Measurement of Caspase 3 activity

Hearts, livers and kidneys from mice treated with DIC were used for determination of Caspase 3 activity. Briefly, samples were prepared as described under the section, “26S Proteasome Activity Assays of H9c2 Cells, Neonatal Cells and Tissue Lysates” and incubated in a 96 black well plate with Caspase 3 buffer (50 mM HEPES, 0.1% CHAPS, 100 mM NaCl, 1 mM EDTA, 10% Glycerol and 10 mM DTT; pH 7.4) in the presence or absence of 10 µM caspase-3 inhibitor, Ac-DEVD-CHO (Biomol). 100 µM caspase-3 substrate, Ac-DEVD-AMC (Biomol) was added to the reaction mixture to initiate the reaction. Caspase-3 activity was measured by the release of free AMC which was recorded every 15 min at an excitation wavelength of 390 nm and an emission wavelength of 460 nm for up to 120 min in a Fluoroskan Ascent fluorometer.