Measurement of CTL-induced cytotoxicity: the caspase 3 assay.
Cytotoxic T lymphocytes (CTL) are critical effector cells of the immune system. Measurement of target cell damage has historically been an important measure of CTL function. CTL kill their target cells predominantly by inducing programmed cell death, or apoptosis. The gold standard for CTL-mediated cytotoxicity has been the (51)Cr release assay. However, measurement of target cell lysis by (51)Cr release does not provide mechanistic information on the fate of target cells, especially at the single cell level. Given the recent advances in our understanding of programmed cell death, newer assays are required which evaluate the status of the apoptotic pathways in target cells. We have developed a flow cytometry-based assay for CTL-mediated cytotoxicity based on specific binding of antibody to activated caspase 3 in target cells. Our assay is convenient and more sensitive than the (51)Cr release assay. The use of this assay should allow mechanistic studies of the intracellular events resulting from CTL attack.
Fred Hutchinson Cancer Research Center, Department of Laboratory Medicine, University of Washington, Seattle, WA 98109, USA. kjerome@fhcrc.org
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Jerome
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Sloan
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Aubert
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D053148:Caspase 3; D020169:Caspases / Q000276:immunology; D002860:Chromium Radioisotopes; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D013602:T-Lymphocytes, Cytotoxic / Q000502:physiology*
D002860:Chromium Radioisotopes; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases