2.7. Annexin V FITC and Propidium Iodide (PI) Apoptosis Detection Assay

Annexin V and propidium iodide staining were conducted employing an annexin V FITC/PI apoptosis detection assay kit (Invitrogen, Carlsbad, CA, USA). Cells were initially plated in 6-well dishes at a density of 5 × 10⁵ cells/well. OPT-0139 was introduced at concentrations of 0, 1, and 10 µM, and the cells were subsequently incubated for an additional 48 h. Following trypsinization for cell detachment, the cells were harvested through centrifugation, washed with 1 mL of PBS, stained in accordance with the manufacturer’s instructions, and subjected to analysis using a flow cytometer (FACSCalibur and BD CellQuest Pro™ software version 5.0, BD Bioscience, San Jose, CA, USA).