Flow cytometric analysis of the cell cycle

For the cell-cycle analysis, a total number of 5×10⁶ breast cancer cells were harvested after a 48h incubation and then washed with cold PBS. The cells were then fixed with 80% ice-cold ethanol at −20°C overnight. After incubation with PBS containing 10mg/ml propidium iodide and 0.5mg/ml RNase A for 15min at 37°C, fixed cells were washed with cold PBS three times. A FACS caliber flow cytometer (BD, Biosciences) was used to measure the DNA content of labeled cells.