Apoptosis Assay by Flow Cytometry

Cell lines were seeded in 6-well plates, added with 2 mL medium/well, and cultured overnight. Afterwards, they were treated with 30 µg/mL of curcumin and 80 µmol/L ginsenoside 20(S)-Rg3 for one hour and exposed to 4 Gy X-rays. After 48 hours, flow cytometry was performed with Annexin V-FITC/PI kit (BioLegend, USD), according to the manufacturer’s instructions. The treated cells were rinsed twice with PBS and trypsinized for three min to obtain a single-cell suspension. Trypsinization was stopped by adding the medium. Cells were rinsed twice in pre-cooled PBS and then resuspended in Annexin V Binding Buffer at a concentration of 1×10⁶ cells/mL. Initially, 100 µL of cell suspension was transferred in a 5 mL test tube, and then 5 µL of FITC Annexin V and 10 µL of PI solution were added. Cells were vortexed and incubated for 15 min at 25 °C in the dark. Next, 400 µL of Annexin V binding buffer was added to each tube. Finally, the samples were analyzed with BD FACSCalibur^TM (Biosciences, San Jose, CA, USA) using flow cytometry. The experiment was repeated three times. ¹⁶