Annexin V-FITC/propidium iodide (PI) apoptosis detection by fluorescence-activated cell sorting (FACS) assay

Cell apoptosis was assessed using annexin-V-FITC and PI staining kit (BD Biosciences). In total, 5×10⁵ cells were collected by trypsinization at 37°C for 5 min, centrifuged under 200 × g at 4°C for 5 min and washed twice with PBS, resuspended in 500 µl 1X binding buffer and incubated in 5 µl Annexin V-FITC and 5 µl PI solution in the dark for 15 min at RT. Cells were analyzed using an integrated BD Accuri™ C6 system (BD Biosciences). The gates for flow cytometry was set using following steps: Target cell population was circled by detecting unstained cells. Annexin V-FITC single positive cells were detected, following which fluorescence compensation was adjusted to ensure no particles were in the upper left (UL) and upper right (UR) quadrants. PI single positive cells were detected and fluorescence compensation was adjusted to ensure no particles were present in the upper right (UR) and lower right (LR) quadrants. A total of four populations of cells were observed: i) Cells that were viable and not undergoing apoptosis are both FITC-Annexin V and PI-negative (LL); ii) cells undergoing apoptosis were FITC-Annexin V-positive and PI-negative (LR); iii) the cells that are undergoing late apoptosis or necrotic are both positive for FITC-Annexin V and PI (UL and UR).