Flow cytometry has evolved since the 1940s into a multidisciplinary field incorporating aspects of laser technology, fluid dynamics, electronics, optics, computer science, physics, chemistry, biology, and mathematics. Innovations in instrumentation, development of small lasers, discovery of new fluorochromes/fluorescent proteins, and implementation of novel methodologies have all contributed to the recent rapid expansion of flow cytometry applications. In this thoroughly revised and updated second edition of Flow Cytometry Protocols, time-proven as well as cutting-edge methods are clearly and comprehensively presented by leading experimentalists. In addition to being a valuable reference manual for experienced flow cytometrists, the editors expect this authoritative up-to-date collection to prove useful to investigators in all areas of the biological and biomedical sciences who are new to the subject. The introductory chapter provides an eloquent synopsis of the principles and diverse uses of flow cytometry, beginning with a historical perspective and ending with a view to the future. Chapters 222 contain step-by-step protocols of highly practical and state-of-the-art techniques. Detailed instructions and helpful tips on experimental design, as well as selection of reagents and data analysis tools, will allow researchers to readily carry out flow cytometric investigations ranging from traditional phenotypic characterizations to emerging genomics and proteomics applications. Complementing these instructive protocols is a chapter that provides a preview of the next generation of solid-state lasers, and one that describes a rapid means to validate containment of infectious aerosols generated during high-speed sorting (Chapters 2324).

Includes supplementary material: sn.pub/extras

Klappentext
In this thoroughly revised and updated edition of a widely used practical guide to flow cytometry, leading experimentalists describe in step-by-step detail an array of time-proven and cutting-edge techniques much needed in today's advanced laboratories. These readily reproducible methods deploy emerging flow cytometry technologies in many new applications, especially in the fields of stem cells, functional genomics and proteomics, and microbiology. Here, the aspiring investigator will find methods for the characterization of stem/progenitor cells by monitoring the efflux of fluorescent dyes and the elucidation of signal transduction pathways using phospho-specific antibodies. There are also techniques for monitoring gene transfer and expression using fluorescent protein technology, high-throughput screening for discovery of novel protein interactions, phenotypic and functional characterization of T-cell subsets and precursors, and microbial flow cytometry, to highlight but some of the many useful procedures. The protocols presented follow the successful Methods in Molecular Biology™ series format, each one offering step-by-step laboratory instructions, an introduction outlining the principle behind the technique, lists of equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.
Comprehensive and highly practical, Flow Cytometry Protocols, Second Edition offers both basic and clinical scientists an up-to-date collection of cutting-edge protocols for solving the widest variety of novel flow cytometry problems.

Inhalt
Flow Cytometry.- Multiparameter Flow Cytometry of Bacteria.- Multiparameter Data Acquisition and Analysis of Leukocytes.- Flow Cytometric Analysis of Kinase Signaling Cascades.- Cytokine Flow Cytometry.- Use of Cell-Tracking Dyes to Determine Proliferation Precursor Frequencies of Antigen-Specific T Cells.- Assessment of Lymphocyte-Mediated Cytotoxicity Using Flow Cytometry.- Multiparametric Analysis of Apoptosis by Flow and Image Cytometry.- Detection and Enrichment of Hematopoietic Stem Cells by Side Population Phenotype.- Hematopoietic Stem Cell Characterization by Hoechst 33342 and Rhodamine 123 Staining.- Phenotypic and Functional Analyses of CD34NEG Hematopoietic Precursors From Mobilized Peripheral Blood.- Multiparameter Flow Cytometry of Fluorescent Protein Reporters.- Analysis of Fluorescent Protein Expressing Cells by Flow Cytometry.- Integrative Flow Cytometric and Microarray Approaches for Use in Transcriptional Profiling.- Flow Cytometric Analysis of Fluorescence Resonance Energy Transfer.- Design of a Fluorescence-Activated Cell Sorting-Based Mammalian Protein-Protein Interaction Trap.- Flow Cytometric Screening of Yeast Surface Display Libraries.- Fluorescence Resonance Energy Transfer-Based HIV-1 Virion Fusion Assay.- Cell-Cycle Analysis of Asynchronous Populations.- Solid Tumor DNA Content Analysis.- Concurrent Flow Cytometric Analysis of DNA and RNA.- Telomere Length Measurement by Fluorescence In Situ Hybridization and Flow Cytometry.- Small Lasers in Flow Cytometry.- Viable Infectious Cell Sorting in a BSL-3 Facility.