Fluorometric analyses of caspase activation. (A) Time course of DEVD-cleaving activity. Cytoplasmic extracts (70 μg) from 1 × 107 neutrophils stimulated with TNF- plus cycloheximide (TNF/CHX) for the indicated time periods were incubated with 100 μmol/L DEVD-MCA for 30 minutes at 37°C. AMC release was measured by a microplate reader as previously described.24(B) Blockade of caspase activation by zVAD-fmk. Neutrophils were preincubated with indicated concentrations of zVAD-fmk before stimulation with TNF- plus cycloheximide for 3 hours. (C and D) Cell-free activation of DEVD-cleaving caspase(s) by recombinant active caspase-8 (C) or cytochrome c (D). Cytoplasmic extracts (70 μg) from nonapoptotic neutrophils were incubated with 0.5 μg purified recombinant caspase-8 (C) or with 1 μmol/L cytochrome c plus 1 mmol/L dATP (D) for 30 minutes at 37°C. (B, C, and D) DEVD-cleaving protease activities in the extracts were analyzed by real-time recordings of AMC release using a fluorometric microplate reader (Fluoroskan Ascent).
