Detection of caspase-activation in intact lymphoid cells using standard caspase substrates and inhibitors
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- 作者：Mack ; Astrid ; Fü ; rmann ; Christine ; Hä ; cker ; Georg
- 关键词：Apoptosis ; Caspase activation ; Intact cells ; Fluorogenic substrate ; Biotin ; Ac-DEVD-AFC ; acetyl-Asp-Glu-Val-Asp-7-amido-4(trifluoromethyl)coumarin ; Ac-DEVD-AMC ; acetyl-Asp-Glu-Val-Asp-7-amido-4methylcoumarin ; Ac-DEVD-cho ; acetyl-Asp-Glu-Val-Asp-aldehyde
- 刊名：Journal of Immunological Methods
- 出版年：2000
- 出版时间：July 31, 2000
- 年：2000
- 卷：241
- 期：1-2
- 页码：19-31
- 全文大小：427 K
文摘
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.