Flow cytometric analysis for apoptosis

To assess apoptosis, an Annexin V-FITC/PI apoptosis kit (Annexin v-FITC Early Detection Kit #6592, Thermo Fisher Sci., MA, USA) was used according to the manufacturer's instructions. Briefly, cells were seeded at a density of 1 × 10⁶ cells per 10 mm culture dish. After attachment overnight, cells were washed twice, collected, suspended with PBS, and cultured in a medium with 5 µM and 10 µM for 24 h or 48 h. All cells were collected and suspended in an ice-cold 1x binding buffer. Approximately 1 × 10⁵ cells were then stained with 1 µL of Annexin V-FITC and 12.5 µL of PI for 15 min and analyzed using a Flow cytometer (BD FACS Aria III; Becton Dickinson, NJ, USA).