Caspase glo 3 7 or 8 reagent

From cell lysates: 25 μl of Caspase‐Glo^®‐3/7 or ‐8 reagent (Promega, Southampton, UK) was added to 5μg (caspase‐3/7 activity) or 10μg (caspase‐8 activity) of cell lysate, made up to 25 μl with PBS in a white‐walled 96‐well plate. Each sample was plated in triplicate. The plate was incubated in the dark at room temperature for 45 min with gentle agitation. After incubation, the plate was read on a Synergy 4 plate reader (Biotek, USA) at a sensitivity of 200. Blank readings were taken in all cases to account for background luminescence. In live cells: Cells were seeded in triplicate at 5 × 10³ per well in a white‐walled 96‐well plate. All drugging was performed in a total volume of 50 μl. After experimental procedures were completed, 50 μl Caspase‐Glo^® reagent (Promega, Southampton, UK) was added to each well and the plate covered in foil to protect the reagent from ambient light. The plate was then incubated at room temperature for 45 min with gentle agitation. After incubation, the plate was read on a Synergy 4 plate reader (Biotek, USA) at a sensitivity of 200. Blank readings were taken in all cases to account for background luminescence.

Twenty-five microliters of Caspase-Glo®-3/7 or -8 reagent (Promega) was added to 5 µg (Caspase-3/7 activity) or 10 µg (Caspase-8 activity) of cell lysate, made up to 25 µL with PBS in a white-walled 96-well plate in duplicate. The plate was incubated in the dark at room temperature for 45 min and subsequently read at 1 s integrated reads in a luminescent plate reader (Biotek Synergy 4 plate reader). All conditions were performed in duplicate.