Abstract
The majority of caspases, cysteine-dependent aspartate-directed proteases, being in their activated state are involved in regulation of apoptosis by cleaving protein substrates harboring specific target motifs. Basically all biochemical and morphological changes in an apoptotic cell, including cell shrinkage, chromatin condensation, DNA fragmentation, and plasma membrane blebbing, are consequence of caspase-mediated proteolysis. Thus, uncovering activities of unique caspases are key determinants of the apoptotic process. This chapter describes a set of experimental protocols available for characterization, quantification and inhibition of caspase activities in mammalian cell cultures, including immunoblotting, usage of synthetic substrates, flow cytometry, and microscopic techniques.
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Abbreviations
- BSA:
-
Bovine serum albumin
- FACS:
-
Fluorescence activated cell sorting
- pAb:
-
Polyclonal antibody
- SDS:
-
Sodium dodecyl sulfate
- Tris:
-
Tris(hydroxymethyl)aminomethane, mAb, monoclonal antibody
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Acknowledgements
Work in the authors’ laboratory was supported by grants from the Swedish and Stockholm Cancer Societies, the Swedish Research Council, and the Swedish Childhood Cancer Foundation.
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Olsson, M., Zhivotovsky, B. (2014). Measurement of Caspase Activation in Mammalian Cell Cultures. In: V. Bozhkov, P., Salvesen, G. (eds) Caspases,Paracaspases, and Metacaspases. Methods in Molecular Biology, vol 1133. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0357-3_10
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DOI: https://doi.org/10.1007/978-1-4939-0357-3_10
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Online ISBN: 978-1-4939-0357-3
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