Caspase 3/7 measurements

The Caspase-Glo 3/7 Assay (Promega) was performed following the manufacturer's protocol. Caspase-Glo 3/7 Buffer and Caspase-Glo 3/7 Substrate were equilibrated to room temperature prior to use. Caspase-Glo 3/7 substrate was reconstituted by transferring the entire Caspase-Glo 3/7 Buffer into the bottle. Cell pellets were resuspended in media and counted as previously described. Cells were diluted to 100,000 cells/ml and 50 μl of cell suspension was plated in an opaque-walled 96-well plate in triplicate. Media alone was added in triplicate to allow the exclusion of background luminescence and Staurosporine (Cell Signaling Technology) treatment was used as a positive control (0.3 μM for 16 h). 50 μl of the reconstituted substrate was added to each well in the dark and the plate was covered in foil and placed on a plate shaker for 30 seconds prior to incubation at room temperature for 1 h. The plate was read on a luminescent plate reader. Triplicate measurements were averaged and the culture media background fluorescence was subtracted from the average of each experimental sample.