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Jurkat cells (T-cell leukemia,human) treated with 10 μM of camptothecin for 4 hours(panel right) or untreated control(panel left). 1. Wash cells twice with cold PBS and then resuspend cells in 1×Binding Buffer at a concentration of 1×106 cells/ml. 2. Transfer 100 μl of the solution (1×105cells) to a 5 ml culture tube. 3. Add 5 μl of Annexin V-FITC. 4. Add 5 μl PI. 5. Gently vortex the cells and incubate for 15 min at RT (25°C) in the dark. 6. Add 400 μl of 1×Binding Buffer to each tube.Analyze by flow cytometry within 1 hr.

Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for PS, and is useful for identifying apoptotic cells with exposed PS. Propidium Iodide (PI) is a standard flow cytometric viability probe and is used to distinguish viable from nonviable cells. Viable cells with intact membranes exclude PI, whereas the membranes of dead and damaged cells are permeable to PI. Cells that stain positive for FITC Annexin V and negative for PI are undergoing apoptosis. Cells that stain positive for both FITC Annexin V and PI are either in the end stage of apoptosis, are undergoing necrosis, or are already dead.