Detection of apoptosis and necrosis by flow cytometry

Cells were harvested by trypsinization, pooled with the culture medium containing floating cells, collected by centrifugation, and incubated with the Annexin V-FITC conjugate and propidium iodide (PI) for 10 minutes using an apoptosis detection kit (APOAF, Sigma-Aldrich), then analyzed by flow cytometry. The percentage of apoptosis and necrosis was evaluated by plotting cell staining by Annexin V (x-axis) vs PI (y-axis). The upper left quadrant (PI positive and Annexin V negative, Q1) represents necrotic/non-viable cells; the upper right quadrant (PI positive and Annexin V positive, Q2) represents necrotic/late apoptotic cells; the lower left quadrant (PI and Annexin V negative, Q3) represents live cells; and the lower right quadrant (PI negative and Annexin V positive, Q4) represents early apoptosis.