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Measurement of Apoptosis by Multiparametric Flow Cytometry

Protocol
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Part of the Methods in Pharmacology and Toxicology book series (MIPT)

Abstract

Apoptosis remains a critical phenomenon in cell biology, playing a regulatory role in virtually every tissue system. It is particular crucial in the immune system, ranging from immature immune cell development and selection to downregulation of the mature immune response. Apoptosis is a primary mechanism in the action of antitumor drugs, and is thus an important phenomenon in pharmacology, drug discovery, and toxicology. Flow cytometry is the primary technique for measuring apoptosis in suspension cells; many flow cytometry assays have been developed to measure the entire apoptotic process, from the earliest signal transduction events to the late morphological changes in cell size, proteolysis, and DNA degradation. These assays become even more powerful when they can be combined into single multiparametric assays that can document the process of apoptosis in a single tube. The ability of flow cytometry to measure multiple structural and fluorescent characteristics in single cells is uniquely suited to this task. In this methods review, we show how multiple individual assays can be combined in this fashion. Combining early biochemical and late morphological assays together gives a comprehensive and detailed picture of the apoptotic process.

Key words

Apoptosis measurement Flow cytometry Apoptosis assays Multiparametric Caspase 3/7 substrate Caspase 3 immunolabeling Annexin V FLICA (fluorescence linked inhibitor of caspase activity) Covalent binding viability probes DNA binding dyes 

Abbreviations

7-AAD

7-aminoactinomycin D

APC

Allophycocyanin

FBS

Fetal bovine serum

FLICA

Fluorescence linked inhibitor of caspase activity

HBSS

Hanks balanced salt solution

PBS

Phosphate buffered saline

PE

Phycoerythrin

TUNEL

Terminal deoxynucleotidyl transferase dUTP nick end labeling

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Experimental Transplantation and Immunology Branch, National Cancer InstituteNational Institutes of HealthBethesdaUSA

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