Summary
Apoptosis is a fundamental process required for proper embryonic development. Various methods have been described to detect apoptosis both in vitro as well as in vivo. Activation of caspases represents the key event in the apoptotic process. To dissect the molecular events leading to caspase activation, we have been using cell-free systems that recapitulate the mitochondrial death pathway. In the cell-free apoptosis assays, we either detect caspase activation in stimulated cells by utilizing subcellular fractions or reconstitute various components in cytosol (or mitochondria) to study molecular mechanisms of caspase activation. In either case, we utilize Western blot and/or substrate cleavage to monitor caspase activation. Using in-vitro reconstitution approach of caspase activation, we have discovered various factors that regulate caspase activity. Therefore, cell-free system not only is an invaluable tool to study apoptosis signaling but also provides molecular insight on caspase activation patterns and inhibitor specificities.
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Acknowledgements
This work was supported in part by an NIH K01 award to DC (7K01CA123142) and by grants from NIH (R01-AG023374, R01-ES015888, and R21-ES015893–01A1), Department of Defense (W81XWH-07–1–0616 and PC073751), and Elsa Pardee Foundation to DGT.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Chandra, D., Tang, D.G. (2009). Detection of Apoptosis in Cell-Free Systems. In: Erhardt, P., Toth, A. (eds) Apoptosis. Methods in Molecular Biology, vol 559. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-017-5_5
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DOI: https://doi.org/10.1007/978-1-60327-017-5_5
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