4.11. Flow Cytometry Analysis of Cell Cycle

For cell cycle analysis, cells were plated in six-well plates at 2 × 10⁵ cells per well for 24 h. The cells were pretreated with 0.3 μM nocodazole (Sigma-Aldrich) for 24 h to synchronize cells at the G2/M boundary. Cells were then harvested, washed twice with cold PBS, and fixed with cold 70% ethanol at −20 °C overnight. The cells were then washed twice with PBS and resuspended with 10 mg/mL RNase A, 400 mg/mL propidium iodide, and 0.1% Triton X in 1 mL PBS at 37 °C for 15 min. Cells were subsequently analyzed by flow cytometry, and DNA content was quantified using ModFit LT software (Verity Software House: Augusta, Topsham, ME, USA).