3.7. Apoptosis Assay

Apoptotic activity was assessed using the Annexin V-FITC/PI staining kit (P-CA-201, Procell, Wuhan, China), containing Annexin V-FITC, propidium iodide (PI), and Annexin V Binding Buffer. MDA-MB-231 cells were plated at a density of 5 × 10⁵ cells per well in 6-well plates. Following treatment with the drug, the cells underwent two PBS washes, followed by harvesting and centrifugation at 300× g for 5 min. Subsequently, the cells were exposed to 500 μL of 1×Annexin V-FITC Buffer along with 5 μL of Annexin-V and 5 μL of PI for 20 min at room temperature in the absence of light. Annexin staining was quantified using a flow cytometer (CytoFLEX S, Beckman, CA, USA).