Annexin V-FITC/PI apoptosis detection

Apoptosis rates were quantified by Annexin V-FITC/PI apoptosis detection kit (KeyGEN, Nanjing, China) according to the manufacturer's protocol. Briefly, after cells were treated with indicated concentrations of parthenolide for 72 h, cells were collected and washed twice with ice-cold PBS. Then 5 × 10⁵ cells were resuspended in 0.5 mL binding buffer containing Annexin-V (1:50) and 40 ng/sample of PI for 30 min at 37°C in the dark. Subsequently, the cells were determined by flow cytometer (Becton Dickinson, NY) and analyzed by CellQuest software. At least 10,000 cells were analyzed for each sample. The apoptosis rate (%) = (the number of apoptotic cells/the number of total cells observed) × 100% [37].