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Detection of caspase-activation in intact lymphoid cells using standard caspase substrates and inhibitors
1Institute for Medical Microbiology, Technische Universität München, Munich, Germany.
Abstract:
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.
Insights
Researchers developed new methods to detect caspase activation, crucial for apoptosis, in intact cells. This allows for single-cell analysis of programmed cell death without cell lysis, advancing apoptosis research.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Caspase proteases are key executioners of apoptotic cell death.
- Caspase activation is typically detected in cell lysates using synthetic substrates.
- Existing methods require cell lysis, potentially altering cellular processes.
Purpose of the Study:
- To develop methods for detecting caspase activation in intact cells.
- To enable single-cell analysis of apoptosis-associated caspase activation.
- To circumvent the need for cell lysis in caspase activity assays.
Main Methods:
- Utilized fluorescent (AMC-labeled) and biotinylated caspase substrates (DEVD-AMC, YVAD-cmk).
- Applied these substrates to intact, apoptosis-induced lymphoid cells.
- Detected substrate cleavage via fluorescence readout and affinity blotting.
- Confirmed results using flow cytometry (FACS) and caspase inhibitors (Ac-DEVD-cho, Z-VAD-fmk).
Main Results:
- Apoptosis-associated caspase activation was successfully detected in intact cells using DEVD-AMC.
- AMC release from DEVD-AMC in intact cells mirrored that in cell lysates.
- Biotinylated peptides enabled detection of active caspases and apoptotic cells via blotting and FACS.
- Caspase inhibitors blocked the observed substrate cleavage, validating the specificity.
Conclusions:
- Novel methods allow for the detection and evaluation of caspase activation in intact cells.
- These techniques facilitate single-cell level analysis of apoptosis without cell lysis.
- The developed assays provide a valuable tool for studying programmed cell death mechanisms.