Annexin V–Fluorescein Isothiocyanate/PI Double-Staining Assay

Cells (approximately 1 × 10⁶ cells per well) were collected and centrifuged at 1,000 × g for 5 min at room temperature, resuspended in ice-cold PBS, centrifuged at 1,000 × g for 5 min, and washed. The cells were resuspended by adding 500 μL of 1 × binding buffer. Subsequently, 5 μL annexin V–fluorescein isothiocyanate (FITC) staining solution and 5 μL PI staining solution were added to the suspension, mixed well, and incubated for 30 min at room temperature. Fluorescence intensity was measured using a BD FACSCalibur cytometer (Becton–Dickinson), and the apoptotic rates of cells were analyzed using the FlowJo software (Tree Star). The cells in Q1 (left upper quadrant), Q2 (right upper quadrant), Q3 (right lower quadrant), and Q4 (left lower quadrant) represent dead cells, late apoptotic cells, early apoptotic cells, and living cells, respectively. Three independent repeats of experiments were performed.