Determination of the cell cycle distribution by flow cytometry and mitotic index

To analyze the cell cycle phase distribution, cells were washed with PBS and fixed with 70% ice‐cold ethanol at −20 °C for at least 2 h. The cells were then stained with propidium iodide from a Muse^® Cell cycle Assay Kit (Merck Millipore, Burlington, MA, USA), according to the manufacturer’s protocol. The cell cycle distribution was analyzed by flow cytometry using a Navios flow cytometer and kaluza analysis 2.1 software (Beckman Coulter, Inc., Brea, CA, USA). The mitotic index was calculated by a number of cells undergoing mitosis divided by a total number of cells, which were manually counted from fluorescence images at least 1000 cells.