Cell-cycle analysis

MDA-MB-231 cells were exposed to graded concentrations of F. carica leaf decoction (0, 4 mg/mL) for 48 or 72 hours and collected. Cell-cycle distribution was evaluated using the cell-cycle-detection kit according to the manufacturer’s instructions. Briefly, cells were washed twice with cold PBS and fixed in 70% ethanol at −20°C overnight. Cells were resuspended in cold PBS containing 1% BSA and 20 μL RNase incubated at 37°C for 30 minutes, and then stained with a propidium iodide solution at 4°C for 30 minutes. Cell cycles were analyzed by flow cytometry (FACScan; BD, Franklin Lakes, NJ, USA). Cell-cycle distribution was calculated using ModFit cell-cycle-analysis software (version 2.01.2; BD).