Annexin V Flow Cytometry for Apoptosis Analysis

Flow cytometry analysis was performed using the Annexin V FITC apoptosis detection kit (KeyGen Biotech, Ltd., Nanjing, China) as described previously [42 (link)]. Briefly, cells (1 × 10⁵) were incubated with cisplatin (33.33 μM), GRN A (14.53 μM) and the combination of the two drugs. After treatment for 24 h, cells were harvested by a centrifugation at 1000 g for 5 min, washed with phosphate-buffered saline (PBS) twice, and resuspended in 500 μL binding buffer (KeyGen Biotech, Ltd., Nanjing, China). Then, Annexin V FITC (5 μL) and PI (5 μL) were added. Cells were gently vortexed, incubated for 10 min at room temperature in the dark, and immediately analyzed by the FACS flow cytometry system (Bection-Dickinson, San Jose, CA, USA). Data are expressed as means ± SD; per group for at least 3 independent experiments.