Caspase activity

Caspase 3 activity was measured using the Caspase-Glo^® 3/7 (Promega Co., Madison, WI), in which 50,000 cells/well were initially seeded in a flat bottom 96-well plate. Cells were incubated with equal volumes of the reagent to the culture medium for 1 h, and a Varioskan Flash Reader (Thermo Fisher Scientific, Waltham, MA) was used to assess relative luminescence. Caspase 9 activity was measured using the Caspase-9 Fluorometric assay (R&D Systems, Inc. Minneapolis, MN). Each sample contained the cell lysate (100 μg of protein in 50 μL), 50 μL of 2X reaction buffer, and 5 μL of caspase 9 fluorometric substrate (LEHD-AFC). The reaction was incubated for 1 h at 37°C in a flat bottom 96-well microplate. Fluorescence signal was indicative of caspase activation, and it was measured on a fluorescent plate reader (Gemini SpectraMax, Molecular Devices, CA, USA).