Quantifying Caspase Activity in Cardiac Tissues

Caspase activity was measured from all experimental cardiac tissues using ApoAlert caspase-3 and –9 Fluorescent Assay Kit (Clontech Laboratories, CA) following manufacturer’s protocol [17] (link). Briefly, tissue samples were homogenized in chilled protein extraction buffer. Then, 50 µl of 2X Reaction Buffer/DTT mix and 1 µl of Caspase-3 Inhibitor DEVD-CHO (for negative control) or 1 µl of DMSO (for other samples) was added to 50 µl of supernatant obtained from each sample. After incubation on ice for 30 min 5 µl of 1 mM Caspase-3 Substrate (DEVD-AFC; 50 µM final conc.) was added to each tube and incubated at 37°C for 1 hr. Fluorescence was measured at 400 nm excitation and 505 nm emission wavelengths (Varioskan Multimode Reader, Thermo Fisher, IL). For Caspase-9 activity, 5 µl of Caspase-9 Substrate (LEHD-AMC; 50 µM final conc.) was added to each tube and after incubation for 1 hr, fluorescence was measured at 380 nm excitation and 460 nm emission wavelength.