Cell Cycle Analysis by Flow Cytometry

Cell cycle analyses were carried out as follows: after treatment, cells were collected by scraping and centrifuging, and the cell pellet was resuspended with phosphate-buffered saline (PBS). The cell suspension was fixed with 1.8 mL of ethanol (70%) at 4 °C, for 1 h. Then, cells were washed twice by centrifugation, using PBS, resuspending the pellet in 300 µL of PBS containing 0.25 mg/mL RNAse (Type I-A, Sigma-Aldrich, Darmstadt, Germany) and 0.1 mg/mL PI. Samples were incubated in the dark at room temperature for 30 min. The PI fluorescence of 10,000 events was analyzed using a FACS Canto II flow cytometer (BD Biosciences, San Jose, CA, USA), with excitation at 488 nm and detection of the emission at 610 nm. Data were analyzed using the software FlowJo v. 7.6.2 (FlowJo, Ashland, OR, USA) [59 ,60 (link)].