Measurement of caspase activity

Caspase 3/7 activity was determined using the Caspase-Glo® 3/7 Assay (Promega, Madison, WI) according to manufacturer's instructions. The assay provides a luminogenic caspase 3/7 substrate, which contains the tetrapeptide sequence DEVD selective for caspases 3/7. Addition of Caspase-Glo® 3/7 reagent results in cell lysis, followed by caspase cleavage of the substrate and release of luminescent signal. The luminescence produced is proportional to the amount of caspase activity present. At the time of apoptosis measurement, 50 microlitres of each samples was transferred into a single well of a 96-well plate and 50 microlitres of Caspase-Glo® 3/7 Assay mixture added. After incubation for 30 minutes at 22°C luminescence was measured using a Berthold luminometer.

Caspase 8 and caspase 9 activity was determined using the Caspase-Glo® 8 and the Caspase-Glo® 9 Assays, respectively, (Promega, Madison, WI) according to manufacturer's instructions and the above protocol.

Caspase 3/7, caspase 8 and caspase 9 activation was measured with Caspase Glo Assay (Promega, Madison, WI) according to the kit protocol and after normalization with ATP content. For Caspase Glo 3/7 and Caspase Glo 9 assays, the intensity of bioluminescence is proportional to the activation of caspases 3/7 and caspase 9 respectively.