4.14. Annexin-V Assay

Cells were seeded in 25-cm² flasks and allowed to attach for 24 h before transfection. After 4 h post-transfection, cells were treated with erlotinib at a single concentration of 1 μg/mL for 6 h, and then, a washout was performed according to the experimental schedule. Cells were harvested, washed once in PBS, and incubated with 25-μg/mL Annexin V-FITC in a binding buffer (Bender MedSystems, Vienna, Austria) for 15 min at 37 °C in a humidified atmosphere in the dark. Cells were then washed in PBS and resuspended in binding buffer. Immediately before the flow cytometric analysis, propidium iodide was added to a final concentration of 5 mg/mL in order to distinguish between the total apoptotic cells and necrotic cells. Experiments were carried out three times, and for each sample, 10,000 events were recorded.