Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.]

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ISSN 1934-9300

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    ABSTRACT: A flow cytometer is made up of many different subsystems that work together to measure the optical properties of individual cells within a sample. The data acquisition system (also called the data system) is one of these subsystems, and it is responsible for converting the electrical signals from the optical detectors into list-mode data. This unit describes the inner workings of the data system, and provides insight into how the instrument functions as a whole. Some of the information provided in this unit is applicable to everyday use of these instruments, and, at minimum, should make it easier for the reader to assemble a specific data system. With the considerable advancement of electronics technology, it becomes possible to build an entirely functional data system using inexpensive hobbyist-level electronics. This unit covers both analog and digital data systems, but the primary focus is on the more prevalent digital data systems of modern flow cytometric instrumentation. © 2015 by John Wiley & Sons, Inc. Copyright © 2015 John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 01/2015; 71:10.19.1-10.19.13. DOI:10.1002/0471142956.cy1019s71
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    ABSTRACT: Using flow cytometry, single-cell measurements of calcium can be made on isolated populations identified by one or more phenotypic characteristics. Most earlier techniques for measuring cellular activation parameters determined the mean value for a population of cells, which did not permit optimal resolution of the responses. The flow cytometer is particularly useful for this purpose because it can measure ion concentrations in large numbers of single cells and thereby allows ion concentration to be correlated with other parameters such as immunophenotype and cell cycle stage. A limitation of flow cytometry, however, is that it does not permit resolution of certain complex kinetic responses such as cellular oscillatory responses. This unit describes the preparation of cells, including labeling with antibodies and with calcium probes, and discusses the principles of data analysis and interpretation. © 2015 by John Wiley & Sons, Inc. Copyright © 2015 John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 01/2015; 72:9.8.1-9.8.21. DOI:10.1002/0471142956.cy0908s72
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    ABSTRACT: Human peripheral blood is often studied by flow cytometry in both the research and clinical laboratories. The methods for collection, storage, and preparation of peripheral blood will vary depending on the cell lineage to be examined as well as the type of assay to be performed. This unit presents protocols for collection of blood, separation of leukocytes from whole blood by lysis of erythrocytes, isolating mononuclear cells by density gradient separation, and assorted non-flow sorting methods, such as magnetic bead separations, for enriching specific cell populations, including monocytes, T lymphocytes, B lymphocytes, neutrophils, and platelets, prior to flow cytometric analysis. A protocol is also offered for cryopreservation of cells, since clinical research often involves retrospective flow cytometric analysis of samples stored over a period of months or years. © 2015 by John Wiley & Sons, Inc. Copyright © 2015 John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 01/2015; 73:5.1.1-5.1.16. DOI:10.1002/0471142956.cy0501s73
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    ABSTRACT: Rapid progress is being made to understand the regulatory mechanisms that underlie the epigenetic control of gene expression through histone modification. It is now recognized that this plays a major role in normal development and disease. This unit describes the application of flow cytometry to the study of epigenetic mechanisms by combining labeling of individual histone modifications and phenotypic markers, and it also discusses practical issues to optimize staining. The focus is on normal blood and samples from leukemia patients, but it can also be applied to cells grown in tissue culture. © 2015 by John Wiley & Sons, Inc. Copyright © 2015 John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 01/2015; 71:6.36.1-9. DOI:10.1002/0471142956.cy0636s71
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    ABSTRACT: The lipophilic cation JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolyl carbocyanine iodide) has been used for more than 20 years as a specific dye for measuring mitochondrial membrane potential (ΔΨm ). In this unit, we revise our original protocol (that made use of a single 488 nm laser for the detection of monomers and aggregates, and where compensation was an important step) to use dual-laser excitation. Moreover, thanks to recently developed multilaser instruments and novel probes for surface and intracellular markers, JC-1 can be utilized by polychromatic flow cytometry to simultaneously detect, without any compensation between fluorescences, ΔΨm along with other biological parameters, such as apoptosis and the production of reactive oxygen species. © 2015 by John Wiley & Sons, Inc. Copyright © 2015 John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 01/2015; 72:7.32.1-7.32.11. DOI:10.1002/0471142956.cy0732s72
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    ABSTRACT: Cell volume is an important parameter in cell adaptation to anisosmotic stress, in the development of apoptosis and necrosis, and in the pathogenesis of several diseases. This unit describes a method for measuring the volume of adherent cells using a standard light microscope. A coverslip with attached cells is placed in a shallow chamber in a medium containing a strongly absorbing and cell-impermeant dye, Acid Blue 9. When such a sample is imaged in transmitted light at a wavelength of maximum dye absorption (630 nm), the resulting contrast quantitatively reflects cell thickness. Once the thickness is known at every point, the volume can be computed as well. Technical details, interpretation of data, and possible artifacts are discussed. Measurements in absolute units require knowledge of the absorption coefficient, and a similar procedure for the measurement of absorption coefficient is described. © 2015 by John Wiley & Sons, Inc. Copyright © 2015 John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 01/2015; 72:12.39.1-9. DOI:10.1002/0471142956.cy1239s72
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    ABSTRACT: Cytokine-producing cells are at the center of the adaptive immune responses, and quantifying these cells is an important aspect to build understanding of the immune response. In particular, Th1 and Th17 cells have been implicated in the pathogenesis of such diseases as inflammatory bowel disease, rheumatoid arthritis, and multiple sclerosis. Quantification of Th1 and Th17 cells can provide important information in research of these diseases and other Th1- and Th17-mediated immune disorders. In vitro stimulation of cells followed by surface and intracellular staining, presented here, has the advantage of detecting the cytokines directly instead of relying exclusively on surrogate surface markers which, although showing enrichment for the effector T cells, are not specific markers for the cytokine-producing cells. © 2015 by John Wiley & Sons, Inc. Copyright © 2015 John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 01/2015; 71:6.35.1-7. DOI:10.1002/0471142956.cy0635s71
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    ABSTRACT: Time-lapse imaging is a rich data source offering potential kinetic information of cellular activity and behavior. Tracking and extracting measurements of objects from time-lapse datasets are challenges that result from the complexity and dynamics of each object's motion and intensity or the appearance of new objects in the field of view. A wide range of strategies for proper data sampling, object detection, image analysis, and post-analysis interpretation are available. Theory and methods for single-particle tracking, spot detection, and object linking are discussed in this unit, as well as examples with step-by-step procedures for utilizing semi-automated software and visualization tools for achieving tracking results and interpreting this output. © 2015 by John Wiley & Sons, Inc. Copyright © 2015 John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 01/2015; 71:12.38.1-12.38.21. DOI:10.1002/0471142956.cy1238s71
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    ABSTRACT: Evidence suggests that extracellular vesicles (EVs) can play roles in physiology and pathology, providing impetus to explore their use as diagnostic and therapeutic targets. However, EVs are also small, heterogeneous, and difficult to measure, and so this potential has not yet been realized. The development of improved approaches to EV detection and characterization will be critical to further understanding their roles in physiology and disease. Flow cytometry has been a popular tool for measuring cell-derived EVs, but has often been used in an uncritical manner in which fundamental principles and limitations of the instrument are ignored. Recent efforts to standardize procedures and document the effects of different methodologies have helped to address this shortcoming, but much work remains. In this paper, I address some of the instrument, reagent, and analysis considerations relevant to measurement of individual EVs in flow, with the aim of clarifying a path to quantitative and standardized measurement of these interesting and potentially important biological nanoparticles. © 2015 by John Wiley & Sons, Inc. Copyright © 2015 John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 01/2015; 73:13.14.1-13.14.16. DOI:10.1002/0471142956.cy1314s73
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    ABSTRACT: Mitochondrial dysfunction has been increasingly implicated as an important mechanism for chemical-induced toxicity. In the present unit, we describe a multi-parametric flow cytometry assay to assess the effects of drug or chemical-induced mitochondrial dysfunction in cells. Cells are cultured in a glucose-supplemented medium and exposed to increasing concentrations of various chemicals. Several key mitochondrial/cellular parameters known to be directly impacted by mitochondrial dysfunction, such as mitochondrial membrane potential (MMP), mitochondrial reactive oxygen species (ROS) production, intracellular reduced glutathione (GSH) level, and cell viability, are simultaneously measured by flow cytometry. © 2015 by John Wiley & Sons, Inc. Copyright © 2015 John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 01/2015; 73:9.48.1-9. DOI:10.1002/0471142956.cy0948s73
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    ABSTRACT: The development of confocal microscopy techniques introduced the ability to optically section fluorescent samples in the axial dimension, perpendicular to the image plane. These approaches, via the placement of a pinhole in the conjugate image plane, provided superior resolution in the axial (z) dimension resulting in nearly isotropic optical sections. However, increased axial resolution, via pinhole optics, comes at the cost of both speed and excitation efficiency. Light sheet fluorescent microscopy (LSFM), a century-old idea made possible with modern developments in both excitation and detection optics, provides sub-cellular resolution and optical sectioning capabilities without compromising speed or excitation efficiency. Over the past decade, several variations of LSFM have been implemented each with its own benefits and deficiencies. Here we discuss LSFM fundamentals and outline the basic principles of several major light-sheet-based imaging modalities (SPIM, inverted SPIM, multi-view SPIM, Bessel beam SPIM, and stimulated emission depletion SPIM) while considering their biological relevance in terms of intrusiveness, temporal resolution, and sample requirements. © 2015 by John Wiley & Sons, Inc. Copyright © 2015 John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 01/2015; 71:12.37.1-12.37.15. DOI:10.1002/0471142956.cy1237s71
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    ABSTRACT: Autophagy is a membrane-trafficking pathway activated to deliver cytosolic material for degradation to lysosomes through a novel membrane compartment, the autophagosome. Fluorescence microscopy is the most common method used to visualize proteins inside cells, and it is widely used in the autophagy field. To distinguish it from the cellular background, the protein of interest (POI) is either fused with a genetically encoded fluorescent protein or stained with an antibody that is conjugated to an inorganic fluorescent compound. Genetic tagging of the POI allows its visualization in live cells, while immunostaining of the POI requires the fixation of cells and the permeabilization of cell membranes. Here we describe detailed protocols on how to visualize autophagy dynamics using fluorescence microscopy in live and fixed cells. We discuss the critical parameters of each technique, their advantages, and why the robustness is increased when they are used in tandem. Curr. Protoc. Cytom. 69:12.34.1-12.34.16. © 2014 by John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 01/2014; 69:12.34.1-12.34.16. DOI:10.1002/0471142956.cy1234s69
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    ABSTRACT: The application of FRET (fluorescence resonance energy transfer) sensors for monitoring protein-protein interactions under vital conditions is attracting increasing attention in molecular and cell biology. Laser-scanning cytometry (LSC), a slide-based sister procedure to flow cytometry, provides an opportunity to analyze large populations of adherent cells or 2-D solid tissues in their undisturbed physiological settings. Here we provide an LSC-based three-laser protocol for high-throughput ratiometric FRET measurements utilizing cyan and yellow fluorescent proteins as a FRET pair. Membrane labeling with Cy5 dye is used for cell identification and contouring. Pixel-by-pixel and single-cell FRET efficiencies are calculated to estimate the extent of the molecular interactions and their distribution in the cell populations examined. We also present a non-high-throughput donor photobleaching FRET application, for obtaining the required instrument parameters for ratiometric FRET. In the biological model presented, HeLa cells are transfected with the ECFP- or EYFP-tagged Fos and Jun nuclear proteins, which heterodimerize to form active AP1 transcription factor. Curr. Protoc. Cytom. 70:2.23.1-2.23.29. © 2014 by John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 01/2014; 70:2.23.1-2.23.29. DOI:10.1002/0471142956.cy0223s70
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    ABSTRACT: Correlative fluorescence and electron microscopy (CFEM) is a multimodal technique that combines dynamic and localization information from fluorescence methods with ultrastructural data from electron microscopy, to give new information about how cellular components change relative to the spatiotemporal dynamics within their environment. In this review, we will discuss some of the basic techniques and tools of the trade for utilizing this attractive research method, which is becoming a very powerful tool for biology labs. The information obtained from correlative methods has proven to be invaluable in creating consensus between the two types of microscopy, extending the capability of each, and cutting the time and expense associated with using each method separately for comparative analysis. The realization of the advantages of these methods in cell biology has led to rapid improvement in the protocols and has ushered in a new generation of instruments to reach the next level of correlation-integration. Curr. Protoc. Cytom. 70:12.36.1-12.36.10. © 2014 by John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 01/2014; 70:12.36.1-12.36.10. DOI:10.1002/0471142956.cy1236s70
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    ABSTRACT: Zebrafish (Danio rerio) embryo assays have recently come into the spotlight as convenient experimental models in both biomedicine and ecotoxicology. As a small aquatic model organism, zebrafish embryo assays allow for rapid physiological, embryo-, and genotoxic tests of drugs and environmental toxins that can be simply dissolved in water. This protocol describes prototyping and application of an innovative, miniaturized, and polymeric chip-based device capable of immobilizing a large number of living fish embryos for real-time and/or time-lapse microscopic examination. The device provides a physical address designation to each embryo during analysis, continuous perfusion of medium, and post-analysis specimen recovery. Miniaturized embryo array is a new concept of immobilization and real-time drug perfusion of multiple individual and developing zebrafish embryos inside the mesofluidic device. The OpenSource device presented in this protocol is particularly suitable to perform accelerated fish embryo biotests in ecotoxicology and phenotype-based pharmaceutical screening. Curr. Protoc. Cytom. 67:9.44.1-9.44.16. © 2014 by John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 01/2014; 67:9.44.1-9.44.16. DOI:10.1002/0471142956.cy0944s67