Detection of caspase-activation in intact lymphoid cells using standard caspase substrates and inhibitors.
Members of the caspase family of proteases are important in the implementation of apoptotic cell death. These caspases are intracellularly activated upon a death stimulus, and exhibit a distinctive proteolytic activity which transmits a death signal and readily detected by measuring the cleavage of synthetic substrates in cell extracts. In this report, we show that apoptosis-associated caspase activation can be recorded not only in cell lysates but also in intact lymphoid cells with commercially available peptides which are either biotinylated or carry an amino-methylcoumarin (AMC) group. Incubation of intact cells induced to undergo apoptosis with Ac-Asp-Glu-Val-Asp-AMC (DEVD-AMC) leads to the release of AMC in amounts very similar to the amounts released when cell extracts are prepared and incubated with DEVD-AMC. This release can be detected by a fluorescence read-out and is blocked by caspase-inhibitors such as Ac-DEVD-cho or Z-VAD-fmk. Similarly, labelling of intact cells with the biotinylated peptides Tyr-Val-Ala-Asp-cmk (YVAD-cmk) or YVAD-faom permits the detection of active caspases by affinity blotting and the detection of apoptotic cells by FACS analysis. These methods enable the investigator to detect at the single-cell level those cells which have activated their caspases and to evaluate such activation without the need for lysis of the cells.
Mack
A
A
Institute for Medical Microbiology, Technische Universität München, Munich, Germany.
Fürmann
C
C
Häcker
G
G
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D001710:Biotin / Q000494:pharmacology; D019921:Biotinylation; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D003374:Coumarins / Q000378:metabolism; D004789:Enzyme Activation; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D019169:Jurkat Cells; D008221:Lymphoid Tissue / Q000166:cytology / Q000201:enzymology*; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology; D013050:Spectrometry, Fluorescence / Q000379:methods
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D003374:Coumarins; C098738:N-acetyl-tyrosyl-valyl-alanyl-aspartyl chloromethyl ketone; D009842:Oligopeptides; C102580:acetyl-aspartyl-glutamyl-valyl-aspartyl-amino-4-methylcoumarin; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D001710:Biotin; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases