Apoptosis and Necrosis Assessment of Breast Cancer Cells

Annexin FITC-conjugated (1 : 500) and propidium iodide (PI) fluorochrome-labeled cells were used to determine the nature of cell death (apoptosis or necrosis). After platting MCF-7 and MDA-MB-231 cells (3.5 × 10⁵ cells/mL) in a 12-well plates, they were incubated with AVME at 10 and 20 µM for MDA-MB-231 cells and 11 and 22 µM for MCF-7, or solvent control (DMSO) for 24 h. Cells were then washed twice with cold PBS and suspended again in a buffer containing 10 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, and 1.8 mM CaCl₂. The pellets were stained on ice in the dark for 15 min with a fluorescent probe solution containing 50 mg/mL PI and 1 mg/mL Annexin. The total percentage of cells experiencing apoptosis was demarcated as the sum of both early and late facets of apoptosis (Annexin V-FITC positive), lower and upper right quadrants in the two-parameter dot plots, as previously described by Robles-Escajeda et al. [26 (link)]. The WinMDI 2.9 software and the flow cytometer BD FACSVerse (Becton Dickinson, Franklin Lakes, NJ, USA) were used for analysis. Each experiment was repeated thrice.