Facsan flow cytometry

In brief, transfected cells were harvested, and washed with PBS (Invitrogen), followed by fixation with ethanol for 1 h. After measurement with FACSan flow cytometry (BD Biosciences) with 5 μL Annexin (V-fluorescein isothiocyanate) V-FITC/Propidium Iodide (PI) (Beyotime, Nantong, China), apoptosis cells were analyzed with Quest software (BD Biosciences). For cell cycle assay, after treatment with ethanol and PBS (Invitrogen), cells were added to RNaseA and PI for 30 min at room temperature. Finally, FACSan flow cytometry (BD Biosciences) and ModFit LT 3.2 software (Verity Software House, Topsham, ME, USA) were utilized to analyze the percentages of cells in the G0-G1 and S phases.

To detect apoptosis, transfected OVCAR3 or SKOV3 cells were harvested, washed twice, and resuspended in binding buffer. Then, the cells were stained using an Annexin V-fluorescein isothiocyanate Apoptosis Detection Kit (BD Biosciences, Franklin Lakes, NJ, USA), following which they were subjected to FACSan flow cytometry (BD Biosciences) to analyze apoptotic distribution.
To determine the distribution of cells in the different phases of the cell cycle, the treated OVCAR3 or SKOV3 cells were washed with PBS and fixed in 70% ice-cold ethanol at 4°C for 24 h. After treating with 0.5 mg/mL RNase A for 30 min at 37°C, cells were stained with propidium iodide for 30 min. The percentage of the cells in G0/G1, S, and G2/M phases was analyzed by FACSan flow cytometry (BD Biosciences) with FlowJo software (Tree Star Corp., Ashland, OR, USA).