Caspillume

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.

Caspase activity assays were performed according to the instructions in the manual (CaspILLUME, Genetex). Cells were seeded at a density of 1×10⁶ cells per culture dish. After being subjected to treatment for 48 and 72 h, the cells were detached with accutase, washed, and resuspended in PBS. Next, 300 μL of each sample was aliquoted into centrifuge tubes, after which 1 μL of fluorescent substrate (FITC-DEVD-FMK for caspase 3 and FITC-LEHD-FMK for caspase 9 activity) was added to each tube and incubated for 1 h at 37°C incubator. At the end of the incubation period, the cells were centrifuged at 3000 rpm for 5 min and the supernatant was removed. After that, the cells were resuspended in 0.5 mL wash buffer (provided in the kit) and centrifuged at 3000 rpm for 5 min. This step was repeated before performing the analysis with flow cytometer (Accuri C6) and a minimum of 10,000 events were acquired for each replicate.