Flow cytometry analysis of cell cycle arrest and apoptosis

CaSki (3.0 × 10⁵ cells/mL) and SiHa (4.0 × 10⁵ cells/mL) cells were seeded in 6-well Petri dishes the day prior to the experiment. Cells were treated with 5, 15, or 20 μM EM23 for 24 h, then harvested, washed twice with ice-cold PBS, and fixed in 70% ethanol at −20°C overnight. Fixed cells were washed once with ice-cold PBS and re-suspended in 1 mL of staining reagent containing 100 mg/mL RNase and 50 mg/mL PI for 25 min in the dark. To assess apoptotic ratio, harvested cells were stained with Annexin-V-FITC/PI (KeyGEN; Nanjing, China) according to the manufacturer's instructions. Cell cycle arrest and apoptosis were analyzed by flow cytometry (BD FACSCalibur; Franklin Lakes, CA, USA). Fluorescence of PI and Annexin-V-FITC was monitored at 630 nm and 525 nm, respectively.