100 μl binding buffer

Flow cytometry was used to detect the cell cycle and apoptosis. Cells were cultured in an incubator under normal conditions, and gastric cancer cells treated with DMSO and Cy at a specific time were collected. To detect the cell cycle, cells were fixed overnight at 4 °C with 75% ethanol. The cells were washed with PBS to remove residual ethanol and incubated with propidium iodide (PI, BD, San Jose, CA, USA) and RNase A (Sigma Aldrich, St Louis, MO, USA) at 37 °C for 1 h. To detect apoptosis, the collected cells were washed with PBS. The cells were then resuspended in 100 μL binding buffer (BD, San Jose, CA, USA). The cells were then incubated with FITC-labeled Annexin V (BD, San Jose, CA, USA) and PI (BD, San Jose, CA, USA) at room temperature for 15 min. All samples were analyzed using FACS C6 (BD, San Jose, CA, USA) with Cell Quest software. Each experiment was repeated three times.

Cells were placed into a cell culture plate and cultured under standard conditions. The adherent cells were incubated with cell-culture medium containing different concentrations of DEH for 2 days for analysis of the cell cycle and apoptosis. For the cell cycle assay, the cells were harvested and fixed in cold 75% ethanol at 4 °C overnight. After washing with PBS to remove the residual alcohol, the cells were incubated with propidium iodide (PI; BD, USA) and RNase A (Sigma Aldrich, USA) at 37 °C for 1 h. For the apoptosis assay, cells were harvested and washed with cold PBS, and then resuspended in 100 μL binding buffer (BD, USA). Thereafter, the cells were incubated with FITC-labeled Annexin V (BD, San Jose, CA, USA) and PI at room temperature for 15 min. All the samples were analyzed by the FACS C6 (BD, USA) using Cell Quest software.