4.4. Determination of Apoptosis by Annexin V and Propidium Iodide (PI) Staining

Induction of apoptosis in OV90 and ES2 cells by eupatilin (0, 10, 25, 40, 50, and 100 μM) and knockdown of SERPINB11 with or without eupatilin (50 μM) was analyzed using the Fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit I (BD Biosciences, Franklin Lakes, NJ, USA). After treatment for 48 h at 37 °C in a CO₂ incubator was applied to the cells (5 × 10⁵ cells) seeded on 6-well plates, supernatants were removed from culture dishes and adherent cells detached with trypsin-EDTA. The cells were collected by centrifugation, washed with PBS, and resuspended using 1× binding buffer at 1 × 10⁶ cells/mL. Then, 100 μL of the cell suspension (1 × 10⁶ cells) was transferred to a 5 mL culture tube and incubated with 5 μL FITC Annexin V and 5 μL PI for 15 min at room temperature in the dark. Then, 400 μL of 1× binding buffer was added in a 5 mL culture tube. The fluorescence intensity was analyzed using the Guava^® easyCyte™ flow cytometer (Merck, Kenilworth, NJ, USA). The data represent three independent experiments.