Detection of cell death by flow cytometry

Cells were labeled with the fluorescent probes Annexin V-APC (Invitrogen-ThermoFisher Scientific, St Laurent, QC) and propidium iodide (PI) (Sigma-Aldrich Canada, Oakville, ON). Cells (10⁶) in a volume of 100 µL, and were incubated with 5 µL of Annexin V-APC (1/20 dilution) for 10 min, according to the manufacturer’s protocol (Invitrogen).⁸⁵ Subsequently, 400 µL of DMEM medium containing 5% FBS were added and then 5 µL of PI (final concentration of 2 µM) was added to the cell mixture for 5 min. At least 10,000 cells were analyzed for each sample by flow cytometry at a flow rate of 35 µL/min (BD Accuri™ C6, BD Biosciences, Mississauga, ON). Annexin V-APC was detected using the FL1 channel and PI with the FL3 channel. For this flow cytometer, the pre-optimizing voltage and gain settings do not reduce the fluorescence detection range. Analysis was carried out using BD Accuri™ C6 software. Doublet exclusion was performed by plotting the height or width against the area for forward scatter or side scatter. Doublets will have double the area and width values of single cells while the height is roughly the same. Therefore, disproportions between height, width, and area can be used to identify doublets (Bio-Rad, flow cytometry guide).