Annexin V-FITC/PI Apoptosis Assay

After trypsin digestion and centrifugation, cells were tested by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection Kit (all BD Company). Cells were suspended in 400 μL of 1 × AnnexinV-binding solution, and the cell density was adjusted to 1 × 10⁶/mL. Then, 5 μL of Annexin V-FITC staining solution was added and incubated in the dark for 15 min. In addition, 10 μL of PI staining solution was added, and cells were tested using a flow cytometer (BD company, model: FACSCalibur). Cells were divided into 4 quadrants with fluorescein isothiocyanate (FITC) rabbit anti-mouse antibody and PI fluorescence as a two-parameter dot plot: viable cells in the lower left quadrant, early apoptotic cells in the lower right quadrant, late apoptotic cells and dead cells in the upper right quadrant, and mechanically damaged cells in the upper left quadrant. Apoptosis rate = early apoptosis + late apoptosis [27 (link)]