Caspase assay

Caspase activity was determined by fluorometric assay using the enzyme substrate IETD-AMC for caspase-8 and LEHD-AMC for caspase-9, which are specifically cleaved by the respective enzymes at the Asp residue to release the fluorescent group, AMC. Cell extracts containing 50 μg of protein were incubated with 100 mM HEPES containing 10% sucrose, 10 mM dithiothreitol, 0.1% 3-[(3-cholamidiopropyl)-dimethylammonio]-1-propane sulfonate and 50 mM caspase substrate in a total reaction volume of 0.25 ml. The reaction mixture was incubated for 60 min at 37 °C, and quantified fluorometrically at the excitation and emission wavelengths of 380 and 460 nm, respectively (Synergy H1 Hybrid Reader, BioTek, Winooski, VT, USA).