4.8. Induction of Apoptosis In Vitro

HeLa cells (1 × 10⁶) were cultured in 35 mm dishes and incubated at 37 °C for 24 h. After adding EA at concentrations of 12.5, 25, and 50 μg/mL, the cells were incubated for 48 h (each concentration was repeated three times). DMSO (0.1%) was used as a control. The treated cells were washed, trypsinized (without EDTA (ethylene diamine tetraacetic acid), and centrifuged. Next, the cells were collected and resuspended in 500 μL of buffer solution loaded with 5 μL of Annexin V-FITC and 5 μL propidium iodide (PI), the cells were incubated for 5–15 min in the dark. Flow cytometry analysis was conducted with a single 488 nm argon laser and 80,000 events with a BD Accuri C6 flow cytometer and software (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) [54].