*3.4. Flow Cytometry for Cell Cycle and Apoptosis Analysis*

HCT116 cells (2 × <sup>10</sup><sup>5</sup> cells/mL) were plated in a 36-mm culture dish and incubated for 24 h. Fresh medium containing the indicated concentration of SA was added to culture dishes. Following a 24 or 48 h incubation, the cells were harvested (via trypsinization and centrifugation), rinsed twice with pre-cooled phosphate buffered saline (PBS), and prepared for apoptosis and cell cycle analysis.

For cell cycle analysis, 1 mL of pre-cooled 70% ethanol was added, and the cells were fixed overnight at −20 ◦C. Next, fixed cells were washed with PBS and incubated with a staining solution containing RNase A (50 μg/mL) and propidium iodide (PI) (50 μg/mL) in PBS for 30 min at room temperature. The cellular DNA content was analyzed with a FACSCalibur® flow cytometer (BD Biosciences, San Jose, CA, USA). At least 10,000 cells were used for each analysis, and the distribution of cells in each phase of the cell cycle was displayed using histograms.

For apoptosis analysis, HCT116 cells were treated with the test compound for 48 h. After incubation, the cells were collected and washed twice with PBS. The cells were stained with Annexin V-FITC and propidium iodide (PI) solution using an Annexin V-FITC apoptosis detection kit (BD Biosciences) according to the manufacturer's instructions. In brief, HCT116 cells were diluted to 1 × 106 cells/mL. A 100 μL aliquot of cell suspension was transferred into a 15 mL round-bottom polystyrene tube, and 5 μL of PI solution were added to the cell suspension, which was further incubated for 20 min at room temperature in the dark. Stained cells were diluted with binding buffer and immediately analyzed by flow cytometry.
