2.6. Flow cytometry analysis

For cell cycle analysis, GC cells (1 × 10⁴ cells/well) were sown into 6-well plates. On the next day, the cells were subjected to 30 µM curcumin (Sigma-Aldrich) for 24 h. Next, the cells were harvested and fixed overnight with 70% ethanol. After that, the fixed cells were washed with PBS (Solarbio, Beijing, China) and resuspended in binding buffer at a concentration of 1.0 × 10⁶ cells/mL followed by incubation with propidium iodide (PI; Beyotime, Shanghai, China) for 30 min at 37°C according to manufacturers’ instructions. Finally, the stained cells were analyzed with a FACScan^® flow cytometer (Beckman Coulter, Atlanta, GA, USA). For cell apoptosis, the cells (1 × 10⁴ cells/well) were plated into 6-well plates and then administered with 30 µM curcumin (Sigma-Aldrich) for 24 h. After that, the cells were collected, washed, resuspended, and then mixed with 5 μL of Annexin V-fluorescein isothiocyanate (Annexin V-FITC; Beyotime) and 5 μL of PI (Beyotime) for 20 min in the dark according to manufacturers’ instructions. The apoptotic cells were estimated using a FACScan^® flow cytometer (Beckman Coulter).