Detection of apoptosis in cell-free systems.

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.