4.8. Annexin V/PI Assay

HepG2 cells were seeded in the 6-well plates (1 × 10⁶ cells/well). After overnight incubation, we treated cells with indicated concentrations of YLE for 24 h. Cells were washed with PBS and harvested using a centrifuge at 2000 rpm for 3 min for the following analysis. To distinguish apoptotic and necrotic cell death in HepG2 cells, we used the BD Annexin V: FITC Apoptosis Detection Kit I (BD Biosciences, San Diego, CA, USA), according to the manufacture’s direction. The cells were analyzed by flow cytometry (BD FACSCalibur, San Jose, CA). Signals were detected in the FL1 channel (for Annexin V) and FL3 channel (for PI), while data were analyzed with Cell Quest Pro software.