Cell cycle assay using flow cytometry

The cells were collected and washed with PBS, fixed with 70% cold ethanol at 4°C for 2 hrs, and passed through 70‐μm Falcon Filters (BD Biosciences, Oxford, UK) to obtain a monodispersed cell suspension. The monodispersed cells were incubated with RNase A at 37°C for 30 min. and stained with PI at 4°C for 30 min. (Cell Cycle Detection Kit; BD, USA). Flow cytometric analysis was performed with a FACSCalibur flow cytometer (Becton Dickinson). Finally, the cell cycle distribution was analysed by Cell Quest software (BD, Franklin Lakes, NJ, USA).