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: 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: 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.
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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.
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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: 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: 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: 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: Optical fluorescence microscopy offers a wide range of technological solutions to address many questions in biomedical research. Spatial resolution has been greatly improved by the use of confocal microscopes, providing a 3-D analysis of the intracellular space. Automation has contributed to make confocal analysis available for high-content image cytometry studies. However, the storage, browsing, and analysis of the amount of data generated can challenge the feasibility of such studies. Presented in this chapter is a multistep acquisition and analysis protocol that can bypass such difficulties by an analysis-driven data collection. Cell-cycle analysis of low-resolution data can be employed to select cell populations of interest that can then be imaged at extremely high resolution and subjected to high-content analysis. Curr. Protoc. Cytom. 70:7.42.1-7.42.14. © 2014 by John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 01/2014; 70:7.42.1-7.42.14.
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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: 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: 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: 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: 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: 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: One of the major limitations of flow cytometry (FCM) is the absence of an intracellular view. Automated microscopy and image analysis, together with technological developments, led to new approaches in cytometry that bypass the above limitation, introducing high resolution, high content, and large statistical sampling. However, few attempts have been made, until now, to translate the wide repertoire of FCM assays into high-content image screening. This unit describes the implementation of an acquisition and analysis protocol for evaluation of the cell cycle by automated microscopy. The approach grants the possibility to perform simultaneous analysis of a high number of different parameters. A large part of this unit is devoted to the description of hardware features that can optimize the recorded information together with the acquisition and analysis procedures employed to produce good-quality data. Curr. Protoc. Cytom. 70:7.41.1-7.41.15. © 2014 by John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 01/2014; 70:7.41.1-7.41.15.
  • 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: 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.