Apoptosis assay

Cultured MDA-MB-231 cells were treated grown to a density of 1x10⁵ cells and treated with different concentrations of metformin for 24 h. After treatment, cells were centrifuged at 500Xg for 5 minutes and the pellet was washed with 1X PBS. Cells in the pellet were then suspended in 1X Annexin binding buffer and stained with fluorescein isothiocyanate [FITC]-conjugated annexin V and propidium iodide [PI] for 30 min at room temperature in the dark following the manufacturer’s protocol (TACS Annexin V-FITC Apoptosis Detection Kit, R&D Systems, 4830-01-K) [20]. After incubation cells were analyzed for apoptotic and necrotic population using BD FACSCalibur (BD Biosciences). The normal healthy cells, early apoptosis, late apoptosis and necrotic populations were represented by annexin V-negative/PI-negative population, annexin V-positive/PI-negative, annexin V-positive/PI-positive and annexin-negative/PI-positive cells, respectively. The data were analysed using Cell Quest program from Becton-Dickinson [21].