01

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

  1. Data Standards for Flow Cytometry

    PubMed Central

    SPIDLEN, JOSEF; GENTLEMAN, ROBERT C.; HAALAND, PERRY D.; LANGILLE, MORGAN; MEUR, NOLWENN LE; OCHS, MICHAEL F.; SCHMITT, CHARLES; SMITH, CLAYTON A.; TREISTER, ADAM S.; BRINKMAN, RYAN R.

    2009-01-01

    Flow cytometry (FCM) is an analytical tool widely used for cancer and HIV/AIDS research, and treatment, stem cell manipulation and detecting microorganisms in environmental samples. Current data standards do not capture the full scope of FCM experiments and there is a demand for software tools that can assist in the exploration and analysis of large FCM datasets. We are implementing a standardized approach to capturing, analyzing, and disseminating FCM data that will facilitate both more complex analyses and analysis of datasets that could not previously be efficiently studied. Initial work has focused on developing a community-based guideline for recording and reporting the details of FCM experiments. Open source software tools that implement this standard are being created, with an emphasis on facilitating reproducible and extensible data analyses. As well, tools for electronic collaboration will assist the integrated access and comprehension of experiments to empower users to collaborate on FCM analyses. This coordinated, joint development of bioinformatics standards and software tools for FCM data analysis has the potential to greatly facilitate both basic and clinical research—impacting a notably diverse range of medical and environmental research areas. PMID:16901228

  2. 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.

  3. Imaging flow cytometry for phytoplankton analysis.

    PubMed

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

    2017-01-01

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

  4. 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.

  5. Multiphoton flow cytometry strategies and applications.

    PubMed

    Tkaczyk, Eric R; Tkaczyk, Alan H

    2011-10-01

    A handful of research teams around the world have recently begun to utilize multiphoton techniques in cytometry, especially for in vivo applications. These approaches offer similar enhancements to flow cytometry as the multiphoton phenomenon brought to the field of microscopy at the turn of the 20th century, with at least six advantages over single-photon excitation. Here, we review the published literature on multiphoton cytometry in vivo or in vitro from the initial experiments in 1999 to present. Multiphoton cytometry instrumentation set-ups vary from adapted multiphoton microscopy to a dedicated system, with laser pulse power and repetition rate serving as important variables. Two-beam geometry enables quantitation of cell size. Labeling strategies include conjugated fluorophore targeting, with folate and/or dendrimer platforms. With two-color measurement, ratiometric labeling is also possible, where one dye serves as a trigger to indicate the amount of excitation a cell receives, and another informs of cellular function. With two-color labeling, geometric fluorophore distribution proves important in theory and experiment for detection sensitivity curves and detected event intensity correlation. The main biological achievements to date using this young technology are reviewed, with emphasis on real-time monitoring of minute-by-minute and long-term cell dynamics as well as the clinically significant surveillance of circulating tumor cells. For this goal, minimally invasive two-photon flow cytometry with a fiber probe may overcome the primary issue of sample volume. The technique of multicolor, multiphoton flow cytometry greatly enhances the capabilities of flow cytometry to investigate the dynamics of circulating cells in cancer and other important diseases, and may in the future benefit from advances in microscopy such as super-resolution imaging, coherent control, and bioluminescence. Copyright © 2011 International Society for Advancement of Cytometry.

  6. 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.

  7. Uses of flow cytometry in virology.

    PubMed Central

    McSharry, J J

    1994-01-01

    This article reviews some of the published applications of flow cytometry for in vitro and in vivo detection and enumeration of virus-infected cells. Sample preparation, fixation, and permeabilization techniques for a number of virus-cell systems are evaluated. The use of flow cytometry for multiparameter analysis of virus-cell interactions for simian virus 40, herpes simplex viruses, human cytomegalovirus, and human immunodeficiency virus and its use for determining the effect of antiviral compounds on these virus-infected cells are reviewed. This is followed by a brief description of the use of flow cytometry for the analysis of several virus-infected cell systems, including blue tongue virus, hepatitis C virus, avian reticuloendotheliosis virus, African swine fever virus, woodchuck hepatitis virus, bovine viral diarrhea virus, feline leukemia virus, Epstein-Barr virus, Autographa californica nuclear polyhedrosis virus, and Friend murine leukemia virus. Finally, the use of flow cytometry for the rapid diagnosis of human cytomegalovirus and human immunodeficiency virus in peripheral blood cells of acutely infected patients and the use of this technology to monitor patients on antiviral therapy are reviewed. Future prospects for the rapid diagnosis of in vivo viral and bacterial infections by flow cytometry are discussed. Images PMID:7530594

  8. Flow cytometry applications in the food industry.

    PubMed

    Comas-Riu, Jaume; Rius, Núria

    2009-08-01

    Flow cytometry has become a valuable tool in food microbiology. By analysing large numbers of cells individually using light-scattering and fluorescence measurements, this technique reveals both cellular characteristics and the levels of cellular components. Flow cytometry has been developed to rapidly enumerate microorganisms; to distinguish between viable, metabolically active and dead cells, which is of great importance in food development and food spoilage; and to detect specific pathogenic microorganisms by conjugating antibodies with fluorochromes, which is of great use in the food industry. In addition, high-speed multiparametric data acquisition, analysis and cell sorting, which allow other characteristics of individual cells to be studied, have increased the interest of food microbiologists in this technique. This mini-review gives an overview of the principles of flow cytometry and examples of the application of this technique in the food industry.

  9. 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.

  10. Near infrared lasers in flow cytometry.

    PubMed

    Telford, William G

    2015-07-01

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

  11. Multinode acoustic focusing for parallel flow cytometry

    PubMed Central

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

    2012-01-01

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

  12. Bacterial characterization by flow cytometry

    SciTech Connect

    Van Dilla, M.A.; Langlois, R.G.; Pinkel, D.; Yajko, D.; Hadley, W.K.

    1983-05-06

    Bacteria were analyzed in a dual-beam flow cytometer after double staining with the fluorescent dyes chromomycin A3 and Hoechst 33258, which bind preferentialy to DNA that is rich in guanine-cytosine and adenine-thymine, respectively. The measurements were indicative of the cellular DNA content and base composition, cell concentration, and proliferative state of the population. The ratio of the chromomycin A3 signal to the Hoechst 33258 signal increased with the guanine-cytosine content of the cellular DNA for the six cultured species measured, following expectation. Bacteria in urine from patients with urinary tract infections were characterized without interference from host cell DNA, debris, or other particulates.

  13. Flow cytometry using spectrally encoded confocal microscopy.

    PubMed

    Golan, Lior; Yelin, Dvir

    2010-07-01

    Flow cytometry techniques often rely on detecting fluorescence from single cells flowing through the cross section of a laser beam, providing invaluable information on vast numbers of cells. Such techniques, however, are often limited in their ability to resolve clusters of cells or parallel cell flow through large vessels. We present a confocal imaging technique that images unstained cells flowing in parallel through a wide channel, using spectrally encoded reflectance confocal microscopy that does not require mechanical scanning. Images of red blood cells from our system are compared to conventional transmission microscopy, and imaging of flowing red blood cells in vitro is experimentally demonstrated.

  14. Flow cytometry in plant breeding.

    PubMed

    Ochatt, Sergio J

    2008-07-01

    Since the first report on the flow cytometric study of plant material 35 years ago, analyzing the nuclear DNA content of field bean, an ever increasing number of applications of FCM has been developed and applied in plant science and industry, but a similar length of time elapsed before the appearance of the first complete volume devoted to FCM of plant cells. Most published information on the uses of FCM addresses various aspects of animal (including human) cell biology, thus failing to provide a pertinent substitute. FCM represents an ideal means for the analysis of both cells and subcellular particles, with a potentially large number of parameters analyzed both rapidly, simultaneously, and quantitatively, thereby furnishing statistically exploitable data and allowing for an accurate and facilitated detection of subpopulations. It is, indeed, the summation of these facts that has established FCM as an important, and sometimes essential, tool for the understanding of fundamental mechanisms and processes underlying plant growth, development, and function. In this review, special attention is paid to FCM as applied to plant cells in the context of plant breeding, and some new and less well-known uses of it for plants will be discussed.

  15. Flow Cytometry Analyses of Adipose Tissue Macrophages

    PubMed Central

    Cho, Kae Won; Morris, David L.; Lumeng, Carey N.

    2014-01-01

    Within adipose tissue, multiple leukocyte interactions contribute to metabolic homeostasis in health as well as to the pathogenesis of insulin resistance with obesity. Adipose tissue macrophages (ATMs) are the predominant leukocyte population in fat and contribute to obesity-induced inflammation. Characterization of ATMs and other leukocytes in the stromal vascular fraction from fat has benefited from the use of flow cytometry and flow-assisted cell sorting techniques. These methods permit the immunophenotyping, quantification, and purification of these unique cell populations from multiple adipose tissue depots in rodents and humans. Proper isolation, quantification, and characterization of ATM phenotypes are critical for understanding their role in adipose tissue function and obesity-induced metabolic diseases. Here, we present the flow cytometry protocols for phenotyping ATMs in lean and obese mice employed by our laboratory. PMID:24480353

  16. Supercontinuum white light lasers for flow cytometry

    PubMed Central

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

    2009-01-01

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

  17. Supercontinuum white light lasers for flow cytometry.

    PubMed

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

    2009-05-01

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

  18. Flow cytometry what you see matters: Enhanced clinical detection using image-based flow cytometry.

    PubMed

    McFarlin, Brian K; Gary, Melody A

    2017-01-01

    Image-based flow cytometry combines the throughput of traditional flow cytometry with the ability to visually confirm findings and collect novel data that would not be possible otherwise. Since image-based flow cytometry borrows measurement parameters and analysis techniques from microscopy, it is possible to collect unique measures (i.e. nuclear translocation, co-localization, cellular synapse, cellular endocytosis, etc.) that would not be possible with traditional flow cytometry. The ability to collect unique outcomes has led many researchers to develop novel assays for the monitoring and detection of a variety of clinical conditions and diseases. In many cases, investigators have innovated and expanded classical assays to provide new insight regarding clinical conditions and chronic disease. Beyond human clinical applications, image-based flow cytometry has been used to monitor marine biology changes, nano-particles for solar cell production, and particle quality in pharmaceuticals. This review article summarizes work from the major scientists working in the field of image-based flow cytometry. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. 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

  20. Future clinical role for flow cytometry.

    PubMed

    Ashcroft, R G

    1988-01-01

    It appears that there is an essential, not just supportive, role for flow cytometry in the clinical context, particularly in providing early information in clinical oncology. High flow rate enumeration and sorting of rare cells, combined with microscopy, offer immediate benefits in the clinical processing of at least some cancers. These benefits would be in diagnosis (perhaps very early detection of metastatic cells in the present prediagnostic phase of solid tumor growth), monitoring, prognosis, and therapy. Importantly, flow cytometric measures can be implemented immediately, and measurement times are short. The value of high flow rate operation of existing facilities in clinical, "supportive" flow cytometry should be better appreciated, if only because shorter measurement times and on-line analysis would make the existing facilities more cost effective: higher throughput for the same overheads. Finally, the wisdom of employing nonsorting cytometers for clinical use should be strongly questioned. Thus, what future impact will the application of flow sorting have in clinical fields old and new, e.g., in bacterial infection measurements in peripheral blood? In particular, nonsorting machines will be unable to adopt the "essential" clinical role I have proposed here.

  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 Central

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

    2014-01-01

    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 murine 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 meiotic prophase I. 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. Application of flow cytometry to wine microorganisms.

    PubMed

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

    2017-04-01

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

  6. Mixture modeling approach to flow cytometry data.

    PubMed

    Boedigheimer, Michael J; Ferbas, John

    2008-05-01

    Flow Cytometry has become a mainstay technique for measuring fluorescent and physical attributes of single cells in a suspended mixture. These data are reduced during analysis using a manual or semiautomated process of gating. Despite the need to gate data for traditional analyses, it is well recognized that analyst-to-analyst variability can impact the dataset. Moreover, cells of interest can be inadvertently excluded from the gate, and relationships between collected variables may go unappreciated because they were not included in the original analysis plan. A multivariate non-gating technique was developed and implemented that accomplished the same goal as traditional gating while eliminating many weaknesses. The procedure was validated against traditional gating for analysis of circulating B cells in normal donors (n = 20) and persons with Systemic Lupus Erythematosus (n = 42). The method recapitulated relationships in the dataset while providing for an automated and objective assessment of the data. Flow cytometry analyses are amenable to automated analytical techniques that are not predicated on discrete operator-generated gates. Such alternative approaches can remove subjectivity in data analysis, improve efficiency and may ultimately enable construction of large bioinformatics data systems for more sophisticated approaches to hypothesis testing.

  7. Microfluidics in flow cytometry and related techniques.

    PubMed

    Béné, M C

    2017-05-01

    Technological advances in laboratory automation are now well understood and applied as they considerably improved the speed and robustness of haematological laboratory data, in the companion fields of blood analyzers and flow cytometry. Still rather confidential is the field of microfluidics, mostly confined so far to academic settings and research laboratories. The literature in the field of microfluidics is growing and applications in hematology range from cell counting to flow cytometry, cell sorting, or ex vivo testing. A literature search allows to identify many innovative solutions developed to master the specific physics of fluid movements in microchips. Miniaturization also dwells on findings that have emerged from different areas such as electronics and nanoengineering. This review proposes an overview of the major principles guiding developments in microfluidics and describes a necessarily limited and nonexhaustive series of specific applications. Readers are strongly encouraged to consult the documents referred to in the references section to learn more about this world knocking at our door and possibly liable to revolutionize our profession of hematology biologists in a not so far future. © 2017 John Wiley & Sons Ltd.

  8. 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

  9. Evaluation of platelet turnover by flow cytometry.

    PubMed

    Salvagno, G L; Montagnana, M; Degan, M; Marradi, P L; Ricetti, M M; Riolfi, P; Poli, G; Minuz, P; Santonastaso, C L; Guidi, G C

    2006-05-01

    The number of circulating newly produced platelets depends on the thrombopoietic capacity of bone marrow as well as platelet removal from the bloodstream. Flow cytometric analysis with thiazole orange (TO), a fluorescent dye that crosses platelet membranes and binds intracellular RNA, has been used to measure circulating reticulated platelets (RPs) with high RNA content as an index of platelet turnover. We first assessed the specificity of TO flow cytometry and then applied this method in the diagnosis of thrombocytopenia caused by impaired platelet production or increased destruction. We also explored the utility of TO flow cytometry to predict thrombocytopoiesis after chemotherapy-induced bone marrow aplasia. Venous blood, anticoagulated with K(2)EDTA, was incubated with 0.6 microg/ml TO plus an anti-GPIIIa monoclonal antibody. The mean percentage of RPs in control subjects (n = 23) was 6.13 +/- 3.09%. RPs were 10.41 +/- 9.02% in patients (n = 10) with hematological malignancies during aplasia induced by chemotherapy and a significant increase in RPs (35.45 +/- 6.11%) was seen in the recovery phase. In 10 patients with idiopathic thrombocytopenic purpura, the percentage of TO positive platelets was 67.81 +/- 18.79 (P < 0.001 vs. controls). In patients with thrombocytopenia associated with hepatic cirrhosis (n = 21; 21.04 +/- 16.21%, P < 0.001 vs. controls) or systemic lupus erythematosus (n = 6, 29.08 +/- 15.57%; P < 0.001 vs. controls) increases in TO-stained platelets were also observed. Measurement of TO positive platelets may be a reliable tool for the laboratory identification of platelet disorders, with a higher sensitivity than measurement of platelet volume. Measurement of RPs may also prove useful to recognize the underlying pathogenetic mechanisms in thrombocytopenia.

  10. Diagnosis of Fanconi's anemia by flow cytometry.

    PubMed

    Miglierina, R; Le Coniat, M; Gendron, M; Berger, R

    1990-01-01

    FA is a progressive bone marrow aplasia genetically transmitted by a recessive autosomal gene or genes. In our laboratory, cytogenetic diagnosis is based on evaluation of the chromosomal breakage of mitotic cell derived from patient blood-cell cultures and sensitized by nitrogen mustard (NM). We have observed, in parallel with this test, fluctuations of the cell cycle of PHA- stimulated peripheral blood lymphocytes from FA patients as compared with controls. FA cells treated with NM show a dramatic and significant increase in G2/M phase after 72 hr in vitro culture, compared with untreated or control cells (normal controls and non-FA patients). This test is rapid and simple, as it consists in staining cells with a DNA dye (propidium iodide), followed by a flow cytometry analysis of the cell cycle phases. Our results in twelve patients are correlated with the cytogenetic results.

  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. © 2015 Society for Laboratory Automation and Screening.

  12. 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

  13. 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

  14. Total Internal Reflection Fluorescence Flow Cytometry

    PubMed Central

    Wang, Jun; Bao, Ning; Paris, Leela L.; Geahlen, Robert L.; Lu, Chang

    2009-01-01

    Total internal reflection fluorescence microscopy (TIRFM) has been widely used to explore biological events that are close to the cell membrane by illuminating fluorescent molecules using the evanescent wave. However, TIRFM is typically limited to the examination of a low number of cells and the results do not reveal potential heterogeneity in the cell population. In this report, we develop an analytical tool referred to as total internal reflection fluorescence flow cytometry (TIRF-FC) to examine the region of the cell membrane with a throughput of ~100–150 cells/s and single cell resolution. We use an elastomeric valve that is partially closed to force flowing cells in contact with the glass surface where the evanescent field resides. We demonstrate that TIRF-FC is able to detect the differences in the subcellular location of an intracellular fluorescent protein. Proper data processing and analysis allows TIRF-FC to be quantitative. With the high throughput, TIRF-FC will be a very useful tool for generating information on cell populations with events and dynamics close to the cell surface. PMID:19007249

  15. Dynamic proliferation assessment in flow cytometry.

    PubMed

    Diermeier-Daucher, Simone; Brockhoff, Gero

    2010-09-01

    Dynamic proliferation assessment via flow cytometry is legitimately supposed to be the most powerful tool for recording cell cycle kinetics in-vitro. The preeminent feature is a single cell-based multi-informative analysis by temporal high-resolution. Flow cytometric approaches are based on labeling of proliferating cells via thymidine substitution by a base analog (e.g., 5-bromo-2'-deoxyuridine, BrdU) that is added to cell cultures either for a short period of time (pulse labeling) or continuously until cell harvesting. This unit describes the alternative use of the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) in place of BrdU for three different applications: (1) dynamic proliferation assessment by EdU pulse cell labeling; (2) the same approach as (1) but in combination with live/dead cell discrimination; and (3) dynamic cell cycle analysis based on continuous cell labeling with EdU and Hoechst fluorochrome quenching. In contrast to the detection of BrdU incorporation, EdU-positive cells can be identified by taking advantage of click chemistry, which facilitates a simplified and fast cell preparation. Further analysis options but also limitations of the utilization of EdU are discussed.

  16. 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.

  17. Immunological techniques: ELISA, flow cytometry, and immunohistochemistry.

    PubMed

    Ford, Pauline J

    2010-01-01

    Techniques to analyze the host immune response elicited by the presence of oral microorganisms and their products are central to our understanding of the local and systemic effects of oral diseases. This immune response has been extensively investigated for periodontal disease. The local response may result in lesions involving the gingival tissues and depending upon host susceptibility and microbial virulence may lead to local tissue destruction. More recently, however, the importance of the systemic inflammatory and immune response to oral organisms has been recognized. These systemic responses have been associated with an increased risk for cardiovascular disease, diabetes, and preterm low birth weight. A number of techniques are used extensively by researchers investigating humoral and cellular immune responses to oral organisms both in local oral tissues and fluids and systemically in peripheral blood. These are enzyme-linked immunosorbent assay (ELISA) to quantify specific antibody and cytokines in serum, gingival crevicular fluid (GCF), and saliva; characterization of T cells from peripheral blood and gingival tissues using flow cytometry; and immunohistological analysis of the inflammatory cell infiltrate in gingival tissues.

  18. Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry.

    PubMed

    Furman, Jennifer L; Holmes, Brandon B; Diamond, Marc I

    2015-12-08

    Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer's disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples. By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as α-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.

  19. 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.

  20. 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

  1. 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

  2. Visible and near infrared fluorescence spectral flow cytometry.

    PubMed

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

    2013-03-01

    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.

  3. 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

  4. Scalable clustering algorithms for continuous environmental flow cytometry.

    PubMed

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

    2016-02-01

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

  5. LED Thermo Flow - Combining Optogenetics with Flow Cytometry.

    PubMed

    Brenker, Kathrin; Osthof, Kerstin; Yang, Jianying; Reth, Michael

    2016-12-30

    Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics. Here, we present a device that combines the power of optogenetics and flow cytometry: the LED Thermo Flow. This device illuminates cells at specific wavelengths, light intensities and temperatures during flow cytometric measurements. It can be built at low cost and be used with most common flow cytometers. To demonstrate its utility, we characterized the photoswitching kinetics of Dronpa proteins in vivo and in real time. This protocol can be adapted to almost all optically controlled substances and substantially expands the set of possible experiments. More importantly, it will greatly simplify the discovery and development of new optogenetic tools.

  6. 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

  7. Human chromosome karyotyping and molecular biology by flow cytometry

    SciTech Connect

    Yu, L.C.; Gray, J.W.; Langlois, R.; Van Dilla, M.A.; Carrano, A.V.

    1982-03-22

    Flow cytometry is a sensitive analytical tool for rapdily measuring the biological, chemical and physical properties of cells and cellular components, such as chromosomes and has become a promising system for automating human chromosomal karyotyping. Unlike traditional approaches based upon chromosomal length, centromeric index, and banding patterns, it is based on the measurement of chromosomal DNA content and base composition and can classify chromosomes more objectively. In this paper we describe flow karyotyping and chromosome sorting and compare flow cytometry with the use of cell hybrids for gene mapping and the construction of chromosome-specific genomic libraries.

  8. 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…

  9. 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…

  10. International Society for Advancement of Cytometry (ISAC) flow cytometry shared resource laboratory (SRL) best practices.

    PubMed

    Barsky, Lora W; Black, Michele; Cochran, Matthew; Daniel, Benjamin J; Davies, Derek; DeLay, Monica; Gardner, Rui; Gregory, Michael; Kunkel, Desiree; Lannigan, Joanne; Marvin, James; Salomon, Robert; Torres, Carina; Walker, Rachael

    2016-11-01

    The purpose of this document is to define minimal standards for a flow cytometry shared resource laboratory (SRL) and provide guidance for best practices in several important areas. This effort is driven by the desire of International Society for the Advancement of Cytometry (ISAC) members in SRLs to define and maintain standards of excellence in flow cytometry, and act as a repository for key elements of this information (e.g. example SOPs/training material, etc.). These best practices are not intended to define specifically how to implement these recommendations, but rather to establish minimal goals for an SRL to address in order to achieve excellence. It is hoped that once these best practices are established and implemented they will serve as a template from which similar practices can be defined for other types of SRLs. Identification of the need for best practices first occurred through discussions at the CYTO 2013 SRL Forum, with the most important areas for which best practices should be defined identified through several surveys and SRL track workshops as part of CYTO 2014. © 2016 International Society for Advancement of Cytometry.

  11. 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.

  12. Solid state yellow and orange lasers for flow cytometry.

    PubMed

    Kapoor, Veena; Karpov, Vladimir; Linton, Claudette; Subach, Fedor V; Verkhusha, Vladislav V; Telford, William G

    2008-06-01

    Diode and DPSS lasers emitting a variety of wavelengths are now commonly incorporated into flow cytometers, greatly increasing our capacity to excite a wide variety of fluorochromes. Until recently, however, virtually no practical technology existed for generating yellow or orange laser light for flow cytometry that was compatible with smaller instrumentation. In this study, we evaluate several new solid state laser systems that emit from the 570 to 600 nm as excitation sources for flow cytometry. DPSS 580, 589, and 592 nm sources were integrated into a cuvette-based flow cytometer (BD LSR II) and a stream-in-air cell sorter (FACSVantage DiVa), and used to excite a variety of yellow, orange, and red excited fluorochromes, including Texas Red, APC, and its tandem conjugates, and the genetically encoded red fluorescent protein HcRed and the more recently developed Katushka. All laser sources were successfully incorporated into the indicated flow cytometry platforms. The yellow and orange sources (particularly 592 nm) were ideal for exciting Texas Red, and provided excitation of APC and its tandems that was comparable to a traditional red laser source, albeit at higher power levels than red sources. Yellow and orange laser light was optimal for exciting HcRed and Katushka. Practical yellow and orange laser sources are now available for flow cytometry. This technology fills an important gap in the laser wavelengths available for flow, now almost any fluorochrome requiring visible light excitation can be accommodated.

  13. Moving pictures: imaging flow cytometry for drug development.

    PubMed

    Elliott, Gary S

    2009-11-01

    As technologies for high throughput and high content screening continue to evolve, new platforms for quantitative cellular imaging will play an increasingly important role in identifying and profiling lead compounds. To gain insight into the effects of a compound on cell morphology or intracellular events, it is necessary to have quality images and the ability to enumerate thousands of data points for statistical relevance. Imaging flow cytometry combines many of the features of flow cytometry, microscopy and imaging as well as a number of unique characteristics. The result is an instrument capable of highly quantitative analysis of cellular behaviors such as receptor internalization, phagocytosis, cell-cell communication, apoptosis and nuclear translocation. This promising new technology and unique type of flow cytometry provides enhanced capabilities for highly multiplexed assays. Here, we review the capabilities of the ImageStream imaging cytometer and discuss several applications relevant to compound screening and profiling.

  14. Engineered viral nanoparticles for flow cytometry and fluorescence microscopy applications.

    PubMed

    Robertson, Kelly L; Liu, Jinny L

    2012-01-01

    Viral nanoparticles (VNPs) are attractive platforms for use in the biotechnology and biomedical fields because of their biological nature. A wide variety of these particles, labeled with fluorescent reporters, have been characterized using flow cytometry and cellular imaging techniques. Fluorescence microscopy allows the direct observation of VNPs on the cell surface or inside the membrane as well as the cellular localization of the nanoparticles while flow cytometry allows the statistical quantification of nanoparticle uptake and targeting specificity. These techniques are essential when characterizing the properties of VNPs and provide information toward the use of VNPs for targeting, imaging, and/or cargo delivery. Copyright © 2012 Wiley Periodicals, Inc.

  15. Cellular Image Analysis and Imaging by Flow Cytometry

    PubMed Central

    Basiji, David A.; Ortyn, William E.; Liang, Luchuan; Venkatachalam, Vidya; Morrissey, Philip

    2007-01-01

    Synopsis Imaging flow cytometry combines the statistical power and fluorescence sensitivity of standard flow cytometry with the spatial resolution and quantitative morphology of digital microscopy. The technique is a good fit for clinical applications by providing a convenient means for imaging and analyzing cells directly in bodily fluids. Examples are provided of the discrimination of cancerous from normal mammary epithelial cells and the high throughput quantitation of FISH probes in human peripheral blood mononuclear cells. The FISH application will be further enhanced by the integration of extended depth of field imaging technology with the current optical system. PMID:17658411

  16. Advantages of full spectrum flow cytometry.

    PubMed

    Sanders, Claire K; Mourant, Judith R

    2013-03-01

    A charge coupled device-based flow-cytometer for the measurement of full spectra was implemented and characterized. The spectral resolution was better than 1.5 nm and the coefficient of variation for fluorescence from flow check beads was 5% or better. Both cell and bead data were analyzed by fitting to measured component spectra. Separation of flow check and align flow beads, which have similar spectra, was nearly identical whether using a spectral analysis or a scatter analysis. After mixing, cells stained with ethidium bromide or propidium iodide were measured at different timepoints. The contribution of these 12 nm separated emission spectra could be separately quantified and the kinetic process of the samples becoming homogeneous due to fluorophor dissociation and rebinding was observed. Principle component analysis was used to reduce noise and alternating least squares (ALS) was used to analyze one set of noise-reduced cell data without knowledge of the component spectra. The component spectra obtained via ALS are very similar to the measured component spectra. The contributions of ethidium bromide and propidium iodide to the individual spectra are also similar to those obtained via the spectral fitting procedure.

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

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-24

    ... workshop include: (1) Overview of Quality control and standardization issues associated with Clinical Flow... monoclonal antibodies and flow cytometry contributed to the automation of lymphocyte subset analysis. Flow... cytometry system devices included flow cytometers, reagents, controls, and associated software for...

  19. Multispectral flow cytometry: The consequences of increased light collection.

    PubMed

    Feher, Kristen; von Volkmann, Konrad; Kirsch, Jenny; Radbruch, Andreas; Popien, Jan; Kaiser, Toralf

    2016-07-01

    In recent years, multispectral flow cytometry systems have come to attention. They differ from conventional flow cytometers in two key ways: a multispectral flow cytometer collects the full spectral information at the single cell level and the detector configuration is fixed and not explicitly tuned to a particular staining panel. This brings about clear hardware advantages, as a closed system should be highly stable, and ease-of-use should be improved if used in conjunction with custom unmixing software. An open question remains: what are the benefits of multispectral over conventional flow cytometry in terms of sensitivity and resolution? To probe this, we use Q (detection efficiency) and B (background) values and develop a novel "multivariate population overlap factor" to characterize the cytometer performance. To verify the usefulness of our factor, we perform representative experiments and compare our overlap factor to Q and B. Finally, we conclude that the increased light collection of multispectral flow cytometry does indeed lead to increased sensitivity, an improved detection limit, and a higher resolution. © 2016 International Society for Advancement of Cytometry.

  20. Standardization, Calibration, and Control in Flow Cytometry.

    PubMed

    Wang, Lili; Hoffman, Robert A

    2017-01-05

    Because flow cytometers are designed to measure particle characteristics, particles are the most common materials used to calibrate, control, and standardize the instruments. Definitions and cautions are provided for common terms to alert the reader to critical distinctions in meaning. This unit presents extensive background on particle types and cautions and describes practical aspects of methods to standardize and calibrate instruments. Procedures are provided to characterize performance in terms of optical alignment, fluorescence and light scatter resolution, and sensitivity. Finally, suggestions follow for analyzing particles used for calibration. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  1. 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

  2. Determination of somatic mutations in human erythrocytes by cytometry

    SciTech Connect

    Jensen, R.H.; Langlois, R.G.; Bigbee, W.L.

    1985-06-21

    Flow cytometric assays of human erythrocytes labeled with monoclonal antibodies specific for glycophorin A were used to enumerate variant cells that appear in peripheral blood as a result of somatic gene-loss mutations in erythrocyte precursor cells. The assay was performed on erythrocytes from 10 oncology patients who had received at least one treatment from radiation or mutagenic chemotherapy at least 3 weeks before being assayed. The patients were suffering from many different malignancies (e.g., breast, renal, bone, colon and lung), and were treated with several different mutagenic therapeutics (e.g., cisplatinum, adriamycin, daunomycin, or cyclophosphamide). The frequency of these variant cells is an indication of the amount of mutagenic damage accumulated in the individual's erythropoietic cell population. Comparing these results to HPRT clonogenic assays, we find similar baseline frequencies of somatic mutation as well as similar correlation with mutagenic exposures. 9 refs., 3 figs., 1 tab.

  3. Phasor plotting with frequency-domain flow cytometry

    PubMed Central

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

    2016-01-01

    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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. Recent advances of flow cytometry in fundamental and applied microbiology.

    PubMed

    Fouchet, P; Jayat, C; Héchard, Y; Ratinaud, M H; Frelat, G

    1993-01-01

    This review focuses on the recent applications of flow cytometry (FCM) in microbiological research (1987-mid 1992). It tries to give a scope of the important breakthroughs which occurred in this field during this period. The technical difficulties of microorganism analysis by flow cytometry is briefly appraised. The significance and the limits of the different microbial cell parameters attainable by flow analyses are systematically evaluated: light scatter for cell size and structure, fluorescence measurements for quantification of cellular components, microbial antigen detection and cell physiological activity estimation. Emphasis is given on the new technological advances which appeared in the last two years. The second part of the review is devoted to the analysis of the usefulness of flow cytometric approach in the different fields of microbiology: fundamental studies in microbial physiology, differentiation, microbial ecology and aquatic sciences, medical microbiology, parasitology, microbial pharmacology and biotechnology.

  9. Analysis of repetitive DNA in chromosomes by flow cytometry.

    PubMed

    Brind'Amour, Julie; Lansdorp, Peter M

    2011-06-01

    We developed a flow cytometry method, chromosome flow fluorescence in situ hybridization (FISH), called CFF, to analyze repetitive DNA in chromosomes using FISH with directly labeled peptide nucleic acid (PNA) probes. We used CFF to measure the abundance of interstitial telomeric sequences in Chinese hamster chromosomes and major satellite sequences in mouse chromosomes. Using CFF we also identified parental homologs of human chromosome 18 with different amounts of repetitive DNA.

  10. Impact of standardization on clinical cell analysis by flow cytometry.

    PubMed

    Keeney, M; Barnett, D; Gratama, J W

    2004-01-01

    The evolution of flow cytometry from a research tool to a pivotal technology for clinical diagnostic purposes has required significant efforts to standardize methods. The great advantage of flow cytometry is that it's applications are highly amenable to standardization. Here, we review the efforts that have been made for flow cytometric applications in four major fields of clinical cell analysis: CD4+ T-cell enumeration, CD34+ hematopoietic stem and progenitor cell enumeration, screening for the HLA-B27 antigen and leukemia/lymphoma immunophenotyping. These standardization efforts have been parallelled by the establishment of external quality assessment (EQA) schemes in many countries worldwide. The goal of these EQA exercises has been primarily educa-tional, but their results will increasingly serve as a basis for laboratory accreditation. This important development requires that the EQA schemes, in particular the quality of the distributed samples and the procedures for evaluating the results, meet the highest standards.

  11. 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

  12. Microfluidic impedance flow cytometry enabling high-throughput single-cell electrical property characterization.

    PubMed

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

    2015-04-29

    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.

  13. 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

  14. 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

  15. 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

  16. Guide to Red Fluorescent Proteins and Biosensors for Flow Cytometry

    PubMed Central

    Piatkevich, Kiryl D.; Verkhusha, Vladislav V.

    2014-01-01

    Since the discovery of the first red fluorescent protein (RFP), named DsRed, 12 years ago, a wide pallet of red-shifted fluorescent proteins has been cloned and biotechnologically developed into monomeric fluorescent probes for optical microscopy. Several new types of monomeric RFPs that change the emission wavelength either with time, called fluorescent timers, or after a brief irradiation with violet light, known as photoactivatable proteins, have been also engineered. Moreover, RFPs with a large Stokes shift of fluorescence emission have been recently designed. Because of their distinctive excitation and fluorescence detection conditions developed specifically for microscopy, these fluorescent probes can be suboptimal for flow cytometry. Here, we have selected and summarized the advanced orange, red, and far-red fluorescent proteins with the properties specifically required for the flow cytometry applications. Their effective brightness was calculated for the laser sources available for the commercial flow cytometers and sorters. Compatibility of the fluorescent proteins of different colors in a multiparameter flow cytometry was determined. Novel FRET pairs, utilizing RFPs, RFP-based intracellular biosensors, and their application to a high-throughput screening, are also discussed. PMID:21704849

  17. Minimal residual disease studies by flow cytometry in acute leukemia.

    PubMed

    Campana, Dario; Coustan-Smith, Elaine

    2004-01-01

    Minimal residual disease (MRD) assays are increasingly important in the clinical management of patients with acute leukemia. Among the methods available for monitoring MRD, flow cytometry holds great promise for clinical application because of its simplicity and wide availability. Several studies have demonstrated strong correlations between MRD levels by flow cytometry during clinical remission and treatment outcome, lending support to the reliability of this approach. Flow-cytometric detection of MRD is based on the identification of immunophenotypic combinations expressed on leukemic cells but not on normal hematopoietic cells. Its sensitivity depends on the specificity of the immunophenotypes used to track leukemic cells and on the number of cells available for study. Immunophenotypes that allow detection of 1 leukemic cell in 10,000 normal cells can be identified in at least 90% of patients with acute lymphoblastic leukemia; immunophenotypes that allow detection of 1 leukemic cell in 1,000-10,000 normal cells can be identified in at least 85% of patients with acute myeloid leukemia. Identification of new markers of leukemia by gene array technology should lead to the design of simple and reliable antibody panels for universal monitoring of MRD. Here we review the relative advantages and disadvantages of flow cytometry for MRD studies, as well as results obtained in correlative studies with treatment outcome. Copyright 2004 S. Karger AG, Basel

  18. 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.

  19. Flow cytometry as a tool to quantify oyster defence mechanisms.

    PubMed

    Goedken, Michael; De Guise, Sylvain

    2004-04-01

    The fast growing oyster aquaculture industry is greatly hindered by Perkinsus marinus and Haplosporidium nelsoni which can kill up to 80% of the production. The relationship between parasites and oyster defence mechanisms is unclear. Two defence mechanisms of the Eastern Oyster (Crassostrea virginica) were quantified at the single cell level utilising flow cytometry. Phagocytosis was measured using fluorescent beads. Respiratory burst activity was quantified as the H2O2-specific increase in dichlorofluorescein-associated fluorescence upon stimulation. These two assays distinguished three populations of haemocytes (granulocytes, hyalinocytes and intermediate cells) with unique functional characteristics. Granulocytes were most active at phagocytosis and H2O2 production while hyalinocytes were relatively inactive. The intermediate cells had moderate phagocytic and respiratory burst activity. Flow cytometry can rapidly, accurately and directly quantify the morphology and function of a large number of individual cells, and will lead to a better understanding of the bivalve immune system.

  20. Data Analysis in Flow Cytometry: The Future Just Started

    PubMed Central

    Lugli, Enrico; Roederer, Mario; Cossarizza, Andrea

    2010-01-01

    In the last 10 years, a tremendous progress characterized flow cytometry in its different aspects. In particular, major advances have been conducted regarding the hardware/instrumentation and reagent development, thus allowing fine cell analysis up to 20 parameters. As a result, this technology generates very complex data sets that demand for the development of optimal tools of analysis. Recently, many independent research groups approached the problem by using both supervised and unsupervised methods. In this paper, we will review the new developments concerning the use of bioinformatics for polychromatic flow cytometry and propose what should be done in order to unravel the enormous heterogeneity of the cells we interrogate each day. PMID:20583274

  1. Step-specific Sorting of Mouse Spermatids by Flow Cytometry.

    PubMed

    Simard, Olivier; Leduc, Frédéric; Acteau, Geneviève; Arguin, Mélina; Grégoire, Marie-Chantal; Brazeau, Marc-André; Marois, Isabelle; Richter, Martin V; Boissonneault, Guylain

    2015-12-31

    The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to the next generation. So far, molecular studies of this morphological transition have been hampered by the lack of a method allowing adequate separation of these important steps of spermatid differentiation for subsequent analyses. Earlier attempts at proper gating of these cells using flow cytometry may have been difficult because of a peculiar increase in DNA fluorescence in spermatids undergoing chromatin remodeling. Based on this observation, we provide details of a simple flow cytometry scheme, allowing reproducible purification of four populations of mouse spermatids fixed with ethanol, each representing a different state in the nuclear remodeling process. Population enrichment is confirmed using step-specific markers and morphological criterions. The purified spermatids can be used for genomic and proteomic analyses.

  2. Analyzing Schizosaccharomyces pombe DNA Content by Flow Cytometry.

    PubMed

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

    2016-06-01

    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.

  3. Using Flow Cytometry to Measure Phagocytic Uptake in Earthworms†

    PubMed Central

    Fuller-Espie, Sheryl L.

    2010-01-01

    This laboratory module familiarizes students with flow cytometry while acquiring quantitative reasoning skills during data analysis. Leukocytes, also known as coelomocytes (including hyaline and granular amoebocytes, and chloragocytes), from Eisenia hortensis (earthworms) are isolated from the coelomic cavity and used for phagocytosis of fluorescent Escherichia coli. Students learn how to set up in vitro cellular assays and become familiar with theoretical principles of flow cytometry. Histograms based on fluorescence and scatter properties combined with gating options permit students to restrict their analyses to particular subsets of coelomocytes when measuring phagocytosis, a fundamentally important innate immune mechanism used in earthworms. Statistical analysis of data is included in laboratory reports which serve as the primary assessment instrument. PMID:23653715

  4. 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.

  5. Ploidy Determination in Agrostis Using Flow Cytometry and Morphological Traits.

    PubMed

    Bonos, Stacy A.; Plumley, Karen A.; Meyer, William A.

    2002-01-01

    The taxonomic classification of the genus Agrostis is one of the most complicated of the grass genera. Classification based upon morphological and anatomical characters is difficult and complicated by the presence of intermediate forms and the misapplication of names. Determining ploidy levels of new germplasm can assist in species determination and is necessary before initiating breeding or genetics studies. The objectives of this study were to (i) evaluate the use of laser flow cytometry as a quick, reliable tool to determine ploidy level and aid in Agrostis species determination, and (ii) identify morphological characters associated with DNA content or ploidy level. The six Agrostis species evaluated were A. canina L. subsp. canina, A. canina L. subsp. montana (Hartm.) Hartm., A. palustris Huds. [= A. stolonifera var. palustris (Huds.) Farw.], A. tenuis Sibth. (= A. capillaris L.), A. castellana Boiss. & Reut., and A. alba L. Ploidy level was determined by flow cytometry and root tip chromosome counts. Plant height, panicle height, flag leaf length, flag leaf width, and highest internode length of mature field-grown spaced plants were measured. Significant differences in 2C DNA content were found between species (P < 0.01) differing in ploidy level. Flow cytometry was effective in differentiating between diploid, tetraploid, and hexaploid species. Chromosome numbers previously reported and those observed in this study were positively correlated with 2C nuclear DNA content (r = 0.98, P < 0.01). Flag leaf length was the only morphological measurement taken that was significantly positively correlated to DNA content (r = 0.98, P < 0.001). The results of this study indicate that laser flow cytometry is a quick, reliable tool to determine ploidy levels and infer certain species of AGROSTIS: This technique will aid breeders to quickly and accurately determine ploidy levels of new germplasm collections.

  6. Analysis of Human and Mouse Neutrophil Phagocytosis by Flow Cytometry.

    PubMed

    Fine, Noah; Barzilay, Oriyah; Glogauer, Michael

    2017-01-01

    Neutrophils are primary phagocytes that recognize their targets through surface chemistry, either through Pattern Recognition Receptor (PPR) interaction with Pathogen-Associated Molecular Patterns (PAMPs) or through immunoglobulin (Ig) or complement mediated recognition. Opsonization can be important for target recognition, and phagocytosis by neutrophils in whole blood can be greatly enhanced due to the presence of blood serum components and platelets. Powerful and sensitive flow cytometry based methods are presented to measure phagocytosis by human blood neutrophils and mouse peritoneal neutrophils.

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

    PubMed

    Sun, Q; Ding, Y; Zhang, J

    2001-01-25

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

  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. 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.

  11. Pitfalls in the use of multicolour flow cytometry in haematology.

    PubMed

    Johansson, Ulrika; Macey, Marion

    2011-07-01

    Multicolour flow cytometry in haematology has developed considerably in recent years. The ability to analyse eight or more colours of fluorescence on millions of cells in a matter of minutes has enabled the provision of rapid and reliable measures of minimal residual disease for clinicians. The use of multicolour analysis has also enabled more specific characterisation of presenting leukaemias and lymphomas. However, there has not been a concomitant increase in the knowledge and experience of the flow cytometrists to deal with certain problems associated with this more complex analysis.

  12. Chromosome classification and purification using flow cytometry and sorting

    SciTech Connect

    Gray, J.W.; Langlois, R.G. )

    1986-01-01

    The authors review here both the techniques and the applications of flow karyotyping and sorting, hereafter called flow cytogenetics. Included in our review of the techniques are the procedures for chromosome isolation and staining, the principles of flow cytometry and sorting, and the analytical procedures for classification of the chromosomes from flow cytometric data. Applications reviewed include classification of normal human chromosomes, identification of disease-linked structural and numerical aberrations, quantification of the frequency of structurally aberrant chromosomes as a measure of the extent of induced genetic damage, and purification of chromosomes of a single type to facilitate interpretation of flow karyotypes, gene mapping, and production of recombinant DNA libraries from chromosomes of a single type.

  13. Quantitative karyotyping of human chromosomes by dual beam flow cytometry

    SciTech Connect

    Langlois, R.G.; Yu, L.C.; Gray, J.W.; Carrano, A.V.

    1982-12-01

    Dual beam flow cytometry of chromosomes stained with Hoechst 33258 and chromomycin A3 has been proposed as a method for quantitative classification of human chromosomes (bivariate flow karotyping). In this paper we investigate the sources and magnitudes of variability in the mean fluorescence intensities of each chromosome group resolved in bivariate flow karyotypes and study the impact of this variablity on chromosome classification. Replicate bivariate flow karyotypes of chromosomes isolated from lymphocyctes from 10 individuals demonstrated that person-to-person variability was significantly greater than run-to-run variability. The total variability was sufficiently small that it did not interfere with classification of normal chromosome types except chromosomes 9 through 12 and chromosomes 14 and 15. Furthermore, the variability was generally smaller than 1/600th of the mitotic genome, so that one-band rearrangements should be detectable in bivariate flow karoyotypes.

  14. Beam shaping in flow cytometry with diffractive optical elements

    NASA Astrophysics Data System (ADS)

    Qu, Weidong; Li, Derong; Jian, Peng

    2016-10-01

    Focusing elements are usually employed in the flow cytometry to focus the input laser beam into elliptically shaped Gaussian beam in order to increase power for excitation of fluorescence for high signal-to-noise ratio (SNR). While in order to ensure repeatable and reliable signal generation for accurate population discrimination - despite slight deviations of the cell from the flow centre, the shaped beam should be a cubic diffraction region with uniform power intensity across the cell flow stream. However, it is hard for beam shaping with refractive optical elements. In this paper, we present a beam shaping system in flow cytometry with diffractive optical elements (DOEs) to shape the input laser beam to a cubic diffraction region with uniform power intensity. The phase distribution of the DOE is obtained by using the inverse Fresnel diffraction based layered holographic stereogram, and the cubic diffraction region with uniform power intensity within the cell flow channel is well reconstructed. Simulation results demonstrate the good performance of the new beam shaping system.

  15. Framework for morphometric classification of cells in imaging flow cytometry.

    PubMed

    Gopakumar, G; Jagannadh, Veerendra Kalyan; Gorthi, Sai Siva; Subrahmanyam, Gorthi R K Sai

    2016-03-01

    Imaging flow cytometry is an emerging technology that combines the statistical power of flow cytometry with spatial and quantitative morphology of digital microscopy. It allows high-throughput imaging of cells with good spatial resolution, while they are in flow. This paper proposes a general framework for the processing/classification of cells imaged using imaging flow cytometer. Each cell is localized by finding an accurate cell contour. Then, features reflecting cell size, circularity and complexity are extracted for the classification using SVM. Unlike the conventional iterative, semi-automatic segmentation algorithms such as active contour, we propose a noniterative, fully automatic graph-based cell localization. In order to evaluate the performance of the proposed framework, we have successfully classified unstained label-free leukaemia cell-lines MOLT, K562 and HL60 from video streams captured using custom fabricated cost-effective microfluidics-based imaging flow cytometer. The proposed system is a significant development in the direction of building a cost-effective cell analysis platform that would facilitate affordable mass screening camps looking cellular morphology for disease diagnosis.

  16. Label-free high-throughput imaging flow cytometry

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  17. Analysis of chromosome damage for biodosimetry using imaging flow cytometry.

    PubMed

    Beaton, L A; Ferrarotto, C; Kutzner, B C; McNamee, J P; Bellier, P V; Wilkins, R C

    2013-08-30

    The dicentric chromosome assay (DCA), which involves counting the frequency of dicentric chromosomes in mitotic lymphocytes and converting it to a dose-estimation for ionizing radiation exposure, is considered to be the gold standard for radiation biodosimetry. Furthermore, for emergency response, the DCA has been adapted for triage by simplifying the scoring method [1]. With the development of new technologies such as the imaging flow cytometer, it may now be possible to adapt this microscope-based method to an automated cytometry method. This technology allows the sensitivity of microscopy to be maintained while adding the increased throughput of flow cytometry. A new protocol is being developed to adapt the DCA to the imaging cytometer in order to further increase the rapid determination of a biological dose. Peripheral blood mononuclear cells (PBMC) were isolated from ex vivo irradiated whole blood samples using a density gradient separation method and cultured with PHA and Colcemid. After 48h incubation, the chromosomes were isolated, stained for DNA content with propidium iodide (PI) and labelled with a centromere marker. Stained chromosomes were then analyzed on the ImageStream(×) (EMD-Millipore, Billerica, MA). Preliminary results indicate that individual chromosomes can be identified and mono- and dicentric chromosomes can be differentiated by imaging cytometry. A dose response curve was generated using this technology. The details of the method and the dose response curve are presented and compared to traditional microscope scoring. Imaging cytometry is a new technology which enables the rapid, automated analysis of fluorescently labelled chromosomes. Adapting the dicentric assay to this technology has the potential for high throughput analysis for mass casualty events.

  18. AutoGate: automating analysis of flow cytometry data

    PubMed Central

    Meehan, Stephen; Walther, Guenther; Moore, Wayne; Orlova, Darya; Meehan, Connor; Parks, David; Ghosn, Eliver; Philips, Megan; Mitsunaga, Erin; Waters, Jeffrey; Kantor, Aaron; Okamura, Ross; Owumi, Solomon; Yang, Yang; Herzenberg, Leonard A.

    2015-01-01

    Nowadays, one can hardly imagine biology and medicine without flow cytometry to measure CD4 T cell counts in HIV, follow bone marrow transplant patients, characterize leukemias, etc. Similarly, without flow cytometry, there would be a bleak future for stem cell deployment, HIV drug development and full characterization of the cells and cell interactions in the immune system. But while flow instruments have improved markedly, the development of automated tools for processing and analyzing flow data has lagged sorely behind. To address this deficit, we have developed automated flow analysis software technology, provisionally named AutoComp and AutoGate. AutoComp acquires sample and reagent labels from users or flow data files, and uses this information to complete the flow data compensation task. AutoGate replaces the manual subsetting capabilities provided by current analysis packages with newly defined statistical algorithms that automatically and accurately detect, display and delineate subsets in well-labeled and well-recognized formats (histograms, contour and dot plots). Users guide analyses by successively specifying axes (flow parameters) for data subset displays and selecting statistically defined subsets to be used for the next analysis round. Ultimately, this process generates analysis “trees” that can be applied to automatically guide analyses for similar samples. The first AutoComp/AutoGate version is currently in the hands of a small group of users at Stanford, Emory and NIH. When this “early adopter” phase is complete, the authors expect to distribute the software free of charge to .edu, .org and .gov users. PMID:24825775

  19. Centromeric index measurement by slit-scan flow cytometry

    SciTech Connect

    Lucas, J.N.; Gray, J.W.; Peters, D.C.; Van Dilla, M.A.

    1983-01-01

    A report is given of the application of slit-scan flow cytometry (SSFCM) in the classification of muntjac, Chinese hamster, and human chromosomes according to centromeric index (CI) and total fluorescence. Chromosomes were isolated from mitotic cells, stained with propidium iodide and processed through the SSFCM where fluorescence profiles were measured. The centromere for each profile was taken as the point of maximum difference between the measured profile and a standard profile having no centromeric dip. The areas under the profile on either side of the centromere were then calculated and the CI was calculated as the ratio of the larger area to the total area under the profile. Relative DNA contents for each chromosome were taken to be proportional to the total fluorescence. Mean CI's for muntjac chromosomes 1, 2, and X + 3 were 0.52, 0.88, and 0.73, respectively; CI's for Chinese hamster M3-1 chromosomes 1, 2, 5, 8, and M2 were 0.53, 0.55, 0.57, 0.77, and 0.86, respectively; and average CI's for chromosome groups 4 + t (X;5), 6 + 7 + Y, 9 + M1, and 10 + 11 were 0.56, 0.82, 0.58, and 0.60, respectively. These results were, on average, within 4.4% of CI measurements made by image cytometry. CI's measured for human chromosomes 9 through 12, were, on average, within 2.0% of those made by image cytometry.

  20. Ultraviolet 320 nm laser excitation for flow cytometry.

    PubMed

    Telford, William; Stickland, Lynn; Koschorreck, Marco

    2017-02-27

    Although multiple lasers and high-dimensional analysis capability are now standard on advanced flow cytometers, ultraviolet (UV) lasers (usually 325-365 nm) remain an uncommon excitation source for cytometry. This is primarily due to their cost, and the small number of applications that require this wavelength. The development of the Brilliant Ultraviolet (BUV fluorochromes, however, has increased the importance of this formerly niche excitation wavelength. Historically, UV excitation was usually provided by water-cooled argon- and krypton-ion lasers. Modern flow cytometers primary rely on diode pumped solid state lasers emitting at 355 nm. While useful for all UV-excited applications, DPSS UV lasers are still large by modern solid state laser standards, and remain very expensive. Smaller and cheaper near UV laser diodes (NUVLDs) emitting at 375 nm make adequate substitutes for 355 nm sources in many situations, but do not work as well with very short wavelength probes like the fluorescent calcium chelator indo-1. In this study, we evaluate a newly available UV 320 nm laser for flow cytometry. While shorter in wavelength that conventional UV lasers, 320 is close to the 325 nm helium-cadmium wavelength used in the past on early benchtop cytometers. A UV 320 nm laser was found to excite almost all Brilliant Ultraviolet dyes to nearly the same level as 355 nm sources. Both 320 nm and 355 nm sources worked equally well for Hoechst and DyeCycle Violet side population analysis of stem cells in mouse hematopoetic tissue. The shorter wavelength UV source also showed excellent excitation of indo-1, a probe that is not compatible with NUVLD 375 nm sources. In summary, a 320 nm laser module made a suitable substitute for conventional 355 nm sources. This laser technology is available in a smaller form factor than current 355 nm units, making it useful for small cytometers with space constraints. © 2017 International Society for Advancement of Cytometry.

  1. 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.

  2. 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

  3. Absolute counting of neutrophils in whole blood using flow cytometry.

    PubMed

    Brunck, Marion E G; Andersen, Stacey B; Timmins, Nicholas E; Osborne, Geoffrey W; Nielsen, Lars K

    2014-12-01

    Absolute neutrophil count (ANC) is used clinically to monitor physiological dysfunctions such as myelosuppression or infection. In the research laboratory, ANC is a valuable measure to monitor the evolution of a wide range of disease states in disease models. Flow cytometry (FCM) is a fast, widely used approach to confidently identify thousands of cells within minutes. FCM can be optimised for absolute counting using spiked-in beads or by measuring the sample volume analysed. Here we combine the 1A8 antibody, specific for the mouse granulocyte protein Ly6G, with flow cytometric counting in straightforward FCM assays for mouse ANC, easily implementable in the research laboratory. Volumetric and Trucount™ bead assays were optimized for mouse neutrophils, and ANC values obtained with these protocols were compared to ANC measured by a dual-platform assay using the Orphee Mythic 18 veterinary haematology analyser. The single platform assays were more precise with decreased intra-assay variability compared with ANC obtained using the dual protocol. Defining ANC based on Ly6G expression produces a 15% higher estimate than the dual protocol. Allowing for this difference in ANC definition, the flow cytometry counting assays using Ly6G can be used reliably in the research laboratory to quantify mouse ANC from a small volume of blood. We demonstrate the utility of the volumetric protocol in a time-course study of chemotherapy induced neutropenia using four drug regimens.

  4. Reticulocyte quantification by flow cytometry, image analysis, and manual counting.

    PubMed

    Schimenti, K J; Lacerna, K; Wamble, A; Maston, L; Iaffaldano, C; Straight, M; Rabinovitch, A; Lazarus, H M; Jacobberger, J W

    1992-01-01

    Reticulocyte counting by flow cytometry with thiazole orange was compared to manual or automated counting of new methylene blue stained blood smears. Forty-nine samples were compared for manual counting from randomly chosen clinical samples. Two hundred and eighty-nine samples from bone marrow transplant patients were compared during the period before and through chemo-irradiation and engraftment. The slopes of correlation plots were less than 1 when flow cytometric data were the dependent variable, suggesting that thiazole orange is less sensitive than new methylene blue. In a third study, 407 samples from bone marrow transplant patients were compared after increasing the thiazole orange concentration. The reticulocyte fluorescence distribution was divided into four groups of the brightest (youngest) 40, 60, 80, and 100% of reticulocytes. The slopes from regression analysis were 0.25, 0.49, 0.78, and 1.14, respectively. This demonstrates that thiazole orange is more sensitive than new methylene blue because the window of analysis includes an increased fraction of mature reticulocytes. In addition, the precision of each assay as measured. The rank order of precision from high to low was flow cytometry > image analysis > manual counting.

  5. Cell-cooling in flow cytometry by Peltier elements.

    PubMed

    Göttlinger, C; Meyer, K L; Weichel, W; Müller, W; Raftery, B; Radbruch, A

    1986-05-01

    We have built a cooling device for cell suspensions in flow cytometry that makes use of the Peltier effect (Barnard RD, Thermo electricity in Metals and Alloys, Taylor and Francis, London; Siemens-Z 34:383-88, 1963). The prototype described here is used for cooling collection tubes during long-duration cell sorting and is capable of maintaining a temperature of 2-5 degrees C in a cell suspension of up to 3 ml. In general, Peltier element-based cooling is useful for equilibrating the temperature of small volumes of fluids. Furthermore, Peltier element-based cooling devices are easy to build and handle.

  6. Measurement and Characterization of Apoptosis by Flow Cytometry.

    PubMed

    Telford, William; Tamul, Karen; Bradford, Jolene

    2016-07-01

    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. Copyright © 2016 John Wiley & Sons, Inc.

  7. Neutrophil aggregation measured in whole blood by flow cytometry

    SciTech Connect

    Simon, S.I.; Sklar, L.A. Los Alamos National Lab., NM )

    1991-03-15

    Flow cytometry has enabled measurement of the kinetics of formyl peptide stimulated neutrophil aggregation and its dependence on the CD11b/CD18 adhesion molecule. The authors are currently measuring aggregation of neutrophils in whole blood using flow cytometry. Fresh whole blood samples were kept at 4C and stained with LDS-751 a vital nucleic stain. Cytometric detection of neutrophil aggregation in whole blood at 37C was achieved by thresholding on LDS-751 fluorescence and then gating on forward and right angle light scatter. Aggregation was up to 10 times more efficient in whole blood than in purified cells, despite the fact that the number of CD11b/CD18 sites was upregulated 5-10 fold in elutriated neutrophil preparations. The time course of whole blood aggregation was often irreversible as compared to elutriated cells. Aggregation was only partially blocked by preincubation with concentrations of antibody to the CD18 integrin effective in blocking aggregation in elutriated cells. Further study is needed to distinguish between the contributions of other cell types, as well as the activity and number of CD11b/CD18 adhesive sites on the kinetics and efficiency of neutrophil collisions in whole blood.

  8. 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

  9. Detection of Significant Bacteriuria by Automated Urinalysis Using Flow Cytometry

    PubMed Central

    Okada, Hiroshi; Sakai, Yutaka; Miyazaki, Shigenori; Arakawa, Soichi; Hamaguchi, Yukio; Kamidono, Sadao

    2000-01-01

    A new flow cytometry-based automated urine analyzer, the UF-50, was evaluated for its ability to screen urine samples for significant bacteriuria. One hundred eighty-six urine specimens from patients attending an outpatient clinic of a university-based hospital were examined. The results obtained with the UF-50 were compared with those obtained by conventional quantitative urine culture. The UF-50 detected significant bacteriuria with a sensitivity of 83.1%, a specificity of 76.4%, a positive predictive value of 62.0%, a negative predictive value of 90.7%, and an accuracy of 78.5%. These results are comparable to those obtained by previously reported screening procedures. Besides detecting significant bacteriuria, the UF-50 can also perform routine urinalysis, including measurement of concentrations of red blood cells, white blood cells, epithelial cells, and casts, within 70 s. This capability renders this new flow cytometry-based urine analyzer superior to previously reported rapid screening methods. PMID:10921941

  10. Thrombocytopenia: diagnosis with flow cytometry and antiplatelet antibodies.

    PubMed

    Guerra, João Carlos de Campos; Kanayama, Ruth Hissae; Nozawa, Sonia Tsukasa; Ioshida, Márcia Regina; Takiri, Irina Yoko; Lazaro, Robson José; Hamerschlak, Nelson; Rosenfeld, Luiz Gastão Mange; Guerra, Celso Carlos de Campos; Bacal, Nydia Strachman

    2011-06-01

    To identify antiplatelet antibodies by flow cytometry (direct method) in patients with thrombocytopenia. Between January 1997 and March 2004 a total of 15100 patients were referred to the Centro de Hematologia de São Paulo for hematological investigation of several diagnoses (anemia, leukopenia, thrombocytopenia, coagulation abnormalities, adenomegaly, leukemia and others). Of those, 1057 were referred because of thrombocytopenia and were divided into two groups: Group Idiopathic thrombocytopenic purpura, with no identifiable cause; and Group Other thrombocytopenia, which included low normal platelet counts cause to be established, hepatitis C and HIV infection, hypersplenism, EDTA-induced artifacts, laboratory error, and other causes. Flow cytometry immunophenotyping was done in 115 cases to identify platelet autoantibodies (direct method). Of the total number of patients, 1057 (7%) presented low platelet counts, 670 were females (63.4%) and age range of one to 75 years. Of the 115 cases (9.7%) submitted to immunophenotyping, the results were positive in 40% and the test was inconclusive in 5%. Idiopathic thrombocytopenic purpura was found in 52% of patients, more often in women. Hepatitis C virus infection was found in 7% and HIV infection in 1%. Low normal platelet counts were found in 17%, laboratory errors in 6%, and laboratory artifacts in 1% of cases. Platelet autoantibodies were found in 76.9% of all idiopathic thrombocytopenic purpura cases. It was negative in 83.3% of the low normal counts. antiplatelet autoantibodies when present help to diagnose idiopathic thrombocytopenic purpura. When absent, suggest other causes of thrombocytopenia.

  11. Zebrafish thrombocyte aggregation by whole blood aggregometry and flow cytometry.

    PubMed

    Sundaramoorthi, Hemalatha; Panapakam, Rekha; Jagadeeswaran, Pudur

    2015-01-01

    Zebrafish has become an excellent model system to study mammalian hemostasis. Despite our extensive efforts to develop technologies to measure zebrafish hemostasis and even with previously established thrombocyte qualitative and quantitative functional assays, quantifying thrombocyte function for high throughput applications has been a challenge. In this paper, we have developed two quantitative methods to estimate thrombocyte aggregation: one by whole blood aggregometry and the other by flow cytometry. We found that it is possible to conduct whole blood aggregometry using only 2 µl of blood and the currently available aggregometer. Each of three agonists, arachidonic acid, ADP, and collagen yielded impedance curves similar to those obtained with human blood. We were also able to use flow cytometry to indirectly quantify the extent of thrombocyte aggregation by labeling whole blood with mepacrine, aggregating in the presence of each of the above agonists, separating the aggregates from the white blood cells by centrifugation, and then sorting the resulting white cell fraction for thrombocyte numbers. These methods have high throughput capabilities and have the potential to be used in large scale screens to detect and characterize mutants with thrombocyte functional defects or to identify genes involved in thrombocyte function by large scale knockdowns.

  12. In vitro hematocrit measurement using spectrally encoded flow cytometry

    PubMed Central

    Zeidan, Adel; Golan, Lior; Yelin, Dvir

    2016-01-01

    Measuring key physiological parameters of small blood samples extracted from patients could be useful for real-time clinical diagnosis at the point of care. An important parameter required from all blood tests is the blood hematocrit, a measure of the fractional volume occupied by the red cells within the blood. In this work, we present a method for in vitro evaluation of hematocrit based on the data acquired using spectrally encoded flow cytometry. Analysis of the reflectance confocal images of blood within a flow chamber resulted in an error as low as 1.7% in the measured hematocrit. The technique could be used as part of an in vitro diagnostic system that measures important blood parameters at the point of care. PMID:27867734

  13. 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

  14. 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.

  15. Bleaching response of Symbiodinium (zooxanthellae): determination by flow cytometry.

    PubMed

    Lee, Co Sin; Yeo, Yin Sheng Wilson; Sin, Tsai Min

    2012-10-01

    Coral bleaching is of increasing concern to reef management and stakeholders. Thus far, quantification of coral bleaching tends to be heavily reliant on the enumeration of zooxanthellae, with less emphasis on assessment of photosynthetic or physiological condition, these being often assessed separately by techniques such as liquid chromatography. Traditional methods of enumeration using microscopy are time consuming, subjected to low precision and great observer error. In this study, we presented a method for the distinction of physoiological condition and rapid enumeration of zooxanthellae using flow cytometry (FCM). Microscopy verified that healthy looking/live versus damaged/dead zooxanthellae could be reliably and objectively distinguished and counted by FCM on the basis of red and green fluorescence and light scatter. Excellent correlations were also determined between FCM and microscopy estimates of cell concentrations of fresh zooxanthellae isolates from Pocillopora damicornis. The relative intensities of chlorophyll and β-carotene fluorescences were shown to be important in understanding the results of increased cell counts in freshly isolated zooxanthellae experimentally exposed to high temperatures (34, 36, and 38°C) over 24 h, with ambient temperature (29°C) used as controls. The ability to simultaneously identify and enumerate subpopulations of different physiological states in the same sample provides an enormous advantage in not just determining bleaching responses, but elucidating adaptive response and mechanisms for tolerance. Therefore, this approach might provide a rapid, convenient, and reproducible methodology for climate change studies and reef management programs. Copyright © 2012 International Society for Advancement of Cytometry.

  16. Assessment of erythrocyte shape by flow cytometry techniques

    PubMed Central

    Piagnerelli, M; Boudjeltia, K Zouaoui; Brohee, D; Vereerstraeten, A; Piro, P; Vincent, J‐L; Vanhaeverbeek, M

    2007-01-01

    Background Red blood cell (RBC) rheology is altered in different diseases, including acute conditions such as patients in intensive care units (ICU) with sepsis or with an inflammatory reaction due to postoperative states or intracerebral haemorrhage, or chronic conditions such as diabetes mellitus or terminal renal failure. Several techniques are available to assess alterations in RBC rheology, especially deformability, but they are too cumbersome to be used on a large number of cells. Objective To develop a new, rapid flow cytometry technique for easy assessment of RBC shape in patients. Methods In flow cytometry, healthy human RBC shape shows a bimodal distribution related to the biconcave form. On this histogram, the second Pearson coefficient of dissymmetry (PCD) representing the asymmetry of this histogram and the spherical index (M2:M1) were calculated, both representing the spherical shape. This technique was used in healthy volunteers (n = 17) and in diseases characterised by abnormalities in RBC rheology, including terminal renal failure requiring haemodialysis (n = 28), diabetes mellitus (n = 18), sepsis (n = 19) and acute inflammatory states (postoperative, intracerebral haemorrhage, chronic obstructive pulmonary disease, epilepsy or severe drug intoxication; n = 21). Multivariate analysis was performed to determine the factors influencing RBC shape. Results Measurement of RBC shape was highly reproducible. A good correlation was observed between the PCD and the spherical index, except in the critically ill patients without sepsis. RBCs were more spherical in patients with terminal renal failure (PCD −0.56 (0.14), p<0.05), diabetes mellitus (PCD −0.59 (0.23), p<0.05), sepsis (PCD −0.58 (0.22), p<0.05) or an acute inflammatory state (PCD −0.65 (0.29), p<0.05) than in healthy volunteers (PCD −0.89 (0.12)). The spherical index was also increased in all populations compared with healthy volunteers (terminal renal failure 2

  17. 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.

  18. 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

  19. A simple diagnostic test for Fanconi anemia by flow cytometry.

    PubMed

    Miglierina, R; Le Coniat, M; Berger, R

    1991-03-01

    A simple diagnostic test for Fanconi anemia (FA) by flow cytometry is proposed. It is based on the cell cycle disturbances of FA cells and their sensitisation by alkylating agents. Following PHA-stimulation of whole blood cell cultures in the presence or absence of nitrogen mustard, the accumulation of cells in G2/M phase was measured. A sharp increase of cells in G2/M was observed in cultures from FA patients when nitrogen mustard was added. This increase allows one to distinguish FA patients from patients with anemias of other origin, healthy controls, and FA heterozygotes, as effectively as chromosome breakage studies. The rapidity of the test and its reliability as demonstrated on the ten FA patients studied, will make the diagnosis of FA easier in centers without cytogenetic laboratory facilities.

  20. Detection and Quantification of Mitochondrial Fusion Using Imaging Flow Cytometry.

    PubMed

    Nascimento, Aldo; Lannigan, Joanne; Kashatus, David

    2017-07-05

    Mitochondria are dynamic organelles that perform several vital cellular functions. Requisite for these functions are mitochondrial fusion and fission. Despite the increasing importance of mitochondrial dynamics in a range of cellular processes, there exist limited methods for robust quantification of mitochondrial fission and fusion. Currently, the most widely used method to measure mitochondrial fusion is the polyethylene glycol (PEG) fusion assay. While this assay can provide useful information regarding fusion activity, the reliance on manual selection of rare fusion events is time consuming and may introduce selection bias. By utilizing the image-capture features and colocalization analysis of imaging flow cytometry in combination with the PEG fusion assay, we are able to develop a high-throughput method to detect and quantify mitochondrial fusion activity. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  1. An improved flow cytometry assay to monitor phagosome acidification.

    PubMed

    Colas, Chloé; Menezes, Shinelle; Gutiérrez-Martínez, Enric; Péan, Claire B; Dionne, Marc S; Guermonprez, Pierre

    2014-10-01

    Phago-lysosome formation is important for cell-autonomous immunity to intracellular pathogens, antigen presentation and metabolism. A hallmark feature of phago-lysosomal compartments is that they undergo progressive luminal acidification controlled by the activation of vacuolar V-ATPase. Acidification is required for many enzymatic processes taking place in phago-lysosomes, like proteolysis, and supports the microbicidal activity of macrophages. Here we present a new quantitative methodology to assess phagosome acidification by flow cytometry based on the use of bi-fluorescent particles. This method relies on the use of UV polystyrene beads labelled with the acid sensor pHrodo-succinimidyl ester (pHrodo(TM) SE red) and enables us to dissociate particle association with phagocytes from their engulfment in acidified compartments. This methodology is well suited to monitor the acidification of phagosomes formed in vivo after fluorescent bead administration.

  2. 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.

  3. [Immunophenotype in multiple myeloma cells detected by multiparameter flow cytometry].

    PubMed

    Cao, Fang-Fang; Chen, Fang; Hu, Yan-Ping; Zhang, Ji-Hong

    2012-06-01

    This study was purposed to investigate the immunophenotypic characteristics in multiple myeloma (MM) cells and their significance. Thirty three cases of MM and 12 cases of reactive plasmacytosis (as control group) were enrolled in the study. The expressions of surface antigens in MM cells were detected with flow cytometry by using direct immunofluorescent technique and gating method of CD38/SSC and were confirmed with morphologic observation of myeloma cells. The results indicated that the proportion of myeloma cells detected by morphologic examination was 6.0% - 76.0%. With CD38/SSC gating method, a cluster of CD38 bright positive cells could be detected in their scatter plot, the proportion ranged from 0.99% to 57.54%. Most phenotype of MM was 38(st+)CD138(+)CD19(-)CD56(+) (78.8%). While the expressions of CD20, CD33, CD117, HLA-DR were seen in some MM patients, the positive rates were 12.1%, 15.2%, 30.3%, 9.1%, respectively; the expression of other antigens was negative. cκ or cλ monoclonal restriction was detected in 27 cases (81.8%) of MM, both cκ and cλ in the remaining cases of MM was negative. It is concluded that detecting the immunophenotype of MM patients by flow cytometry with CD38/SSC gating method and basing on the heterogeneity of cell antigens can discriminate myeloma cells from normal plasma cells, which provides evidence for targeted therapy and prognosis evaluation.

  4. Determination of DNA Content of Aquatic Bacteria by Flow Cytometry

    PubMed Central

    Button, D. K.; Robertson, Betsy R.

    2001-01-01

    The distribution of DNA among bacterioplankton and bacterial isolates was determined by flow cytometry of DAPI (4′,6′-diamidino-2-phenylindole)-stained organisms. Conditions were optimized to minimize error from nonspecific staining, AT bias, DNA packing, changes in ionic strength, and differences in cell permeability. The sensitivity was sufficient to characterize the small 1- to 2-Mb-genome organisms in freshwater and seawater, as well as low-DNA cells (“dims”). The dims could be formed from laboratory cultivars; their apparent DNA content was 0.1 Mb and similar to that of many particles in seawater. Preservation with formaldehyde stabilized samples until analysis. Further permeabilization with Triton X-100 facilitated the penetration of stain into stain-resistant lithotrophs. The amount of DNA per cell determined by flow cytometry agreed with mean values obtained from spectrophotometric analyses of cultures. Correction for the DNA AT bias of the stain was made for bacterial isolates with known G+C contents. The number of chromosome copies per cell was determined with pure cultures, which allowed growth rate analyses based on cell cycle theory. The chromosome ratio was empirically related to the rate of growth, and the rate of growth was related to nutrient concentration through specific affinity theory to obtain a probe for nutrient kinetics. The chromosome size of a Marinobacter arcticus isolate was determined to be 3.0 Mb by this method. In a typical seawater sample the distribution of bacterial DNA revealed two major populations based on DNA content that were not necessarily similar to populations determined by using other stains or protocols. A mean value of 2.5 fg of DNA cell−1 was obtained for a typical seawater sample, and 90% of the population contained more than 1.1 fg of DNA cell−1. PMID:11282616

  5. 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...

  6. 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...

  7. Identification of inflammatory cells in bovine milk by flow cytometry.

    PubMed

    Redelman, D; Butler, S; Robison, J; Garner, D

    1988-09-01

    Cells recovered from normal or mastitic bovine milk were examined by flow cytometry. All milk samples contained particulate material that was heterogeneous in size and that produced a right-angle light-scatter signal equal to or greater than that produced by human or bovine neutrophils. Although this material labeled with Hoechst 33342, it produced fluorescence intensities below that of intact bovine cells, suggesting that it consisted of cell fragments. Mastitic milk additionally contained other populations of cells that were poorly resolved from the normal particulate material by size (electronic volume sensor) and right-angle light scatter. In order to improve this resolution, the milk cells were incubated with carboxydimethylfluorescein diacetate (CMFDA) to label intact cells. When milk samples labeled with CMFDA were examined by dual-parameter analysis using green fluorescence and right-angle light scatter, five or more populations of cells could be identified in mastitic milk. These populations included intact and degenerate neutrophils, lymphocytes, including both small and activated cells, monocytes, and large activated macrophages containing many vacuoles and phagocytosed particles. Using this procedure, all the animals in the University of Nevada-Reno Holstein dairy herd were tested once a month for 6 months. In addition, individual animals with mastitis were examined one or more times each day during the course of the inflammatory process. In the routine screening, the flow cytometric examination detected mastitis before overt symptoms developed. In cows identified to have mastitis, the flow cytometric examination provided prognostic information regarding the success of treatments.

  8. 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

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

    PubMed Central

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

    2016-01-01

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

  10. How fruit developmental biology makes use of flow cytometry approaches.

    PubMed

    Pirrello, Julien; Bourdon, Matthieu; Cheniclet, Catherine; Bourge, Mickaël; Brown, Spencer C; Renaudin, Jean-Pierre; Frangne, Nathalie; Chevalier, Christian

    2014-02-01

    Fleshy fruit species such as tomato are important because of their nutritional and economic value. Several stages of fruit development such as ovary formation, fruit set, and fruit maturation have already been the subject of many developmental studies. However, fruit growth per se has been much less addressed. Fruit growth like all plant organs depends upon the developmental processes of cell division and cell expansion. The activity of cell divisions sets the number of cells that will compose the fruit; the cell expansion activity then determines its final size. Among the various mechanisms that may influence the determination of cell size, endopolyploidy by the means of endoreduplication, i.e. genome amplification in the absence of mitosis, appears to be of great importance in fleshy fruits. In tomato fruit, endoreduplication is associated with DNA-dependent cell expansion: cell size can reach spectacular levels such as hundreds of times its initial size (e.g. >0.5 mm in diameter), with as much as a 256-fold increase in nuclear DNA content. Using tomato fruit development as a model, recent investigations combining the use of flow cytometry, cellular imaging and molecular analyses have provided new data in favor of the long-standing karyoplasmic ratio theory, stating that cells tend to adjust their cytoplasmic volume to the nuclear DNA content. By establishing a highly structured cellular system where multiple physiological functions are integrated, endoreduplication acts as a morphogenetic factor supporting cell growth during tomato fruit development. In the context of plant breeding, deciphering the mechanisms controlling fruit growth, in particular those connecting the process of nuclear endoreduplication with modulation of gene expression, the regulation of cell size and final fruit size and composition, is necessary to understand better the establishment of fleshy fruit quality traits. © 2013 International Society for Advancement of Cytometry.

  11. In Vivo Flow Cytometry of Circulating Tumor-Associated Exosomes

    PubMed Central

    Sarimollaoglu, Mustafa; Nedosekin, Dmitry A.; Jamshidi-Parsian, Azemat; Kore, Rajshekhar A.

    2016-01-01

    Circulating tumor cells (CTCs) demonstrated the potential as prognostic markers of metastatic development. However, the incurable metastasis can already be developed at the time of initial diagnosis with the existing CTC assays. Alternatively, tumor-associated particles (CTPs) including exosomes can be a more valuable prognostic marker because they can be released from the primary tumor long before CTCs and in larger amount. However, little progress has been made in high sensitivity detection of CTPs, especially in vivo. We show here that in vivo integrated photoacoustic (PA) and fluorescence flow cytometry (PAFFC) platform can provide the detection of melanoma and breast-cancer-associated single CTPs with endogenously expressed melanin and genetically engineered proteins or exogenous dyes as PA and fluorescent contrast agents. The two-beam, time-of-light PAFFC can measure the sizes of CTCs and CTPs and identify bulk and rolling CTCs and CTC clusters, with no influence on blood flow instability. This technique revealed a higher concentration of CTPs than CTCs at an early cancer stage. Because a single tumor cell can release many CTPs and in vivo PAFFC can examine the whole blood volume, PAFFC diagnostic platform has the potential to dramatically improve (up to 105-fold) the sensitivity of cancer diagnosis. PMID:27965916

  12. Biology and Flow Cytometry of Proangiogenic Hematopoietic Progenitors Cells

    PubMed Central

    Rose, Jonathan A.; Erzurum, Serpil; Asosingh, Kewal

    2015-01-01

    During development hematopoiesis and neovascularization are closely linked to each other via a common bipotent stem cell called the hemangioblast that gives rise to both hematopoietic cells and endothelial cells. In postnatal life this functional connection between the vasculature and hematopoiesis is maintained by a subset of hematopoietic progenitor cells endowed with the capacity to differentiate into potent proangiogenic cells. These proangiogenic hematopoietic progenitors comprise a specific subset of bone marrow-derived cells that homes to sites of neovascularization and possess potent paracrine angiogenic activity. There is emerging evidence that this subpopulation of hematopoietic progenitors plays a critical role in vascular health and disease. Their angiogenic activity is distinct from putative “endothelial progenitor cells” that become structural cells of the endothelium by differentiation into endothelial cells. Proangiogenic hematopoietic progenitor cell research requires multi-disciplinary expertise in flow cytometry, hematology and vascular biology. This review provides a comprehensive overview of proangiogenic hematopoietic progenitor cell biology and flow cytometric methods to detect these cells in the peripheral blood circulation and bone marrow. PMID:25418030

  13. Microchip-based flow cytometry for effective detection and count

    NASA Astrophysics Data System (ADS)

    Mu, Canjun; Zhang, Zhiyi; Lin, Min; Cao, Xudong; Zhang, Feiling

    2009-06-01

    High-throughput detection and identification of foodborne pathogens are in increasing demand for rapid bacteria detections in food safety and quality monitoring. As an effective method, microchip-based flow cytometry (microcytometery) has a potential to be less expensive and high throughout, and requires less bulky instrumentation than conventional methods. In this work, a low-cost and robust microcytometer with a simple optical setup was developed for demonstrating the high-throughput identification of foodborne bacterial pathogens that integrate sample flow focusing and detection into one testing procedure. High performance identification capability was achieved through simultaneously detecting the fluorescence and scatter light emitted from micro-fabricated channel, after designing and optimizing the laser shaping optical system and the micro-channel structure to improve the excitation light intensity as well as the detection sensitivity. In our configuration, the simple testing configuration with the collection angle of 42° in the orthogonal plane to micro chip presents the best SNR for both signals through simulation and systematic measurements. As a result, the maximum throughput of 83particles/s for the fluorescence-labelled bead with diameter of 1.013μm was obtained as well as the high detection efficiency (above 99%) and the correlation percentage (above 99.5%). Apart from the high detection sensitivity and identification power, this microcytometer also has the advantages of simple optical structure, compactness and ease in building.

  14. Diagnostic flow cytometry for low-grade myelodysplastic syndromes.

    PubMed

    Ogata, Kiyoyuki

    2008-12-01

    It has long been considered that flow cytometry (FCM) has little role in clinical practice in the diagnosis of myelodysplastic syndromes (MDS). However, recent advances in the analytical method and knowledge of MDS FCM are changing this stereotype. This paper reviews the concept and current status of FCM in the diagnosis of low-grade MDS. The diagnosis of low-grade MDS in the absence of ringed sideroblasts and chromosomal aberration is not always straightforward, and a report from a recent international working conference has proposed FCM as an adjunctive diagnostic test for such cases. Currently, only a limited number of laboratories are applying FCM to the diagnosis of MDS. Furthermore, standard analytical methods in FCM for MDS have not been established, and no single FCM parameter is sufficiently sensitive and specific to make the diagnosis of MDS. To establish MDS FCM as a widely accepted, dependable diagnostic tool, prospective studies should increase flow parameters that can be analysed reproducibly and determine their sensitivity and specificity, either alone or in combination. CD34+ cell-related parameters that are applicable for diagnosing low-grade MDS in many laboratories are introduced here.

  15. Modeling flow cytometry data for cancer vaccine immune monitoring.

    PubMed

    Frelinger, Jacob; Ottinger, Janet; Gouttefangeas, Cécile; Chan, Cliburn

    2010-09-01

    Flow cytometry (FCM) is widely used in cancer research for diagnosis, detection of minimal residual disease, as well as immune monitoring and profiling following immunotherapy. In all these applications, the challenge is to detect extremely rare cell subsets while avoiding spurious positive events. To achieve this objective, it helps to be able to analyze FCM data using multiple markers simultaneously, since the additional information provided often helps to minimize the number of false positive and false negative events, hence increasing both sensitivity and specificity. However, with manual gating, at most two markers can be examined in a single dot plot, and a sequential strategy is often used. As the sequential strategy discards events that fall outside preceding gates at each stage, the effectiveness of the strategy is difficult to evaluate without laborious and painstaking back-gating. Model-based analysis is a promising computational technique that works using information from all marker dimensions simultaneously, and offers an alternative approach to flow analysis that can usefully complement manual gating in the design of optimal gating strategies. Results from model-based analysis will be illustrated with examples from FCM assays commonly used in cancer immunotherapy laboratories.

  16. 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.

  17. Toward lower contrast computer vision in vivo flow cytometry.

    PubMed

    Markovic, Stacey; Siyuan Li; Tianxue Zhang; Niedre, Mark

    2014-01-01

    There are many applications in biomedical research where detection and enumeration of circulating cells (CCs) is important. Existing techniques involve drawing and enriching blood samples and analyzing them ex vivo. More recently, small animal "in vivo flow cytometry" (IVFC) techniques have been developed, where fluorescently-labeled cells flowing through small arterioles (ear, retina) are detected and counted. We recently developed a new high-sensitivity IVFC technique termed "Computer Vision(CV)-IVFC". Here, large circulating blood volumes were monitored in the ears of mice with a wide-field video-rate near-infrared (NIR) fluorescent camera. Cells were labeled with a membrane dye and were detected and tracked in noisy image sequences. This technique allowed enumeration of CCs in vivo with overall sensitivity better than 10 cells/mL. However, an ongoing area of interest in our lab is optimization of the system for lower-contrast imaging conditions, e.g. when CCs are weakly labeled, or in the case higher background autofluorescence with visible dyes. To this end, we developed a new optical flow phantom model to control autofluorescence intensity and physical structure to better mimic conditions observed in mice. We acquired image sequences from a series of phantoms with varying levels of contrast and analyzed the distribution of pixel intensities, and showed that we could generate similar conditions to those in vivo. We characterized the performance of our CV-IVFC algorithm in these phantoms with respect to sensitivity and false-alarm rates. Use of this phantom model in optimization of the instrument and algorithm under lower-contrast conditions is the subject of ongoing work in our lab.

  18. 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.

  19. Click Chemistry for Analysis of Cell Proliferation in Flow Cytometry.

    PubMed

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

    2017-10-02

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

  20. Detecting endotoxin with a flow cytometry-based magnetic aptasensor.

    PubMed

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

    2014-12-01

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

  1. Diagnostic Utility of Flow Cytometry in Myelodysplastic Syndromes

    PubMed Central

    Della Porta, Matteo G.; Picone, Cristina

    2017-01-01

    The pathological hallmark of myelodysplastic syndromes (MDS) is marrow dysplasia, which represents the basis of the WHO classification of these disorders. This classification provides clinicians with a useful tool for defining the different subtypes of MDS and individual prognosis. The WHO proposal has raised some concern regarding minimal diagnostic criteria particularly in patients with normal karyotype without robust morphological markers of dysplasia (such as ring sideroblasts or excess of blasts). Therefore, there is clearly need to refine the accuracy to detect marrow dysplasia. Flow cytometry (FCM) immunophenotyping has been proposed as a tool to improve the evaluation of marrow dysplasia. The rationale for the application of FCM in the diagnostic work up of MDS is that immunophenotyping is an accurate method for quantitative and qualitative evaluation of hematopoietic cells and that MDS have been found to have abnormal expression of several cellular antigens. To become applicable in clinical practice, FCM analysis should be based on parameters with sufficient specificity and sensitivity, data should be reproducible between different operators, and the results should be easily understood by clinicians. In this review, we discuss the most relevant progresses in detection of marrow dysplasia by FCM in MDS PMID:28293405

  2. 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

  3. Quantitative assessment of Mycoplasma hemadsorption activity by flow cytometry.

    PubMed

    García-Morales, Luis; González-González, Luis; Costa, Manuela; Querol, Enrique; Piñol, Jaume

    2014-01-01

    A number of adherent mycoplasmas have developed highly complex polar structures that are involved in diverse aspects of the biology of these microorganisms and play a key role as virulence factors by promoting adhesion to host cells in the first stages of infection. Attachment activity of mycoplasma cells has been traditionally investigated by determining their hemadsorption ability to red blood cells and it is a distinctive trait widely examined when characterizing the different mycoplasma species. Despite the fact that protocols to qualitatively determine the hemadsorption or hemagglutination of mycoplasmas are straightforward, current methods when investigating hemadsorption at the quantitative level are expensive and poorly reproducible. By using flow cytometry, we have developed a procedure to quantify rapidly and accurately the hemadsorption activity of mycoplasmas in the presence of SYBR Green I, a vital fluorochrome that stains nucleic acids, allowing to resolve erythrocyte and mycoplasma cells by their different size and fluorescence. This method is very reproducible and permits the kinetic analysis of the obtained data and a precise hemadsorption quantification based on standard binding parameters such as the dissociation constant K d. The procedure we developed could be easily implemented in a standardized assay to test the hemadsorption activity of the growing number of clinical isolates and mutant strains of different mycoplasma species, providing valuable data about the virulence of these microorganisms.

  4. Impedance Flow Cytometry: A Novel Technique in Pollen Analysis

    PubMed Central

    Lambalk, Joep; Ottiger, Marcel

    2016-01-01

    Introduction An efficient and reliable method to estimate plant cell viability, especially of pollen, is important for plant breeding research and plant production processes. Pollen quality is determined by classical methods, like staining techniques or in vitro pollen germination, each having disadvantages with respect to reliability, analysis speed, and species dependency. Analysing single cells based on their dielectric properties by impedance flow cytometry (IFC) has developed into a common method for cellular characterisation in microbiology and medicine during the last decade. The aim of this study is to demonstrate the potential of IFC in plant cell analysis with the focus on pollen. Method Developing and mature pollen grains were analysed during their passage through a microfluidic chip to which radio frequencies of 0.5 to 12 MHz were applied. The acquired data provided information about the developmental stage, viability, and germination capacity. The biological relevance of the acquired IFC data was confirmed by classical staining methods, inactivation controls, as well as pollen germination assays. Results Different stages of developing pollen, dead, viable and germinating pollen populations could be detected and quantified by IFC. Pollen viability analysis by classical FDA staining showed a high correlation with IFC data. In parallel, pollen with active germination potential could be discriminated from the dead and the viable but non-germinating population. Conclusion The presented data demonstrate that IFC is an efficient, label-free, reliable and non-destructive technique to analyse pollen quality in a species-independent manner. PMID:27832091

  5. 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.

  6. Quantification of cellular viability by automated microscopy and flow cytometry

    PubMed Central

    Sauvat, Allan; Wang, Yidan; Segura, Florian; Spaggiari, Sabrina; Müller, Kevin; Zhou, Heng; Galluzzi, Lrenzo; Kepp, Oliver; Kroemer, Guido

    2015-01-01

    Cellular viability is usually determined by measuring the capacity of cells to exclude vital dyes such as 4′,6-diamidino-2-phenylindole (DAPI), or by assessing nuclear morphology with chromatinophilic plasma membrane-permeant dyes, such as Hoechst 33342. However, a fraction of cells that exclude DAPI or exhibit normal nuclear morphology have already lost mitochondrial functions and/or manifest massive activation of apoptotic caspases, and hence are irremediably committed to death. Here, we developed a protocol for the simultaneous detection of plasma membrane integrity (based on DAPI) or nuclear morphology (based on Hoechst 33342), mitochondrial functions (based on the mitochondrial transmembrane potential probe DiOC6(3)) and caspase activation (based on YO-PRO®-3, which can enter cells exclusively upon the caspase-mediated activation of pannexin 1 channels). This method, which allows for the precise quantification of dead, dying and healthy cells, can be implemented on epifluorescence microscopy or flow cytometry platforms and is compatible with a robotized, high-throughput workflow. PMID:25816366

  7. Applications of flow cytometry in environmental microbiology and biotechnology.

    PubMed

    Bergquist, Peter L; Hardiman, Elizabeth M; Ferrari, Belinda C; Winsley, Tristrom

    2009-05-01

    Flow cytometry (FCM) is a technique for counting, examining and sorting microscopic particles suspended in a stream of fluid. It uses the principles of light scattering, light excitation and the emission from fluorescent molecules to generate specific multiparameter data from particles and cells. The cells are hydrodynamically focussed in a sheath solution before being intercepted by a focused light source provided by a laser. FCM has been used primarily in medical applications but is being used increasingly for the examination of individual cells from environmental samples. It has found uses in the isolation of both culturable and hitherto non-culturable bacteria present infrequently in environmental samples using appropriate growth conditions. FCM lends itself to high-throughput applications in directed evolution for the analysis of single cells or cell populations carrying mutant genes. It is also suitable for encapsulation studies where individual bacteria are compartmentalised with substrate in water-in-oil-in-water emulsions or with individual genes in transcriptional/translational mixtures for the production of mutant enzymes. The sensitivity of the technique has allowed the examination of gene optimisation by a procedure known as random or neutral drift where screening and selection is based on the retention of some predetermined level of activity through multiple rounds of mutagenesis.

  8. Reevaluating multicolor flow cytometry to assess microbial viability.

    PubMed

    Buysschaert, Benjamin; Byloos, Bo; Leys, Natalie; Van Houdt, Rob; Boon, Nico

    2016-11-01

    Flow cytometry is a rapid and quantitative method to determine bacterial viability. Although different stains can be used to establish viability, staining protocols are inconsistent and lack a general optimization approach. Very few "true" multicolor protocols, where dyes are combined in one sample, have been developed for microbiological applications. In this mini-review, the discrepancy between protocols for cell-permeant nucleic acid and functional stains are discussed as well as their use as viability dyes. Furthermore, optimization of staining protocols for a specific setup are described. Original data using the red-excitable SYTO dyes SYTO 59 to 64 and SYTO 17, combined with functional stains, for double and triple staining applications is also included. As each dye and dye combination behaves differently within a certain combination of medium matrix, microorganism, and instrument, protocols need to be tuned to obtain reproducible results. Therefore, single, double, and triple stains are reviewed, including the different parameters that influence staining such as stain kinetics, optimal stain concentration, and the effect of the chelator EDTA as membrane permeabilizer. In the last section, we highlight the need to investigate the stability of multicolor assays to ensure correct results as multiwell autoloaders are now commonly used.

  9. Impedance Flow Cytometry: A Novel Technique in Pollen Analysis.

    PubMed

    Heidmann, Iris; Schade-Kampmann, Grit; Lambalk, Joep; Ottiger, Marcel; Di Berardino, Marco

    2016-01-01

    An efficient and reliable method to estimate plant cell viability, especially of pollen, is important for plant breeding research and plant production processes. Pollen quality is determined by classical methods, like staining techniques or in vitro pollen germination, each having disadvantages with respect to reliability, analysis speed, and species dependency. Analysing single cells based on their dielectric properties by impedance flow cytometry (IFC) has developed into a common method for cellular characterisation in microbiology and medicine during the last decade. The aim of this study is to demonstrate the potential of IFC in plant cell analysis with the focus on pollen. Developing and mature pollen grains were analysed during their passage through a microfluidic chip to which radio frequencies of 0.5 to 12 MHz were applied. The acquired data provided information about the developmental stage, viability, and germination capacity. The biological relevance of the acquired IFC data was confirmed by classical staining methods, inactivation controls, as well as pollen germination assays. Different stages of developing pollen, dead, viable and germinating pollen populations could be detected and quantified by IFC. Pollen viability analysis by classical FDA staining showed a high correlation with IFC data. In parallel, pollen with active germination potential could be discriminated from the dead and the viable but non-germinating population. The presented data demonstrate that IFC is an efficient, label-free, reliable and non-destructive technique to analyse pollen quality in a species-independent manner.

  10. Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

    PubMed Central

    Inglis, Heather; Norris, Philip; Danesh, Ali

    2015-01-01

    Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal–to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes. PMID:25867010

  11. Hardware-software complex for diagnostics of breast cancer on the basis of flow cytometry

    NASA Astrophysics Data System (ADS)

    Selchuk, V. Y.; Shamilov, F. A.; Beznos, O. A.; Vorotnikov, I. K.; Polyakov, E. V.; Tupitsyn, N. N.

    2017-01-01

    The method of multicolor flow cytometry makes it possible to quantify the disseminated tumor cells (DTC) in patients with solid tumors. Application of the method multi-color flow cytometry showed its efficiency and accuracy in the detection of DTC in patients with breast cancer.

  12. 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.

  13. Alkaline unwinding flow cytometry assay to measure nucleotide excision repair.

    PubMed

    Thyagarajan, Bharat; Anderson, Kristin E; Lessard, Christopher J; Veltri, Gregory; Jacobs, David R; Folsom, Aaron R; Gross, Myron D

    2007-03-01

    Nucleotide excision repair (NER), one of the DNA repair pathways, is the primary mechanism for repair of bulky adducts caused by physical and chemical agents, such as UV radiation, cisplatin and 4-nitroquinolones. Variations in DNA repair may be a significant risk factor for several cancers, but its measurement in epidemiological studies has been hindered by the high variability, complexity and laborious nature of currently available assays. An alkaline unwinding flow cytometric assay using UV-C radiation as a DNA-damaging agent was adapted for measurement of NER-mediated breaks. This assay was based on the principle of alkaline unwinding of strand breaks in double-stranded DNA to yield single-stranded DNA with the number of strand breaks being proportional to the amount of DNA damage. This assay measured 50,000 events per sample with several samples being analyzed per specimen, thereby providing very reliable measurements, which can be performed on a large-scale basis. Using area under the curve (AUC) to quantitate amount of NER-mediated breaks, this assay was able to detect increased NER-mediated breaks with increasing doses of UV-C radiation. The assay detected NER-mediated breaks in lymphocytes from normal donors and not in xeroderma pigmentosum lymphoblastoid cell lines indicating specificity for the detection of NER-mediated breaks. The assay measured NER-mediated breaks within G(1), S and G(2)/M phases of the cell cycle; thereby decreasing variability in measurements of NER-mediated breaks due to differences in cell cycle phases. Intraindividual variability for AUC after 120 min of repair was 15% with interindividual variability being approximately 43% for cells in the G(1) phase, indicating substantial between-subject variation and relatively low within-subject variation. Thus, the alkaline unwinding flow cytometry-based assay provides a high-throughput method for the specific measurement of NER-mediated breaks in lymphocytes.

  14. Laser flow cytometry as a tool for the advancement of clinical medicine.

    PubMed

    Aebisher, David; Bartusik, Dorota; Tabarkiewicz, Jacek

    2017-01-01

    Flow cytometry is a classic laser technology. With the discovery of the cytometer, flow cytometry has become a primary tool in biodiagnostic research. This review focuses on current applications of flow cytometry to the diagnosis of disease and treatment monitoring at the single-cell level. A description of the principles of flow cytometry and a brief overview of the major applications are presented. Our criteria for selecting research papers for this review are those that show advances in biomedicine and pharmacotherapy achieved by using non-invasive flow cytometry. New concepts for diagnosis and classification based on quantitative measurements of cellular parameters and the expression of specific differentiation antigens on the surface of cells will be discussed herein. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  15. Improved sensitivity for solid-support invasive cleavage reactions with flow cytometry analysis.

    PubMed

    Stevens, P Wilkins; Rao, K V N; Hall, J G; Lyamichev, V; Neri, B P; Kelso, D M

    2003-01-01

    A new configuration of the solid-support invasive cleavage reaction provides a small reaction-volume format for high-sensitivity discrimination of nucleic acid targets with single nucleotide differences. With target concentrations as low as 2 amol/assay, the solid-support invasive cleavage reaction clearly distinguishes single base mutations. Two oligonucleotides tethered to the solid support hybridize to the target nucleic acid, forming a tripartite substrate that can be recognized and cleaved by Cleavase, a structure-specific 5'-nuclease. Each cleavage event yields fluorescence signal on the surface. When microspheres serve as the solid-support surface, analysis by fluorometer imparts real-time information about change in the reaction signal over time. Flow cytometry provides an alternative detection technology that collects endpoint information about the reaction signal on individual microspheres. A reaction volume of 10 microL with as few as 3000 microspheres is sufficient to distinguish single nucleotide differences at target concentrations less than 200 fM. This sensitivity level is within the range required for analysis of SNPs in genomic DNA. In addition, the flow cytometry format has multiplexing potential, making the microsphere-based invasive cleavage assay attractive for high-throughput genomic applications.

  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. Aequorea green fluorescent protein analysis by flow cytometry.

    PubMed

    Ropp, J D; Donahue, C J; Wolfgang-Kimball, D; Hooley, J J; Chin, J Y; Hoffman, R A; Cuthbertson, R A; Bauer, K 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 (Chalfie et al.: Science 263: 802-805, 1994). 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. (Nature 373:663-664, 1995) 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.

  18. Accurate Assessment of Cell Death by Imaging Flow Cytometry.

    PubMed

    Rieger, Aja M; Barreda, Daniel R

    2016-01-01

    The number of investigators using cell death analysis applications has greatly expanded since the introduction of flow cytometry. The Annexin V/propidium iodide (PI) method is among the most commonly used procedures and allows users to determine if cells are viable, apoptotic, or necrotic, based on changes in membrane lipid composition, integrity, and permeability. Unfortunately, PI can intercalate into RNA, in addition to DNA, which contributes to a large number of events showing PI staining within the cytoplasmic compartment. We show that this occurs across a broad range of animal primary cells and commonly used cell lines, and is most prevalent in large cells (nuclear:cytoplasmic ratio <0.5). Any cellular system where RNA levels change throughout an experiment will be particularly affected, such as those that utilize virally infected cells. As two examples, we highlight our recent work on cells infected with vesicular stomatitis virus (VSV), an RNA virus, and herpes simplex virus-1 (HSV-1), a DNA virus. Similarly, these issues are relevant to experimental systems where cells have increased RNA content such as during genotoxic stress, following exposure to cell cycle arrest drugs such as thymidine or hydroxyurea, or where developmental progression promotes discrete changes in cellular RNA synthesis. This chapter outlines a modified Annexin V/PI method that addresses cytoplasmic RNA staining issues to allow for accurate assessment of cell death. This protocol takes advantage of an additional cellular permeability caused by fixation to promote RNase A entry into the cell. Based on our observations, cell morphological parameters are well maintained and less than 5 % of total cellular events exhibit cytoplasmic PI staining under this protocol.

  19. Staging and monitoring of childhood rhabdomyosarcoma with flow cytometry

    PubMed Central

    SHEN, HONGQIANG; TANG, YONGMIN; DONG, AO; LI, HUAMEI; SHEN, DIYING; YANG, SHILONG; TANG, HONGFENG; GU, WEIZHONG; SHU, QIANG

    2014-01-01

    Patients with metastatic rhabdomyosarcoma (RMS) have a poor prognosis. The detection of contaminating RMS cells in the bone marrow (BM) is important in clinical staging and risk assessment. The cytological examination of the BM remains the gold standard for the diagnosis of RMS, but has a limited sensitivity. In the present study, 32 BM and two cerebrospinal fluid (CSF) samples from 11 patients with suspected metastasis were analyzed by flow cytometry (FCM) with ganglioside D2 (GD2) conjugated with fluorescein isothiocyanate, cluster of differentiation (CD)90-phycoerythrin, CD45-peridinin chlorophyll protein and CD56-allophycocyanin monoclonal antibody cocktail in parallel to morphological examination at diagnosis or during treatment. Five samples (14.7%) were positive for RMS onup morphological examination. By FCM, 16 samples (47.1%) were positive for RMS. A significant difference was identified between the two methods. The four-color FCM assay successfully detected RMS cells in BM samples to a level of 0.01% (1 per 104 cells). RMS cells demonstrated a phenotype with CD56+/CD90+/CD45−/GD2− expression, which is different from the CD56+/CD90+/CD45−/GD2+ expression phenotype in neuroblastoma cells. The follow-up of four patients by FCM demonstrated that two patients became minimal residual disease-negative following two and four cycles of chemotherapy, respectively, and survived. The other two cases remained FCM-positive despite receiving four courses of chemotherapy and consequently succumbed to progressive disease. In addition, FCM analysis of the CSF samples from one patient confirmed a diagnosis of CSF metastasis with RMS. In conclusion, FCM may have a role not only in staging and monitoring the effects of therapy, but also in providing diagnostic confirmation of CSF metastasis with RMS. PMID:24944652

  20. 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

  1. Applications of imaging flow cytometry in the diagnostic assessment of acute leukaemia.

    PubMed

    Grimwade, Lizz F; Fuller, Kathryn A; Erber, Wendy N

    2017-01-01

    Automated imaging flow cytometry integrates flow cytometry with digital microscopy to produce high-resolution digital imaging with quantitative analysis. This enables cell identification based on morphology (cell size, shape), antigen expression, quantification of fluorescence signal intensity and localisation of detected signals (i.e. surface, cytoplasm, nuclear). We describe applications of imaging flow cytometry for the diagnostic assessment of acute leukaemia. These bone marrow malignancies are traditionally diagnosed and classified by cell morphology, phenotype and cytogenetic abnormalities. Traditionally morphology is assessed by light microscopy, phenotyping by conventional flow cytometry and genetics by karyotype and fluorescence in situ hybridisation (FISH) on interphase nuclei/metaphase spreads of cells on slides. Imaging flow cytometry adds a new dimension to the diagnostic assessment of these neoplasms. We describe three specific applications: From this we conclude that imaging flow cytometry offers benefits over conventional diagnostic methods. Specifically the ability to visualise the cells of interest, the pattern and localisation of expressed antigens and assess cytogenetic abnormalities in one integrated automated high-throughput test. Imaging flow cytometry presents a new paradigm for the diagnostic assessment of leukaemia.

  2. Good Cell, Bad Cell: Flow Cytometry Reveals T-cell Subsets Important in HIV Disease

    PubMed Central

    Chattopadhyay, Pratip K.; Roederer, Mario

    2010-01-01

    Flow cytometry is a key technology in the study of HIV disease. In this article, we review various cellular markers that can be measured in the setting of pathogenesis or vaccination studies, including markers of activation, differentiation, senescence, immune suppression, and function. In addition, we discuss important considerations for making these measurements. Finally, we examine how flow cytometry studies have taught researchers about the disease process, and the potential for flow cytometry technology to guide treatment decisions and evaluate vaccine candidates in the future. PMID:20583275

  3. Preparing a Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) compliant manuscript using the International Society for Advancement of Cytometry (ISAC) FCS file repository (FlowRepository.org).

    PubMed

    Spidlen, Josef; Breuer, Karin; Brinkman, Ryan

    2012-07-01

    FlowRepository.org is a Web-based flow cytometry data repository provided by the International Society for Advancement of Cytometry (ISAC). It supports storage, annotation, analysis, and sharing of flow cytometry datasets. A fundamental tenet of scientific research is that published results should be open to independent validation and refutation. With FlowRepository, researchers can annotate their datasets in compliance with the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard, thus greatly facilitating third-party interpretation of their data. In this unit, we will mainly focus on the deposition, sharing, and annotation of flow cytometry data.

  4. flowVS: channel-specific variance stabilization in flow cytometry

    DOE PAGES

    Azad, Ariful; Rajwa, Bartek; Pothen, Alex

    2016-07-28

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

  5. Visualization of pulmonary clearance mechanisms via noninvasive optical imaging validated by near-infrared flow cytometry.

    PubMed

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

    2015-05-01

    In vivo optical imaging with near-infrared (NIR) probes is an established method of diagnostics in preclinical and clinical studies. However, the specificities of these probes are difficult to validate ex vivo due to the lack of NIR flow cytometry. To address this limitation, we modified a flow cytometer to include an additional NIR channel using a 752 nm laser line. The flow cytometry system was tested using NIR microspheres and cell lines labeled with a combination of visible range and NIR fluorescent dyes. The approach was verified in vivo in mice evaluated for immune response in lungs after intratracheal delivery of the NIR contrast agent. Flow cytometry of cells obtained from the lung bronchoalveolar lavage demonstrated that the NIR dye was taken up by pulmonary macrophages as early as 4-h post-injection. This combination of optical imaging with NIR flow cytometry extends the capability of imaging and enables complementation of in vivo imaging with cell-specific studies.

  6. 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

  7. Flow cytometry in the exploration of the physiopathology of occupational lung disease

    PubMed Central

    Curran, A. D.

    1999-01-01

    Flow cytometry is a simple analytical technique used for the investigation of cells taken from various sources. Cells are identified by both their physical characteristics and the presence or absence of specific molecules on the cell surface. These molecules may be either phenotypic, or induced by a specific stimulus. Flow cytometry has been used to identify the nature and extent of the immune response in several occupational respiratory conditions including occupational asthma, irritant induced respiratory problems, and asbestos related lung disease. Also, it may be of value in monitoring workplace exposure to some hazardous materials. Although of limited diagnostic value at present, the technique has provided an insight into the modulation of immune cells, and their function, in people exposed to hazardous materials in the workplace. In this review, the principals of flow cytometry will be explored and the use of flow cytometry to investigate occupational respiratory disease will be discussed.   PMID:10658559

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

    PubMed

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

    2017-09-18

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

  9. Implementation of erythroid lineage analysis by flow cytometry in diagnostic models for myelodysplastic syndromes

    PubMed Central

    Cremers, Eline M.P.; Westers, Theresia M.; Alhan, Canan; Cali, Claudia; Visser-Wisselaar, Heleen A.; Chitu, Dana A.; van der Velden, Vincent H.J.; te Marvelde, Jeroen G.; Klein, Saskia K.; Muus, Petra; Vellenga, Edo; de Greef, Georgina E.; Legdeur, Marie-Cecile C.J.C.; Wijermans, Pierre W.; Stevens-Kroef, Marian J.P.L.; da Silva-Coelho, Pedro; Jansen, Joop H.; Ossenkoppele, Gert J.; van de Loosdrecht, Arjan A.

    2017-01-01

    Flow cytometric analysis is a recommended tool in the diagnosis of myelodysplastic syndromes. Current flow cytometric approaches evaluate the (im)mature myelo-/monocytic lineage with a median sensitivity and specificity of ~71% and ~93%, respectively. We hypothesized that the addition of erythroid lineage analysis could increase the sensitivity of flow cytometry. Hereto, we validated the analysis of erythroid lineage parameters recommended by the International/European LeukemiaNet Working Group for Flow Cytometry in Myelodysplastic Syndromes, and incorporated this evaluation in currently applied flow cytometric models. One hundred and sixty-seven bone marrow aspirates were analyzed; 106 patients with myelodysplastic syndromes, and 61 cytopenic controls. There was a strong correlation between presence of erythroid aberrancies assessed by flow cytometry and the diagnosis of myelodysplastic syndromes when validating the previously described erythroid evaluation. Furthermore, addition of erythroid aberrancies to two different flow cytometric models led to an increased sensitivity in detecting myelodysplastic syndromes: from 74% to 86% for the addition to the diagnostic score designed by Ogata and colleagues, and from 69% to 80% for the addition to the integrated flow cytometric score for myelodysplastic syndromes, designed by our group. In both models the specificity was unaffected. The high sensitivity and specificity of flow cytometry in the detection of myelodysplastic syndromes illustrates the important value of flow cytometry in a standardized diagnostic approach. The trial is registered at www.trialregister.nl as NTR1825; EudraCT n.: 2008-002195-10 PMID:27658438

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

    PubMed

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

    2017-08-01

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

  11. Image and flow cytometry: companion techniques for adherent and non-adherent cell analysis and sorting.

    PubMed

    Métézeau, P

    1993-01-01

    Flow cytometry (FMC) is an analytical and preparative technique whereas image analysis is only applied to cell analysis. Recently, image analysis has been adapted as a preparative method using a new technique: image cytometry for analysis and sorting (ICAS). FCM and ICAS are complementary. Flow cytometry allows rapid, quantitative and precise study of fluorescence and light scattering in a large number of cells in suspension, while ICAS analyses fewer cells (adherent cells or tissue) on the basis of fluorescence, morphology and size. ICAS can use these criteria to destroy unwanted cells and hence sort selected cells. ICAS can also be used for confocal microscopy and laser surgery.

  12. [Assessment of bactericidal and growth-inhibiting activity of blood serum using flow cytometry and photometry].

    PubMed

    Budikhina, A S; Mikhaĭlova, N A; Bitkova, E E; Khvatov, V B; Pinegin, B V

    2007-01-01

    Method of measurement of biological fluids bactericidal activity against Staphylococcus aureus using laser flow cytometry has been developed and proposed for clinical use. Overall bactericidal activity of sera of healthy donors has been assessed by this method. Strong positive correlation between bactericidal activity measured by flow cytometry and ability of the sera of healthy donors to inhibit bacterial growth assessed by photometric method was determined. High degree of positive correlation between results of cytometry and classical microbiological method of measurement of mentioned parameters has been shown.

  13. 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

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

    PubMed

    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 routine

  15. Imaging Flow Cytometry for the Study of Erythroid Cell Biology and Pathology

    PubMed Central

    Samsel, Leigh; McCoy, J Philip

    2015-01-01

    Erythroid cell maturation and diseases affecting erythrocytes are frequently accompanied by morphologic and immunophenotypic changes to these cells. In the past, these changes have been assessed primarily through the use of manual microscopy, which substantially limits the statistical rigor, throughput, and objectivity of these studies. Imaging flow cytometry provides a technology to examine both the morphology of cells as well as to quantify the staining intensity and signal distribution of numerous fluorescent markers on a cell-by-cell basis with high throughput in a statistically robust manner, and thus is ideally suited to studying erythroid cell biology. To date imaging flow cytometry has been used to study erythrocytes in three areas: 1) erythroid cell maturation, 2) sickle cell disease, and 3) infectious diseases such as malaria. In the maturation studies, imaging flow cytometry can closely recapitulate known stages of maturation and has led to the identification of a new population of erythroid cell precursors. In sickle cell disease, imaging flow cytometry provides a robust method to quantify sickled erythrocytes and to identify cellular aggregates linked to morbidities, and in malaria, imaging flow cytometry has been used to screen for new chemotherapeutic agents. These studies have demonstrated the value of imaging flow cytometry for investigations of erythrocyte biology and pathology. PMID:25858229

  16. 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. © 2016 International Society for Advancement of Cytometry.

  17. 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...

  18. 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...

  19. Diagnosis of chronic lymphoproliferative disorders by flow cytometry using four-color combinations for immunophenotyping: A proposal of the brazilian group of flow cytometry (GBCFLUX).

    PubMed

    Sales, M M; Ferreira, S I A C P; Ikoma, M R V; Sandes, A F; Beltrame, M P; Bacal, N S; Silva, M C A; Malvezzi, M; Lorand-Metze, I G H; Orfao, A; Yamamoto, M

    2017-09-01

    Multiparametric flow cytometry (MFC) is a powerful tool for the diagnosis of hematological malignancies and has been useful for the classification of chronic lymphoproliferative disorders (CLPD) according to the WHO criteria. Following the purposes of the Brazilian Group of Flow Cytometry (GBCFLUX), the aim of this report was to standardize the minimum requirements to achieve an accurate diagnosis in CLPDs, considering the different economic possibilities of the laboratories in our country. Most laboratories in Brazil work with 4-fluorescence flow cytometers, which is why the GBCFLUX CLPD Committee has proposed 4-color monoclonal antibody (MoAb) panels. Panels for screening and diagnosis in B, T and NK lymphoproliferative disorders were developed based on the normal differentiation pathways of these cells and the most frequent phenotypic aberrations. Important markers for prognosis and for minimal residual disease (MRD) evaluation were also included. The MoAb panels presented here were designed based on the diagnostic expertise of the participating laboratories and an extensive literature review. The 4-color panels presented to aid in the diagnosis of lymphoproliferative neoplasms by GBCFLUX aim to provide clinical laboratories with a systematic, step-wise, cost-effective, and reproducible approach to obtain an accurate immunophenotypic diagnosis of the most frequent of these disorders. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

  20. flowVS: channel-specific variance stabilization in flow cytometry.

    PubMed

    Azad, Ariful; Rajwa, Bartek; Pothen, Alex

    2016-07-28

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

  1. Multiplexed microbead immunoassays by flow cytometry for molecular profiling: Basic concepts and proteomics applications.

    PubMed

    Krishhan, V V; Khan, Imran H; Luciw, Paul A

    2009-01-01

    Flow cytometry was originally established as an automated method for measuring optical or fluorescence characteristics of cells or particles in suspension. With the enormous increase in development of reliable electronics, lasers, micro-fluidics, as well as many advances in immunology and other fields, flow cytometers have become user-friendlier, less-expensive instruments with an increasing importance for both basic research and clinical applications. Conventional uses of flow cytometry include immunophenotyping of blood cells and the analysis of the cell cycle. Importantly, methods for labeling microbeads with unique combinations of fluorescent spectral signatures have made multiplex analysis of soluble analytes (i.e. the ability to detect multiple targets in a single test sample) feasible by flow cytometry. The result is a rapid, high-throughput, sensitive, and reproducible detection technology for a wide range of biomedical applications requiring detection of proteins (in cells and biofluids) and nucleic acids. Thus, novel methods of flow cytometry are becoming important for diagnostic purposes (e.g. identifying multiple clinical biomarkers for a wide range of diseases) as well as for developing novel therapies (e.g. elucidating drug mechanisms and potential toxicities). In addition, flow cytometry for multiplex analysis, coupled with automated sample handling devices, has the potential to significantly enhance proteomics research, particularly analysis of post-translational modifications of proteins, on a large scale. Inherently, flow cytometry methods are strongly rooted in the laws of the physics of optics, fluidics, and electromagnetism. This review article describes principles and early sources of flow cytometry, provides an introduction to the multiplex microbead technology, and discusses its applications and advantages in comparison to other methods. Anticipated future directions, particularly for translational research in medicine, are also discussed.

  2. 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

  3. FuGEFlow: data model and markup language for flow cytometry.

    PubMed

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

    2009-06-16

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

  4. Use of flow cytometry for high-throughput cell population estimates in brain tissue

    PubMed Central

    Young, Nicole A.; Flaherty, David K.; Airey, David C.; Varlan, Peter; Aworunse, Feyi; Kaas, Jon H.; Collins, Christine E.

    2012-01-01

    The large size of primate brains is an impediment to obtaining high-resolution cell number maps of the cortex in humans and non-human primates. We present a rapid, flow cytometry-based cell counting method that can be used to estimate cell numbers from homogenized brain tissue samples comprising the entire cortical sheet. The new method, called the flow fractionator, is based on the isotropic fractionator (IF) method (Herculano-Houzel and Lent, 2005), but substitutes flow cytometry analysis for manual, microscope analysis using a Neubauer counting chamber. We show that our flow cytometry-based method for total cell estimation in homogenized brain tissue provides comparable data to that obtained using a counting chamber on a microscope. The advantages of the flow fractionator over existing methods are improved precision of cell number estimates and improved speed of analysis. PMID:22798947

  5. Use of flow cytometry for high-throughput cell population estimates in brain tissue.

    PubMed

    Young, Nicole A; Flaherty, David K; Airey, David C; Varlan, Peter; Aworunse, Feyi; Kaas, Jon H; Collins, Christine E

    2012-01-01

    The large size of primate brains is an impediment to obtaining high-resolution cell number maps of the cortex in humans and non-human primates. We present a rapid, flow cytometry-based cell counting method that can be used to estimate cell numbers from homogenized brain tissue samples comprising the entire cortical sheet. The new method, called the flow fractionator, is based on the isotropic fractionator (IF) method (Herculano-Houzel and Lent, 2005), but substitutes flow cytometry analysis for manual, microscope analysis using a Neubauer counting chamber. We show that our flow cytometry-based method for total cell estimation in homogenized brain tissue provides comparable data to that obtained using a counting chamber on a microscope. The advantages of the flow fractionator over existing methods are improved precision of cell number estimates and improved speed of analysis.

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

    NASA Astrophysics Data System (ADS)

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

    2017-02-01

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

  7. Nonselective enrichment for yeast adenine mutants by flow cytometry

    NASA Technical Reports Server (NTRS)

    Bruschi, C. V.; Chuba, P. J.

    1988-01-01

    The expression of certain adenine biosynthetic mutations in the yeast Saccharomyces cerevisiae results in a red colony color. This phenomenon has historically provided an ideal genetic marker for the study of mutation, recombination, and aneuploidy in lower eukaryotes by classical genetic analysis. In this paper, it is reported that cells carrying ade1 and/or ade2 mutations exhibit primary fluorescence. Based on this observation, the nonselective enrichment of yeast cultures for viable adenine mutants by using the fluorescence-activated cell sorter has been achieved. The advantages of this approach over conventional genetic analysis of mutation, recombination, and mitotic chromosomal stability include speed and accuracy in acquiring data for large numbers of clones. By using appropriate strains, the cell sorter has been used for the isolation of both forward mutations and chromosomal loss events in S. cerevisiae. The resolving power of this system and its noninvasiveness can easily be extended to more complex organisms, including mammalian cells, in which analogous metabolic mutants are available.

  8. Nonselective enrichment for yeast adenine mutants by flow cytometry

    NASA Technical Reports Server (NTRS)

    Bruschi, C. V.; Chuba, P. J.

    1988-01-01

    The expression of certain adenine biosynthetic mutations in the yeast Saccharomyces cerevisiae results in a red colony color. This phenomenon has historically provided an ideal genetic marker for the study of mutation, recombination, and aneuploidy in lower eukaryotes by classical genetic analysis. In this paper, it is reported that cells carrying ade1 and/or ade2 mutations exhibit primary fluorescence. Based on this observation, the nonselective enrichment of yeast cultures for viable adenine mutants by using the fluorescence-activated cell sorter has been achieved. The advantages of this approach over conventional genetic analysis of mutation, recombination, and mitotic chromosomal stability include speed and accuracy in acquiring data for large numbers of clones. By using appropriate strains, the cell sorter has been used for the isolation of both forward mutations and chromosomal loss events in S. cerevisiae. The resolving power of this system and its noninvasiveness can easily be extended to more complex organisms, including mammalian cells, in which analogous metabolic mutants are available.

  9. Multiplexed labeling of viable cells for high-throughput analysis of glycine receptor function using flow cytometry.

    PubMed

    Gilbert, Daniel F; Wilson, John C; Nink, Virginia; Lynch, Joseph W; Osborne, Geoffrey W

    2009-05-01

    Flow cytometry is an important drug discovery tool because it permits high-content multiparameter analysis of individual cells. A new method dramatically enhanced screening throughput by multiplexing many discrete fixed cell populations; however, this method is not suited to assays requiring functional cellular responses. HEK293 cells were transfected with unique mutant glycine receptors. Mutant receptor expression was confirmed by coexpression of yellow fluorescent protein (YFP). Commercially available cell-permeant dyes were used to label each glycine receptor expressing mutant with a unique optical code. All encoded cell lines were combined in a single tube and analyzed on a flow cytometer simultaneously before and after the addition of glycine receptor agonist. We decoded multiplexed cells that expressed functionally distinct glycine receptor chloride channels and analyzed responses to glycine in terms of chloride-sensitive YFP expression. Here, data provided by flow cytometry can be used to discriminate between functional and nonfunctional mutations in the glycine receptor, a process accelerated by the use of multiplexing. Further, this data correlates to data generated using a microscopy-based technique. The present study demonstrates multiplexed labeling of live cells, to enable cell populations to be subject to further cell culture and experimentation, and compares the results with those obtained using live cell microscopy. (c) 2009 International Society for Advancement of Cytometry.

  10. In vitro micronucleus assay for the analysis of total particulate matter in cigarette smoke: comparison of flow cytometry and laser scanning cytometry with microscopy.

    PubMed

    Yao, Jianhua; Gao, Qian; Mi, Qili; Li, Xuemei; Miao, Mingming; Cheng, Peng; Luo, Ying

    2013-08-15

    The possible genotoxicity of the total particulate matter (TPM) in cigarette smoke has typically been evaluated using the in vitro micronucleus assay. In recent years, automated scoring techniques have been developed to replace the manual counting process in this assay. However, these automated scoring techniques have not been applied in routine genotoxicity assays for the analysis of TPM to improve the assay efficiency. Chinese hamster ovary (CHO) cells were treated with TPM produced from 14 types of cigarettes at five concentrations (25-200μg/ml) without exogenous metabolic activation. The three following methods were used to score the micronucleus (MN) frequency: (a) flow cytometry with SYTOX and EMA dyes, which differentially stain micronuclei and apoptotic/necrotic chromatin to enhance assay reliability; (b) laser scanning cytometry with FITC and PI dyes, which is a system that combines the analytical capabilities of flow and image cytometry; and (c) visual microcopy with Giemsa dye. The test results obtained using the three methods were compared using correlation analysis. The key findings for this set of compounds include the following: (a) both flow cytometry- and laser scanning cytometry-based methods were effective for MN identification, (b) the three scoring methods could detect dose-dependent micronucleus formation for the 14 types of TPM, and (c) the MN frequencies that were measured in the same samples by flow cytometry, laser scanning cytometry, and visual microscopy were highly correlated, and there were no significant differences (p>0.05). In conclusion, both flow cytometry and laser scanning cytometry can be used to evaluate the MN frequency induced by TPM without exogenous metabolic activation. The simpler and faster processing and the high correlation of the results make these two automatic methods appropriate tools for use in in vitro micronucleus assays for the analysis of TPM using CHO cells.

  11. Flow cytometry immunophenotypic analysis of Philadelphia-negative myeloproliferative neoplasms: Correlation with histopathologic features.

    PubMed

    Ouyang, Juan; Zheng, Wenli; Shen, Qi; Goswami, Maitrayee; Jorgensen, Jeffrey L; Medeiros, L Jeffrey; Wang, S A

    2015-01-01

    Compared with the proven utility of flow cytometry immunophenotyping (FCI) analysis in the workup of myelodysplastic syndromes (MDS), immunophenotypic alterations in myeloproliferative neoplasms (MPN) have been less studied and the potential utility of FCI is not defined. Bone marrow (BM) samples of 83 Philadelphia-negative MPN patients were assessed by multicolor FCI including 27 with essential thrombocythemia (ET); 17 polycythemia vera (PV); 33 primary myelofibrosis (PMF) and 6 MPN-unclassifiable (MPN-U). The time interval from initial diagnosis of MPN to FCI analysis was 18 months (0-370). Ninety-five age-matched MDS patients with a similar BM blast count were included for comparison. Immunophenotypic alterations, either in CD34(+) cells or myelomonocytic cells, were detected in 82 of 83 (99%) MPN cases. FCI abnormalities were more frequently observed in cases with substantial myelofibrosis but not different between PMF and fibrotic stage of ET/PV. Furthermore, FCI abnormalities were more frequent in cases with ≥5% BM blasts and/or circulating blasts (P = 0.006); as well as cases with an abnormal karyotype (P = 0.036); but not associated with morphologic dysplasia or JAK2 mutation status. Comparing with MDS, FCI abnormalities were overall less pronounced in MPN cases (P = 0.001). MPNs exhibit frequent immunophenotypic alterations, more pronounced in cases with adverse histopathologic features. These findings illustrate that immunophenotypic alterations are a part of constellational findings in MPN, and correlate progressively with disease stage. The study results also suggest a role of FCI in diagnosis of MPN and monitoring disease over time and after therapy. © 2014 International Clinical Cytometry Society.

  12. Flow cytometry immunophenotypic analysis of Philadelphia-negative myeloproliferative neoplasms: Correlation with histopathologic features.

    PubMed

    Ouyang, Juan; Zheng, Wenli; Shen, Qi; Goswami, Maitrayee; Jorgensen, Jeffrey L; Medeiros, L Jeffrey; Wang, Sa A

    2014-12-11

    Background: Compared with the proven utility of flow cytometry immunophenotyping (FCI) analysis in the workup of myelodysplastic syndromes (MDS), immunophenotypic alterations in myeloproliferative neoplasms (MPN) have been less studied and the potential utility of FCI is not defined. Methods: Bone marrow (BM) samples of 83 Philadelphia-negative MPN patients were assessed by multicolor FCI including 27 with essential thrombocythemia (ET); 17 polycythemia vera (PV); 33 primary myelofibrosis (PMF) and 6 MPN-unclassifiable (MPN-U). The time interval from initial diagnosis of MPN to FCI analysis was 18 months (0-370). Ninety-five age-matched MDS patients with a similar BM blast count were included for comparison. Results: Immunophenotypic alterations, either in CD34(+) cells or myelomonocytic cells, were detected in 82 of 83 (99%) MPN cases. FCI abnormalities were more frequently observed in cases with substantial myelofibrosis but not different between PMF and fibrotic stage of ET/PV. Furthermore, FCI abnormalities were more frequent in cases with ≥5% BM blasts and/or circulating blasts (p=0.006); as well as cases with an abnormal karyotype (p=0.036); but not associated with morphologic dysplasia or JAK2 mutation status. Comparing with MDS, FCI abnormalities were overall less pronounced in MPN cases (p=0.001). Conclusions: MPNs exhibit frequent immunophenotypic alterations, more pronounced in cases with adverse histopathologic features. These findings illustrate that immunophenotypic alterations are a part of constellational findings in MPN, and correlate progressively with disease stage. The study results also suggest a role of FCI in diagnosis of MPN and monitoring disease over time and after therapy. This article is protected by copyright. All rights reserved. Copyright © 2014 Clinical Cytometry Society.

  13. 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

  14. First proposed panels on acute leukemia for four-color immunophenotyping by flow cytometry from the Brazilian group of flow cytometry-GBCFLUX.

    PubMed

    Ikoma, Maura R V; Sandes, Alex F; Thiago, Leandro S; Cavalcanti Júnior, Geraldo B; Lorand-Metze, Irene G H; Costa, Elaine S; Pimenta, Glicinia; Santos-Silva, Maria C; Bacal, Nydia S; Yamamoto, Mihoko; Souto, Elizabeth X

    2015-01-01

    Multiparameter flow cytometry is a highly sensitive, fast, and specific diagnostic technology with a wide range of applicability in hematology. Although well-established eight-color immunophenotyping panels are already available, most Brazilian clinical laboratories are equipped with four-color flow cytometer facilities. Based on this fact, the Brazilian Group of Flow Cytometry (Grupo Brasileiro de Citometria de Fluxo, GBCFLUX) for standardization of clinical flow cytometry has proposed an antibody panel designed to allow precise diagnosis and characterization of acute leukemia (AL) within resource-restricted areas. Morphological analysis of bone marrow smears, together with the screening panel, is mandatory for the primary identification of AL. The disease-oriented panels proposed here are divided into three levels of recommendations (mandatory, recommendable, and optional) in order to provide an accurate final diagnosis, as well as allow some degree of flexibility based on available local resources and patient-specific needs. The proposed panels will be subsequently validated in an interlaboratory study to evaluate its effectiveness on the diagnosis and classification of AL. (Assoc editor comm. 2).

  15. Amphiphilic mediated sample preparation for micro-flow cytometry

    DOEpatents

    Clague, David S [Livermore, CA; Wheeler, Elizabeth K [Livermore, CA; Lee, Abraham P [Irvine, CA

    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.

  16. 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.

  17. A liposome-based size calibration method for measuring microvesicles by flow cytometry.

    PubMed

    Simonsen, J B

    2016-01-01

    ESSENTIALS: A gold standard to determine the sizes of microvesicles by flow cytometry is needed. We used fluorescently labeled liposomes to estimate the size of microvesicles with flow cytometry. We suggest that liposomes are more accurate size calibrators than the commonly used polystyrene beads. The liposome-based size calibrators improve the size assessment of microvesicle made with flow cytometry. During the past years, the need for a gold standard to determine the sizes of extracellular vesicles including microvesicles by flow cytometry has been emphasized. This work suggests that artificial vesicles can be used as calibrators to estimate the size of microvesicles from the side scattering (SSC) measured with flow cytometry. We prepared fluorescently labeled liposomes with different maximum sizes defined by the pore size (200, 400, 800, and 1000 nm) of the membrane used for the extrusion. The fluorescence strengths from the largest liposomes pertaining to each pore size enabled us to verify the correlation between the SSC from a liposome and the corresponding size. This study indicates that artificial vesicles are more accurate size calibrators compared to the commonly used polystyrene calibrator beads illustrated by the SSC from 110 nm polystyrene beads corresponds to the scattering from ~400 nm vesicle-like particles. We also show that this method of size assessment based on SSC has a low resolution that is roughly estimated to be between 60 and 200 nm, dependent on the vesicle size. © 2015 International Society on Thrombosis and Haemostasis.

  18. Use of flow cytometry for rapid and accurate enumeration of live pathogenic Leptospira strains.

    PubMed

    Fontana, Célia; Crussard, Steve; Simon-Dufay, Nathalie; Pialot, Daniel; Bomchil, Natalia; Reyes, Jean

    2017-01-01

    Enumeration of Leptospira, the causative agent of leptospirosis, is arduous mainly because of its slow growth rate. Rapid and reliable tools for numbering leptospires are still lacking. The current standard for Leptospira cultures is the count on Petroff-Hausser chamber under dark-field microscopy, but this method remains time-consuming, requires well-trained operators and lacks reproducibility. Here we present the development of a flow-cytometry technique for counting leptospires. We showed that upon addition of fluorescent dyes, necessary to discriminate the bacterial population from debris, several live Leptospira strains could be enumerated at different physiologic states. Flow cytometry titers were highly correlated to counts with Petroff-Hausser chambers (R(2)>0.99). Advantages of flow cytometry lie in its rapidity, its reproducibility significantly higher than Petroff-Hausser method and its wide linearity range, from 10(4) to 10(8)leptospires/ml. Therefore, flow cytometry is a fast, reproducible and sensitive tool representing a promising technology to replace current enumeration techniques of Leptospira in culture. We were also able to enumerate Leptospira in artificially infected urine and blood with a sensitivity limit of 10(5)leptospires/ml and 10(6)leptospires/ml, respectively, demonstrating the feasibility to use flow cytometry as first-line tool for diagnosis or bacterial dissemination studies.

  19. Detection of Intracellular ADAMTS13, a Secreted Zinc-metalloprotease, via Flow Cytometry

    PubMed Central

    S., Geetha; Allen, Courtni E.; Hunt, Ryan; Plum, Elizabeth; Garfield, Susan; Friedman, Scott L.; Soejima, Kenji; Sauna, Zuben E.; Kimchi-Sarfaty, Chava

    2009-01-01

    Background ADAMTS13 is a secreted metalloprotease that cleaves von Willebrand Factor multimers and maintains proper homeostasis. A severe deficiency in ADAMTS13 triggers a disorder known as thrombotic thrombocytopenic purpura (TTP). At present, ADAMTS13 expression levels are determined by immunoblotting. Methods We established a flow cytometry methodology to detect intracellular ADAMTS13 in liver and kidney cells using a polyclonal antibody, BL154G, and several monoclonal antibodies previously used to detect ADAMTS13 by immunoblotting. Results were validated using confocal microscopy, immunoblotting and an activity assay (FRETS-VWF73). Results We show that labeling ADAMTS13 with specific antibodies and detection by flow cytometry yields results that are comparable to previously established methods for ADAMTS13 detection. Specifically, we compared the endogenous expression levels of ADAMTS13 in various liver cell lines using flow cytometry and obtained results that parallel immunoblot analysis. Knock-down of ADAMTS13 expression via targeted siRNA resulted in significantly reduced median signal, displaying the sensitivity of this detection method. A further analysis of reliability and specificity was achieved through plasmid DNA and transfection reagent dose response studies. Conclusions The flow cytometry method described here is useful in determining the expression of both endogenous and recombinant forms of intracellular ADAMTS13. Flow cytometry is a convenient, efficient and cost effective way to measure the expression levels of ADAMTS13. PMID:19526483

  20. 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

  1. Flow Cytometry, a Versatile Tool for Diagnosis and Monitoring of Primary Immunodeficiencies

    PubMed Central

    Aubert, Geraldine

    2016-01-01

    Genetic defects of the immune system are referred to as primary immunodeficiencies (PIDs). These immunodeficiencies are clinically and immunologically heterogeneous and, therefore, pose a challenge not only for the clinician but also for the diagnostic immunologist. There are several methodological tools available for evaluation and monitoring of patients with PIDs, and of these tools, flow cytometry has gained prominence, both for phenotyping and functional assays. Flow cytometry allows real-time analysis of cellular composition, cell signaling, and other relevant immunological pathways, providing an accessible tool for rapid diagnostic and prognostic assessment. This minireview provides an overview of the use of flow cytometry in disease-specific diagnosis of PIDs, in addition to other broader applications, which include immune phenotyping and cellular functional measurements. PMID:26912782

  2. Methodology and Application of Flow Cytometry for Investigation of Human Malaria Parasites

    PubMed Central

    Grimberg, Brian T.

    2011-01-01

    Historically, examinations of the inhibition of malaria parasite growth/invasion, whether using drugs or antibodies, have relied on the use of microscopy or radioactive hypoxanthine uptake. These are considered gold standards for measuring the effectiveness of antimalarial treatments, however, these methods have well known shortcomings. With the advent of flow cytometry coupled with the use of fluorescent DNA stains allowed for increased speed, reproducibility, and qualitative estimates of the effectiveness of antibodies and drugs to limit malaria parasite growth which addresses the challenges of traditional techniques. Because materials and machines available to research facilities are so varied, different methods have been developed to investigate malaria parasites by flow cytometry. This review is intended to serve as a reference guide for advanced users and importantly, as a primer for new users, to support expanded use and improvements to malaria flow cytometry, particularly in endemic countries. PMID:21296083

  3. An integrated, multiparametric flow cytometry chip using "microfluidic drifting" based three-dimensional hydrodynamic focusing.

    PubMed

    Mao, Xiaole; Nawaz, Ahmad Ahsan; Lin, Sz-Chin Steven; Lapsley, Michael Ian; Zhao, Yanhui; McCoy, J Philip; El-Deiry, Wafik S; Huang, Tony Jun

    2012-06-01

    In this work, we demonstrate an integrated, single-layer, miniature flow cytometry device that is capable of multi-parametric particle analysis. The device integrates both particle focusing and detection components on-chip, including a "microfluidic drifting" based three-dimensional (3D) hydrodynamic focusing component and a series of optical fibers integrated into the microfluidic architecture to facilitate on-chip detection. With this design, multiple optical signals (i.e., forward scatter, side scatter, and fluorescence) from individual particles can be simultaneously detected. Experimental results indicate that the performance of our flow cytometry chip is comparable to its bulky, expensive desktop counterpart. The integration of on-chip 3D particle focusing with on-chip multi-parametric optical detection in a single-layer, mass-producible microfluidic device presents a major step towards low-cost flow cytometry chips for point-of-care clinical diagnostics.

  4. Quantifying autophagy: Measuring LC3 puncta and autolysosome formation in cells using multispectral imaging flow cytometry.

    PubMed

    Pugsley, Haley R

    2017-01-01

    The use of multispectral imaging flow cytometry has been gaining popularity due to its quantitative power, high throughput capabilities, multiplexing potential and its ability to acquire images of every cell. Autophagy is a process in which dysfunctional organelles and cellular components that accumulate during growth and differentiation are degraded via the lysosome and recycled. During autophagy, cytoplasmic LC3 is processed and recruited to the autophagosomal membranes; the autophagosome then fuses with the lysosome to form the autolysosome. Therefore, cells undergoing autophagy can be identified by visualizing fluorescently labeled LC3 puncta and/or the co-localization of fluorescently labeled LC3 and lysosomal markers. Multispectral imaging flow cytometry is able to collect imagery of large numbers of cells and assess autophagy in an objective, quantitative, and statistically robust manner. This review will examine the four predominant methods that have been used to measure autophagy via multispectral imaging flow cytometry.

  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. Flow cytometry data analysis: comparing large multivariate data sets using classification trees

    SciTech Connect

    Norman, J.

    1994-12-31

    This paper describes a method to compare flow cytometry data sets, which typically contain 50,000 six-parameter measurements each. By this method, the data points in two such data sets are divided into subpopulations using a binary classification tree generated from the data. The {chi}{sup 2} test is then used to establish the homogeneity of the two data sets based on how their data are distributed across these subpopulations. Preliminary results indicate that this comparison method is sufficiently sensitive to detect differences between flow cytometry data sets that are too subtle for human investigators to notice.

  7. Nuclear DNA content analysis of plant seeds by flow cytometry.

    PubMed

    Sliwinska, Elwira

    2006-02-01

    Procedures describing the utilization of seeds or their parts for flow cytometric determination of plant ploidy and endopolyploidy, genome size, and cell cycle activity are presented. The methods have been developed for a single-fluorescence-parameter flow cytometer, equipped with light sources for 488-nm and UV-light illumination. The procedures presented in this unit utilize the two most widely used fluorochromes for plant DNA content analysis, propidium iodide (PI) and 4',6-diamidino-2-phenylindole (DAPI). These methods provide an alternative to estimation of DNA content based on the fluorescence of DNA in cell nuclei isolated from plant leaves. In some instances seeds are more suitable for analysis than leaves, e.g., when plant material must be transported for a long distances or stored for prolonged periods before flow cytometric analysis, or when leaves contain fluorochrome-staining inhibitors. In addition, flow cytometric determination of nuclear replication stages in seeds gives information about their physiological status (e.g., maturity, advancement of germination), which is valuable to seed producers and technologists.

  8. Statistical mixture modeling for cell subtype identification in flow cytometry.

    PubMed

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

    2008-08-01

    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. 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 were fitted with a mixture of multivariate Gaussians using standard Bayesian statistical approaches and Markov chain Monte Carlo computations. 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. This article describes the 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.

  9. Report of the results of the International Clinical Cytometry Society and American Society for Clinical Pathology workload survey of clinical flow cytometry laboratories.

    PubMed

    Wolniak, Kristy; Goolsby, Charles; Choi, Sarah; Ali, Asma; Serdy, Nina; Stetler-Stevenson, Maryalice

    2016-07-01

    Thorough review of current workload, staffing, and testing practices in clinical laboratories allows for optimization of laboratory efficiency and quality. This information is largely missing with regard to clinical flow cytometry laboratories. The purpose of this survey is to provide comprehensive, current, and accurate data on testing practices and laboratory staffing in clinical laboratories performing flow cytometric studies. Survey data was collected from flow cytometry laboratories through the ASCP website. Data was collected on the workload during a 1-year time period of full-time and part-time technical and professional (M.D./D.O./Ph.D. or equivalent) flow cytometry employees. Workload was examined as number of specimens and tubes per full time equivalent (FTE) technical and professional staff. Test complexity, test result interpretation, and reporting practices were also evaluated. There were 205 respondent laboratories affiliated predominantly with academic and health system institutions. Overall, 1,132 FTE employees were reported with 29% professional FTE employees and 71% technical. Fifty-one percent of the testing performed was considered high complexity and 49% was low complexity. The average number of tubes per FTE technologist was 1,194 per year and the average number of specimens per FTE professional was 1,659 per year. The flow cytometry reports were predominantly written by pathologists (57%) and were typically written as a separate report (58%). This survey evaluates the overall status of the current practice of clinical flow cytometry and provides a comprehensive dataset as a framework to help laboratory departments, directors, and managers make appropriate, cost-effective staffing decisions. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

  10. 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.

  11. Quantifying spore viability of the honey bee pathogen Nosema apis using flow cytometry.

    PubMed

    Peng, Yan; Lee-Pullen, Tracey F; Heel, Kathy; Millar, A Harvey; Baer, Boris

    2014-05-01

    Honey bees are hosts to more than 80 different parasites, some of them being highly virulent and responsible for substantial losses in managed honey bee populations. The study of honey bee pathogens and their interactions with the bees' immune system has therefore become a research area of major interest. Here we developed a fast, accurate and reliable method to quantify the viability of spores of the honey bee gut parasite Nosema apis. To verify this method, a dilution series with 0, 25, 50, 75, and 100% live N. apis was made and SYTO 16 and Propidium Iodide (n = 35) were used to distinguish dead from live spores. The viability of spores in each sample was determined by flow cytometry and compared with the current method based on fluorescence microscopy. Results show that N. apis viability counts using flow cytometry produced very similar results when compared with fluorescence microscopy. However, we found that fluorescence microscopy underestimates N. apis viability in samples with higher percentages of viable spores, the latter typically being what is found in biological samples. A series of experiments were conducted to confirm that flow cytometry allows the use of additional fluorescent dyes such as SYBR 14 and SYTOX Red (used in combination with SYTO 16 or Propidium Iodide) to distinguish dead from live spores. We also show that spore viability quantification with flow cytometry can be undertaken using substantially lower dye concentrations than fluorescence microscopy. In conclusion, our data show flow cytometry to be a fast, reliable method to quantify N. apis spore viabilities, which has a number of advantages compared with existing methods. © 2013 International Society for Advancement of Cytometry.

  12. 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.

  13. Ultrasonic analyte concentration and application in flow cytometry

    DOEpatents

    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.

  14. 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.

  15. 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.

  16. 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.

  17. Computational prediction of manually gated rare cells in flow cytometry data1

    PubMed Central

    Qiu, Peng

    2015-01-01

    Rare cell identification is an interesting and challenging question in flow cytometry data analysis. In the literature, manual gating is a popular approach to distill flow cytometry data and drill down to the rare cells of interest, based on prior knowledge of measured protein markers and visual inspection of the data. Several computational algorithms have been proposed for rare cell identification. To compare existing algorithms and promote new developments, FlowCAP-III put forward one computational challenge that focused on this question. The challenge provided flow cytometry data for 202 training samples and two manually gated rare cell types for each training sample, roughly 0.02% and 0.04% of the cells, respectively. In addition, flow cytometry data for 203 testing samples were provided, and participants were invited to computationally identify the rare cells in the testing samples. Accuracy of the identification results was evaluated by comparing to manual gating of the testing samples. We participated in the challenge, and developed a method that combined the Hellinger divergence, a downsampling trick and the ensemble SVM. Our method achieved the highest accuracy in the challenge. PMID:25755118

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2016-01-01

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

  1. 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

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

    PubMed

    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

    2015-07-01

    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.

  3. Cell-cycle analysis of fission yeast cells by flow cytometry.

    PubMed

    Knutsen, Jon Halvor Jonsrud; Rein, Idun Dale; Rothe, Christiane; Stokke, Trond; Grallert, Beáta; Boye, Erik

    2011-02-28

    The cell cycle of the fission yeast, Schizosaccharomyces pombe, does not easily lend itself to analysis by flow cytometry, mainly because cells in G(1) and G(2) phase contain the same amount of DNA. This occurs because fission yeast cells under standard growth conditions do not complete cytokinesis until after G(1) phase. We have devised a flow cytometric method exploiting the fact that cells in G(1) phase contain two nuclei, whereas cells in G(2) are mononuclear. Measurements of the width as well as the total area of the DNA-associated fluorescence signal allows the discrimination between cells in G(1) and in G(2) phase and the cell-cycle progression of fission yeast can be followed in detail by flow cytometry. Furthermore, we show how this method can be used to monitor the timing of cell entry into anaphase. Fission yeast cells tend to form multimers, which represents another problem of flow cytometry-based cell-cycle analysis. Here we present a method employing light-scatter measurements to enable the exclusion of cell doublets, thereby further improving the analysis of fission yeast cells by flow cytometry.

  4. Detection of an early cytomegalovirus antigen with two-color quantitative flow cytometry.

    PubMed

    Elmendorf, S; McSharry, J; Laffin, J; Fogleman, D; Lehman, J M

    1988-05-01

    An early cytomegalovirus (CMV) antigen was detected with a monoclonal antibody by two-color fluorescent flow cytometry. With the aid of a human diploid fibroblast cell strain, FLOW 2000, infected with the AD169 strain of CMV, the viral antigen and the DNA content of infected or uninfected cells were measured. There was no evidence of change in the cell-cycle distribution of the infected cells. The viral antigen was detected within 30 minutes following virus adsorption at 0.1 and 1.0 plaque-forming units/cells; and the percentage of positive cells increased with time and viral dosage. All stages of the cell cycle were susceptible to viral infection and the average fluorescence was greater than the background fluorescence. Flow cytometry detected the viral antigen earlier than conventional immunofluorescent microscopy and cell culture for CMV cytopathological effect (CPE). Ten bronchoalveolar lavages assayed by flow cytometry and conventional diagnostic procedures demonstrated that flow cytometry might be useful in early diagnosis for CMV infection.

  5. Validation of a Flow Cytometry Based Binding Assay for Evaluation of Monoclonal Antibody Recognizing EGF Receptor

    PubMed Central

    Cedeño-Arias, Mercedes; Sánchez-Ramírez, Javier; Blanco-Santana, Rancés; Rengifo-Calzado, Enrique

    2011-01-01

    An ideal test used to characterize a product must be appropriate for the measurement of product quality, manufacturing consistency, product stability, and comparability studies. Flow cytometry has been successfully applied to the examination of antibodies and receptors on membrane surfaces; however, to date, the analytical validation of cytometry based assays is limited. Here we report on the validation of a flow cytometry-based assay used in the evaluation of nimotuzumab binding to cells over-expressing EGFR on cell surface. The assay was validated by examining, assay robustness, specificity, repeatability and intermediate precision. The assay was highly specific, robust for all studied factors except for cell fixation with 1% paraformaldehyde and met criteria for precision with RSD < 2%. In addition the assay has stability-indicating properties evidenced by the ability to detect changes in mAb degraded samples. Most importantly, the assay demonstrated to be useful for its intended use. PMID:21886904

  6. Receptor occupancy assessment by flow cytometry as a pharmacodynamic biomarker in biopharmaceutical development

    PubMed Central

    Schwickart, Martin; Schneider, Amy K.; Vainshtein, Inna; Del Nagro, Christopher; Standifer, Nathan; Roskos, Lorin K.

    2015-01-01

    Receptor occupancy (RO) assays are designed to quantify the binding of therapeutics to their targets on the cell surface and are frequently used to generate pharmacodynamic (PD) biomarker data in nonclinical and clinical studies of biopharmaceuticals. When combined with the pharmacokinetic (PK) profile, RO data can establish PKPD relationships, which are crucial for informing dose decisions. RO is commonly measured by flow cytometry on fresh blood specimens and is subject to numerous technical and logistical challenges. To ensure that reliable and high quality results are generated from RO assays, careful assay design, key reagent characterization, data normalization/reporting, and thorough planning for implementation are of critical importance during development. In this article, the authors share their experiences and perspectives in these areas and discuss challenges and potential solutions when developing and implementing a flow cytometry‐based RO method in support of biopharmaceutical drug development. © 2015 The Authors Cytometry Part B: Clinical Cytometry Published by Wiley Periodicals, Inc. PMID:26054054

  7. Cell cycle analysis by flow cytometry: principles and applications.

    PubMed

    Jayat, C; Ratinaud, M H

    1993-01-01

    Numerous flow cytometric analyses are based on DNA content studies. We have considered firstly monoparametric cell cycle analyses, which only take DNA content into account, but are sometimes of limited interest. Then, we have presented multiparametric analyses, which can be used to improve cycle phase identification by taking simultaneously into account DNA and other cellular components, or by considering some events occurring during cell cycle. Finally, we have discussed monoparametric and multiparametric cell cycle analysis interest in various application fields, particularly in pharmacology, toxicology, tumoral pathology and higher plant system studies.

  8. 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.

  9. Sex-sorting sperm using flow cytometry/cell sorting.

    PubMed

    Garner, Duane L; Evans, K Michael; Seidel, George E

    2013-01-01

    The sex of mammalian offspring can be predetermined by flow sorting relatively pure living populations of X- and Y-chromosome-bearing sperm. This method is based on precise staining of the DNA of sperm with the nucleic acid-specific fluorophore, Hoechst 33342, to differentiate between the subpopulations of X- and Y-sperm. The fluorescently stained sperm are then sex-sorted using a specialized high speed sorter, MoFlo(®) SX XDP, and collected into biologically supportive media prior to reconcentration and cryopreservation in numbers adequate for use with artificial insemination for some species or for in vitro fertilization. Sperm sorting can provide subpopulations of X- or Y-bearing bovine sperm at rates in the 8,000 sperm/s range while maintaining; a purity of 90% such that it has been applied to cattle on a commercial basis. The sex of offspring has been predetermined in a wide variety of mammalian species including cattle, swine, horses, sheep, goats, dogs, cats, deer, elk, dolphins, water buffalo as well as in humans using flow cytometric sorting of X- and Y-sperm.

  10. Prognostic value of ZAP-70 expression in chronic lymphocytic leukemia as assessed by quantitative polymerase chain reaction and flow cytometry.

    PubMed

    Adams, Rebecca L C; Cheung, Catherine; Banh, Raymond; Saal, Russell; Cross, Donna; Gill, Devinder; Self, Marlene; Klein, Kerenaftali; Mollee, Peter

    2014-03-01

    Chronic lymphocytic leukemia (CLL) is a disorder in which the tempo of disease progression is highly variable, and prognostic markers that can be utilized at diagnosis are regarded as clinically important. Currently, there are several prognostic factors, such as immunoglobulin heavy chain (IgVH) mutational status, and ZAP-70 protein expression in neoplastic B-cells, that have demonstrated significant discriminative power in the prognostication of CLL. They are, however, largely unavailable in the routine diagnostic laboratory setting. In this study, we characterized the IgVH status and ZAP-70 expression by molecular techniques in a cohort of 108 patients with CLL, and correlated these results with three different methods of ZAP-70 expression by flow cytometry. We then assessed the results of these methods in terms of prognostic power as characterized by time to first treatment (TTFT). By comparing three different flow cytometry methods using receiver–operator curve (ROC) analysis, we identified that by utilizing a corrected mean fluorescence intensity (CorrMFI) algorithm for assessing ZAP-70 expression, there was good correlation with both IgVH mutational status, and ZAP-70 expression as assessed by qPCR. We were also able to show that ZAP-70 expression, as assessed by both qPCR and the CorrMFI method, was prognostic of TTFT. While confirmation in a larger patient cohort, with longer follow-up is required, we believe that the CorrMFI represents the most promising method currently available in a routine diagnostic setting for the assessment of ZAP-70 expression in CLL patients. © 2013 International Clinical Cytometry Society.

  11. Flow cytometry-based DNA hybridization and polymorphism analysis

    SciTech Connect

    Cai, H.; Kommander, K.; White, P.S.; Nolan, J.P.

    1998-07-01

    Functional analysis of the humane 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. The authors are developing a suite of microsphere-based approaches employing fluorescence detection to screen and analyze genomic sequence. The 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 advances 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.

  12. 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.

  13. Comparison of clustering methods for high-dimensional single-cell flow and mass cytometry data.

    PubMed

    Weber, Lukas M; Robinson, Mark D

    2016-12-01

    Recent technological developments in high-dimensional flow cytometry and mass cytometry (CyTOF) have made it possible to detect expression levels of dozens of protein markers in thousands of cells per second, allowing cell populations to be characterized in unprecedented detail. Traditional data analysis by "manual gating" can be inefficient and unreliable in these high-dimensional settings, which has led to the development of a large number of automated analysis methods. Methods designed for unsupervised analysis use specialized clustering algorithms to detect and define cell populations for further downstream analysis. Here, we have performed an up-to-date, extensible performance comparison of clustering methods for high-dimensional flow and mass cytometry data. We evaluated methods using several publicly available data sets from experiments in immunology, containing both major and rare cell populations, with cell population identities from expert manual gating as the reference standard. Several methods performed well, including FlowSOM, X-shift, PhenoGraph, Rclusterpp, and flowMeans. Among these, FlowSOM had extremely fast runtimes, making this method well-suited for interactive, exploratory analysis of large, high-dimensional data sets on a standard laptop or desktop computer. These results extend previously published comparisons by focusing on high-dimensional data and including new methods developed for CyTOF data. R scripts to reproduce all analyses are available from GitHub (https://github.com/lmweber/cytometry-clustering-comparison), and pre-processed data files are available from FlowRepository (FR-FCM-ZZPH), allowing our comparisons to be extended to include new clustering methods and reference data sets. © 2016 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.

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

    PubMed

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

    2017-06-28

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

  15. Detection of micrometastases by flow cytometry in sentinel lymph nodes from patients with renal tumours.

    PubMed

    Hartana, Ciputra Adijaya; Kinn, Johan; Rosenblatt, Robert; Anania, Stefan; Alamdari, Farhood; Glise, Hans; Sherif, Amir; Winqvist, Ola

    2016-10-11

    Stage is an important prognostic factor in renal tumours and dissemination to regional lymph nodes is associated with poor outcomes. Lymph nodes are routinely assessed by immunohistochemistry and microscopic evaluation, a time-consuming process where micrometastases might go undiagnosed. We evaluate an alternative method for detecting metastatic cells in sentinel nodes (SNs) by flow cytometry. A total of 15 nodes from 5 patients diagnosed with renal tumours were analysed by flow cytometry. Staining for the intracellular marker cytokeratin 18 (CK18) with the surface markers carbonic anhydrase IX (CA9) and Cadherin 6 were used in flow cytometry analysis. Peripheral blood mononuclear cells (PBMCs) with the addition of known concentrations of cancer cell lines were analysed to investigate the sensitivity of micrometastasis detection. Stability of the assay was marked by low intra-assay variability (coefficient of variance ⩽16%) and low inter-assay variability (R(2)=0.9996-1). Eight nodes in four patients were positive for metastasis; six of them were considered being micrometastatic. These metastases were undetected by routine pathology and the patients were restaged from pN0 to pN1. Flow cytometry is able to detect micrometastases in lymph nodes of renal tumour patients that were undetected under H&E examination.

  16. 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…

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

    EPA Science Inventory

    Rationale: The evaluation of sputum leukocytes by flow cytometry is an opportunity to assess characteristics of cells residing in the central airways, yet it is hampered by certain inherent properties of sputum including mucus and large amounts of contaminating cells and debris. ...

  18. Evaluating the spore genome sizes of ferns and lycophytes: a flow cytometry approach.

    PubMed

    Kuo, Li-Yaung; Huang, Yi-Jia; Chang, JenYu; Chiou, Wen-Liang; Huang, Yao-Moan

    2017-03-01

    Ferns and lycophytes produce spores to initiate the gametophyte stage for sexual reproduction. Approximately 10% of these seedless vascular plants are apomictic, and produce genomic unreduced spores. Genome size comparisons between spores and leaves are a reliable, and potentially easier way to determine their reproductive mode compared to traditional approaches. However, estimation of the spore genome sizes of these plants has not been attempted. We attempted to evaluate the spore genome sizes of ferns and lycophytes using flow cytometry, collected spores from selected species representing different spore physical properties and taxonomic groups, and sought to optimize bead-vortexing conditions. By evaluating the spore and sporophyte genome sizes, we examined whether reproductive modes could be ascertained from these flow cytometry results. We proposed two separate sets of optimized bead-vortexing conditions for the nuclear extraction of green and nongreen spores. We further successfully extracted spore nuclei of 19 families covering most orders, and the qualities and quantities of these extractions satisfied the C-value criteria. These evaluated genome sizes further supported the reproductive modes reported previously. In the current study, flow cytometry was used for the first time to evaluate the spore genome sizes of ferns and lycophytes. This use of spore flow cytometry provides a new, efficient approach to ascertaining the reproductive modes of these plants.

  19. 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.

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

    SciTech Connect

    Kovtun, Oleg; Ross, Emily J.; Tomlinson, Ian D.; Rosenthal, Sandra J.

    2012-04-05

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

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

    PubMed

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

    2017-03-23

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

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

    EPA Science Inventory

    Rationale: The evaluation of sputum leukocytes by flow cytometry is an opportunity to assess characteristics of cells residing in the central airways, yet it is hampered by certain inherent properties of sputum including mucus and large amounts of contaminating cells and debris. ...

  3. 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

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

    PubMed

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

    2000-08-01

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

  5. Evaluation of Cellular Wound Healing Using Flow Cytometry and Expanded Polytetrafluroethylene Implants

    PubMed Central

    Tsuji, Joyce M.; Whitney, JoAnne D.; Tolentino, Ernesto J.; Perrin, Margot E.; Swanson, Paul E.

    2010-01-01

    In wound healing studies that investigate therapeutic interventions, it is important to characterize cellular responses. In a randomized trial enrolling patients at risk for surgical infection, one goal is to phenotype cells within a polytetrafluoroethylene implant using flow cytometry and immunohistochemistry, together with standard hematoxylin and eosin based histology. Subcutaneous implants are removed 8–9 days postoperatively. To obtain single cells associated with the mechanism of wound healing, we initially used a mouse skin digestion protocol. We optimized this to increase cell yield and isolate sufficient cells for flow cytometry. The modifications increased the total cells recovered per subject from an average of 5.3×104 to 41×104 with an average viability of 80%.The immunoflourescent staining assay was verified for our samples, which have smaller cell sample numbers than tissue biopsies. Thirty-two samples were stained. Cells from the polytetrafluoroethylene tubes were isolated and stained positively with fluorescent-labeled antibodies to CD3, CD20, CD31, CD34, CD68, CD133 and VEGFR2. Flow cytometry data correlated with IHC data especially with respect to CD68. This antigen was the most prevalent in both cell analysis methods. Our findings demonstrate flow cytometry can be used with polytetrafluoroethylene samples as an additional evaluation method to document and describe cellular wound healing responses. PMID:20459506

  6. 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.

  7. 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…

  8. Fluorescence intensity analysis through simplex optimization in flow cytometry.

    PubMed

    Takase, K; Iwaki, K; Gunji, T; Yata, J

    1989-03-10

    Fluorescence intensity analysis in flow cytometric surface immunophenotyping has recently been appreciated in clinical applications. A curve fitting method to estimate the mean and SD values of fluorescence intensity is described in this report. A Gaussian distribution is aimed to be adapted for a specified distribution in logarithmically scaled histogram data through the simplex optimization, one of the non-linear least squares methods. In comparison with the conventional methods which include the detection of peak point and the direct calculation, this fitting method has demonstrated exceeding precisions in the estimation of both parameters with limited involved cell counts in typical lymphocytic phenotyping. The actual estimation for a precise SD value will develop the quality control approaches based on the fluorescence intensity analysis. While this method is not suitable for distributions that involve extremely small cell counts or that deviate markedly from a symmetric Gaussian, it has additional advantages of loose requirements, namely, narrow fitting regions, ordinarily small cell counts, practical computational periods and a simple programming.

  9. Considerations for the control of background fluorescence in clinical flow cytometry.

    PubMed

    Hulspas, Ruud; O'Gorman, Maurice R G; Wood, Brent L; Gratama, Jan W; Sutherland, D Robert

    2009-11-01

    Accurate measurement of antigen-positive cells by flow cytometry can be hampered by background fluorescence of antigen-negative cells and other particles (e.g., debris). This article focuses on three major causes of background (autofluorescence, spectral overlap, and undesirable antibody binding) by reviewing individual aspects of flow cytometric measurements that contribute to these causes. The appropriate use of controls facilitates a thorough understanding of these contributing factors as well as the development of robust cell labeling protocols intended for routine flow cytometric analysis. We present a set of recommendations that enables the user to develop an optimized cell labeling protocol that minimizes background and maximizes the ability to reliably distinguish between a positive and a negative population of cells. These recommendations are also intended to augment existing guidelines designed to aid in the formulation of a consensus regarding the utility of flow cytometry for the analysis of clinical samples.

  10. 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

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

    PubMed

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

    2015-05-29

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

  12. Two-step protocol for preparing adherent cells for high-throughput flow cytometry.

    PubMed

    Kaur, Mandeep; Esau, Luke

    2015-09-01

    We have developed a simple, cost-effective, and labor-efficient two-step protocol for preparing adherent cells for high-throughput flow cytometry. Adherent cells were grown on microplates, detached with 2.9 mM EDTA (pH 6.14) added directly to wells containing cell culture medium, stained, and then analyzed on a flow cytometer. This protocol bypasses washing, centrifugation, and transfer between plates, reducing the cell loss that occurs in standard multistep protocols. The method has been validated using six adherent cell lines, four commercially available dyes, and two antibodies; the results have been confirmed using two different flow cytometry (FC) instruments. Our approach has been used for estimating apoptosis, mitochondrial membrane potential, reactive oxygen species, and autophagy in response to exposure to pure compounds as well as plant and bacterial extracts.

  13. 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

  14. 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

    2016-02-11

    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.

  15. Measurement of DNA-Dependent Protein Kinase Phosphorylation Using Flow Cytometry Provides a Reliable Estimate of DNA Repair Capacity.

    PubMed

    Abramenkovs, Andris; Stenerlöw, Bo

    2017-09-27

    Uncontrolled generation of DNA double-strand breaks (DSBs) in cells is regarded as a highly toxic event that threatens cell survival. Radiation-induced DNA DSBs are commonly measured by pulsed-field gel electrophoresis, microscopic evaluation of accumulating DNA damage response proteins (e.g., 53BP1 or γ-H2AX) or flow cytometric analysis of γ-H2AX. The advantage of flow cytometric analysis is that DSB formation and repair can be studied in relationship to cell cycle phase or expression of other proteins. However, γ-H2AX is not able to monitor repair kinetics within the first 60 min postirradiation, a period when most DSBs undergo repair. A key protein in non-homologous end joining repair is the catalytic subunit of DNA-dependent protein kinase. Among several phosphorylation sites of DNA-dependent protein kinase, the threonine at position 2609 (T2609), which is phosphorylated by ataxia telangiectasia mutated (ATM) or DNA-dependent protein kinase catalytic subunit itself, activates the end processing of DSB. Using flow cytometry, we show here that phosphorylation at T2609 is faster in response to DSBs than γ-H2AX. Furthermore, flow cytometric analysis of T2609 resulted in a better representation of fast repair kinetics than analysis of γ-H2AX. In cells with reduced ligase IV activity, and wild-type cells where DNA-dependent protein kinase activity was inhibited, the reduced DSB repair capacity was observed by T2609 evaluation using flow cytometry. In conclusion, flow cytometric evaluation of DNA-dependent protein kinase T2609 can be used as a marker for early DSB repair and gives a better representation of early repair events than analysis of γ-H2AX.

  16. European canine lymphoma network consensus recommendations for reporting flow cytometry in canine hematopoietic neoplasms.

    PubMed

    Comazzi, S; Avery, P R; Garden, O A; Riondato, F; Rütgen, B; Vernau, W

    2017-09-01

    Flow cytometry (FC) is assuming increasing importance in diagnosis in veterinary oncology. The European Canine Lymphoma Network (ECLN) is an international cooperation of different institutions working on canine lymphoma diagnosis and therapy. The ECLN panel of experts on FC has defined the issue of reporting FC on canine lymphoma and leukemia as their first hot topic, since a standardized report that includes all the important information is still lacking in veterinary medicine. The flow cytometry panel of the ECLN started a consensus initiative using the Delphi approach. Clinicians were considered the main target of FC reports. A panel of experts in FC was interrogated about the important information needed from a report. Using the feedback from clinicians and subsequent discussion, a list of information to be included in the report was made, with four different levels of recommendation. The final report should include both a quantitative part and a qualitative or descriptive part with interpretation of the salient results. Other items discussed included the necessity of reporting data regarding the quality of samples, use of absolute numbers of positive cells, cutoff values, the intensity of fluorescence, and possible aberrant patterns of antigen expression useful from a clinical point of view. The consensus initiative is a first step toward standardization of diagnostic approach to canine hematopoietic neoplasms among different institutions and countries. This harmonization will improve communication and patient care and also facilitate the multicenter studies necessary to further our knowledge of canine hematopoietic neoplasms. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

  17. 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.

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

    PubMed

    Zhu, Hongying; Ozcan, Aydogan

    2013-04-11

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

  19. 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.

  20. Flow cytometry of ALK-negative anaplastic large cell lymphoma of breast implant-associated effusion and capsular tissue.

    PubMed

    Wu, David; Allen, Camilla T; Fromm, Jonathan R

    2014-06-28

    Background: Anaplastic large cell lymphoma (ALCL) of the breast capsule is a rare lymphoma involving capsular tissues and/or effusions associated with breast implants. While several studies have detailed the histological and immunohistochemical features of these tumors, no study has yet described flow cytometry features of the neoplastic cells of this entity. Here, we report two cases from our institution in which multi-parametric flow cytometry was performed. Methods: The immunophenotype of ALCL in association with breast implant was evaluated by flow cytometry. Results: We show that much like CD30+ tumor cells of classical Hodgkin lymphoma (CHL) and ALCL of non-breast implant tumors, the neoplastic cells of this entity can be readily identified by flow cytometry. The neoplastic cells of both cases were largely devoid of T-cell antigens, but had expression of weak CD15, strong CD30, and expression of CD40. These results are correlated with routine morphologic and immunohistochemical analysis, supporting the flow cytometry immunophenotype. Conclusions: Flow cytometry can aid in the diagnostic evaluation of effusions or tissue samples in association with breast implant/prostheses. © 2014 Clinical Cytometry Society. Copyright © 2014 Clinical Cytometry Society.

  1. 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

  2. A bead-assisted flow cytometry method for the semi-quantitative analysis of Extracellular Vesicles.

    PubMed

    Suárez, Henar; Gámez-Valero, Ana; Reyes, Raquel; López-Martín, Soraya; Rodríguez, María Josefa; Carrascosa, José L; Cabañas, Carlos; Borràs, Francesc E; Yáñez-Mó, María

    2017-09-12

    Most experimental approaches commonly employed for the characterization and quantitation of EVs are time consuming, require of specialized instrumentation and often are rather inaccurate. To circumvent the caveats imposed by EV small size, we used general and specific membrane markers in bead assisted flow cytometry, to provide a semi-quantitative measure of EV content in a given sample. EVs were isolated from in vitro cultured cells-conditioned medium and biological fluids by size exclusion chromatography and coupled to latex beads to allow their detection by standard flow cytometers. Our analyses demonstrate a linear correlation between EV concentration and Mean Fluorescence Intensity values in samples cleared of protein contaminants. Comparison with one of the most widespread method such as NTA, suggests a similar linear range and reliable accuracy to detect saturation. However, although detection of the different biomarkers is feasible when tested on ultracentrifugation-enriched samples, protein contamination impairs quantitation of this type of samples by bead-based flow cytometry. Thus, we provide evidence that bead-assisted flow cytometry method is an accurate and reliable method for the semiquantitative bulk analysis of EVs, which could be easily implemented in most laboratories.

  3. Quantification of total T-cell receptor diversity by flow cytometry and spectratyping.

    PubMed

    Ciupe, Stanca M; Devlin, Blythe H; Markert, Mary Louise; Kepler, Thomas B

    2013-08-06

    T-cell receptor diversity correlates with immune competency and is of particular interest in patients undergoing immune reconstitution. Spectratyping generates data about T-cell receptor CDR3 length distribution for each BV gene but is technically complex. Flow cytometry can also be used to generate data about T-cell receptor BV gene usage, but its utility has not been compared to or tested in combination with spectratyping. Using flow cytometry and spectratype data, we have defined a divergence metric that quantifies the deviation from normal of T-cell receptor repertoire. We have shown that the sample size is a sensitive parameter in the predicted flow divergence values, but not in the spectratype divergence values. We have derived two ways to correct for the measurement bias using mathematical and statistical approaches and have predicted a lower bound in the number of lymphocytes needed when using the divergence as a substitute for diversity. Using both flow cytometry and spectratyping of T-cells, we have defined the divergence measure as an indirect measure of T-cell receptor diversity. We have shown the dependence of the divergence measure on the sample size before it can be used to make predictions regarding the diversity of the T-cell receptor repertoire.

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

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

  5. 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

  6. 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

  7. Determination of the time of death in a homicide-suicide case using flow cytometry.

    PubMed

    Di Nunno, N; Costantinides, F; Melato, M

    1999-09-01

    The study of the biologic processes involved in determining the postmortem interval also can be useful for determining the time of death. In this instance, we investigated the time of death in a case of homicide-suicide using circumstantial findings and thanatochronological and flow cytometric data. The flow cytometric data confirmed the time of death as calculated from the police investigations. This method has proven more reliable than thanatochronological techniques and can be used to supplement these observations. This case indicates that the use of flow cytometry "on the spot" is useful and reliable; however, more evidence is needed to support this new, although to date very sound, method of investigation.

  8. Flow cytometry as a useful tool for process development: rapid evaluation of expression systems.

    PubMed

    Patkar, Anant; Vijayasankaran, Natarajan; Urry, Dan W; Srienc, Friedrich

    2002-02-28

    Flow cytometry is an established tool in fundamental studies of single-cell microbial physiology. Here we show that it can also provide valuable information for process development. Using recombinant Escherichia coli strains, which express the protein-based polymer (GVGIP)(260)GVGVP, the utility of flow cytometry in monitoring and optimization of fermentations is demonstrated. Single cell right angle light scatter was found to be significantly affected by intracellular product formation possibly due to the formation of inclusion bodies. Translational fusions with green fluorescent protein (GFP) enabled monitoring of product accumulation, as well as plasmid free cell fraction (PFCF). Such fusions also allowed rapid evaluation of induction strategies and three different expression systems based on the T7 promoter, T7-lac promoter and the P(BAD) promoter. The expression system based on the P(BAD) promoter was found to be superior to the T7-based system.

  9. 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.

  10. Establishment and application of a flow cytometry-based method for detecting histone acetylation levels.

    PubMed

    Jianhui, Li; Chunfu, Wang; Fan, Bai; Yan, Zhuang; Zhuojun, Mao; Yongtao, Sun

    2016-06-20

    Histone deacetylase inhibitors, which have also received attention in AIDS and other diseases, are a new class of anticancer drugs developed in recent years. However, there is still a lack of a unified and reliable method for detecting histone acetylation levels in basic and clinical research. In this study, we developed a flow cytometry-based method to detect histone acetylation levels by comparing different sample processing temperature (on ice vs. room temperature), permeabilization method (intracellular vs. nuclear), antibody dose (antibody titration) and antibody incubation time (time gradient) using whole blood and peripheral blood mononuclear cells. In addition, we applied this optimized method in in vitro experiment and clinical trial of Chidamide (the only China FDA approved HDACi), the result of which confirmed that the flow cytometry-based method for detecting histone acetylation levels is a reliable, fast and convenient method which can be used in basic and clinical research.

  11. 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.

  12. Fixation and long-term storage of human lymphocytes for surface marker analysis by flow cytometry.

    PubMed

    Lal, R B; Edison, L J; Chused, T M

    1988-05-01

    A method to preserve stained human lymphocytes for subsequent cell surface analysis by flow cytometry (FCM) is described. Cells stained with fluorescein isothiocyanate (FITC) and phycoerythrin (PE)-conjugated monoclonal antibodies and then fixed in 1% paraformaldehyde, followed by extensive washing and resuspension in 1% BSA medium, could be stored at 4 degrees C for at least 2 weeks prior to FCM analysis without significant alteration in the light scatter or fluorescence properties of the cells. Furthermore, the method was also suitable for analyzing lymphocytes that express T-cell activation markers in certain disease conditions. In addition, we have identified monoclonal antibody combinations that discriminate different lymphocyte subsets that are satisfactory for multiparameter analysis after 2 weeks of storage. This method should prove useful for enumerating lymphocyte subsets in field study sites remote from flow cytometry laboratories.

  13. Validation of algal viability treated with total residual oxidant and organic matter by flow cytometry.

    PubMed

    Lee, Junghyun; Choi, Eun Joo; Rhie, Kitae

    2015-08-15

    Algal cell growth after starch and oxidant treatments in seawater species (Isochrysis galbana and Phaeodactylum tricornutum) and freshwater species (Selenastrum capricornutum and Scenedesmus obliquus) were evaluated by flow cytometry with fluorescein diacetate (FDA) staining to determine algal viability. Growth of algal cell was found to be significantly different among groups treated with NaOCl, starch and/or sodium thiosulfate, which are active substance (Total Residual Oxidant; TRO as Cl2), organic compound to meet efficacy testing standard and neutralizer of TRO by Ballast Water Management Convention of International Maritime Organization, respectively. The viability of algal cell treated with TRO in starch-add culture of 5days after treatment and neutralization was decreased significantly. ATP contents of the treated algal cells corresponded to the FL1 fluorescent signal of flow cytometry with FDA staining. I. galbana was the most sensitive to TRO-neutralized cultures during viability analysis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Genome size determination in chagas disease transmitting bugs (hemiptera-triatominae) by flow cytometry.

    PubMed

    Panzera, Francisco; Ferrandis, Inés; Ramsey, Janine; Salazar-Schettino, Paz M; Cabrera, Margarita; Monroy, Carlota; Bargues, María D; Mas-Coma, Santiago; O'Connor, José E; Angulo, Víctor M; Jaramillo, Nicolás; Pérez, Ruben

    2007-03-01

    Because information about genome size in triatomines is scarce and contradictory, we performed DNA quantification by flow cytometry in 13 species belonging to five genera (Dipetalogaster, Eratyrus, Panstrongylus, Rhodnius, and Triatoma) to infer overall tendencies and phylogenetic associations. The results show that the haploid DNA content of the subfamily Triatominae varies nearly 4-fold, from<0.7 pg in Rhodnius species (0.6x10(9) bp) to 2.7 pg in Triatoma delpontei (2.6x10(9) bp). Considering that triatomines present similar chromosome numbers, we suggest that genome size differences are the result of variation in the quantity of repetitive DNA sequences localized in hetero and euchromatin. Changes in heterochromatin are particularly important when considering populations or closely related species; in more distant taxa, euchromatic changes also play a role. Our analyses indicate that flow cytometry is a useful tool for population, taxonomic, and evolutionary studies in this subfamily.

  15. Detection of Intracellular Factor VIII Protein in Peripheral Blood Mononuclear Cells by Flow Cytometry

    PubMed Central

    Pandey, Gouri Shankar; Tseng, Sandra C.; Howard, Tom E.; Sauna, Zuben E.

    2013-01-01

    Flow cytometry is widely used in cancer research for diagnosis, detection of minimal residual disease, as well as immune monitoring and profiling following immunotherapy. Detection of specific host proteins for diagnosis predominantly uses quantitative PCR and western blotting assays. In this study, we optimized a flow cytometry-based detection assay for Factor VIII protein in peripheral blood mononuclear cells (PBMCs). An indirect intracellular staining (ICS) method was standardized using monoclonal antibodies to different domains of human Factor VIII protein. The FVIII protein expression level was estimated by calculating the mean and median fluorescence intensities (MFI) values for each monoclonal antibody. ICS staining of transiently transfected cell lines supported the method's specificity. Intracellular FVIII protein expression was also detected by the monoclonal antibodies used in the study in PBMCs of five blood donors. In summary, our data suggest that intracellular FVIII detection in PBMCs of hemophilia A patients can be a rapid and reliable method to detect intracellular FVIII levels. PMID:23555096

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

    SciTech Connect

    Wallner, G.; Amann, R. |

    1995-12-31

    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 microorganisms. Combined with flow cytometry, up to several thousand cells per second can be classified. In addition to the identification and specific enumeration of microorganisms, further information on the distribution of cell size, DNA and ribosome content -- and therefore an assessment of activity -- within the entire community of 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 labelled oligonucleotides and counterstained with the DNA-specific dye Hoechst 33342 are presented as examples for its applicability to complex microbial communities.

  17. Study of planarian stem cell proliferation by means of flow cytometry.

    PubMed

    Ermakov, Artem M; Ermakova, Olga N; Kudravtsev, Andrei A; Kreshchenko, Natalia D

    2012-03-01

    The stem cells in freshwater flatworms (planarian) are called neoblasts. Neoblasts are capable of proliferation and differentiation into every cell type, including the gametes. For the investigation of the mechanisms of stem cells proliferation and differentiation the proper evaluation of changes in the cell cycle of neoblasts in different physiological conditions of planarian is necessary. In the present study the possibility of qualitative and quantitative characteristics of the neoblasts population were investigated using flow cytometry. In the cell suspension prepared from planarian tissue proliferating neoblasts have been observed in heterogenic cell population. Quantitative estimation of the cell cycle related changes of planarian stem cells system have been performed in various physiological conditions (intact and regenerating animals) and under the influence of physical (ionizing radiation) and chemical (melatonin and colchicine) factors. The modified protocol for planarian stem cells isolation proved to be effective and reproducible and can be recommended for flow cytometry analyses of human and animal proliferating cells.

  18. Analysis of polyethylene glycol (PEG) fusion in cultured neuroblastoma cells via flow cytometry: Techniques & optimization.

    PubMed

    Hoffman, Ashley N; Bamba, Ravinder; Pollins, Alonda C; Thayer, Wesley P

    2017-02-01

    Polyethylene glycol (PEG) has long been used as a membrane fusogen, but recently it has been adopted as a technique for peripheral nerve repair. Vertebrate models using PEG fusion have shown improved outcomes when PEG is applied during repair of severed peripheral nerves. The cellular mechanism of PEG fusion in the peripheral nerve repair model has not previously been assessed via flow cytometry. PEG fusion was assessed in this experiment by dying B35 rat neuroblastoma cells with different color fluorescent labels. The different color cells were combined and PEG was applied in concentrations of 50%, 75% and 100%. The amount of cell fusion was assessed via flow cytometry as the percentage of double positive cells. Results showed increasing fusion and decreasing viability with increasing concentrations of PEG.

  19. 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.

  20. In Vivo Photoswitchable Flow Cytometry for Direct Tracking of Single Circulating Tumor Cells

    PubMed Central

    Nedosekin, Dmitry A.; Verkhusha, Vladislav V.; Melerzanov, Alexander V.; Zharov, Vladimir P.; Galanzha, Ekaterina I.

    2014-01-01

    SUMMARY Photoswitchable fluorescent proteins (PSFPs) that change their color in response to light have led to breakthroughs in studying static cells. However, using PSFPs to study cells in dynamic conditions is challenging. Here we introduce a method for in vivo ultrafast photoswitching of PSFPs that provides labeling and tracking of single circulating cells. Using in vivo multicolor flow cytometry, this method demonstrated the capability for studying recirculation, migration, and distribution of circulating tumor cells (CTCs) during metastasis progression. In tumor-bearing mice, it enabled monitoring of real-time dynamics of CTCs released from primary tumor, identifying dormant cells, and imaging of CTCs colonizing a primary tumor (self-seeding) or existing metastasis (reseeding). Integration of genetically encoded PSFPs, fast photoswitching, flow cytometry, and imaging makes in vivo single cell analysis in the circulation feasible to provide insights into the behavior of CTCs and potentially immune-related and bacterial cells in circulation. PMID:24816228

  1. QUAliFiER: An automated pipeline for quality assessment of gated flow cytometry data

    PubMed Central

    2012-01-01

    Background Effective quality assessment is an important part of any high-throughput flow cytometry data analysis pipeline, especially when considering the complex designs of the typical flow experiments applied in clinical trials. Technical issues like instrument variation, problematic antibody staining, or reagent lot changes can lead to biases in the extracted cell subpopulation statistics. These biases can manifest themselves in non–obvious ways that can be difficult to detect without leveraging information about the study design or other experimental metadata. Consequently, a systematic and integrated approach to quality assessment of flow cytometry data is necessary to effectively identify technical errors that impact multiple samples over time. Gated cell populations and their statistics must be monitored within the context of the experimental run, assay, and the overall study. Results We have developed two new packages, flowWorkspace and QUAliFiER to construct a pipeline for quality assessment of gated flow cytometry data. flowWorkspace makes manually gated data accessible to BioConductor’s computational flow tools by importing pre–processed and gated data from the widely used manual gating tool, FlowJo (Tree Star Inc, Ashland OR). The QUAliFiER package takes advantage of the manual gates to perform an extensive series of statistical quality assessment checks on the gated cell sub–populations while taking into account the structure of the data and the study design to monitor the consistency of population statistics across staining panels, subject, aliquots, channels, or other experimental variables. QUAliFiER implements SVG–based interactive visualization methods, allowing investigators to examine quality assessment results across different views of the data, and it has a flexible interface allowing users to tailor quality checks and outlier detection routines to suit their data analysis needs. Conclusion We present a pipeline constructed from two

  2. Strategies for the synthesis and screening of glycoconjugates. 2. Covalent immobilization for flow cytometry.

    PubMed

    Vetter, D; Tate, E M; Gallop, M A

    1995-01-01

    Glycosylamines are readily available carbohydrate derivatives that undergo acylation reactions with homobifunctional N-hydroxysuccinimidyl esters. The product glycosylamides carry a spacer group equipped with one active ester functionality. This route provides well-defined glycoconjugates, which may be cross-linked to various amino-functionalized resins. Carbohydrate recognition of the resulting sugar-bead conjugates is probed by lectin immunostaining or flow cytometry using a fluorescently labeled lectin.

  3. Assessment of Granulocyte Subset Activation: New Information from Image-Based Flow Cytometry.

    PubMed

    McFarlin, Brian K; Venable, Adam S; Henning, Andrea L; Williams, Randall R; Prado, Eric A

    2016-01-01

    Analysis of granulocyte function can provide important information about the state of the body's innate immune system. Existing flow cytometry methods that lack image-based analysis capabilities fail to fully evaluate granulocyte function. In the present method, we combine simultaneous detection of phagocytosis and oxidative burst in granulocytes to identify unique subsets of activated granulocytes. This analysis method provides novel information about granulocytes that allows our lab and others to evaluate the effectiveness of nutritional and lifestyle countermeasures, designed to improve immunity.

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

    DTIC Science & Technology

    1993-01-01

    Coombs’ Sera from neutropenic patients with a variety of associated positive hemolytic anemia , chronic hepatitis, and lympho- clinical conditions...Liss, Inc.* for enhanced binding of IgG to normal target Key words: neutrophils, immunology, flow cytometry, autoimmunity , irnunoglobulins, neutro- A...however, can be time-consuming, te- autoimmune neutropenia and from normal volunteers by ven- dious, difficult to quantitate, and of limited

  5. 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. © 2016 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Rapid parallel flow cytometry assays of active GTPases using effector beads.

    PubMed

    Buranda, Tione; BasuRay, Soumik; Swanson, Scarlett; Agola, Jacob; Bondu, Virginie; Wandinger-Ness, Angela

    2013-11-15

    We describe a rapid assay for measuring the cellular activity of small guanine triphosphatases (GTPases) in response to a specific stimulus. Effector-functionalized beads are used to quantify in parallel multiple GTP-bound GTPases in the same cell lysate by flow cytometry. In a biologically relevant example, five different Ras family GTPases are shown for the first time to be involved in a concerted signaling cascade downstream of receptor ligation by Sin Nombre hantavirus.

  7. An automated analysis of highly complex flow cytometry-based proteomic data.

    PubMed

    Stuchlý, Jan; Kanderová, Veronika; Fišer, Karel; Cerná, Daniela; Holm, Anders; Wu, Weiwei; Hrušák, Ondřej; Lund-Johansen, Fridtjof; Kalina, Tomáš

    2012-02-01

    The combination of color-coded microspheres as carriers and flow cytometry as a detection platform provides new opportunities for multiplexed measurement of biomolecules. Here, we developed a software tool capable of automated gating of color-coded microspheres, automatic extraction of statistics from all subsets and validation, normalization, and cross-sample analysis. The approach presented in this article enabled us to harness the power of high-content cellular proteomics. In size exclusion chromatography-resolved microsphere-based affinity proteomics (Size-MAP), antibody-coupled microspheres are used to measure biotinylated proteins that have been separated by size exclusion chromatography. The captured proteins are labeled with streptavidin phycoerythrin and detected by multicolor flow cytometry. When the results from multiple size exclusion chromatography fractions are combined, binding is detected as discrete reactivity peaks (entities). The information obtained might be approximated to a multiplexed western blot. We used a microsphere set with >1,000 subsets, presenting an approach to extract biologically relevant information. The R-project environment was used to sequentially recognize subsets in two-dimensional space and gate them. The aim was to extract the median streptavidin phycoerythrin fluorescence intensity for all 1,000+ microsphere subsets from a series of 96 measured samples. The resulting text files were subjected to algorithms that identified entities across the 24 fractions. Thus, the original 24 data points for each antibody were compressed to 1-4 integrated values representing the areas of individual antibody reactivity peaks. Finally, we provide experimental data on cellular protein changes induced by treatment of leukemia cells with imatinib mesylate. The approach presented here exemplifies how large-scale flow cytometry data analysis can be efficiently processed to employ flow cytometry as a high-content proteomics method.

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

    PubMed Central

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

    2011-01-01

    A new flow cytometry method that uses an optimized DNA and RNA staining strategy to monitor the growth and development of the Plasmodium falciparum strain W2mef has been used in a pilot study and has identified Bay 43-9006 1, SU 11274 2, and TMC 125 5 as compounds that exhibit potent (<1 μM) overall and ring stage in vitro antimalarial activity. PMID:19666223

  9. Flow cytometry applications for the analysis of chemokine receptor expression and function.

    PubMed

    Anselmo, Achille; Mazzon, Cristina; Borroni, Elena Monica; Bonecchi, Raffaella; Graham, Gerard J; Locati, Massimo

    2014-04-01

    Chemokine receptors play an important role in leukocyte migration, both in physiological and pathological conditions, and the interest in new methodologies for their detection is increasing. In this review, we focused on chemokine receptors detection through flow cytometric approaches, including the use of specific antibodies and fluorescent chemokines, and on approaches aimed at the analysis of their functions, from intracellular trafficking to signaling activities. © 2014 International Society for Advancement of Cytometry.

  10. Application of flow cytometry to monitor assimilable organic carbon (AOC) and microbial community changes in water.

    PubMed

    Elhadidy, Ahmed M; Van Dyke, Michele I; Peldszus, Sigrid; Huck, Peter M

    2016-11-01

    Flow cytometry is an efficient monitoring tool for rapid cell counting, and can be applied to research on water quality and treatment. In this study, a method that employs flow cytometry and a natural microbial inoculum to determine assimilable organic carbon (AOC) was adapted for use with challenging surface waters that have a high organic and particle content, and subsequently applied in a long term river water study. AOC method optimization showed that river water bacteria could pass through a 0.2μm membrane filter, and therefore membrane filtration combined with heat treatment was required for sample sterilization. Preparation of the natural river inoculum with an acceptable yield value could only be achieved when grown using the natural water source, since growth was limited on different types of inorganic minimal media and in natural spring water. The resulting flow cytometry AOC method was reliable and reproducible, and results were comparable to the standard plate count AOC method. Size exclusion chromatography showed that both high and low molecular weight organic matter fractions were utilized by the natural AOC inoculum. Flow cytometry was used to measure both AOC levels and total cell counts in a long term study to monitor the water quality of a river which was used as a drinking water source. The method could distinguish between high nucleic acid (HNA) and low nucleic acid (LNA) groups of bacteria, and HNA bacteria were found to respond faster than LNA bacteria to seasonal changes in nutrients and water temperature. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2016-01-01

    To discuss the implementation of technical advances in laboratory diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria for validation of high-sensitivity flow cytometry protocols. A retrospective study based on analysis of laboratory data from 745 patient samples submitted to flow cytometry for diagnosis and/or monitoring of paroxysmal nocturnal hemoglobinuria. Implementation of technical advances reduced test costs and improved flow cytometry resolution for paroxysmal nocturnal hemoglobinuria clone detection. High-sensitivity flow cytometry allowed more sensitive determination of paroxysmal nocturnal hemoglobinuria clone type and size, particularly in samples with small clones. Discutir as melhorias técnicas no diagnóstico e no acompanhamento laboratorial de hemoglobinúria paroxística noturna para a validação da técnica de citometria de fluxo de alta sensibilidade. Estudo retrospectivo, que envolveu a análise de dados laboratoriais de 745 pacientes com hipótese diagnóstica e/ou acompanhamento de hemoglobinúria paroxística noturna por citometria de fluxo. Os avanços técnicos não só reduziram o custo do ensaio, mas também melhoraram a identificação e a resolução da citometria de fluxo para a detecção de clone hemoglobinúria paroxística noturna. A citometria de fluxo de alta sensibilidade possibilitou a identificação do tipo e do tamanho de clone de hemoglobinúria paroxística noturna, especialmente em amostras com pequeno clone.

  12. RBC aggregation in dextran solutions can be measured by flow cytometry.

    PubMed

    Zhao, Lian; Kaewprayoon, Waraporn; Zhou, Hong; Georgieva, Radostina; Bäumler, Hans

    2017-01-01

    The impact of macromolecules on RBC aggregation continues to be of interest, nevertheless present measurements still have limitations and need improvement. We applied flow cytometry to measure RBC aggregation in dextran T500 (Dx500) solution. The samples were fixed in the aggregated state by glutaraldehyde. Fixed RBC exhibit auto fluorescence, which can be detected by flow cytometry. Single cells, doublets, triplets and larger aggregates can be distinguished quantitatively and quickly due to the correlation between auto fluorescence intensity and number of RBC per measured event. With the increase in concentration of Dx500, percentages of all aggregates and bigger aggregates increased significantly at concentration of 2%, 4% and 6%, while decreased when the concentration reached 8% and 10%. The percentage of bigger aggregates in concentration of 4% was higher than that in 2% and 6%. The data of flow cytometry was confirmed by microscopic observation and are in good agreement with the literature. The method provide additional advantages to the conventional measurement of RBC aggregation. It gets the distribution of single cells and aggregates as derived from the microscopic observation with hematocrit of physiological level. It uses sample volume as 1/5∼1/10 as needed in sendimentation and photometricmethods.

  13. Subcellular localization-dependent changes in EGFP fluorescence lifetime measured by time-resolved flow cytometry

    PubMed Central

    Gohar, Ali Vaziri; Cao, Ruofan; Jenkins, Patrick; Li, Wenyan; Houston, Jessica P.; Houston, Kevin D.

    2013-01-01

    Intracellular protein transport and localization to subcellular regions are processes necessary for normal protein function. Fluorescent proteins can be fused to proteins of interest to track movement and determine localization within a cell. Currently, fluorescence microscopy combined with image processing is most often used to study protein movement and subcellular localization. In this contribution we evaluate a high-throughput time-resolved flow cytometry approach to correlate intracellular localization of human LC3 protein with the fluorescence lifetime of enhanced green fluorescent protein (EGFP). Subcellular LC3 localization to autophagosomes is a marker of the cellular process called autophagy. In breast cancer cells expressing native EGFP and EGFP-LC3 fusion proteins, we measured the fluorescence intensity and lifetime of (i) diffuse EGFP (ii) punctate EGFP-LC3 and (iii) diffuse EGFP-ΔLC3 after amino acid starvation to induce autophagy-dependent LC3 localization. We verify EGFP-LC3 localization with low-throughput confocal microscopy and compare to fluorescence intensity measured by standard flow cytometry. Our results demonstrate that time-resolved flow cytometry can be correlated to subcellular localization of EGFP fusion proteins by measuring changes in fluorescence lifetime. PMID:24010001

  14. Characterisation of the green turtle's leukocyte subpopulations by flow cytometry and evaluation of their phagocytic activity.

    PubMed

    Muñoz, F A; Franco-Noguez, S Y; Gonzalez-Ballesteros, E; Negrete-Philippe, A C; Flores-Romo, L

    2014-06-01

    Phagocytosis is a fundamental aspect of innate immunity that is conserved across many species making it a potentially useful health-assessment tool for wildlife. In non-mammalian vertebrates, heterophils, monocytes, macrophages, melanomacrophages, and thrombocytes all have phagocytic properties. Recently, B lymphocytes from fish, amphibians, and aquatic turtles have also showed phagocytic capacity. Phagocytes can be studied by flow cytometry; however, the use of this tool is complicated in reptiles partly because nucleated erythrocytes complicate the procedure. We separated green turtle leukocytes by density gradient centrifugation and identified subpopulations by flow cytometry and confocal microscopy. Additionally, we assessed their ability to phagocytize Fluorspheres and Ovoalbumin-Alexa. We found that heterophils and lymphocytes but not monocytes could be easily identified by flow cytometry. While heterophils from adults and juvenile turtles were equally able to phagocytize fluorspheres, adults had significantly more phagocytic ability for OVA-Alexa. Lymphocytes had a mild phagocytic activity with fluorospheres (27-38 %; 39-45 %) and OVA-Alexa (35-46 %; 14-22 %) in juvenile and adult green turtles, respectively. Confocal microscopy confirmed phagocytosis of fluorospheres in both heterophils and lymphocytes. This provides the first evidence that green turtle lymphocytes have phagocytic activity and that this assay could potentially be useful to measure one aspect of innate immunity in this species.

  15. 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).

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

    PubMed

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

    2017-10-01

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

  17. In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

    PubMed Central

    Lelliott, Patrick M.; McMorran, Brendan J.; Foote, Simon J.; Burgio, Gaetan

    2015-01-01

    During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection. PMID:25867202

  18. Unfold High-Dimensional Clouds for Exhaustive Gating of Flow Cytometry Data.

    PubMed

    Qiu, Peng

    2014-01-01

    Flow cytometry is able to measure the expressions of multiple proteins simultaneously at the single-cell level. A flow cytometry experiment on one biological sample provides measurements of several protein markers on or inside a large number of individual cells in that sample. Analysis of such data often aims to identify subpopulations of cells with distinct phenotypes. Currently, the most widely used analytical approach in the flow cytometry community is manual gating on a sequence of nested biaxial plots, which is highly subjective, labor intensive, and not exhaustive. To address those issues, a number of methods have been developed to automate the gating analysis by clustering algorithms. However, completely removing the subjectivity can be quite challenging. This paper describes an alternative approach. Instead of automating the analysis, we develop novel visualizations to facilitate manual gating. The proposed method views single-cell data of one biological sample as a high-dimensional point cloud of cells, derives the skeleton of the cloud, and unfolds the skeleton to generate 2D visualizations. We demonstrate the utility of the proposed visualization using real data, and provide quantitative comparison to visualizations generated from principal component analysis and multidimensional scaling.

  19. 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.

  20. 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.

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

    PubMed Central

    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.

    2012-01-01

    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. The principles of integrated photoacoustic and fluorescence flow cytometry using positive contrast for detection of strongly absorbing and fluorescent cells and negative contrast for detection of weakly absorbing and fluorescent cells in blood absorption and autofluorescence background, respectively. PMID:22903924

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

    PubMed

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

    2003-06-01

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

  3. 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

  4. Comparison of nucleolar organiser regions and DNA flow cytometry in the evaluation of pleural effusion.

    PubMed Central

    Huang, M. S.; Tsai, M. S.; Hwang, J. J.; Wang, T. H.

    1994-01-01

    BACKGROUND--In conventional cytological diagnosis of pleural effusions the assessment of morphological features plays an important part. However, false negative and false positive results may occur. In this study conventional cytology was compared with flow cytometric DNA analysis and the argyrophil staining technique for nucleolar organiser regions (AgNOR) to characterise benign and malignant effusions. METHODS--Pleural effusions from 71 patients (38 with benign lung disease, 33 with proven adenocarcinoma of lung) were studied by conventional cytology, flow cytometric DNA analysis, and the AgNOR technique. Tumour cell ploidy was determined by flow cytometry. In an attempt to detect the cell proliferative state, flow cytometric S phase fraction and the AgNOR technique were used. The correlations among conventional cytology, flow cytometric DNA ploidy, S phase fraction analysis, and nucleolar organiser regions were investigated. RESULTS--All the 38 benign pleural effusions were diploid. There were 17 (52%) aneuploid and 16 (48%) diploid malignant pleural effusions. Based on these results this type of DNA analysis had a sensitivity of 52% and a specificity of 100%. The mean (SD) numbers of flow cytometric S phase fractions of benign and malignant cases were 5.32 (1.67)% and 12.45 (3.93)% respectively. The mean numbers of S phase fractions of diploid malignant cases were higher than diploid benign cases. In each case the number of AgNORs was counted in 100 cells. The mean number of AgNOR dots per nucleus was 12.57 (3.64) for malignant pleural effusion cells and 3.96 (1.39) for benign pleural effusion cells. The mean number of AgNOR dots was 14.45 (3.36) for aneuploid malignant pleural effusion cells and 10.57 (2.82) for diploid malignant pleural effusion cells. The AgNOR numbers were higher in diploid malignant cells than in diploid benign cells. There was a significant correlation between the S phase fraction determined by flow cytometry and the mean number of Ag

  5. Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry.

    PubMed

    Schulz, Danae; Mugnier, Monica R; Boothroyd, Catherine E; Papavasiliou, F Nina

    2016-10-19

    Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

  6. Plug flow cytometry extends analytical capabilities in cell adhesion and receptor pharmacology.

    PubMed

    Edwards, B S; Kuckuck, F W; Prossnitz, E R; Okun, A; Ransom, J T; Sklar, L A

    2001-03-01

    Plug flow cytometry is a recently developed system for the automated delivery of multiple small boluses or "plugs" of cells or particles to the flow cytometer for analysis. Important system features are that sample plugs are of precisely defined volume and that the sample vessel need not be pressurized. We describe how these features enable direct cell concentration determinations and novel ways to integrate flow cytometers with other analytical instruments. Adhesion assays employed human polymorphonuclear neutrophils (PMNs) loaded with Fura Red and Chinese hamster ovary (CHO) cells cotransfected with genes for green fluorescent protein (GFP) and human P-selectin. U937 cells expressing the human 7-transmembrane formyl peptide receptor were loaded with the fluorescent probe indo-1 for intracellular ionized calcium determinations. A computer-controlled syringe or peristaltic pump loaded the sample into a sample loop of the plug flow coupler, a reciprocating eight-port valve. When the valve position was switched, the plug of sample in the sample loop was transported to the flow cytometer by a pressure-driven fluid line. In stirred mixtures of PMNs and CHO cells, we used plug flow cytometry to directly quantify changes in concentrations of nonadherent singlet PMNs. This approach enabled accurate quantification of adherent PMNs in multicell aggregates. We constructed a novel plug flow interface between the flow cytometer and a cone-plate viscometer to enable real-time flow cytometric analysis of cell-cell adhesion under conditions of uniform shear. The High Throughput Pharmacology System (HTPS) is an instrument used for automated programming of complex pharmacological cell treatment protocols. It was interfaced via the plug flow coupling device to enable rapid (< 5 min) flow cytometric characterization of the intracellular calcium dose-response profile of U937 cells to formyl peptide. By facilitating the coupling of flow cytometers to other fluidics-based analytical

  7. 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.

  8. 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

  9. Flow cytometry of ALK-negative anaplastic large cell lymphoma of breast implant-associated effusion and capsular tissue.

    PubMed

    Wu, David; Allen, Camilla T; Fromm, Jonathan R

    2015-01-01

    Anaplastic large cell lymphoma (ALCL) of the breast capsule is a rare lymphoma involving capsular tissues and/or effusions associated with breast implants. While several studies have detailed the histological and immunohistochemical (IHC) features of these tumors, no study has yet described flow cytometry features of the neoplastic cells of this entity. Here, we report two cases from our institution in which multi-parametric flow cytometry was performed. The immunophenotype of ALCL in association with breast implant was evaluated by flow cytometry. We show that much like CD30+ tumor cells of classical Hodgkin lymphoma (CHL) and ALCL of non-breast implant tumors, the neoplastic cells of this entity can be readily identified by flow cytometry. The neoplastic cells of both cases were largely devoid of T-cell antigens, but had expression of weak CD15, strong CD30, and expression of CD40. These results are correlated with routine morphologic and IHC analysis, supporting the flow cytometry immunophenotype. Flow cytometry can aid in the diagnostic evaluation of effusions or tissue samples in association with breast implant/prostheses. © 2014 Clinical Cytometry Society.

  10. [Research progress on multiple myeloma immunophenotyping and minimal residual disease detected by flow cytometry].

    PubMed

    Li, Han-Qing; Zhai, Yong-Ping

    2015-02-01

    Multiple myeloma (MM) is a haematological malignancy characterized by the accumulation of monoclonal plasma cells in the bone marrow and remained incurable. Flow cytometry has been widely used in the detection of immunophenotype and minimal residual disease, diagnosis, monitoring and prognosis of MM. Normal plasma cells and malignant plasma cells can be distinguished according to different cell surface antigen expression. The clinical significane of many immune markes has been elucidated. However, the clinical significance of some phenotype remains controversial, the detection scheme and gating strategy are not unified. This review discusses the recent research progress on detection of MM immunophenotype and minimal residual disease by flow cytovetry.

  11. Best practices in performing flow cytometry in a regulated environment: feedback from experience within the European Bioanalysis Forum.

    PubMed

    der Strate, Barry van; Longdin, Robin; Geerlings, Marie; Bachmayer, Nora; Cavallin, Maria; Litwin, Virginia; Patel, Minesh; Passe-Coutrin, Wilfried; Schoelch, Corinna; Companjen, Arjen; Fjording, Marianne Scheel

    2017-08-01

    Flow cytometry is a powerful tool that can be used for the support of (pre)clinical studies. Although various white papers are available that describe the set-up and validation of the instrumentation (the flow cytometer) and validation of flow cytometry methods, to date no guidelines exist that address the requirements for performing flow cytometry in a regulated environment. In this manuscript, the European Bioanalysis Forum presents additional practice guidance on the use of flow cytometry in the support of drug development programs and addresses areas that are not covered in the previous publications. The concepts presented here are based on the consensus of discussions in the European Bioanalysis Forum Topic Team 32, in meetings in Barcelona, Limelette and multiple telephone conferences.

  12. Feature-guided clustering of multi-dimensional flow cytometry datasets.

    PubMed

    Zeng, Qing T; Pratt, Juan Pablo; Pak, Jane; Ravnic, Dino; Huss, Harold; Mentzer, Steven J

    2007-06-01

    Flow cytometry produces large multi-dimensional datasets of the physical and molecular characteristics of individual cells. The objective of this study was to simplify the cytometry datasets by arranging or clustering "objects" (cells) into a smaller number of relatively homogeneous groups (clusters) on the basis of interobject similarities and dissimilarities. The algorithm was designed to be driven by histogram features; that is, the relevant single parameter histogram features were used to guide multidimensional k-means clustering without an a priori estimate of cluster number. To test this approach, we simulated cell-derived datasets using protein-coated microspheres (artificial "cells"). The microspheres were constructed to provide 119 populations in 40 samples. The feature-guided (FG) approach accurately identified 100% of the predetermined cluster combinations. In contrast, an approach based on the partition index (PI) cluster validity measure accurately identified 83.2% of the clusters. Direct comparisons of the two methods indicated that the FG method was significantly more accurate than PI in identifying both the number of clusters and the number of objects within the clusters (p<.0001). We conclude that parameter feature analysis can be used to effectively guide k-means clustering of flow cytometry datasets.

  13. Detection of Sp110 by Flow Cytometry and Application to Screening Patients for Veno-occlusive Disease with Immunodeficiency.

    PubMed

    Marquardsen, Florian A; Baldin, Fabian; Wunderer, Florian; Al-Herz, Waleed; Mikhael, Raymond; Lefranc, Gérard; Baz, Zeina; Rezaee, Fariba; Hanna, Rabi; Kfir-Erenfeld, Shlomit; Stepensky, Polina; Meyer, Benedikt; Jauch, Annaise; Bigler, Marc B; Burgener, Anne-Valérie; Higgins, Rebecca; Navarini, Alexander A; Church, Joeseph A; Chou, Janet; Geha, Raif; Notarangelo, Luigi D; Hess, Christoph; Berger, Christoph T; Bloch, Donald B; Recher, Mike

    2017-08-21

    Mutations in Sp110 are the underlying cause of veno-occlusive disease with immunodeficiency (VODI), a combined immunodeficiency that is difficult to treat and often fatal. Because early treatment is critically important for patients with VODI, broadly usable diagnostic tools are needed to detect Sp110 protein deficiency. Several factors make establishing the diagnosis of VODI challenging: (1) Current screening strategies to identify severe combined immunodeficiency are based on measuring T cell receptor excision circles (TREC). This approach will fail to identify VODI patients because the disease is not associated with severe T cell lymphopenia at birth; (2) the SP110 gene contains 17 exons, making it a challenge for Sanger sequencing. The recently developed next-generation sequencing (NGS) platforms that can rapidly determine the sequence of all 17 exons are available in only a few laboratories; (3) there is no standard functional assay to test for the effects of novel mutations in Sp110; and (4) it has been difficult to use flow cytometry to identify patients who lack Sp110 because of the low level of Sp110 protein in peripheral blood lymphocytes. We report here a novel flow cytometric assay that is easily performed in diagnostic laboratories and might thus become a standard assay for the evaluation of patients who may have VODI. In addition, the assay will facilitate investigations directed at understanding the function of Sp110.

  14. [Establishment of Method for Detecting Red Blood Cell Osmotic Fragility by Flow Cytometry].

    PubMed

    Zhu, Hong-Yan; Meng, Qiang; Ouyan, Hong-Mei; Dong, Ting; Zhang, Qiong-Yue; Zhou, You-Quan; Ping, Zhu-Xian

    2016-02-01

    To establish a new method for detection of red blood cell osmotic fragility by using flow cytometry. The hypotension salt solution of different concentrations (0.70 ml normal saline+0.3 ml deionized water, 0.60 ml normal saline+0.40 ml deionized water and 0.55 ml normal saline+0.45 ml deionized water) were prepared with normal saline and deionized water, in which the red blood cells were suspended, and the residual red blood cells were detected by flow cytometer. There was no significant difference in percentage of residual red blood cells between different time points detected by flow cytometer in 3 different hypotonic salt solutions. The percentage of residual red blood cells in B+C+D+E+F+G detected time region was different among 3 NaCl dilution groups. The percentage of residual red blood cells in normal control was lower than that in hemoglobinopathy group. The percentage of residual red blood cells in hereditary spherocytosis (HS) group was obviously lower than that in hemoglobinopathy and normal control groups. The comparison of 3 different dilution concentrations found that the second concentration (0.60 ml normal saline+0.40 ml deionized water) is more suitable to screen HS by FC500 flow cytometer. The detection of red cell osmotic fragility by using flow cytometry is a simple, rapid, objective and economic way that can be an effective screening method for diagnose the HS.

  15. 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.

  16. Flow Cytometry as a Tool for Quality Control of Fluorescent Conjugates Used in Immunoassays

    PubMed Central

    de Almeida Santiago, Marta; de Paula Fonseca e Fonseca, Bruna; da Silva Marques, Christiane de Fátima; Domingos da Silva, Edimilson

    2016-01-01

    The use of antibodies in immunodiagnostic kits generally implies the conjugation of these proteins with other molecules such as chromophores or fluorochromes. The development of more sensitive quality control procedures than spectrophotometry is essential to assure the use of better fluorescent conjugates since the fluorescent conjugates are critical reagents for a variety of immunodiagnostic kits. In this article, we demonstrate a new flow cytometric protocol to evaluate conjugates by molecules of equivalent soluble fluorochromes (MESF) and by traditional flow cytometric analysis. We have coupled microspheres with anti-IgG-PE and anti-HBSAg-PE conjugates from distinct manufactures and/or different lots and evaluated by flow cytometry. Their fluorescence intensities were followed for a period of 18 months. Our results showed that there was a great difference in the fluorescence intensities between the conjugates studied. The differences were observed between manufactures and lots from both anti-IgG-PE and anti-HBSAg-PE conjugates. Coefficients of variation (CVs) showed that this parameter can be used to determine better coupling conditions, such as homogenous coupling. The MESF analysis, as well as geometric mean evaluation by traditional flow cytometry, showed a decrease in the values for all conjugates during the study and were indispensable tools to validate the results of stability tests. Our data demonstrated the feasibility of the flow cytometric method as a standard quality control of immunoassay kits. PMID:27936034

  17. 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

  18. Improved signal recovery for flow cytometry based on ‘spatially modulated emission’

    NASA Astrophysics Data System (ADS)

    Quint, S.; Wittek, J.; Spang, P.; Levanon, N.; Walther, T.; Baßler, M.

    2017-09-01

    Recently, the technique of ‘spatially modulated emission’ has been introduced (Baßler et al 2008 US Patent 0080181827A1; Kiesel et al 2009 Appl. Phys. Lett. 94 041107; Kiesel et al 2011 Cytometry A 79A 317-24) improving the signal-to-noise ratio (SNR) for detecting bio-particles in the field of flow cytometry. Based on this concept, we developed two advanced signal processing methods which further enhance the SNR and selectivity for cell detection. The improvements are achieved by adapting digital filtering methods from RADAR technology and mainly address inherent offset elimination, increased signal dynamics and moreover reduction of erroneous detections due to processing artifacts. We present a comprehensive theory on SNR gain and provide experimental results of our concepts.

  19. flowPeaks: a fast unsupervised clustering for flow cytometry data via K-means and density peak finding.

    PubMed

    Ge, Yongchao; Sealfon, Stuart C

    2012-08-01

    For flow cytometry data, there are two common approaches to the unsupervised clustering problem: one is based on the finite mixture model and the other on spatial exploration of the histograms. The former is computationally slow and has difficulty to identify clusters of irregular shapes. The latter approach cannot be applied directly to high-dimensional data as the computational time and memory become unmanageable and the estimated histogram is unreliable. An algorithm without these two problems would be very useful. In this article, we combine ideas from the finite mixture model and histogram spatial exploration. This new algorithm, which we call flowPeaks, can be applied directly to high-dimensional data and identify irregular shape clusters. The algorithm first uses K-means algorithm with a large K to partition the cell population into many small clusters. These partitioned data allow the generation of a smoothed density function using the finite mixture model. All local peaks are exhaustively searched by exploring the density function and the cells are clustered by the associated local peak. The algorithm flowPeaks is automatic, fast and reliable and robust to cluster shape and outliers. This algorithm has been applied to flow cytometry data and it has been compared with state of the art algorithms, including Misty Mountain, FLOCK, flowMeans, flowMerge and FLAME. The R package flowPeaks is available at https://github.com/yongchao/flowPeaks. yongchao.ge@mssm.edu Supplementary data are available at Bioinformatics online.

  20. Clinical utility of flow cytometry in the study of erythropoiesis and nonclonal red cell disorders.

    PubMed

    Chesney, Alden; Good, David; Reis, Marciano

    2011-01-01

    Erythropoiesis involves proliferation and differentiation of small population of hematopoietic stem cells resident in the bone marrow into mature red blood cells. The determination of the cellular composition of the blood is a valuable tool in the diagnosis of diseases and monitoring of therapy. Flow cytometric analysis is increasingly being used to characterize the heterogeneous cell populations present in the blood and the hematopoietic cell differentiation and maturation pathways of the bone marrow. Here we discuss the role of flow cytometry in the study of erythropoiesis and nonclonal red blood cell disorders. First, we discuss flow cytometric analysis of reticulocytes. Next, we review salient quantitative methods that can be used for detection of fetal-maternal hemorrhage (FMH). We also discuss flow cytometric analysis of high hemoglobin F (HbF) in Sickle Cell Disease (SCD), hereditary spherocytosis (HS), red cell survival and red cell volume. We conclude by discussing cell cycle of erythroid cells.

  1. 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

  2. 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

  3. Single and multi-subject clustering of flow cytometry data for cell-type identification and anomaly detection.

    PubMed

    Pouyan, Maziyar Baran; Jindal, Vasu; Birjandtalab, Javad; Nourani, Mehrdad

    2016-08-10

    Measurement of various markers of single cells using flow cytometry has several biological applications. These applications include improving our understanding of behavior of cellular systems, identifying rare cell populations and personalized medication. A common critical issue in the existing methods is identification of the number of cellular populations which heavily affects the accuracy of results. Furthermore, anomaly detection is crucial in flow cytometry experiments. In this work, we propose a two-stage clustering technique for cell type identification in single subject flow cytometry data and extend it for anomaly detection among multiple subjects. Our experimentation on 42 flow cytometry datasets indicates high performance and accurate clustering (F-measure > 91 %) in identifying main cellular populations. Furthermore, our anomaly detection technique evaluated on Acute Myeloid Leukemia dataset results in only <2 % false positives.

  4. MRT letter: light sheet based imaging flow cytometry on a microfluidic platform.

    PubMed

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

    2013-11-01

    We propose a light sheet based imaging flow cytometry technique for simultaneous counting and imaging of cells on a microfluidic platform. Light sheet covers the entire microfluidic channel and thus omits the necessity of flow focusing and point scanning based technology. Another advantage lies in the orthogonal detection geometry that totally cuts-off the incident light, thereby substantially reducing the background in the detection. Compared to the existing state-of-art techniques the proposed technique shows marked improvement. Using fluorescently-coated Saccharomyces cerevisiae cells we have recorded cell counting with throughput as high as 2,090 cells/min in the low flow rate regime and were able to image the individual cells on-the-go. Overall, the proposed system is cost-effective and simple in channel geometry with the advantage of efficient counting in operational regime of low laminar flow. This technique may advance the emerging field of microfluidic based cytometry for applications in nanomedicine and point of care diagnostics. Copyright © 2013 Wiley Periodicals, Inc.

  5. Detection of serine 473 phosphorylated Akt in acute myeloid leukaemia blasts by flow cytometry.

    PubMed

    Tazzari, Pier Luigi; Cappellini, Alessandra; Grafone, Tiziana; Mantovani, Irina; Ricci, Francesca; Billi, Anna Maria; Ottaviani, Emanuela; Conte, Roberto; Martinelli, Giovanni; Martelli, Alberto M

    2004-09-01

    The phosphoinositide 3-kinase/Akt signalling pathway is a recently recognized important parameter in the prognosis and the response to treatment of acute myeloid leukaemia (AML). Akt kinase is activated by phosphorylation on Thr 308 and Ser 473. Active Akt promotes cell growth and survival to apoptotic insults. Thus, it seems important to evaluate Akt phosphorylation in AML blasts. This work aimed to establish whether it was possible to detect Akt phosphorylation on Ser 473 of AML blasts by means of flow cytometry. High levels of Akt activity and phosphorylation were detected in 13 of 15 cases of AML. Flow cytometric analysis revealed similar patterns of Ser 473 expression as was observed with Akt kinase activity and Western blot analysis of Thr 308 and Ser 473 phosphorylation. Double immunostaining enabled the simultaneous flow cytometric detection of an AML-associated antigen (CD33) and Ser 473 phosphorylated Akt in leukaemic blast populations. Our results indicate that flow cytometry enabled the rapid and quantitative assessment of Ser 473 phosphorylated Akt of AML blasts that, when used in combination with cell surface staining, can provide more accurate phenotyping of AML blasts.

  6. 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

  7. 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

    2014-12-18

    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.

  8. 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.

  9. 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

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

    PubMed

    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; Bacon, Joanna

    2016-07-01

    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.

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

    PubMed Central

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

    2009-01-01

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

  12. 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

  13. 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

  14. Polyploidy in the Olive Complex (Olea europaea): Evidence from Flow Cytometry and Nuclear Microsatellite Analyses

    PubMed Central

    Besnard, G.; Garcia-Verdugo, C.; Rubio De Casas, R.; Treier, U. A.; Galland, N.; Vargas, P.

    2008-01-01

    Background Phylogenetic and phylogeographic investigations have been previously performed to study the evolution of the olive tree complex (Olea europaea). A particularly high genomic diversity has been found in north-west Africa. However, to date no exhaustive study has been addressed to infer putative polyploidization events and their evolutionary significance in the diversification of the olive tree and its relatives. Methods Representatives of the six olive subspecies were investigated using (a) flow cytometry to estimate genome content, and (b) six highly variable nuclear microsatellites to assess the presence of multiple alleles at co-dominant loci. In addition, nine individuals from a controlled cross between two individuals of O. europaea subsp. maroccana were characterized with microsatellites to check for chromosome inheritance. Key Results Based on flow cytometry and genetic analyses, strong evidence for polyploidy was obtained in subspp. cerasiformis (tetraploid) and maroccana (hexaploid), whereas the other subspecies appeared to be diploids. Agreement between flow cytometry and genetic analyses gives an alternative approach to chromosome counting to determine ploidy level of trees. Lastly, abnormalities in chromosomes inheritance leading to aneuploid formation were revealed using microsatellite analyses in the offspring from the controlled cross in subsp. maroccana. Conclusions This study constitutes the first report for multiple polyploidy in olive tree relatives. Formation of tetraploids and hexaploids may have played a major role in the diversification of the olive complex in north-west Africa. The fact that polyploidy is found in narrow endemic subspecies from Madeira (subsp. cerasiformis) and the Agadir Mountains (subsp. maroccana) suggests that polyploidization has been favoured to overcome inbreeding depression. Lastly, based on previous phylogenetic analyses, we hypothesize that subsp. cerasiformis resulted from hybridization between ancestors

  15. Cytology, immunopathology and flow cytometry in the diagnosis of pleural and peritoneal effusions.

    PubMed

    Croonen, A M; van der Valk, P; Herman, C J; Lindeman, J

    1988-06-01

    There were 106 pleural and peritoneal effusions studied in order to investigate the contribution of immunocytochemistry and flow cytometry to routine cytologic diagnosis. A panel of antibodies (to cytokeratin, vimentin, human milk fat globule, epithelial membrane antigen and carcinoembryonic antigen) was applied to aceton-fixed slides, using immunoperoxydase and immunofluorescence methods. Flow cytometry was performed using a double labeling method, i.e., propidium iodide for DNA staining and keratin for labeling of epithelial cells. In this way DNA aneuploidy was more evident in the histograms when the fluid contained many reactive nonepithelial cells (lymphocytes). A designation of marker profiles was made for the three most frequently occurring diagnoses, i.e., reactive mesothelial proliferation (I), adenocarcinoma (II), and malignant mesothelioma (III). For the differentiation between adenocarcinoma and malignant mesothelioma, carcinoembryonic antigen was the most useful marker as 75% of the adenocarcinomas was carcinoembryonic antigen-positive and the malignant mesotheliomas were consistently negative. Furthermore, evident DNA-aneuploidy strongly supported the diagnosis of adenocarcinoma, as most malignant mesotheliomas were DNA-euploid, even though occasional DNA-aneuploidy was found in malignant mesotheliomas when different effusions from the same patient were examined. For the differentiation between reactive mesothelial cells and malignant mesothelioma human milk fat globule and/or epithelial membrane antigen, in this study proved to be most reliable, their presence strongly indicating malignancy. It is stressed that the method used (fixation, antibodies, washing procedures) can influence these findings. In 16 patients (17%) performing immunopathology and/or flow cytometry meant an important contribution to diagnosis.

  16. Optimization of buffer solutions to analyze inflammatory cytokines in gingival crevicular fluid by multiplex flow cytometry

    PubMed Central

    Ríos-Lugo, María-Judith; Martin, Conchita; Alarcón, José-Antonio; Esquifino, Ana; Solano, Patricia; Sanz, Mariano

    2015-01-01

    Objective: the aim of this study was to test two buffer solutions in order to attain a reliable and reproducible analysis of inflammatory cytokines (IL-1β, IL-6, TNF-α, OPG, OPN and OC), in gingival crevicular fluid (GCF) by flow cytometry. Material and Methods: GCF samples from healthy volunteers were collected with perio-paper strips and diluted either in phosphate buffered saline (PBS) or Tris-HCl buffer, with and without protease inhibitors (PI). Cytokine immunoassays were carried out by flow cytometry (Luminex Xmap 200) generating standard curves. Results: standards curves generated with the use of phosphate-buffered saline (PBS) demonstrated best adjustment for cytokines IL-1ß, IL-6 and TNF- α levels, when using Tris-HCl (p<0.05). Conclusions: The use of PBS buffer with the addition of PI provided reliable measurements of inflammatory biomarkers in GCF samples of healthy volunteers. Key words:Curve fitting, flow cytometer, immunoassay buffer, crevicular fluid, cytokines. PMID:24880451

  17. Quantitative analysis of gold and carbon nanoparticles in mammalian cells by flow cytometry light scattering

    NASA Astrophysics Data System (ADS)

    Zhou, Gang; Liu, Naicheng; Wang, Zhenheng; Shi, Tongguo; Gan, Jingjing; Wang, Zhenzhen; Zhang, Junfeng

    2017-02-01

    Nanoparticle-based applications for diagnostics and therapeutics have been extensively studied. These applications require a profound understanding of the fate of nanoparticles (NPs) in cellular environments. However, until now, few analytical methods are available and most of them rely on fluorescent properties or special elements of NPs; therefore, for NPs without observable optical properties or special elements, the existing methods are hardly applicable. In this study, we introduce a flow cytometry light scattering (FCLS)-based approach that quantifies in situ NPs accurately in mammalian cells. Continuous cells of heterogeneous human epithelial colorectal adenocarcinoma (Caco-2 cells), mouse peritoneal macrophages (MPM), and human adenocarcinomic alveolar basal epithelia (A549 cells) were cultured with NPs with certain concentrations and size. The intensity of the flow cytometric side scattered light, which indicates the quantity of NPs in the cells, was analyzed. The result shows an accurate size- and dose-dependent uptake of Au NPs (5, 30, 250 nm) in Caco-2 cells. The size- and dose- dependence of Au NPs (5, 30, 250 nm) and carbon NPs (50, 500 nm) in cells was validated by transmission electron microscope (TEM). This paper demonstrates the great potential of flow cytometry light scattering in the quantitative study of the size and dose effect on in situ metallic or non-metallic NPs in mammalian cells.

  18. Characterization of porcine peripheral blood leukocytes by light-scattering flow cytometry.

    PubMed Central

    Wang, F I; Williams, T J; el-Awar, F Y; Pang, V F; Hahn, E C

    1987-01-01

    As a basis for other experiments using flow cytometry of porcine peripheral blood leukocytes, cell fractions were isolated by various methods and analyzed by forward angle light scatter and 90 degree light scatter. Cytospin smears of cell samples were also studied by leukocyte differential counts and nonspecific esterase staining. Three main populations of peripheral blood leukocytes [lymphocytes, monocytes, and granulocytes (primarily neutrophils)], were defined in the log 90 degree light scatter by forward angle light scatter histogram. Partial overlap was observed between lymphocyte and monocyte, and between monocyte and granulocyte domains. Correlation between leukocyte differential counts and flow cytometric quantification based on bitmap statistics of appropriate domains was between r = 0.872-0.892 for lymphocyte and granulocyte. Percoll density gradients were used for subfractionation of leukocyte populations, especially for the enrichment of granulocytes. The specific densities were calculated for lymphocytes (1.0585-1.0819 g/cc), monocytes (1.0585-1.0702 g/cc), granulocyte (1.0819-1.0936 g/cc), and erythrocytes (greater than 1.0952 g/cc). We suggest that light scatter characterization is a basis for future studies of porcine blood by flow cytometry. PMID:3453262

  19. Detection and capture of breast cancer cells with photoacoustic flow cytometry

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

  20. Detection and capture of breast cancer cells with photoacoustic flow cytometry

    PubMed Central

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

    2016-01-01

    Abstract. According to the Centers for Disease Control and Prevention, breast cancer is the most common cancer and the second leading cause of cancer related deaths among women. Metastasis—the presence of secondary tumors caused by the spread of cancer cells via the circulatory or lymphatic systems—significantly worsens the prognosis of any breast cancer patient. A technique is developed to detect circulating breast cancer cells in human blood using a photoacoustic flow cytometry method. A Q-switched laser is used to interrogate thousands of blood cells with one pulse as they flow through the beam path. Cells that are optically absorbing, either naturally or artificially, emit an ultrasound wave as a result of the photoacoustic (PA) effect. Breast cancer cells are targeted with chromophores through immunochemistry in order to enhance optical absorption. After which, the PA cytometry device is calibrated to demonstrate the ability to detect single cells. Cultured breast cancer cells are added to whole blood to reach a biologically relevant concentration of about 25 to 45 breast cancer cells per 1 mL of blood. An in vitro PA flow cytometer is used to detect and isolate these cells followed by capture with the use of a micromanipulator. This method can not only be used to determine the disease state of the patient and the response to therapy but also it can be used for genetic testing and in vitro drug trials since the circulating cell can be captured and studied. PMID:27580367

  1. 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.

  2. Novel quantitative autophagy analysis by organelle flow cytometry after cell sonication.

    PubMed

    Degtyarev, Michael; Reichelt, Mike; Lin, Kui

    2014-01-01

    Autophagy is a dynamic process of bulk degradation of cellular proteins and organelles in lysosomes. Current methods of autophagy measurement include microscopy-based counting of autophagic vacuoles (AVs) in cells. We have developed a novel method to quantitatively analyze individual AVs using flow cytometry. This method, OFACS (organelle flow after cell sonication), takes advantage of efficient cell disruption with a brief sonication, generating cell homogenates with fluorescently labeled AVs that retain their integrity as confirmed with light and electron microscopy analysis. These AVs could be detected directly in the sonicated cell homogenates on a flow cytometer as a distinct population of expected organelle size on a cytometry plot. Treatment of cells with inhibitors of autophagic flux, such as chloroquine or lysosomal protease inhibitors, increased the number of particles in this population under autophagy inducing conditions, while inhibition of autophagy induction with 3-methyladenine or knockdown of ATG proteins prevented this accumulation. This assay can be easily performed in a high-throughput format and opens up previously unexplored avenues for autophagy analysis.

  3. A Case of Blastic Plasmacytoid Dendritic Cell Neoplasm Extensively Studied by Flow Cytometry and Immunohistochemistry

    PubMed Central

    Cesana, Clara; Cittone, Micol Giulia; Bandiera, Laura; Scarpati, Barbara; Mancini, Valentina; Soriani, Silvia; Veronese, Silvio; Truini, Mauro; Rossini, Silvano; Cairoli, Roberto

    2017-01-01

    Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy with aggressive clinical course and poor prognosis. Diagnosis is based on detection of CD4+ CD56+, CD123high, TCL-1+, and blood dendritic cell antigen-2/CD303+ blasts, together with the absence of lineage specific antigens on tumour cells. In this report we present a case of BPDCN presenting with extramedullary and bone marrow involvement, extensively studied by flow cytometry and immunohistochemistry, who achieved complete remission after acute lymphoblastic leukemia like chemotherapy and allogeneic hematopoietic stem cell transplantation. PMID:28409040

  4. A flow cytometry-based assay for measuring invasion of red blood cells by Plasmodium falciparum.

    PubMed

    Bei, Amy K; Desimone, Tiffany M; Badiane, Aida S; Ahouidi, Ambroise D; Dieye, Tandakha; Ndiaye, Daouda; Sarr, Ousmane; Ndir, Omar; Mboup, Souleymane; Duraisingh, Manoj T

    2010-04-01

    Variability in the ability of the malaria parasite Plasmodium falciparum to invade human erythrocytes is postulated to be an important determinant of disease severity. Both the parasite multiplication rate and erythrocyte selectivity are important parameters that underlie such variable invasion. We have established a flow cytometry-based method for simultaneously calculating both the parasitemia and the number of multiply-infected erythrocytes. Staining with the DNA-specific dye SYBR Green I allows quantitation of parasite invasion at the ring stage of parasite development. We discuss in vitro and in vivo applications and limitations of this method in relation to the study of parasite invasion.

  5. 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

  6. Micro Flow Cytometry Miniaturisation - Towards in-situ Optical Phytoplankton Analysis

    NASA Astrophysics Data System (ADS)

    Zmijan, R.; Abi Kaed Bey, S.; Mowlem, M. C.; Morgan, H.

    2012-04-01

    The use of flow cytometry for studies of temporal and spatial variability of phytoplankton populations is a valuable tool contributing to research relating carbon biogeochemistry and climate change. Early designs and marine deployments of such devices started over two decades ago [1-3]. Miniaturisation and cost reduction without sacrificing performance remains a major challenge but would enable mass production and deployment. Large numbers of measurement nodes (e.g. as part of a global ocean observation system) would be possible which would increase data available over both spatial and temporal scales. This research presents two different design approaches for miniaturisation and integration of optics into a microfluidic cytometer chip. The proposed solutions are suitable for micro cytometers with external components coupled with optical fibres and were simulated and optimised using ray tracing software (Zemax). The two designs address light delivery for excitation of particles within the measurement region of the cytometer. One uses an integrated micro lens (fabricated in the chip) and the other a ball shaped micro lens manufactured separately and then inserted into the chip. Both approaches collimate the excitation light beam (from an off chip diode laser coupled with an optical fibre) into the fluidic channel. The predicted (by ray tracing) excitation beam widths are 70 and 80 µm for the integrated and the ball lens respectively, and are in agreement with experimental data presented. The proposed cytometer chip design is compatible with low cost materials (acrylic glass, cyclo-olefines) and manufacturing methods (micro milling, hot embossing, injection moulding). 1. Dubelaar, G.B.J. and P.L. Gerritzen, CytoBuoy: a step forward towards using flow cytometry in operational oceanography. Scientia Marina, 2000. 64(2): p. 255-265. 2. Peeters, J.C.H., et al., Optical Plankton Analyzer - a Flow Cytometer for Plankton Analysis .1. Design Considerations. Cytometry, 1989

  7. A structured population modeling framework for quantifying and predicting gene expression noise in flow cytometry data.

    PubMed

    Flores, Kevin B

    2013-07-01

    We formulated a structured population model with distributed parameters to identify mechanisms that contribute to gene expression noise in time-dependent flow cytometry data. The model was validated using cell population-level gene expression data from two experiments with synthetically engineered eukaryotic cells. Our model captures the qualitative noise features of both experiments and accurately fit the data from the first experiment. Our results suggest that cellular switching between high and low expression states and transcriptional re-initiation are important factors needed to accurately describe gene expression noise with a structured population model.

  8. Stem and Progenitor Cell Subsets Are Affected by JAK2 Signaling and Can Be Monitored by Flow Cytometry

    PubMed Central

    Iida, Ryuji; Welner, Robert S.; Zhao, Wanke; Alberola-lla, José; Medina, Kay L.; Zhao, Zhizhuang Joe; Kincade, Paul W.

    2014-01-01

    Although extremely rare, hematopoietic stem cells (HSCs) are divisible into subsets that differ with respect to differentiation potential and cell surface marker expression. For example, we recently found that CD86− CD150+ CD48− HSCs have limited potential for lymphocyte production. This could be an important new tool for studying hematological abnormalities. Here, we analyzed HSC subsets with a series of stem cell markers in JAK2V617F transgenic (Tg) mice, where the mutation is sufficient to cause myeloproliferative neoplasia with lymphocyte deficiency. Total numbers of HSC were elevated 3 to 20 fold in bone marrow of JAK2V617F mice. Careful analysis suggested the accumulation involved multiple HSC subsets, but particularly those characterized as CD150HI CD86− CD18L°CD41+ and excluding Hoechst dye. Real-Time PCR analysis of their HSC revealed that the erythropoiesis associated gene transcripts Gata1, Klf1 and Epor were particularly high. Flow cytometry analyses based on two differentiation schemes for multipotent progenitors (MPP) also suggested alteration by JAK2 signals. The low CD86 on HSC and multipotent progenitors paralleled the large reductions we found in lymphoid progenitors, but the few that were produced functioned normally when sorted and placed in culture. Either of two HSC subsets conferred disease when transplanted. Thus, flow cytometry can be used to observe the influence of abnormal JAK2 signaling on stem and progenitor subsets. Markers that similarly distinguish categories of human HSCs might be very valuable for monitoring such conditions. They could also serve as indicators of HSC fitness and suitability for transplantation. PMID:24699465

  9. Cell synchronization by inhibitors of DNA replication induces replication stress and DNA damage response: analysis by flow cytometry

    PubMed Central

    Darzynkiewicz, Zbigniew; Halicka, H. Dorota; Zhao, Hong

    2010-01-01

    Cell synchronization is often achieved by inhibition of DNA replication. The cells cultured in the presence of such inhibitors as hydroxyurea, aphidicolin or thymidine become arrested at the entrance to S-phase and upon release from the block they synchronously progress through S, G2 and M. We recently reported that exposure of cells to these inhibitors at concentrations commonly used to synchronize cell populations led to phosphorylation of histone H2AX on Ser139 (induction of γH2AX) through activation of ataxia telangiectasia mutated and Rad3-related protein kinase (ATR). These findings imply that the induction of DNA replication stress by these inhibitors activates the DNA damage response cell signaling pathways and caution about interpreting data obtained with the use of cells synchronized such way as representing unperturbed cells. The protocol presented in this chapter describes the methodology of assessment of phosphorylation of histone H2AX-Ser139, ATM/ATR substrate on Ser/Thr at SQ/TQ cluster domains as well as ataxia telangiectasia mutated (ATM) protein kinase in cells treated with inhibitors of DNA replication. Phosphorylation of these proteins is detected in individual cell immunocytochemically with phospho-specific Ab and measured by flow cytometry. Concurrent measurement of cellular DNA content and phosphorylated proteins followed by multiparameter cytometric analysis allows one to correlate extent of their phosphorylation with cell cycle phase. PMID:21755443

  10. 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

  11. In search for cross-reactivity to immunophenotype equine mesenchymal stromal cells by multicolor flow cytometry.

    PubMed

    De Schauwer, Catharina; Piepers, Sofie; Van de Walle, Gerlinde R; Demeyere, Kristel; Hoogewijs, Maarten K; Govaere, Jan L J; Braeckmans, Kevin; Van Soom, Ann; Meyer, Evelyne

    2012-04-01

    During recent years, cell-based therapies using mesenchymal stem cells (MSC) are reported in equine veterinary medicine with increasing frequency. In most cases, the isolation and in vitro differentiation of equine MSC are described, but their proper immunophenotypic characterization is rarely performed. The lack of a single marker specific for MSC and the limited availability of monoclonal antibodies (mAbs) for equine MSC in particular, strongly hamper this research. In this study, 30 commercial mAbs were screened with flow cytometry for recognizing equine epitopes using the appropriate positive controls to confirm their specificity. Cross-reactivity was found and confirmed by confocal microscopy for CD45, CD73, CD79α, CD90, CD105, MHC-II, a monocyte marker, and two clones tested for CD29 and CD44. Unfortunately, none of the evaluated CD34 clones recognized the equine epitopes on positive control endothelial cells. Subsequently, umbilical cord blood-derived undifferentiated equine MSC of the fourth passage of six horses were characterized using multicolor flow cytometry based on the selected nine-marker panel of both cell surface antigens and intracytoplasmatic proteins. In addition, appropriate positive and negative controls were included, and the viable single cell population was analyzed by excluding dead cells using 7-aminoactinomycin D. Isolated equine MSC of the fourth passage were found to be CD29, CD44, CD90 positive and CD45, CD79α, MHC-II, and a monocyte marker negative. A variable expression was found for CD73 and CD105. Successful differentiation towards the osteogenic, chondrogenic, and adipogenic lineage was used as additional validation. We suggest that this selected nine-marker panel can be used for the adequate immunophenotyping of equine MSC. Copyright © 2012 International Society for Advancement of Cytometry.

  12. 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

  13. Use of flow cytometry for analysis of phage-mediated killing of Enterobacter aerogenes.

    PubMed

    Verthé, Kristof; Verstraete, Willy

    2006-09-01

    In this study, the use of flow cytometry to analyze phage-mediated killing of Enterobacter aerogenes under varying conditions of temperature and nutrient availability was assessed. Bacteriophage UZ1, specific for an E. aerogenes strain, was applied at a multiplicity of infection (MOI) of 1 and 1000 to a Teflon surface, artificially infected with its host at a level of 4.5 log cells. After incubation for 20 h, bacteriophages were quantified using the soft agar layer method. For the quantification of bacterial cells, plate counting and flow cytometric analysis of live/dead stained cells were performed in parallel. At an MOI of 1, phage treatment was successful only after incubation under nutrient-rich conditions at 37 degrees C: E. aerogenes cells were not detected and a tenfold increase in phage UZ1 was observed. At a MOI of 1000, no E. aerogenes cells could be cultured after incubation at 37 and 4 degrees C. However, flow cytometric analysis revealed that lysis did not occur at 4 degrees C but was achieved during subsequent plate culture. In conclusion, the use of flow cytometry enabled identification of culture-based bias during plate culture. The flow cytometric assay used in this study proved to be rapid, as this culture-independent method does not require lengthy incubation periods post-sampling. The bacteriophage-mediated killing of E. aerogenes cells on Teflon surfaces indicated that disinfection of E. aerogenes with bacteriophage UZ1 can be successful when high MOIs are achieved, while at low multiplicities of infection conditions favorable for phage replication are required.

  14. 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

  15. Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

    PubMed Central

    Pickles, Sarah; Arbour, Nathalie; Vande Velde, Christine

    2014-01-01

    Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver. PMID:25285411

  16. 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

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

    PubMed

    Lee, Denise; Grigoriadis, George; Westerman, David

    2015-12-01

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

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

    PubMed

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

    2014-01-01

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

  19. Quantitative studies of chicken somatotrophs during growth and development by morphometry, immunocytochemistry, and flow cytometry.

    PubMed

    Malamed, S; Deaver, D; Perez, F; Radecki, S; Gibney, J; Scanes, C G

    1997-10-01

    Changes in the male chicken somatotroph during growth and maturation have been examined by morphometric and immunocytochemical (ICC) analysis of serial sections of the anterior pituitary gland and by flow cytometry of dispersed anterior pituitary cells. ICC showed that somatotrophs are confined to the middle and caudal thirds of the anterior pituitary gland at all ages from 5 to 26 weeks. At a given age somatotrophs are of equal size at all positions along the cephalocaudal axis of the anterior pituitary gland. However, there are age-related changes: from 5 to 11 weeks rises occur in both the mean total somatotroph volume per gland (64%) and the mean number of somatotrophs (78%), while the mean volume of the single somatotroph is unchanged. From 11 to 18 weeks the mean volume of the single somatotroph decreases 41%. From 18 to 26 weeks the mean volume of the somatotroph, the mean total somatotroph volume, and the mean number per gland do not change. Flow cytometry studies suggested that somatotrophs from adults have less growth hormone (GH) than somatotrophs from young birds. The increases in total somatotroph volume and number from 5 to 11 weeks are consistent with the rise in anterior pituitary GH reported previously. Basic quantitative morphological information about age-related changes in somatotrophs is reported here. When combined with additional facts from future work, they may explain the well-documented sharp decline in circulating GH from 5 to 11 weeks. Copyright 1997 Academic Press.

  20. 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

  1. 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.

  2. Characterisation of cryoinjury in Euglena gracilis using flow-cytometry and cryomicroscopy.

    PubMed

    Fleck, Roland A; Pickup, Roger W; Day, John G; Benson, Erica E

    2006-04-01

    Flow-cytometry and cryomicroscopy elucidated that the unicellular algal protist Euglena gracilis was undamaged by cryoprotectant added at 0 degree C, and super-cooling in the absence of ice. Cryoinjuries were however induced by: osmotic shock resulting from excessive cryodehydration, intracellular ice, and fracturing of the frozen medium on thawing. Suboptimal cooling at -0.3 degrees C min(-1) to -60 degrees C and osmotic shock invariably resulted in damage to the organism's pellicle and osmoregulatory system causing, a significant (P > 0.005) increase in cell size. Cell damage was not repairable and led to death. The responses of E. gracilis to cryopreservation as visualised by flow-cytometry and cryomicroscopy assisted the development of an improved storage protocol. This comprised: cryoprotection with methanol [10%(v/v)] at 0 degree C, cooling at 0.5 degrees C min(-1) to -60 degrees C, isothermal hold for 30 min, and direct immersion in liquid nitrogen. Highest post-thaw viability (>60%) was obtained using two-step thawing, which involved initial slow warming to -130 degrees C followed by relatively rapid warming (approximately 90 degrees C min(-1)) to ambient temperature (ca. 25 degrees C).

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

    PubMed

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

    2015-10-01

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

  4. 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

  5. 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

  6. Assessment of ploidy stability of the somatic embryogenesis process in Quercus suber L. using flow cytometry.

    PubMed

    Loureiro, J; Pinto, G; Lopes, T; Dolezel, J; Santos, C

    2005-08-01

    Flow cytometry analyses were used to verify the ploidy stability of Quercus suber L. somatic embryogenesis process. Leaf explants of two adult cork oak trees (QsG0 and QsG5) of the North of Portugal were inoculated on MS medium with 2,4-D and zeatin. After 3 months, calluses with embryogenic structures were isolated and transferred to fresh MS medium without growth regulators and somatic embryo evolution was followed. Morphologically normal somatic embryos (with two cotyledons) and abnormal somatic embryos (with one or three cotyledons) were used in this assay. Flow cytometry combined with propidium iodide staining was employed to estimate DNA ploidy levels and nuclear DNA content of somatic embryos and leaves from mother plants. No significant differences (P< or =0.05) were detected among embryos, and between the embryos and the mother plants. Also, after conversion of these embryos, no significant morphological differences were observed among the somatic embryo-derived plants. These results and further studies using converted plantlet leaves and embryogenic callus tissue indicate that embryo cultures and converted plantlets were stable with regard to ploidy level. As no major somaclonal variation was detected our primary goal of "true-to-type" propagation of cork oak using somatic embryogenesis was assured at this level. The estimation of the 2C nuclear DNA content for this species is similar to the previously obtained value.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. Viability and membrane potential analysis of Bacillus megaterium cells by impedance flow cytometry.

    PubMed

    David, F; Hebeisen, M; Schade, G; Franco-Lara, E; Di Berardino, M

    2012-02-01

    Single cell analysis is an important tool to gain deeper insights into microbial physiology for the characterization and optimization of bioprocesses. In this study a novel single cell analysis technique was applied for estimating viability and membrane potential (MP) of Bacillus megaterium cells cultured in minimal medium. Its measurement principle is based on the analysis of the electrical cell properties and is called impedance flow cytometry (IFC). Comparatively, state-of-the-art fluorescence-based flow cytometry (FCM) was used to verify the results obtained by IFC. Viability and MP analyses were performed with cells at different well-defined growth stages, focusing mainly on exponential and stationary phase cells, as well as on dead cells. This was done by PI and DiOC(2)(3) staining assays in FCM and by impedance measurements at 0.5 and 10 MHz in IFC. In addition, transition growth stages of long-term cultures and agar plate colonies were characterized with both methods. FCM and IFC analyses of all experiments gave comparable results, quantitatively and qualitatively, indicating that IFC is an equivalent technique to FCM for the study of physiological cell states of bacteria. Copyright © 2011 Wiley Periodicals, Inc.

  12. 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.

  13. Analysis of molecular assemblies by flow cytometry: determinants of Gi1 and by binding

    NASA Astrophysics Data System (ADS)

    Sarvazyan, Noune A.; Neubig, Richard R.

    1998-05-01

    We report here a novel application of flow cytometry for the quantitative analysis of the high affinity interaction between membrane proteins both in detergent solutions and when reconstituted into lipid vesicles. The approach is further advanced to permit the analysis of binding to expressed protein complexes in native cell membranes. The G protein heterotrimer signal transduction function links the extracellularly activated transmembrane receptors and intracellular effectors. Upon activation, (alpha) and (beta) (gamma) subunits of G protein undergo a dissociation/association cycle on the cell membrane interface. The binding parameters of solubilized G protein (alpha) and (beta) (gamma) subunits have been defined but little is known quantitatively about their interactions in the membrane. Using a novel flow cytometry approach, the binding of low nanomolar concentrations of fluorescein-labeled G(alpha) i1 (F- (alpha) ) to (beta) (gamma) both in detergent solution and in a lipid environment was quantitatively compared. Unlabeled (beta) $gama reconstituted in biotinylated phospholipid vesicles bound F-(alpha) tightly (Kd 6 - 12 nM) while the affinity for biotinylated-(beta) (gamma) in Lubrol was even higher (Kd of 2.9 nM). The application of this approach to proteins expressed in native cell membranes will advance our understanding of G protein function in context of receptor and effector interaction. More generally, this approach can be applied to study the interaction of any fluorescently labeled protein with a membrane protein which can be expressed in Sf9 plasma membranes.

  14. 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.

  15. Determination of aneuploids in hop (Humulus lupulus L.) using flow cytometry.

    PubMed

    Šesek, Predrag; Šuštar-Vozlič, Jelka; Bohanec, Borut

    2000-01-01

    In order to study the possibility that high-resolution flow cytometry can be used for determination of aneuploids, different genotypes of Humulus lupulus were analyzed. Triploid cultivars are bred by hybridization between diploid and tetraploid lines, and as the result of this process, some aneuploids are occasionally also formed. We analyzed eight triploid cultivars and seven putative aneuploids. Triploid cultivars Cerera, Cicero, Celeia, Cekin, Blisk, Mt. Hood, Huller Bit. and Willamette (3x = 30) were measured for nuclear DNA content using Trifolium repens as reference. No significant differences among peak positions of triploid cultivars (having an average CV value per peak of 1.94%) were found. Measurement of nuclear DNA content was also performed for seven lines: 175/75, 89/113, 89/154, 91/215, 175/17, 89/87 and 91/74 previously determined by chromosome counting to be aneuploids (CV per peak was 1.41%). A statistically lower DNA content was found for line 175/75 and higher values were measured for lines 89/154, 89/113 and 91/215. Repeated chromosome counting revealed that the number of chromosomes in line 175/75 was 29, while lines 89/154, 89/113 and 91/215 possessed 31 chromosomes. The other lines were identified as triploids. We conclude that flow cytometry can be efficiently used for determination of aneuploidy in Humulus lupulus.

  16. A novel flow cytometry-based assay for the quantification of antibody-dependent pneumococcal agglutination

    PubMed Central

    van der Gaast—de Jongh, Christa E.; Diavatopoulos, Dimitri A.; de Jonge, Marien I.

    2017-01-01

    The respiratory pathogen Streptococcus pneumoniae is a major cause of diseases such as otitis media, pneumonia, sepsis and meningitis. The first step towards infection is colonization of the nasopharynx. Recently, it was shown that agglutinating antibodies play an important role in the prevention of mucosal colonization with S. pneumoniae. Here, we present a novel method to quantify antibody-dependent pneumococcal agglutination in a high-throughput manner using flow cytometry. We found that the concentration of agglutinating antibodies against pneumococcal capsule are directly correlated with changes in the size and complexity of bacterial aggregates, as measured by flow cytometry and confirmed by light microscopy. Using the increase in size, we determined the agglutination index. The cutoff value was set by measuring a series of non-agglutinating antibodies. With this method, we show that not only anti-polysaccharide capsule antibodies are able to induce agglutination but that also anti-PspA protein antibodies have agglutinating capabilities. In conclusion, we have described and validated a novel method to quantify pneumococcal agglutination, which can be used to screen sera from murine or human vaccination studies, in a high-throughput manner. PMID:28288168

  17. 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

  18. Suitability of Flow Cytometry for Estimating Bacterial Biovolume in Natural Plankton Samples: Comparison with Microscopy Data▿

    PubMed Central

    Felip, Marisol; Andreatta, Stefan; Sommaruga, Ruben; Straskrábová, Viera; Catalan, Jordi

    2007-01-01

    The relationship between flow cytometry data and epifluorescence microscopy measurements was assessed in bacterioplankton samples from 80 lakes to estimate bacterial biovolume and cell size distribution. The total counts of 4′,6′-diamidino-2-phenylindole-stained cells estimated by both methods were significantly related, and the slope of their linear regression was not significantly different from 1, indicating that both methods produce very similar estimates of bacterial abundance. The relationships between side scatter (SSC) and 4′,6′-diamidino-2-phenylindole fluorescence and cell volume (microscopy values) were improved by binning of the data in three frequency classes for each, but further increases in the number of classes did not improve these relationships. Side scatter was the best cell volume predictor, and significant relationships were observed between the SSC classes and the smallest (R2 = 0.545, P < 0.001, n = 80) and the largest (R2 = 0.544, P < 0.001, n = 80) microscopy bacterial-size classes. Based on these relationships, a reliable bacterial biomass estimation was obtained from the SSC frequency classes. Our study indicates that flow cytometry can be used to properly estimate bacterioplankton biovolume, with an accuracy similar to those of more time-consuming microscopy methods. PMID:17513595

  19. 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

  20. DNA ploidy pattern in choroidal melanoma: correlation with survival. A flow cytometry study on archival material

    PubMed Central

    Toti, P.; Greco, G.; Mangiavacchi, P.; Bruni, A.; Palmeri, M. L.; Luzi, P.

    1998-01-01

    BACKGROUND/AIMS—Paraffin embedded samples have provided an important source of material for retrospective cytofluorimetric studies, useful in establishing the predictive value of DNA content measurements. The aim of this study was to investigate the incidence and type of aneuploidy in choroidal malignant melanomas (CMM) and the significance in the clinical outcome (median follow up 55 months).
METHODS—DNA content was quantified by flow cytometry in 61 CMM from archival material. Non-tumour ocular tissue was used as the reference diploid standard. Cases in which the coefficient of variation (CV) of the diploid peak was >8% were excluded. The CMM were classified as spindle A, spindle B, mixed spindle and epithelioid, epithelioid, and necrotic.
RESULTS—The frequency of the aneuploid DNA pattern was 38%. Necrotic tumours showed a worse clinical outcome independent of the ploidy pattern. Spindle A tumours were found to be diploid. Spindle B and mixed tumours showed a prevalent diploid and near diploid aneuploid pattern (DI <1.3), yet aneuploidy was not correlated with a worse prognosis. The epithelioid tumours were prevalently diploid. However, 83% of the aneuploid tumours were hypodiploid (DI <0.95), and showed the worst prognosis.
CONCLUSION—These results indicate that increasing DNA abnormalities in CMM, especially in the epithelioid histotype, were associated with an increasing mortality.

 Keywords: ocular melanoma; flow cytometry; DNA PMID:9930278

  1. 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

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

    PubMed

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

    2015-10-14

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

  3. Enhanced red and near infrared detection in flow cytometry using avalanche photodiodes.

    PubMed

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

    2008-08-01

    Polychromatic flow cytometry enables detailed identification of cell phenotype using multiple fluorescent parameters. The photomultiplier tubes (PMTs) 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 (APDs), which have improved red sensitivity and a working fluorescence detection range beyond 1,000 nm. A comparison of the wavelength-dependent performance of the APD and PMT 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 PMTs and APD detectors. The APD used an additional amplifier stage to match the internal gain of the PMT. The resolution of the APD and PMT was compared for flow cytometry applications using a pulsed light-emitting diode source over the 500-1060 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 APD and PMTs were used to measure the signal-to-noise response for a set of six peak calibration beads over the 530-800 nm wavelength range. CD4-positive cells labeled with antibody-conjugated phycoerythrin or 800 nm quantum dots were identified by simultaneous detection using the APD and the PMT. 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 APD was able to separate these populations at wavelengths above 800 nm. These measurements illustrate the differences in APD and PMT performance at different wavelengths and signal intensity levels. While the APD and PMT show similar signal-to-noise performance in the visible spectral range, the dark noise of the APD detector reduces the sensitivity at low signal levels. At wavelengths longer than 650 nm, the high quantum efficiency

  4. 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

  5. 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).

  6. Flow cytometry methods for the study of cell-cycle parameters of planarian stem cells.

    PubMed

    Kang, Hara; Sánchez Alvarado, Alejandro

    2009-05-01

    Due to their characteristic inaccessibility and low numbers, little is known about the cell-cycle dynamics of most stem cells in vivo. A powerful, established methodology to study cell-cycle dynamics is flow cytometry, which is used routinely to study the cell-cycle dynamics of proliferating cells in vitro. Its use in heterogeneous mixtures of cells obtained from whole animals, however, is complicated by the relatively low abundance of cycling to non-cycling cells. We report on flow cytometric methods that take advantage of the abundance of proliferating stem cells in the planarian Schmidtea mediterranea. The optimized protocols allow us to measure cell-cycle dynamics and follow BrdU-labeled cells specifically in complex mixtures of cells. These methods expand on the growing toolkit being developed to study stem cell biology in planarians, and open the door to detailed cytometric studies of a collectively totipotent population of adult stem cells in vivo.

  7. Artificial neural network study of whole-cell bacterial bioreporter response determined using fluorescence flow cytometry.

    PubMed

    Busam, Sirisha; McNabb, Maia; Wackwitz, Anke; Senevirathna, Wasana; Beggah, Siham; Meer, Jan Roelof van der; Wells, Mona; Breuer, Uta; Harms, Hauke

    2007-12-01

    Genetically engineered bioreporters are an excellent complement to traditional methods of chemical analysis. The application of fluorescence flow cytometry to detection of bioreporter response enables rapid and efficient characterization of bacterial bioreporter population response on a single-cell basis. In the present study, intrapopulation response variability was used to obtain higher analytical sensitivity and precision. We have analyzed flow cytometric data for an arsenic-sensitive bacterial bioreporter using an artificial neural network-based adaptive clustering approach (a single-layer perceptron model). Results for this approach are far superior to other methods that we have applied to this fluorescent bioreporter (e.g., the arsenic detection limit is 0.01 microM, substantially lower than for other detection methods/algorithms). The approach is highly efficient computationally and can be implemented on a real-time basis, thus having potential for future development of high-throughput screening applications.

  8. A rapid and robust tri-color flow cytometry assay for monitoring malaria parasite development.

    PubMed

    Malleret, Benoît; Claser, Carla; Ong, Alice Soh Meoy; Suwanarusk, Rossarin; Sriprawat, Kanlaya; Howland, Shanshan Wu; Russell, Bruce; Nosten, Francois; Rénia, Laurent

    2011-01-01

    Microscopic examination of Giemsa-stained thin blood smears remains the gold standard method used to quantify and stage malaria parasites. However, this technique is tedious, and requires trained microscopists. We have developed a fast and simple flow cytometry method to quantify and stage, various malaria parasites in red blood cells in whole blood or in vitro cultured Plasmodium falciparum. The parasites were stained with dihydroethidium and Hoechst 33342 or SYBR Green I and leukocytes were identified with an antibody against CD45. Depending on the DNA stains used, samples were analyzed using different models of flow cytometers. This protocol, which does not require any washing steps, allows infected red blood cells to be distinguished from leukocytes, as well as allowing non-infected reticulocytes and normocytes to be identified. It also allows assessing the proportion of parasites at different developmental stages. Lastly, we demonstrate how this technique can be applied to antimalarial drug testing.

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

    PubMed Central

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

    2010-01-01

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

  10. 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

  11. flowPeaks: a fast unsupervised clustering for flow cytometry data via K-means and density peak finding

    PubMed Central

    Ge, Yongchao; Sealfon, Stuart C.

    2012-01-01

    Motivation: For flow cytometry data, there are two common approaches to the unsupervised clustering problem: one is based on the finite mixture model and the other on spatial exploration of the histograms. The former is computationally slow and has difficulty to identify clusters of irregular shapes. The latter approach cannot be applied directly to high-dimensional data as the computational time and memory become unmanageable and the estimated histogram is unreliable. An algorithm without these two problems would be very useful. Results: In this article, we combine ideas from the finite mixture model and histogram spatial exploration. This new algorithm, which we call flowPeaks, can be applied directly to high-dimensional data and identify irregular shape clusters. The algorithm first uses K-means algorithm with a large K to partition the cell population into many small clusters. These partitioned data allow the generation of a smoothed density function using the finite mixture model. All local peaks are exhaustively searched by exploring the density function and the cells are clustered by the associated local peak. The algorithm flowPeaks is automatic, fast and reliable and robust to cluster shape and outliers. This algorithm has been applied to flow cytometry data and it has been compared with state of the art algorithms, including Misty Mountain, FLOCK, flowMeans, flowMerge and FLAME. Availability: The R package flowPeaks is available at https://github.com/yongchao/flowPeaks. Contact: yongchao.ge@mssm.edu Supplementary information: Supplementary data are available at Bioinformatics online PMID:22595209

  12. 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

  13. Use of flow cytometry and monochlorobimane to quantitate intracellular glutathione concentrations in feline leukocytes.

    PubMed

    Webb, Craig; Bedwell, Cathy; Guth, Amanda; Avery, Paul; Dow, Steven

    2006-08-15

    Oxidative stress and abnormal glutathione metabolism is thought to play an important role in various diseases of cats. However, current assays for the reduced form of glutathione (GSH) are time-consuming and semi-quantitative and do not allow assessment of GSH concentrations in individual cell populations. Therefore, we developed a flow cytometric assay for rapid determination of intracellular GSH concentrations in feline blood leukocytes. The assay was based on the ability of the non-fluorescent substrate monochlorobimane (mBCl) to form fluorescent adducts with GSH in a reaction catalyzed by the enzyme glutathione-S-transferase. Using flow cytometry, we found that mBCl was sensitive and specific for intracellular detection of the reduced form of GSH in feline leukocytes. Intracellular GSH concentrations were also stable for at least 24h in EDTA preserved whole blood samples stored at 4 degrees C. Neutrophils and monocytes from normal cats had significantly higher intracellular concentrations of GSH than T cells and B cells. The effects of FIV infection on intracellular GSH concentrations in cats were assessed using flow cytometry. We found that neutrophils from FIV-infected cats had significantly increased GSH concentrations, whereas intracellular GSH concentrations were significantly decreased in CD4(+) and CD8(+) lymphocytes from FIV-infected cats, compared to age-matched control animals. We conclude that a flow cytometric assay based on mBCl may be used to accurately and rapidly assess the effects of various disease states and treatments on GSH concentration in cat leukocytes and to help assess intracellular oxidative stress.

  14. Evaluation of primary mediastinal large B cell lymphoma by flow cytometry.

    PubMed

    Cherian, Sindhu; Fromm, Jonathan R

    2017-07-21

    Primary mediastinal large B cell lymphoma (PMLBCL) is a B cell non-Hodgkin lymphoma (B-NHL) that shows morphologic, immunophenotypic, and genetic similarities to classical Hodgkin lymphoma (CHL). We evaluated the neoplastic and reactive infiltrate of PMLBCL by flow cytometry (FC). A total of 24 cases of PMLBCL were retrospectively characterized using FC combinations for B-NHL, T-NHL, and CHL. The CHL assay identified the neoplastic population (NP) in all cases, while the B-NHL assay identified the NP in 18 of 24 cases. In four cases, the neoplastic population was retrospectively identified in the B-NHL tube, given the CHL tube-derived immunophenotype. Neoplastic cells of PMLBCL displayed a B cell immunophenotype (CD19/CD20(+) ), with CD40 consistently and brightly expressed. Most cases lacked immunoglobulin light chains, CD10, and CD15; CD30 was frequently expressed. All cases showed expression of CD71 and CD95. The reactive infiltrate in PMLBCL showed increases in T cells relative to reactive tissues. Lower CD4/CD8 ratios in PMLBCL relative to CHL and reactive tissues were also observed. Although not seen as commonly as with CHL, 41% of cases showed a reactive CD3/CD4/CD7(bright) /CD45(bright+) T cell population. Reactive populations seen in PMLBCL were similar to that seen in diffuse large B cell lymphoma, not otherwise specified. FC can detect the NP of PMLBCL and a FC assay for CHL performed better than a B-NHL assay in this regard. The neoplastic cells showed a B cell immunophenotype with over-expression of CD40. The reactive infiltrate in PMLBCL shows unique features including a prominent CD8(+) T cell infiltrate. © 2017 International Clinical Cytometry Society. © 2017 International Clinical Cytometry Society.

  15. Measurement of lipid accumulation in Chlorella vulgaris via flow cytometry and liquid-state ¹H NMR spectroscopy for development of an NMR-traceable flow cytometry protocol.

    PubMed

    Bono, Michael S; Garcia, Ravi D; Sri-Jayantha, Dylan V; Ahner, Beth A; Kirby, Brian J

    2015-01-01

    In this study, we cultured Chlorella vulgaris cells with a range of lipid contents, induced via nitrogen starvation, and characterized them via flow cytometry, with BODIPY 505/515 as a fluorescent lipid label, and liquid-state 1H NMR spectroscopy. In doing so, we demonstrate the utility of calibrating flow cytometric measurements of algal lipid content using triacylglyceride (TAG, also known as triacylglycerol or triglyceride) content per cell as measured via quantitative 1H NMR. Ensemble-averaged fluorescence of BODIPY-labeled cells was highly correlated with average TAG content per cell measured by bulk NMR, with a linear regression yielding a linear fit with r2 = 0.9974. This correlation compares favorably to previous calibrations of flow cytometry protocols to lipid content measured via extraction, and calibration by NMR avoids the time and complexity that is generally required for lipid quantitation via extraction. Flow cytometry calibrated to a direct measurement of TAG content can be used to investigate the distribution of lipid contents for cells within a culture. Our flow cytometry measurements showed that Chlorella vulgaris cells subjected to nitrogen limitation exhibited higher mean lipid content but a wider distribution of lipid content that overlapped the relatively narrow distribution of lipid content for replete cells, suggesting that nitrogen limitation induces lipid accumulation in only a subset of cells. Calibration of flow cytometry protocols using direct in situ measurement of TAG content via NMR will facilitate rapid development of more precise flow cytometry protocols, enabling investigation of algal lipid accumulation for development of more productive algal biofuel feedstocks and cultivation protocols.

  16. 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

  17. Study of inertial hydrodynamic focusing in sheath-driven flows for lab-on-a-chip flow cytometry

    NASA Astrophysics Data System (ADS)

    Panwar, Nishtha; Song, Peiyi; Yong, Ken-Tye; Tjin, Swee Chuan

    2017-05-01

    Miniature flow cytometer models enable fast and cost-effective management of diseases in vulnerable and low-end settings. The single-line focusing of cell or particle samples is achieved using hydrodynamic forces in the microfluidic channels. The two common configurations among them are the single-sheath and dual-sheath flows wherein the sample is directed through the main channel, and the surrounding sheath fluids are directed into the main channel through inlets on either side of the main channel. Most models predict the width of the focused sample stream based on hydrodynamic focusing in the low Reynolds number regime (Re << 1), where the viscous forces dominate the inertial forces. In this work, we present comparative analysis of particle focusing by single-sheath and dual-sheath configurations for focusing of micron-sized cells/particles in the range 2 to 20 μm in the higher Re (10 < Re < 80) laminar regime. A quantitative analysis of the relative focused stream width (wf/wch) as a function of flow rate ratio (FRR = Sample flow rate/Sheath flow rate) for the two configurations is presented. The particle tracing results are also compared with the experimental fluorescent microscopy results at various FRR. The deviations of the results from the theoretical predictions of hydrodynamic focusing at Re << 1, are explained analytically. These findings clearly outline the range of flow parameters and relative particle sizes that can be used for cytometry studies for a given channel geometry. This is a highly predictive modeling method as it provides substantial results of particle positions across the microchannel width according to their size and FRR for single-line focusing of particles. Such information is crucial for one to engineer miniaturized flow cytometry for screening of desired cells or particles.

  18. Characterization of viability and proliferation of alginate-poly-L-lysine-alginate encapsulated myoblasts using flow cytometry.

    PubMed

    Thakur, Ajit; Sengupta, Ruchira; Matsui, Hideto; Lillicrap, David; Jones, Kim; Hortelano, Gonzalo

    2010-08-01

    Genetically modified cells encapsulated in alginate-poly-L-lysine-alginate (APA) are being developed to deliver therapeutic products to treat a variety of diseases. The characterization of the encapsulated cells thus becomes paramount. This study reports a novel method to assess the viability, granularity and proliferation of encapsulated cells based on flow cytometry. The in vitro viability of encapsulated G8 murine myoblasts secreting canine FVIII (cFVIII) measured by flow cytometry was comparable to the traditional trypan blue exclusion method and both correlated with cFVIII secretion levels. In contrast, after implantation into mice, only viability measured by flow cytometry correlated with cFVIII secretion. Further, flow cytometry analysis of encapsulated cells maintained in vitro and in vivo revealed a greater fraction of granular cells compared to free cells, suggesting that encapsulation influences the morphology (cytoplasmic composition) of cells within APA microcapsules. Interestingly, the proliferation study showed that encapsulated cells proliferate faster, on average, and were more heterogeneous in vivo compared to in vitro culture conditions, suggesting that encapsulated cell proliferation is complex and environment-dependent. In conclusion, we show that flow cytometry analysis allows for a more consistent and comprehensive examination of encapsulated cells to aid in the development of cell therapy protocols.

  19. 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.

  20. 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.

  1. An open-source solution for advanced imaging flow cytometry data analysis using machine learning.

    PubMed

    Hennig, Holger; Rees, Paul; Blasi, Thomas; Kamentsky, Lee; Hung, Jane; Dao, David; Carpenter, Anne E; Filby, Andrew

    2017-01-01

    Imaging flow cytometry (IFC) enables the high throughput collection of morphological and spatial information from hundreds of thousands of single cells. This high content, information rich image data can in theory resolve important biological differences among complex, often heterogeneous biological samples. However, data analysis is often performed in a highly manual and subjective manner using very limited image analysis techniques in combination with conventional flow cytometry gating strategies. This approach is not scalable to the hundreds of available image-based features per cell and thus makes use of only a fraction of the spatial and morphometric information. As a result, the quality, reproducibility and rigour of results are limited by the skill, experience and ingenuity of the data analyst. Here, we describe a pipeline using open-source software that leverages the rich information in digital imagery using machine learning algorithms. Compensated and corrected raw image files (.rif) data files from an imaging flow cytometer (the proprietary .cif file format) are imported into the open-source software CellProfiler, where an image processing pipeline identifies cells and subcellular compartments allowing hundreds of morphological features to be measured. This high-dimensional data can then be analysed using cutting-edge machine learning and clustering approaches using "user-friendly" platforms such as CellProfiler Analyst. Researchers can train an automated cell classifier to recognize different cell types, cell cycle phases, drug treatment/control conditions, etc., using supervised machine learning. This workflow should enable the scientific community to leverage the full analytical power of IFC-derived data sets. It will help to reveal otherwise unappreciated populations of cells based on features that may be hidden to the human eye that include subtle measured differences in label free detection channels such as bright-field and dark-field imagery

  2. Quantitative data analysis methods for bead-based DNA hybridization assays using generic flow cytometry platforms.

    PubMed

    Corrie, S R; Lawrie, G A; Battersby, B J; Ford, K; Rühmann, A; Koehler, K; Sabath, D E; Trau, M

    2008-05-01

    Bead-based assays are in demand for rapid genomic and proteomic assays for both research and clinical purposes. Standard quantitative procedures addressing raw data quality and analysis are required to ensure the data are consistent and reproducible across laboratories independent of flow platform. Quantitative procedures have been introduced spanning raw histogram analysis through to absolute target quantitation. These included models developed to estimate the absolute number of sample molecules bound per bead (Langmuir isotherm), relative quantitative comparisons (two-sided t-tests), and statistical analyses investigating the quality of raw fluorescence data. The absolute target quantitation method revealed a concentration range (below probe saturation) of Cy5-labeled synthetic cytokeratin 19 (K19) RNA of c.a. 1 x 10(4) to 500 x 10(4) molecules/bead, with a binding constant of c.a. 1.6 nM. Raw hybridization frequency histograms were observed to be highly reproducible across 10 triplex assay replicates and only three assay replicates were required to distinguish overlapping peaks representing small sequence mismatches. This study provides a quantitative scheme for determining the absolute target concentration in nucleic acid hybridization reactions and the equilibrium binding constants for individual probe/target pairs. It is envisaged that such studies will form the basis of standard analytical procedures for bead-based cytometry assays to ensure reproducibility in inter- and intra-platform comparisons of data between laboratories. (c) 2008 International Society for Advancement of Cytometry.

  3. Impedance flow cytometry gauges proliferative capacity by detecting TRPC1 expression.

    PubMed

    Crocetti, Sara; Beyer, Christian; Unternährer, Silvio; Benavides Damm, Tatiana; Schade-Kampmann, Grit; Hebeisen, Monika; Di Berardino, Marco; Fröhlich, Jürg; Franco-Obregón, Alfredo

    2014-06-01

    When examined, the expansion of many stem cell classes has been shown to be facilitated by mechanically-regulated calcium entry from the extracellular space that also helps direct their developmental programs towards mechanosensitive tissues such as muscle, bone, and connective tissues. Cation channels of the transient receptor potential C class (TRPC) are the predominant conduit for calcium entry into proliferating myoblasts. Nonetheless, methods to non-invasively study this calcium-entry pathway are still in their infancy. Here we show that a microfluidic configuration of impedance-based flow cytometry (IFC) provides a method to detect TRP channel expression in cells at high throughput. Using this technology we discern changes in the IFC signal that correlates with the functional expression of TRPC1 channels and coincides with cell proliferation. Pharmacological agents, mechanical conditions or malignant states that alter the expression of TRPC1 channels are reflected in the IFC signal accordingly, whereas pharmacological agents that alter cation-permeation through TRPC1 channels, or ionophores that independently increase calcium entry across the membrane, have little effect. Our results suggest that IFC detects changes in whole-cell membrane organization associated with TRPC1 activation and surface expression, rather than cation permeation through the channel per se. IFC-based technologies thus have the potential to identify living stem cells in their earliest stages of expansion without staining or chemical fixation. © 2014 International Society for Advancement of Cytometry.

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

    PubMed

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

    2016-01-01

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

  5. NetFCM: a semi-automated web-based method for flow cytometry data analysis.

    PubMed

    Frederiksen, Juliet; Buggert, Marcus; Karlsson, Annika C; Lund, Ole

    2014-11-01

    Multi-parametric flow cytometry (FCM) represents an invaluable instrument to conduct single cell analysis and has significantly increased our understanding of the immune system. However, due to new techniques allowing us to measure an increased number of phenotypes within the immune system, FCM data analysis has become more complex and labor-intensive than previously. We have therefore developed a semi-automatic gating strategy (NetFCM) that uses clustering and principal component analysis (PCA) together with other statistical methods to mimic manual gating approaches. NetFCM is an online tool both for subset identification as well as for quantification of differences between samples. Additionally, NetFCM can classify and cluster samples based on multidimensional data. We tested the method using a data set of peripheral blood mononuclear cells collected from 23 HIV-infected individuals, which were stimulated with overlapping HIV Gag-p55 and CMV-pp65 peptides or medium alone (negative control). NetFCM clustered the virus-specific CD8+ T cells based on IFNγ and TNF responses into distinct compartments. Additionally, NetFCM was capable of identifying HIV- and CMV-specific responses corresponding to those obtained by manual gating strategies. These data demonstrate that NetFCM has the potential to identify relevant T cell populations by mimicking classical FCM data analysis and reduce the subjectivity and amount of time associated with such analysis. © 2014 International Society for Advancement of Cytometry.

  6. Evaluation of microparticles in whole blood by multicolour flow cytometry assay.

    PubMed

    Christersson, Christina; Johnell, Matilda; Siegbahn, Agneta

    2013-04-01

    To develop and evaluate a multicolour flow cytometry method for analysis of microparticles (MPs) in fresh whole blood without any centrifugation steps or freezing/thawing procedure. Flow cytometry was performed using a FC500 MPL cytometer. The compensation in the protocol was performed based on the platelet population. Polystyrene microspheres 0.50-1.27 μm were used for size position, and the MP gate was set as particles 0.5-1.0 μm. Whole blood was incubated with annexin V and antibodies to tissue factor (TF), platelets (CD41 and CD62P), monocyte (CD14) and endothelial cells (CD144). For comparison, MPs from platelet free supernatant was used. The TF activity was evaluated by Calibrated Automated Thrombogram. Annexin V was used to distinguish true events from background noise. For standardization, each analysis included 10,000 events in the gate of platelets. There were 622(462-1001) MP(annV+)/10,000 platelets and of these, 66 (49-82)/10,000 platelets expressed TF. After correction for the individual platelet counts, the amount of circulating MP(annV+) was 17.1 (12.1-24.9) × 10(9)/L in whole blood, and of these, 10% (6-12%) expressed TF. The majority of the MPs expressed CD41, and 5.6% (2.2-6.9%) of these co-expressed TF. The amount of CD41 + MP(annV+) tended to correlate to the TF activity in whole blood. There was no correlation between the MP(annV+) in whole blood and MPs derived from platelet free supernatant. Patients with pulmonary arterial hypertension and stable coronary artery disease had increased concentrations of CD41 + MP(annV+) in whole blood. This multicolour flow cytometry assay in whole blood mimics the in vivo situation by avoiding several procedure steps interfering with the MP count. By standardized quantification of MPs a reference interval of MPs can be created.

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

    PubMed

    Petrunkina, A M; Harrison, R A P

    2010-04-15

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

  8. Novel flow cytometry approach to identify bronchial epithelial cells from healthy human airways

    PubMed Central

    Maestre-Batlle, Danay; Pena, Olga M.; Hirota, Jeremy A.; Gunawan, Evelyn; Rider, Christopher F.; Sutherland, Darren; Alexis, Neil E.; Carlsten, Chris

    2017-01-01

    Sampling various compartments within the lower airways to examine human bronchial epithelial cells (HBEC) is essential for understanding numerous lung diseases. Conventional methods to identify HBEC in bronchoalveolar lavage (BAL) and wash (BW) have throughput limitations in terms of efficiency and ensuring adequate cell numbers for quantification. Flow cytometry can provide high-throughput quantification of cell number and function in BAL and BW samples, while requiring low cell numbers. To date, a flow cytometric method to identify HBEC recovered from lower human airway samples is unavailable. In this study we present a flow cytometric method identifying HBEC as CD45 negative, EpCAM/pan-cytokeratin (pan-CK) double-positive population after excluding debris, doublets and dead cells from the analysis. For validation, the HBEC panel was applied to primary HBEC resulting in 98.6% of live cells. In healthy volunteers, HBEC recovered from BAL (2.3% of live cells), BW (32.5%) and bronchial brushing samples (88.9%) correlated significantly (p = 0.0001) with the manual microscopy counts with an overall Pearson correlation of 0.96 across the three sample types. We therefore have developed, validated, and applied a flow cytometric method that will be useful to interrogate the role of the respiratory epithelium in multiple lung diseases. PMID:28165060

  9. 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.

  10. Study of low speed flow cytometry for diffraction imaging with different chamber and nozzle designs.

    PubMed

    Sa, Yu; Feng, Yuanming; Jacobs, Kenneth M; Yang, Jun; Pan, Ran; Gkigkitzis, Ioannis; Lu, Jun Q; Hu, Xin-Hua

    2013-11-01

    Achieving effective hydrodynamic focusing and flow stability at low speed presents a challenging design task in flow cytometry for studying phenomena such as cell adhesion and diffraction imaging of cells with low-cost cameras. We have developed different designs of flow chamber and sheath nozzle to accomplish the above goal. A 3D computational model of the chambers has been established to simulate the fluid dynamics in different chamber designs and measurements have been performed to determine the velocity and size distributions of the core fluid from the nozzle. Comparison of the simulation data with experimental results shows good agreement. With the computational model significant insights were gained for optimization of the chamber design and improvement of the cell positioning accuracy for study of slow moving cells. The benefit of low flow speed has been demonstrated also by reduced blurring in the diffraction images of single cells. Based on these results, we concluded that the new designs of chamber and sheath nozzle produce stable hydrodynamic focusing of the core fluid at low speed and allow detailed study of cellular morphology under various rheological conditions using the diffraction imaging method.

  11. Implementation of flow cytometry for quality control in four Danish bull studs.

    PubMed

    Christensen, P; Hansen, C; Liboriussen, T; Lehn-Jensen, H

    2005-02-01

    A flow cytometric method for simultaneous determination of sperm concentration and viability has recently been developed. In 2001, four Danish bull studs purchased flow cytometers and eight technicians were trained for routine analysis of raw and frozen-thawed semen. After initial training of the technicians, an experiment was carried out to document the precision of the system. The aim was also to assess if flow cytometric determination of sperm concentration could result in a more uniform production of semen doses. Results of this experiment showed high precision in the determination of sperm concentration and coefficients of variation were 3.5 and 2.4% for raw and frozen-thawed semen, respectively. Sperm viability was also assessed with high precision and coefficients of variation were 0.9% for raw semen and 1.7% for frozen-thawed semen. Furthermore, the experiment showed that package of semen doses after flow cytometric determination of sperm concentration in the raw semen results in a significantly smaller variation in the number of sperm per dose. In the second experiment, frozen semen was exchanged between the participating studs and were analysed by flow cytometry as well as by microscopic assessments by the eight technicians. Results show that the average correlation between technicians were 0.38 for motility assessments while flow cytometric agreement between technicians was significantly higher (average correlation was 0.86 for sperm viability and 0.92 for sperm concentration). The experiment also showed very high agreements between assessments within lab technician (correlations r=0.98 (sperm concentration) and r=0.99 (sperm viability)). Experiment 3 revealed that straws from the same batch varies in both concentration and viability. It is concluded that flow cytometric determination of sperm concentration and viability can be used to improve semen assessment by AI studs and result in a better quality control.

  12. 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

  13. 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.

  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. FRET and Flow Cytometry Assays to Measure Proteopathic Seeding Activity in Biological Samples.

    PubMed

    Furman, Jennifer L; Diamond, Marc I

    2017-01-01

    Transcellular propagation of protein aggregates-or seeds-is increasingly implicated as a mechanism for disease progression in many neurodegenerative disorders, including Alzheimer's disease and the related tauopathies. While neuropathology generally originates in one discrete brain region, pathology progresses as disease severity advances, often along discrete neural networks. The stereotypical spread of tau pathology suggests that cell-to-cell transfer of toxic protein aggregates could underlie disease progression, and recent studies implicate seeding as a proximal marker of disease, as compared to standard histological and biochemical analyses. Commonly used metrics for protein aggregation detection, however, lack sensitivity, are not quantitative, and/or undergo subjective classification. Here, we describe a FRET and flow cytometry cell-based assay that allows for rapid and quantitative detection of protein aggregates from human and rodent biological specimens.

  16. Analysis of DNA-guided self-assembly of microspheres using imaging flow cytometry.

    PubMed

    Tang, Hao; Deschner, Ryan; Allen, Peter; Cho, Younjin; Sermas, Patrick; Maurer, Alejandro; Ellington, Andrew D; Willson, C Grant

    2012-09-19

    Imaging flow cytometry was used to analyze the self-assembly of DNA-conjugated polystyrene microspheres. This technique enables quantitative analysis of the assembly process and thereby enables detailed analysis of the effect of structural and process variables on the assembly yield. In a demonstration of the potential of this technique, the influence of DNA strand base pair (bp) length was examined, and it was found that 50 bp was sufficient to drive the assembly of microspheres efficiently, forming not only dimers but also chainlike structures. The effect of stoichiometry on the yield was also examined. The analysis demonstrated that self-assembly of 50 bp microspheres can be driven nearly to completion by stoichiometric excess in a manner similar to Le Chatelier's principle in common chemical equilibrium.

  17. Quality control in the application of flow cytometry to studies of environmentally-induced genetic damage

    SciTech Connect

    McCreedy, C.D.; Robinson, J.P.; Dallas, C.E.; Jagoe, C.H.

    1999-07-01

    Flow cytometry (FCM) has been used to demonstrate altered DNA content in fish, reptiles, birds and mammals exposed to radionuclides, PAHs and other contaminants. However, artifacts resulting from sample preparation, handling, variations in instrument parameters or other factors may confound such measurements. Some artifacts resemble genotoxic responses and so could lead to erroneous positive conclusions. As part of ongoing studies of effects of various pollutants on DNA content in fishes, the authors tested sample handling and preparation methods for the induction of artifacts. The authors describe QA/QC methods, including control of staining, conditions, doublet discrimination by comparison of peak versus integral fluorescence, internal DNA standards, and the use of time versus fluorescence plots. Consistent application of these practices is essential to obtain valid measurements of DNA content in environmental samples, and neglect of these can result in poor quality data and the acceptance of incorrect hypotheses.

  18. Hematogones Detected by Flow Cytometry in a Child with Vitamin B12 Deficiency.

    PubMed

    Sutton, Lisa; Mba, Nkechi

    2017-01-01

    Vitamin B12 deficiency is a known cause of megaloblastic anemia and bone marrow failure. Bone marrow biopsies are not frequently performed as part of the diagnostic workup and can demonstrate morphologic features that overlap with myelodysplastic syndrome (MDS) and acute leukemia. We describe a case of a dysplastic bone marrow with increased bone marrow hematogones detected by flow cytometry in a child with vitamin B12 deficiency. Hematogones are normal B cell precursors, and hyperplasia has been described in a variety of often reactive conditions and also disease. Hematogones are not typically seen in MDS. The presence of hematogones may help differentiate the dysplastic changes seen in vitamin B12 deficiency from MDS.

  19. Hematogones detected by flow cytometry in a child with vitamin B12 deficiency: A case report.

    PubMed

    Sutton, Lisa; Mba, Nkechi

    2016-06-10

    Vitamin B12 deficiency is a known cause of megaloblastic anemia and bone marrow failure. Bone marrow biopsies are not frequently performed as part of the diagnostic workup and can demonstrate morphologic features that overlap with myelodysplastic syndrome (MDS) and acute leukemia. We describe a case of a dysplastic bone marrow with increased bone marrow hematogones detected by flow cytometry in a child with vitamin B12 deficiency. Hematogones are normal B cell precursors, and hyperplasia has been described in a variety of often reactive conditions and also disease. Hematogones are not typically seen in MDS. The presence of hematogones may help differentiate the dysplastic changes seen in vitamin B12 deficiency from MDS.

  20. Flow cytometry evidence of human granulocytes interaction with polyhedral oligomeric silsesquioxanes: effect of nanoparticle charge

    NASA Astrophysics Data System (ADS)

    Renò, Filippo; Carniato, Fabio; Rizzi, Manuela; Olivero, Francesco; Pittarella, Pamela; Marchese, Leonardo

    2013-05-01

    Nanoparticles (NPs) entering the human body are immediately confronted with the innate part of human immune system. In particular, monocyte and neutrophil granulocytes readily clear particles by phagocytosis, even if in the case of NPs the uptake mechanism may be classified as macropinocytosis. Among engineered nanoparticles, in the last years, siliceous materials have emerged as promising materials for several applications ranging from catalysis to biomedical. The polyhedral oligomeric silsesquioxanes (POSS) are nanodimensional, easily synthesizable molecular compounds and POSS-based systems are promising carriers for biological molecules. In this work, the ability of human granulocytes to uptake positively and negatively charged POSS was measured using a simple flow cytometry analysis based on cell size modifications. The data obtained showed that after a 30 min exposure only positive NPs were uptaken by human granulocyte using both macropinocytosis and clathrin-mediated mechanisms as demonstrated by uptake inhibition mediated by amiloride and chlorpromazine.

  1. Flow cytometry analysis of drug transport mechanisms in Haemonchus contortus susceptible or resistant to anthelmintics.

    PubMed

    Kerboeuf, D; Chambrier, P; Le Vern, Y; Aycardi, J

    1999-02-01

    The role of membrane drug-transport mechanisms in resistance to anthelmintics was examined using a flow cytometry method. This method was adapted from assays developed for the study of similar mechanisms in tumor cells. Rhodamine 123, a P-glycoprotein transport probe, associated with the reversal agent verapamil gave a significantly higher level of green fluorescence in Haemonchus contortus-resistant eggs as compared with that of susceptible eggs. In the same way, verapamilbodipy, a new fluorescent probe for the detection of multidrug resistance in cells, showed a significantly higher degree of binding to resistant eggs. The results confirm those obtained with biological drug assays using both anthelmintics and verapamil and provide a quantitative and effective methodology for the functional study of multidrug resistance in nematodes.

  2. 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.

  3. 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

  4. [Multi-parametric Flow Cytometry for Neuroblastoma, a new and possible diagnostic tool: case report].

    PubMed

    Manrique, Belén; López Marti, Jessica; Cacciavillano, Walter; Rossi, Jorge

    2016-04-01

    Neuroblastoma is the most frequent extracranial solid tumor in childhood, representing 5.6% according to the "Registro Oncopediatrico Hospitalario Argentino". For its diagnosis, several complementary methods (radiological, biological and biochemical) are required, and Multi-parametric Flow Cytometry (MFC) arises as a potential diagnostic method, despite not having been so far extensively explored. MFC is a method that allows to obtain several information about size, internal complexity and antigenic expression by the use of a laser and fluorescent monoclonal antibodies. There are an increasing number of reports in the literature, which reveal the importance of using MFC for diagnosis and monitoring of solid tumors. The aim in this presentation is to highlight the fundamental role that MFC had in the case of a patient affected by neuroblastoma, in which an early diagnosis using this methodology allowed prompt administration of adequate treatment.

  5. Expression of Bruton's tyrosine kinase in B-cell neoplasms evaluated by flow cytometry.

    PubMed

    Marcondes, Natália Aydos; Fernandes, Flavo Beno; Alegretti, Ana Paula; Faulhaber, Gustavo Adolpho Moreira

    2016-12-27

    Bruton's tyrosine kinase (BTK) is a cytoplasmatic protein that is part of the B-cell antigen receptor signaling pathway. Our aim was to evaluate the expression of BTK in B-cell neoplasms and compare it to normal B-cell lymphocytes. After surface staining with CD19 and CD45, flow cytometry staining for intracellular BTK was performed in leukemic or mature B-cells from bone marrow or peripheral blood samples. No differences in BTK expression were identified between groups, or in comparison to control samples, there was no association between BTK expression and the clinical variables evaluated. BTK expression in B-cell neoplasms was similar to that of normal B-cell lymphocytes.

  6. 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.

  7. 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.

  8. CASP3 protein expression by flow cytometry in Down's syndrome subjects.

    PubMed

    Salemi, Michele; Condorelli, Rosita A; Romano, Corrado; Concetta, Barone; Romano, Carmelo; Salluzzo, Maria Grazia; Bosco, Paolo; Calogero, Aldo E

    2014-01-01

    Down's syndrome (DS), the most common chromosomal disorder, is caused by 21 trisomy and is featured by intellectual disability. Subjects with DS can develop some traits of Alzheimer disease (AD) at an earlier age than subjects without trisomy 21. Apoptosis is a programmed cell death process under both normal physiological and pathological conditions. Caspase-3 (CASP3) plays an important role in neuronal death during nervous system development and under certain pathological conditions. Furthermore, in vitro and in vivo studies report elevated expression and activation of CASP3 in models of AD. On this account, the expression of CASP3 gene was evaluated in cultures of fibroblasts of DS and normal subjects by flow cytometry. CASP3 protein was up-regulated in fibroblasts of DS. The data obtained from this study strengthen the hypothesis that the over-expression of CASP3 gene could have a role in the activation of the apoptotic pathways acting in the neurodegenerative processes in DS.

  9. Rapid flow cytometry analysis of antimicrobial properties of nettle powder and cranberry powder

    NASA Astrophysics Data System (ADS)

    Hattuniemi, Maarit; Korhonen, Johanna; Jaakkola, Mari; Räty, Jarkko; Virtanen, Vesa

    2010-11-01

    Both nettle (Urtica dioica) and cranberry (Vaccinium oxycoccus) are widely known to have good influence on health. The aim of this study was to investigate antimicrobial properties of nettle powder and cranberry powder against Escherichia coli (E. coli) and monitor the growth of the bacteria by a rapid flow cytometry (FCM) method. For FCM measurements samples were stained with fluorescent dyes. The inhibitory effects of plant material on growth of E. coli were estimated by comparing the results of control sample (E. coli) to E. coli samples with plant material. FCM offers both a brilliant tool to investigate the kinetics of the growth of bacterium, since subsamples can be taken from the same liquid medium during the growing period and with fluorescent dyes a rapid method to investigate viability of the bacterium.

  10. 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

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

    PubMed Central

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

    2015-01-01

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

  12. 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

  13. Heterokaryotic nuclear conditions and a heterogeneous nuclear population are observed by flow cytometry in Phytophthora infestans.

    PubMed

    Catal, Mursel; King, Louis; Tumbalam, Pavani; Wiriyajitsomboon, Prissana; Kirk, William W; Adams, Gerard C

    2010-08-01

    A simple and reliable method for preparation of whole nuclei of a common oomycete, Phytophthora infestans, is described for laser flow cytometry. The ease of preparation, the absence of detectable debris and aggregates, and the precision in determinations of DNA content per nucleus improve interpretation and understanding of the genetics of the organism. Phytophthora infestans is the pathogen that causes potato and tomato late blight. The genetic flexibility of P. infestans and other oomycete pathogens has complicated understanding of the mechanisms of variation contributing to shifts in race structure and virulence profiles on important agricultural crops. Significant phenotypic and genotypic changes are being reported in the apparent absence of sexual recombination in the field. Laser flow cytometry with propidium iodide is useful in investigating the nuclear condition of the somatic colony of field strains of P. infestans. The majority of the studied strains contain a single population of nuclei in nonreplicated diplophase. However, mean DNA content per nucleus varies considerably among isolates confirming the heterogeneity of the nuclear population in regard to C-value, for field isolates. Nuclear DNA content varies from 1.75x to 0.75x that of nuclei in a standard strain from central Mexico. Some strains contain two to three populations of nuclei with differing DNA contents in the mycelium and are heterokaryons. Such a range in DNA content suggests DNA-aneuploidy, but direct confirmation of aneuploidy will require microscopy of chromosomes. Heterokaryosis and populations of nuclei of differing DNA content necessarily confound standardized assays used worldwide in crop breeding programs for determination of race profiles and virulence phenotypes of this important pathogen.

  14. Kinetic Measurements Using Flow Cytometry: New Methods for Monitoring Intracellular Processes

    PubMed Central

    Szalay, Balázs; Toldi, Gergely; Kaposi, Ambrus; Vásárhelyi, Barna; Treszl, András

    2012-01-01

    Abstract The aim of our work was to establish flow cytometry methods for the characterization of mitochondrial Ca2+ levels, plasma membrane potential, and superoxide generation and to relate kinetics to that of cytoplasmic Ca2+ levels during short-term activation of T-lymphocytes. We monitored the change of fluorescence absorbance of sequentially measured Jurkat cells for 12 min. The cells were stained with the fluorescent dyes Fluo3-AM, Rhod2/AM, di-BA-C4-(5), or dihydroethidium and then were stimulated with increasing doses of phytohemagglutinin (PHA) or were treated with rotenone. Double-logistic function was fitted to cytoplasmic Ca2+ signal and mitochondrial Ca2+ levels, whereas logistic function was fitted to plasma membrane potential and superoxide levels. The calculated function parameters were area under the curve (AUC), maximum (Max), time to reach maximum (tmax), slope at the first 50% value of Max (Slope), and ending (End) values, respectively. We found significant dose–response relationship between PHA dose and cytoplasmic Ca2+ signals (AUC, Max, Slope: P<0.05), mitochondrial Ca2+ levels (AUC and Max: P<0.05), and plasma membrane potential (AUC and End values: P<0.05). In rotenone-treated cells, superoxide generation increased in a dose-dependent manner (P<0.05 for AUC and End values, respectively). The present methodology provides an opportunity for monitoring and characterizing mitochondrial Ca2+ levels, plasma membrane potential, and superoxide generation in PHA-activated or rotenone-treated Jurkat cells with flow cytometry. PMID:21919740

  15. A new flow cytometry method to measure oxidative status: the Peroxidation of Leukocytes Index Ratio (PLIR).

    PubMed

    Peluso, Ilaria; Adorno, Gaspare; Raguzzini, Anna; Urban, Lourdes; Ghiselli, Andrea; Serafini, Mauro

    2013-04-30

    A complex relationship between immune system and metabolic pathway exists and can induce oxidative stress. The objective of this study was to design a new methodology allowing the measurement of oxidative status of leukocytes. We developed a flow cytometry technique, based on C11-BODIPY 581/591 staining, to evaluate peroxidation in leukocytes. We defined the Peroxidation of Leukocytes Index Ratio (PLIR) as the ratio between the damage after AAPH-induced and PMA-induced peroxidation, using Trolox as standard antioxidant. Sensitivity of the method was assessed by correlating results with plasma antioxidant capacity (TRAP and FRAP), levels of endogenous antioxidants (uric acid and sulfhydryls) and markers of metabolic status (cholesterol, triglycerides, glucose and insulin). PLIR measures the ratio between the resistance to exogenous and endogenous ROS injury, independently from baseline level of oxidation, which was directly correlated with plasma cholesterol on lymphocytes (0.738, p=0.029), monocytes (0.691, p=0.047) and neutrophils (0.690, p=0.047). PLIR of lymphocytes was inversely correlated with uric acid (-0.810, p=0.009) and FRAP (-0.738, p=0.029) levels. On the other hand, PLIR of monocytes was directly correlated with the total scavenger antioxidant capacity attributable to nutritional antioxidants (0.738, p=0.029), calculated as the difference between TRAP and the contribution of uric acid and sulfhydryls to its value. This study reports a feasible and reproducible new flow cytometry assay for assessing the leukocytes redox status. PLIR discriminates between reducing and scavenger activities and is able to appreciate the potentially dangerous effect of uric acid on innate immune response. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Standardization of microparticle enumeration across different flow cytometry platforms: results of a multicenter collaborative workshop.

    PubMed

    Cointe, S; Judicone, C; Robert, S; Mooberry, M J; Poncelet, P; Wauben, M; Nieuwland, R; Key, N S; Dignat-George, F; Lacroix, R

    2017-01-01

    Essentials The clinical enumeration of microparticles (MPs) is hampered by a lack of standardization. A new strategy to standardize MP counts by flow cytometry was evaluated in a multicenter study. No difference was found between instruments using forward or side scatter as the trigger parameter. This study demonstrated that beads can be used as a standardization tool for MPs. Click to hear the ISTH Academy's webinar on microvesicles SUMMARY: Background Microparticles (MPs) are extracellular vesicles resulting from the budding of cellular membranes that have a high potential as emergent biomarkers; however, their clinical relevance is hampered by methodological enumeration concerns and a lack of standardization. Flow cytometry (FCM) remains the most commonly used technique with the best capability to determine the cellular origin of single MPs. However, instruments behave variably depending on which scatter parameter (forward (FSC) or side scatter (SSC)) provides the best resolution to discriminate submicron particles. To overcome this problem, a new approach, based on two sets of selected beads adapted to FSC or SSC-optimized instruments, was recently proposed to reproducibly enumerate platelet-derived MP counts among instruments with different optical systems. Objective The objective was to evaluate this strategy in an international workshop that included 44 laboratories accounting for 52 cytometers of 14 types. Methods/Results Using resolution capability and background noise level as criteria to qualify the instruments, the standardization strategy proved to be compatible with 85% (44/52) of instruments. All instruments correctly ranked the platelet MP (PMP) levels of two platelet-free plasma samples. The inter-laboratory variability of PMP counts was 37% and 28% for each sample. No difference was found between instruments using forward or side-scattered light as the relative sizing parameter. Conclusions Despite remaining limitations, this study is the first to

  17. 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

  18. Biomass measurement by flow cytometry during solid-state fermentation of basidiomycetes.

    PubMed

    Steudler, Susanne; Böhmer, Ulrike; Weber, Jost; Bley, Thomas

    2015-02-01

    Solid-state fermentation (SSF) is a robust process that is well suited to the on-site cultivation of basidiomycetes that produce enzymes for the treatment of lignocellulosics. Reliable methods for biomass quantification are essential for the analysis of fungal growth kinetics. However, direct biomass determination is not possible during SSF because the fungi grow into the substrate and use it as a nutrient source. This necessitates the use of indirect methods that are either very laborious and time consuming or can only provide biomass measurements during certain growth periods. Here, we describe the development and optimization of a new rapid method for fungal biomass determination during SSF that is based on counting fungal nuclei by flow cytometry. Fungal biomass was grown on an organic substrate and its concentration was measured by isolating the nuclei from the fungal hyphae after cell disruption, staining them with SYTOX(®) Green, and then counting them using a flow cytometer. A calibration curve relating the dry biomass of the samples to their concentrations of nuclei was established. Multiple buffers and disruption methods were tested. The results obtained were compared with values determined using the method of ergosterol determination, a classical technique for fungal biomass measurement during SSF. Our new approach can be used to measure fungal biomass on a range of different scales, from 15 mL cultures to a laboratory reactor with a working volume of 10 L (developed by the Research Center for Medical Technology and Biotechnology (fzmb GmbH)). © 2014 International Society for Advancement of Cytometry.

  19. Gram-typing of mastitis bacteria in milk samples using flow cytometry.

    PubMed

    Langerhuus, S N; Ingvartsen, K L; Bennedsgaard, T W; Røntved, C M

    2013-01-01

    Fast identification of pathogenic bacteria in milk samples from cows with clinical mastitis is central to proper treatment. In Denmark, time to bacterial diagnosis is typically 24 to 48 h when using traditional culturing methods. The PCR technique provides a faster and highly sensitive identification of bacterial pathogens, although shipment of samples to diagnostic laboratories delays treatment decisions. Due to the lack of fast on-site tests that can identify the causative pathogens, antibiotic treatments are often initiated before bacterial identification. The present study describes a flow cytometry-based method, which can detect and distinguish gram-negative and gram-positive bacteria in mastitis milk samples. The differentiation was based on bacterial fluorescence intensities upon labeling with biotin-conjugated wheat germ agglutinin and acridine orange. Initially 19 in-house bacterial cultures (4 gram-negative and 15 gram-positive strains) were analyzed, and biotin-conjugated wheat germ agglutinin and acridine orange florescence intensities were determined for gram-negative and gram-positive bacteria, respectively. Fluorescence cut-off values were established based on receiver operating characteristic curves for the 19 bacterial cultures. The method was then tested on 53 selected mastitis cases obtained from the department biobank (milk samples from 6 gram-negative and 47 gram-positive mastitis cases). Gram-negative bacteria in milk samples were detected with a sensitivity of 1 and a specificity of 0.74, when classification was based on the previously established cut-off values. However, when receiver operating characteristic curves were constructed for the 53 mastitis cases, results indicate that a sensitivity and specificity of 1 could be reached if cut-off values were reduced. This flow cytometry-based technique could potentially provide dairy farmers and attending veterinarians with on-site information on bacterial gram-type and prevent ineffective

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

    DOE PAGES

    Azad, Ariful; Rajwa, Bartek; Pothen, Alex

    2016-08-31

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

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

    SciTech Connect

    Azad, Ariful; Rajwa, Bartek; Pothen, Alex

    2016-08-31

    We describe algorithms for discovering immunophenotypes from large collections of flow cytometry samples and using them to organize the samples into a hierarchy based on phenotypic similarity. The hierarchical organization is helpful for effective and robust cytometry data mining, including the creation of collections of cell populations’ characteristic of different classes of samples, robust classification, and anomaly detection. We summarize a set of samples belonging to a biological class or category with a statistically derived template for the class. Whereas individual samples are represented in terms of their cell populations (clusters), a template consists of generic meta-populations (a group 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. 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.

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