Caspase 9 colorimetric kit

Caspase-8 and caspase-9 activity in cell lysates were measured using the fluorogenic Ac-VETDAMC peptide substrate (Sigma) or the caspase-9 colorimetric kit (Invitrogen) according to manufacturer’s instructions. For these experiments, cells were cultured on 6-well tissue culture plates. Cells were washed with serum-free medium and incubated for 16 hours in serum-free medium containing the peptides at final concentrations as indicated. Following incubation, cells were washed with PBS and lysed in 200 μl of lysis buffer ( 20 mM HEPES, 50 mM NaCl, pH7.2 10 mM DTT containing 1% CHAPS, 1 mM EDTA, 2 mM PMSF, leupeptin (10 μg/ml; Sigma) and pepstatin A (10 μg/ml; Sigma) for 30 min on ice. After centrifugation (7,000 xg, 10 min), the protein concentration of the supernatant was determined using the BCA Protein assay reagent (Bio-Rad). Subsequently, 20 μg of each sample was diluted to a final volume of 200 μl in assay buffer (20 mM HEPES, 50 mM NaCl, pH7.2 10 mM DTT, 0.1% CHAPS containing either the caspase-8 or caspase-9 substrate) in a 96-well plate. For caspase-8, fluorescence was determined (excitation 405 nm, emission 538 nm) with a Cary Eclipse fluorescence spectrophotometer (Varian, Palo Alto, Ca). For caspase-9 activity, absorbance at 450 nm was measured.