Assay for activation of Caspase-3/8/9

Caspases are key mediators of cell death. Caspase activity assay was performed using Caspases-3, -8 and -9 Staining Kit (CaspILLUME, Genetex). Briefly, cells at a density of 1×10⁶ were cultured in 60 mm petri dish. The cells were then treated with FKC in 0.5% DMSO at concentrations equivalent to; and also two and three times higher than their IC₅₀ values for 48 hours. After incubation, the cells were washed and incubated with 1μl of in situ marker (FITC-DEVD-FMK for caspase-3, FITC-IETD-FMK for caspase-8 and FITC-LEHD-FMK for caspase-9) for 20 minutes in 5% CO₂ at 37°C before being analyzed by flow cytometry (Accuri C6) and BD CFlow software. The results were analyzed by determining the percentage of activated caspase-3, -8 and -9 in comparison to the control. Untreated cells in 0.5% DMSO served as the control.