Cell Cycle Analysis of HCT-116 Cells

For cell cycle analysis, 1 × 10⁵ HCT-116 cells were seeded in 12-well plates. On the second day, either OPT-A or DMSO (vehicle) was administrated. Treated cells continued to be cultured for 48 h. Then, all adherent cells were trypsinized, collected and fixed in 80% ethanol for 2 h at −20 °C. After being treated with 0.25% Triton X-100 for 5 min, the cells were resuspended in 50 µL of PI/RNase staining reagent (Becton Dickinson, San Diego, CA, USA), incubated in the dark for 20 min at room temperature, and finally counted with a FACS Canto flow cytometer [18 (link)]. At least 10,000 cells were read for each measurement.