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

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Publications in this journal

  • [Show abstract] [Hide abstract]
    ABSTRACT: In this unit, we describe a multiplex microsphere quantitative PCR. The system is based on the use of two additional oligonucleotides within a single tube PCR reaction. The first oligonucleotide is modified with a single base pair mismatch and is otherwise equivalent to a universal sequence added to the forward PCR primer. Further, this first extra oligonucleotide is coupled to Luminex microspheres. The second additional oligonucleotide is designed to be complementary to the universal sequence, and is modified with the fluorescent dye Cy3. As the PCR reaction proceeds, the second oligonucleotide is able to bind to the microspheres. Thus, quantitative monitoring of PCR progress takes place. The microsphere-mediated Cy3-detection is measured using flow cytometry directly after the PCR reaction. This allows a flow cytometer analysis from up to 150 different spheres and, therefore, multiple genes in one reaction. The multiplex microsphere qPCR is demonstrated using three target genes from Influenza A and Neisseria meningitidis. The multiplex microsphere system will enable a higher degree of multiplexing than is possible with currently available qPCR systems. Curr. Protoc. Cytom. 69:13.13.1-13.13.10. © 2014 by John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 01/2014; 69:13.13.1-13.13.10.
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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.
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    ABSTRACT: In recent years, flow cytometry has been used to detect the presence of autophagy mainly by the fluorescent antibody labeling of the autophagy marker, the microtubule associated protein LC3-II. Here we describe the indirect antibody labeling of LC3-II in cells displaying drug-induced autophagy by the use of rapamycin and chloroquine, as well as cells undergoing serum starvation. Although the mechanism of action of LysoTracker dyes is not fully understood, lysosomal mass increases during the autophagic process to enable the cell to produce autolysosomes. Given that LC3-II and LysoTracker are measuring different biological events in the autophagic process, they surprisingly both up-regulated during autophagic process. This approach shows that although LysoTracker dyes do not specifically label lysosomes or autophagosomes within the cell, they allow the simultaneous measurement of an autophagy related process and other live cell functions, which is not possible with the standard LC3-II antibody technique. Curr. Protoc. Cytom. 68:9.45.1-9.45.10. © 2014 by John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 01/2014; 68:9.45.1-9.45.10.
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    ABSTRACT: We combined fluorogen-activating protein (FAP) technology with high-throughput flow cytometry to detect real-time protein trafficking to and from the plasma membrane in living cells. The hybrid platform allows drug discovery for trafficking receptors, such as G protein-coupled receptors, receptor tyrosine kinases, and ion channels, which were previously not suitable for high-throughput screening by flow cytometry. The system has been validated using the β2-adrenergic receptor (β2AR) system and extended to other GPCRs. When a chemical library containing ∼1200 off-patent drugs was screened against cells expressing FAP-tagged β2AR, all known β2AR active ligands in the library were successfully identified, together with a few compounds that were later confirmed to regulate receptor internalization in a nontraditional manner. The unexpected discovery of new ligands by this approach indicates the potential of using this protocol for GPCR de-orphanization. In addition, screens of multiplexed targets promise improved efficiency with minor protocol modification. Curr. Protoc. Cytom. 67:9.43.1-9.43.11. © 2014 by John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 01/2014; 67:9.43.1-9.43.11.
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    ABSTRACT: Stereological methods for tissue cell counting, specifically for neuron quantification, decrease systematic error and sampling bias; however, they are tedious, labor intensive, and time consuming. Approaches for cell (neuron) quantification in vitro are not accurate, sensitive, or subsequently reproducible. Neuronal phenotype is related to alterations in cell morphology and neurite pattern. The techniques currently available for quantification of these features present several limitations. In this unit, we provide validated automated procedures for in vivo and in vitro quantification of cell number, morphological cell changes, and neurite morphometry in a fast, simple, and reliable manner. Our method counts up to 8 times as many neurons in less than 5% to 10% of the time required for stereological analysis (optical fractionator). In summary, this technology offers an unparalleled opportunity to examine features of cells at high resolution in a complex three-dimensional environment. These techniques provide an exceptional in vivo and in vitro system for neurotoxicity studies, disease modeling, and drug discovery. Curr. Protoc. Cytom. 68:12.33.1-12.33.22. © 2014 by John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 01/2014; 68:12.33.1-12.33.22.
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    ABSTRACT: Non-antibody commercial fluorescent reagents for imaging of cytoskeletal structures have been limited primarily to tubulin and actin, with the main factor in choice based mainly on whether cells are live or fixed and permeabilized. A wider range of options exist for cell membrane dyes, and the choice of reagent primarily depends on the preferred localization in the cell (i.e., all membranes or only the plasma membrane) and usage (i.e., whether the protocol involves fixation and permeabilization). For whole-cell or cytoplasmic imaging, the choice of reagent is determined mostly by the length of time that the cells need to be visualized (hours or days) and by fixation status. Presented here is a discussion on choosing commercially available reagents for these cellular structures, with an emphasis on use for microscopic imaging, with a featured reagent for each structure, a recommended protocol, troubleshooting guide, and example image. Curr. Protoc. Cytom. 67:12:32.1-12:32.17. © 2014 by John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 01/2014; 67:12.32.1-12.32.17.
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    ABSTRACT: Standardization in fluorescence microscopy involves calibration of intensity in reproducible units and correction for spatial nonuniformity of illumination (flat-field or shading correction). Both goals can be achieved using concentrated solutions of fluorescent dyes. When a drop of a highly concentrated fluorescent dye is placed between a slide and a coverslip it produces a spatially uniform field, resistant to photobleaching and with reproducible quantum yield; it can be used as a brightness standard for wide-field and confocal microscopes. For wide-field microscopes, calibration can be further extended to absolute molecular units. This can be done by imaging a solution of known concentration and known depth; the latter can be prepared by placing a small spherical lens in a diluted solution of the same fluorophore that is used in the biological specimen. Curr. Protoc. Cytom. 68:10.14.1-10.14.10. © 2014 by John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 01/2014; 68:10.14.1-10.14.10.
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    ABSTRACT: Described is an in vitro model of premature senescence in pulmonary adenocarcinoma A549 cells induced by persistent DNA replication stress in response to treatment with the DNA damaging drug mitoxantrone (Mxt). The degree of cellular senescence, based on characteristic changes in cell morphology, is measured by laser scanning cytometry. Specifically, the flattening of cells grown on slides (considered the hallmark of cellular senescence) is measured as the decline in local intensity of DNA-associated DAPI fluorescence (represented by maximal pixels). This change is paralleled by an increase in nuclear area. Thus, the ratio of mean intensity of maximal pixels to nuclear area provides a very sensitive morphometric biomarker for the degree of senescence. This analysis is combined with immunocytochemical detection of senescence markers, such as overexpression of cyclin kinase inhibitors (e.g., p21(WAF1) ) and phosphorylation of ribosomal protein S6 (rpS6), a key marker associated with aging/senescence that is detected using a phospho-specific antibody. These biomarker indices are presented in quantitative terms defined as a senescence index (SI), which is the fraction of the marker in test cultures relative to the same marker in exponentially growing control cultures. This system can be used to evaluate the anti-aging potential of test agents by assessing attenuation of maximal senescence. As an example, the inclusion of berberine, a natural alkaloid with reported anti-aging properties and a long history of use in traditional Chinese medicine, is shown to markedly attenuate the Mxt-induced SI and phosphorylation of rpS6. The multivariate analysis of senescence markers by laser scanning cytometry offers a promising tool to explore the potential anti-aging properties of a variety agents. Curr. Protoc. Cytom. 69:9.47.1-9.47.10. © 2014 by John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 01/2014; 69:9.47.1-9.47.10.
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    ABSTRACT: The sensitivity of filter-based fluorescence microscopy techniques is limited by autofluorescence background. Time-gated detection is a practical way to suppress autofluorescence, enabling higher contrast and improved sensitivity. In the past few years, three groups of authors have demonstrated independent approaches to build robust versions of time-gated luminescence microscopes. Three detailed, step-by-step protocols are provided here for modifying standard fluorescent microscopes to permit imaging time-gated luminescence. Curr. Protoc. Cytom. 67:2.22.1-2.22.36. © 2014 by John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 01/2014; 67:2.22.1-2.22.36.
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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.
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    ABSTRACT: This method makes it possible to measure the fluorescence of a DNA probe in cells with known division number and targeted surface antigen. In fact, this method is a combination or consistent application of three other methods: cell tracking by vital dye, surface immunophenotyping, and flow-FISH. The idea in developing this method was to study telomere length changes in cells with known surface antigen after every new cell division. First, the in vitro cell culturing and staining with CFSE vital dye are performed. Then, cells are stained with surface MAbs labeled with biotin, followed by incubation with streptavidin-labeled fluorochrome. After that, cells are fixed with BS(3) reagent followed by the flow-FISH procedure with PNA-probe complementary to telomere DNA repeats. Finally, in one tube, it is possible to determine telomere length in surface antigen-labeled cells that have made the exact same number of divisions after incubation. Curr. Protoc. Cytom. 69:8.14.1-8.14.10. © 2014 by John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 01/2014; 69:8.14.1-8.14.10.
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    ABSTRACT: Generating loss of protein function is a powerful investigatory tool particularly if carried out on a physiologically relevant timescale in a live-cell fluorescent imaging experiment. KillerRed mediated chromophore assisted light inactivation (CALI) uses genetic encoding for specificity and light for acute inactivation that can also be spatially restricted. This unit provides protocols for setting up and carrying out properly controlled KillerRed experiments during live-cell imaging of cultured cells. Curr. Protoc. Cytom. 69:12.35.1-12.35.10. © 2014 by John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 01/2014; 69:12.35.1-12.35.10.
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    ABSTRACT: Fluorescent labeling of vesicular structures in cultured cells, particularly for live cells, can be challenging for a number of reasons. The first challenge is to identify a reagent that will be specific enough where some structures have a number of potential reagents and others very few options. The emergence of BacMam constructs has allowed more easy-to-use choices. Presented here is a discussion of BacMam constructs as well as a review of commercially-available reagents for labeling vesicular structures in cells, including endosomes, peroxisomes, lysosomes, and autophagosomes, complete with a featured reagent for each structure, recommended protocol, troubleshooting guide, and example image. Curr. Protoc. Cytom. 65:12.30.1-12.30.27. © 2013 by John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 07/2013; Chapter 12:Unit12.30.
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    ABSTRACT: In this post-genomic era, the predominant focus of biomedical research has moved towards elucidating the functionality of molecules at the cellular and subcellular level in integrated living systems. As such, biologic imaging has moved beyond the collection of static "snapshots" of the cellular state to the real-time visualization of cellular and molecular behavior in three-dimensional (3D) space. Live-cell imaging techniques can be used to assess protein abundance, as well as determine the functional role(s) and interactions of multiple unique molecules concurrently within the cellular environment, and determine the effects of these molecules on cell development, organization, and fate over extended periods of time. Such studies require advanced systems that allow multiparametric analysis of cells while maintaining their functional viability. This unit will focus on the design and implementation of modern live-cell imaging systems. We will examine the principle problems facing live-cell microscopists including: focus drift, cell viability, photo-toxicity, and data acquisition, and suggest appropriate solutions to these issues. Our goal in this unit is to provide individual scientists with the information required to implement live-cell imaging solutions within their own laboratories. Curr. Protoc. Cytom. 65:2.21.1-2.21.10. © 2013 by John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 07/2013; Chapter 2:Unit2.21.
  • Article: Fluidics.
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    ABSTRACT: The use of fluidics is implicit in a technology named "flow cytometry," which flows a cell or particle through a sensing volume to obtain serial analysis of particles on a one by one basis. This flow of particles enables flow cytometry to collect information on multiple particle populations, giving it a distinct advantage over bulk analysis approaches. Moreover, flow cytometers can analyze thousands of particles per second in a single flowing stream. Additionally, use of volumetric sample delivery makes it possible for flow cytometers to accurately count cells and particles. Furthermore, the analysis results can be coupled with a fluidic diversion mechanism to sort and collect particles based on desired properties. Finally, when high-throughput sampling technologies are employed to rapidly change the input of the sample stream, a flow cytometer can become an integral tool for high-throughput screening. The above properties have made flow cytometry useful in a wide range of biomedical applications. In this unit we will present an overview of fluidic systems that make flow cytometry possible. This will introduce historical approaches, explanations of the commonly implemented current fluidics, and brief discussions of potential future fluidics where appropriate. Curr. Protoc. Cytom. 65:1.2.1-1.2.14. © 2013 by John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 07/2013; Chapter 1:Unit1.2.
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    ABSTRACT: The technique described in this unit uses the intracellular fluorescent label carboxyfluorescein diacetate succinimidyl ester (CFSE) to track proliferating cells. Covalently bound CFSE is divided equally between daughter cells, allowing discrimination of successive rounds of cell division. The technique is applicable to in vitro cell division, as well as to in vivo division of adoptively transferred cells and can resolve eight or more successive generations. CFSE is long lived, permitting analysis for several months after cell transfer, and has the same spectral characteristics as fluorescein, so monoclonal antibodies conjugated to phycoerythrin or other compatible fluorochromes may be used to immunophenotype the dividing cells. In addition, information is given on a second-generation dye, Cell Trace Violet (CTV), excited by 405-nm blue laser light. CTV is chemically related to CFSE, but allows the 488-nm line of the Argon laser to be used for other probes. Curr. Protoc. Cytom. 64:9.11.1-9.11.12. © 2013 by John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 04/2013; Chapter 9:Unit9.11.
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    ABSTRACT: Time-lapse microscopy can be described as the repeated collection of an image (in n-dimensions; x, y, z, λ) or field of view from a microscope at discrete time intervals. The duration of the time interval defines the temporal resolution, which in turn characterizes the type of event detected. This unit describes the implementation of time-lapse microscopy to link initial cell cycle position during acute exposures to anti-cancer agents with anti-proliferative consequences for individual cells. The approach incorporates fundamental concepts arising from the ability to capture simple video sequences of cells from which it is possible to extract kinetic descriptors that reflect the interplay of mitosis and cell death in the growth of an unsynchronized tumor population. Utilizing a multi-well format enables the user to screen different drug derivatives, multiple dose ranges, or cell cultures with unique genetic backgrounds. The objective of this unit is to present the basic methodology for capturing time-lapse sequences and touch upon subsequent mining of the data for deriving event curves and possible cell lineage maps. Curr. Protoc. Cytom. 64:12.4.1-12.4.13. © 2013 by John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 04/2013; Chapter 12:Unit12.4.

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