2.4. Apoptotic cell death assay

To conduct an apoptosis assay, MCF-7 and MDA-MB-231 cell lines were grown on 6-well growth plates in medium containing various concentrations genistein, 1,25(OH)₂D₃, and genistein+1,25(OH)₂D₃ as described above for 48 h. Then, the cells were trypsinized, centrifuged at 3000 rpm for 5 min and rinsed twice with phosphate-buffered saline (PBS). The supernatant was discarded, and the cells were resuspended in 200 μl of annexin V-FITC binding buffer. Then, 5 μl of Annexin-V FITC was added in the dark, followed by incubation for 15 min at room temperature and then for 30 min at 4 °C. Cells were centrifuged for 5 min at 3000 rpm and then resuspended in 200 μl of annexin V-FITC binding buffer. Ten microlitres of propidium iodide (PI) was added in a cold bath, and then flow cytometry analysis was performed [18,19].