Caspase-3 assay

Cells were incubated with inhibitors for 30 min at 37 °C, followed by 4 h stimulation with 100 ng/ml TNF under cell culture conditions. Cells were then sedimented and lysed (10 mM HEPES [pH 7.4], 5 mM MgCl₂, 1 mM EGTA, 0.2% NP40, 2 mM AEBSF/Pefabloc, 1 mM DTT (freshly added). 5 μg total cell lysate was then incubated with 100 μl assay buffer (20 mM PIPES [pH 7.2], 100 mM NaCl, 1 mM EDTA, 0.1% CHAPS, 10% Sucrose, 10 mM DTT (freshly added) containing 100 μM zDEVD-AFC (#13420, AAT Bioquest). Increase in fluorescence intensity was monitored at ex: 405 nm/em: 505 nm using an Infinite M200 (Tecan) plate reader at 37 °C.