Quantifying Caspase Activity Assays

Activities of caspase-3/7 and caspase-9 were assessed using an Apo-ONE™ homogeneous caspase-3/7 assay kit (Promega, Madison, WI, USA) and a Caspase-Glo™ 9 assay kit (Promega), respectively, according to the manufacturer’s protocols [16 (link), 17 (link)]. Briefly, cells were seeded into 96-well plates (2 × 10⁴ cells/well) and incubated overnight. On the following day, the cells were treated with the indicated concentrations of the appropriate drug for varying times. After the treatment, 100 μL of the Apo-ONE™ caspase-3/7 reagent was added to each well. The plates were incubated at room temperature for 1 h, and the luminescence of each well was measured using an EnSpire™ multimode plate reader (PerkinElmer, Waltham, MA, USA). Caspase activity was calculated by comparing the levels of luminescence of the treated cells with that of the control cell population incubated without drugs, which was defined as 100%.