Annexin V and propidium iodide (PI) staining for apoptosis assay

To assess the apoptosis, the cells were harvested and centrifuged at 3000 rpm for 3 min. cell proportions were detected using FITC Annexin V Apoptosis Detection Kit I (BD pharmingen, La Jolla, CA) according to the manufacturer’s instructions. The cell pellets were washed with 1X PBS and resuspended in 100 μl of Annexin V binding buffer. Cells were labeled with 5 μl of Annexin V-Alexa Fluor 488 and/or PI. After 15 min incubation in a dark, 400 μl of Annexin V binding buffer was added to wash the Annexin/PI stained cells. A minimum of 10,000 cells per sample was analyzed using BD Accuri™ C6 Plus (BD FACS, San Jose, CA).