Current Protocols in Cytometry最新文献

Multiphoton intravital calcium imaging is a powerful technique that enables high-resolution longitudinal monitoring of cellular and subcellular activity hundreds of microns deep in the living organism. This unit addresses the application of 2-photon microscopy to imaging of genetically encoded calcium indicators (GECIs) in the mouse brain. The protocols in this unit enable real-time intravital imaging of intracellular calcium concentration simultaneously in hundreds of neurons, or at the resolution of single synapses, as mice respond to sensory stimuli or perform behavioral tasks. Protocols are presented for implantation of a cranial imaging window to provide optical access to the brain and for 2-photon image acquisition. Protocols for implantation of both open skull and thinned skull windows for single or multi-session imaging are described. © 2018 by John Wiley & Sons, Inc.

Since its commercialization in the late 1980's, confocal laser scanning microscopy (CLSM) has since become one of the most prevalent fluorescence microscopy techniques for three-dimensional structural studies of biological cells and tissues. The flexibility of the approach has enabled its application in a diverse array of studies, from the fast imaging of dynamic processes in living cells, to meticulous morphological analyses of tissues, and co-localization of protein expression patterns. In this chapter, we introduce the principles of confocal microscopy and discuss how the approach has become a mainstay in the biological sciences. We describe the components of a CLSM system and assess how modern implementations of the approach have further expanded the use of the technique. Finally, we briefly outline some practical considerations to take into account when acquiring data using a CLSM system. © 2018 by John Wiley & Sons, Inc.

Although in recent years flow cytometry has become commonplace in hematology and immunology laboratories, application of the technology to microbiology remains largely unrealized. This overview presents the historical background, discusses applications in various areas of the field, and speculates on the directions of future developments. The availability of high-quality methods should be a prime factor in convincing microbiologists that flow cytometry may have certain advantages over traditional methods and that it does indeed have much to contribute to microbiology. © 2018 by John Wiley & Sons, Inc.

Large population-based cohort studies, through their prospective collection of a broad range of health information, represent an invaluable resource for novel insights into the pathogenesis of human diseases. Collection and cryopreservation of viable cells from blood samples is becoming increasingly common in large cohorts as these cells are a valuable resource for immunophenotyping and functional studies. The cryopreservation of peripheral blood mononuclear cells (PBMCs), thawing, and immunophenotyping protocols used to immunophenotype 9938 participants in the Health and Retirement Study (HRS) are described. The extensive quality control involved in a large-scale immunophenotyping epidemiological study is also outlined. The existing literature on the effect of cryopreservation on various immune cell subsets including T, B, NK cells, monocytes, and dendritic cells is provided. © 2018 by John Wiley & Sons, Inc.

Acoustic cytometry uses radiation pressure forces instead of or in addition to hydrodynamic focusing to position cells or particles in a flowing stream for analysis. Commercial implementations to date combine both hydrodynamic and acoustic focusing together to enable high precision analysis of a broad dynamic range of volumetric sample input rates up to an order of magnitude higher than is practical with hydrodynamic focus alone. This capability allows great flexibility in reducing assay time or modifying or eliminating concentration requirements or concentration steps in sample preparation protocols. It also provides a practical method for processing sub-microliter volumes using sample dilution. In order to take full advantage of this dynamic range, it is necessary to understand the fundamental benefits and limitations of acoustic focusing as applied to flow cytometry. © 2018 by John Wiley & Sons, Inc.

Human leukocyte antigen (HLA) antibodies with the ability to activate complement are associated with an increased risk of early antibody-mediated graft rejection in kidney transplantation (KTx). Detection of these potentially harmful complement-fixing HLA antibodies is commonly performed via the complement-dependent cytotoxicity (CDC) assay according to protocols that were developed as early as 40 years ago. The read-out for this assay is based on manual scoring by visual inspection of cells under a fluorescence microscope. CDC is often used in combination with the flow cytometry–based lymphocyte crossmatch assay (FCXM), which, with high sensitivity, detects HLA antibody binding. Here we describe a new approach wherein both cytotoxicity and antibody binding can be simultaneously assessed with flow cytometry. Two strategies are described, using either magnetic bead–enriched T and B lymphocytes or bulk peripheral blood mononuclear cells (PBMC) as donor target cells. Curr. Protoc. Cytom. 66:6.34.1-6.34.11. © 2013 by John Wiley & Sons, Inc.

A wide range of fluorescent dyes and reagents exist for labeling organelles in live and fixed cells. Choosing between them can sometimes be confusing, and optimization for many of them can be challenging. Presented here is a discussion on the commercially-available reagents that have shown the most promise for each organelle of interest, including endoplasmic reticulum/nuclear membrane, Golgi apparatus, mitochondria, nucleoli, and nuclei, with an emphasis on localization of these structures for microscopy. Included is a featured reagent for each structure with a recommended protocol, troubleshooting guide, and example image. Curr. Protoc. Cytom. 66:12.31.1-12.34.24. © 2013 by John Wiley & Sons, Inc.

The age of microfluidic flow cytometry (µFCM) is fast becoming a reality. One of the most exciting applications of miniaturized chip-based cytometers is multivariate analysis using sampling volumes as small as 10 µl while matching the multiparameter data collection of conventional flow cytometers. We outline several innovative protocols for analyzing caspase-dependent cell death and cell cycle (DNA-content) profile using a fully integrated microfluidic flow cytometry system, Fishman-R. The first protocol describes the use of a new plasma membrane–permeability marker, DRAQ7, and the fluorogenic caspase substrate PhiPhiLux to track caspase activation during programmed cell death. Also outlined is the use of DRAQ7 fluorochrome in conjunction with the mitochondrial membrane potential–sensitive probe TMRM to track dissipation of inner mitochondrial cross-membrane potential. Another protocol adds the ability to measure dissipation of mitochondrial inner membrane potential (using TMRM probe) in relation to the cell cycle profile (using DRAQ5 probe) in living leukemic cells. Finally, we describe the combined use of fluorogenic caspases substrate PhiPhiLux with DRAQ5 probe to measure caspase activation in relation to the cell cycle profile in living tumor cells. Curr. Protoc. Cytom. 66:9.42.1-9.42.15. © 2013 by John Wiley & Sons, Inc.

This unit describes protocols for labeling tissue sections using combinations of metal-tagged antibodies and an iridium-containing DNA intercalator for analysis by imaging mass cytometry. Imaging mass cytometry (IMC) allows the labeling of up to 40 individual markers simultaneously using antibody cocktails. We discuss labeling of both cryostat sections and sections from formalin-fixed, paraffin-embedded (FFPE) tissue blocks. The protocols are similar to those used for optical microscopy techniques, while allowing much higher complexity of analysis. © 2017 by John Wiley & Sons, Inc.