Flow cytometry apoptosis assay in Jurkat cells

ABT-737 is a small molecule drug that inhibits Bcl2, an apoptosis regulator. Jurkat cells were treated with a dilution series of ABT-737 and incubated for either 2, 4 or 6 hours, following which they were stained with Propidium Iodide and Annexin-V FITC for identification of apoptotic cells. Propidium Iodide is a dead cell marker, and Annexin-V binds to phosphatidylserine (exposed on outer membrane during apoptosis).

Figure 1: ABT-737 induces apoptosis in Jurkat cells. Following treatment with ABT-737, Jurkat cells were stained with Propidium Iodide and Annexin-V FITC and assessed by flow cytometry. Cells were initially gated based on FSC-A vs SSC-A, with doublets excluded using FSC-A vs FSC-H (data not shown). Cells were then examined using Annexin-V FITC vs Propidium Iodide (bottom panel), with a quadrant gate set using untreated and single stained controls. Apoptotic cells were classified as Annexin-V+Propidium Iodide– with live cells Annexin-V-Propidium Iodide–, with percentages plotted against ABT-737 concentration for generation of concentration-response curves (top panel).

Results shows that ABT-737 induces apoptosis in Jurkat cells, with a 4-hour incubation period determined as optimal for running the assay, as determined by the percentage of Jurkat cells identified as apoptotic.