Annexin V-PE/7-AAD Double Staining Assay

For flow cytometry analysis of annexin V externalization, cells were seeded on a 6-well plate (2 × 10⁵ cells per well) to achieve 70% confluence overnight before assay. AGS cells were transfected with Cav-1 overexpression vector or empty vector for 24 h, followed by incubation with 20 μg/ml cisplatin for 12 and 24 h, respectively. MGC803 cells were transfected with shCav-1 vector or negative control vector for 48 h, followed by incubation with 8 μg/ml cisplatin for 12 and 24 h, respectively. Annexin V levels were determined using an Annexin V-PE/7-AAD staining kit (BD Pharmingen, San Diego, CA, USA) according to the manufacturer's protocol. All cells were analyzed by flow cytometry using a BD FACSCanto II flow cytometry (BD Pharmingen) to detect the fluorescence signal.