Caspase-3/7 as well as caspase-8 and -9 activity was measured using flow cytometry. Briefly, cells were cultured and plated in a concentration of 200,000 cells/ml. We then treated the cells either with ABT263 (5 μM) (intrinsic apoptotic stimulus) or Birinapant (50 nM) and hTNF (10 ng/ml) (B + T) (extrinsic apoptotic stimulus) for 1 to 30 hours. The cell suspension was centrifugated and resuspended in RPMI 1640 to stop the reaction. Using FAM-FLICA Caspase Assay Kit for caspase-8 (catalog no. 910; ImmunoChemistry Technologies), for caspase-9 (catalog no. 912; ImmunoChemistry Technologies), and for caspase-3/7 (catalog no. 93; ImmunoChemistry Technologies), we incubated the cells in 1:30 of 1:5-diluted FLICA staining buffer for 30 min protected from light. Fluorescence was detected by flow cytometry (FAM-FLICA excites at 492 nm and emits at 520 nm).

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