Apoptosis detection.

Apoptosis was determined by the phycoerythrin (PE)-annexin V apoptosis detection kit (559763; BD Biosciences) or the FITC-annexin V apoptosis detection kit (556547; BD Biosciences) according to the kit guidelines. Briefly, floating and attached cells were collected and resuspended in 1× binding buffer at a concentration of 1 × 10⁶ cells/ml. Then, 100 μl of cell suspension was stained with 5 μl PE-annexin V/FITC-annexin V and 5 μl 7-AAD/propidium iodide (PI) for 15 min at room temperature in the dark. Samples were then analyzed by the FACSCalibur flow cytometer (BD Biosciences) within 1 h to determine the percentages of apoptotic cells (annexin V positive).