2.7. Cell cycle analysis

Cells were suspended in PBS, and then, 100% ethanol was added to be the final concentration of 70% ethanol while gently vortexing. The fixed cells were permeabilized with 0.25% Triton X‐100 in PBS on ice for 15 minutes. The cells were incubated with anti‐H3‐pS10 (Millipore; 06‐570) antibody for 2 hours and then incubated with FITC‐conjugated goat anti‐rabbit IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA; 111‐095‐144) at room temperature in the dark for 1 hour. Cells were incubated with DNase‐free RNase A at 37°C for 30 minutes and then with propidium iodide (PI) at 37°C in the dark for another 30 minutes. The percentage of cells in each cell cycle phase and H3‐pS10‐positive cells were determined by flow cytometry.