Annexin V/PI assay for apoptosis

Apoptotic MCF-7 and MDA-MB 231 cells were investigated using the Annexin V/propidium iodide (PI) apoptosis detection kit (Beyotime Institute of Biotechnology) followed by flow cytometry, according to the manufacturer's protocol. MCF-7 and MDA-MB 231 cells (5×10³ cells/well) were cultured in 6-well plates and treated with different concentrations (15, 30 and 60 µM) of Rosmanol for 48 h at 37°C. Cells were harvested by trypsinization with no EDTA and washed twice with PBS and then stained with 5 µl Annexin V-FITC (Beyotime Institute of Biotechnology) and 10 µl PI in 500 µl binding buffer for 15 min at room temperature (RT) in the dark. Apoptotic cells were determined by using Cytomics FC 500 flow cytometer (Beckman Coulter Inc.) and the data were analysed using Cellquest analysis software v.3.0 (BD Biosciences).