Apoptosis assay

Cell apoptosis was assessed using the Annexin V-FITC Apoptosis Detection Kit I. Jurkat cells were collected and resuspended to adjust the cell density to 2x10⁵ cells/ml. This cell suspension was added to a 6-well plate at 2 ml/well and different concentrations (0, 3, 5, 6, and 9 µM) of hirsutanol A and/or PFTα (10 µM) were added. After 48 h of incubation at 37˚C, the cells were collected, washed twice with cold PBS and resuspended with 1X binding buffer. Of this suspension, 100 µl (~1x10⁵ cells/ml) were placed in a 5-ml culture tube and propidium iodide (PI; 5 µl) and Annexin V-FITC (5 µl) were added. Following incubation for 15 min in the dark at room temperature, 200 µl 1X binding buffer was added per tube and apoptotic cells were detected using a BD FACScan™ flow cytometer (BD Biosciences) within 1 h. The results were analyzed using BD FACSuite™ (BD Biosciences). Early and late apoptosis were assessed.