Detecting Cleaved Caspase-3 in Apoptotic Cells by Flow Cytometry.
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.
Apoptosis and Cytotoxicity Laboratory, Mater Research, Translational Research Institute, Woolloongabba, Brisbane, Queensland 4102, Australia.
Lisa C
LC
Crowley
Apoptosis and Cytotoxicity Laboratory, Mater Research, Translational Research Institute, Woolloongabba, Brisbane, Queensland 4102, Australia.|Flow Cytometry and Imaging, QIMR Berghofer Medical Research Institute, Herston, Brisbane, Queensland 4006, Australia.|School of Medicine, University of Queensland, St. Lucia, Brisbane, Queensland 4072, Australia.
Nigel J
NJ
Waterhouse
D000906:Antibodies / Q000276:immunology; D017209:Apoptosis; D053148:Caspase 3 / Q000032:analysis* / Q000276:immunology; D005057:Eukaryotic Cells; D005434:Flow Cytometry / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*
D000906:Antibodies; D053148:Caspase 3