Detecting cleaved caspase-3 in apoptotic cells by flow cytometry

Crowley, Lisa C. and Waterhouse, Nigel J. (2016) Detecting cleaved caspase-3 in apoptotic cells by flow cytometry. Cold Spring Harbor protocols, 2016 11: 958-962. doi:10.1101/pdb.prot087312


Author Crowley, Lisa C.
Waterhouse, Nigel J.
Title Detecting cleaved caspase-3 in apoptotic cells by flow cytometry
Journal name Cold Spring Harbor protocols   Check publisher's open access policy
ISSN 1559-6095
1940-3402
Publication date 2016-11-01
Sub-type Article (original research)
DOI 10.1101/pdb.prot087312
Open Access Status Not yet assessed
Volume 2016
Issue 11
Start page 958
End page 962
Total pages 5
Place of publication Cold Spring Harbor, United States
Publisher Cold Spring Harbor Laboratory Press
Collection year 2017
Language eng
Abstract Apoptosis is orchestrated by caspases, a family of cysteine proteases that cleave their substrates on the carboxy-terminal side of specific aspartic acid residues. These proteases are generally present in healthy cells as inactive zymogens, but when stimulated they undergo autolytic cleavage to become fully active. They subsequently cleave their substrates at one or two specific sites, which can result in activation, inactivation, relocalization, or remodeling of the substrate. Consequently, many of the cleaved fragments remain intact during apoptosis and can be detected using substrate-specific antibodies. These fragments are most commonly detected by western blotting, which resolves proteins and their fragments based on molecular mass. However, antibodies that only recognize cleaved fragments can be used to specifically label cells in which caspase cleavage has occurred. It is then possible to quantify these cells by flow cytometry. A number of antibodies that specifically recognize caspase-cleaved fragments have been generated, including antibodies that recognize the cleaved form of caspase-3. This caspase is responsible for the majority of proteolysis during apoptosis, and detection of cleaved caspase-3 is therefore considered a reliable marker for cells that are dying, or have died by apoptosis. This protocol outlines the quantification of apoptosis by flow cytometric detection of cleaved caspase-3.
Q-Index Code CX
Q-Index Status Provisional Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Mater Research Institute-UQ (MRI-UQ)
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Created: Tue, 15 Nov 2016, 00:54:37 EST by Julia McCabe on behalf of Learning and Research Services (UQ Library)