Caspase Activity Analysis in U-2 OS Cells

Flow cytometry was used to measure the activities of caspase-3, -8m and -9 in U-2 OS cells after they were exposed to bufalin. Briefly, U-2 OS cells (1 × 10⁵ cells/well) were cultured onto 12-well plates and were incubated with 200 nM of bufalin for 0, 12, 24, and 48 h, and then cells were harvested and re-suspended in 25 μL of 10 μM substrate solution (PhiPhiLux-G1D1 for caspase-3, CaspaLux8-L₁D₂ for caspase-8 and CaspaLux9-M1D2 for caspase-9) and incubated at 37 °C for 60 min. After incubations, all samples were harvested, washed with PBS, and analyzed by flow cytometry for caspase-3, -8, and -9 activities, as described previously [59 (link)].