Cell cycle analysis using flow cytometry

HCT116 (p21^−/−) cells were plated in 60-mm tissue culture dishes at a density of 1.2 × 10⁵ cells/mL. At 50% confluency, cells were treated with different concentrations of GT and incubated for 48 hours. Cells were centrifuged at 4°C for 5 minutes at 1200 rpm. After that, pellets were washed by 1 × phosphate buffered saline (PBS) then fixed with 70% ice-cold ethanol and stored at –20°C. After 24 hours, cells were washed with 1 × PBS and incubated with 200 µg/mL RNase for 1.5 hours at 37°C. A 6.25 µg/mL propidium iodide was used for cells staining for at least 15 minutes. Fluorescence Activated Cell Sorter flow cytometer (Becton Dickinson, Research Triangle, NC) was used to measure the cellular fluorescence. Cell cycle analysis was then performed using the Cell Quest (BD Biosciences, USA) program and the percentages of cells distributed, according to their DNA content, were calculated. Cells in pre-G1 have DNA content <2n and represent apoptotic or necrotic cells.