4.10. Cell Cycle Analysis

HT29 cells (2 × 10⁵ cells/6-cm² culture dish) were fixed in cold 75% ethanol at 4 °C overnight and washed twice with PBS. The cells were then stained with 0.5 μL of 10 mg/mL propidium iodide (PI) containing 0.1 mg/mL RNase A for 30 min at room temperature. DNA content in the cells was analyzed by flow cytometry (BD Biosciences, San Diego, CA, USA) using Modfit LT 3.0 software (Verity Software House, San Diego, CA, USA).