Annexin 5 pi kit

Cells were seeded into culture plates and cultured for 24 h to allow cell attachment. Then proliferating cells were determined using the Edu Cell Proliferation Kit (Beyotime Biotechnology, Shanghai, China), and the positive signals were captured under a fluorescence microscopy (Bio‐Rad, CA, USA). The percentage of proliferating cells was calculated through dividing the number of Edu⁺ cells by the number of Hoechst⁺ cells. Apoptotic cells were directly determined using the Annexin V/PI kit (Vazyme Biotech, Nanjing, China) according to the manufacturer's instructions, and the cells were analyzed by flow cytometry.

The A549/DDP cell cycle and apoptosis were estimated using flow cytometry. In brief, 48 h after transfection, cells were harvested for DDP treatment for additional 24 h. For the cell cycle assay, A549/DDP cells were fixed in 75% alcohol for 30 min, and then stained with propidium idoide (Sigma, MO, USA) for 15 min in dark. For cell apoptosis analysis, A549/DDP cells were washed with PBS followed by staining with Annexin V/PI kit (Vazyme, Nanjing, China) following the instructions of manufacturer. Finally, a BD Biosciences FACSCalibur Flow Cytometer (BD Biasciences, USA) was applied to detect the cell cycle and apoptosis.

Transfected EOC cells were harvested after 48 h and then the cell supernatant and washed cold PBS was collected in a tube. Cells were digested using EDTA-free trypsin (Invitrogen) and were collected in a tube. The collected cells were treated with an Annexin V/PI kit (Vazyme Biotech Co., Ltd, Nanjing, China) according to the manufacturer’s instructions and analyzed by flow cytometry (Beckman, FL, USA).