*4.6. Flow Cytometry and Cell Proliferation Assays*

Cells were trypsinized and washed twice with PBS prior to being fixed with 80% ethanol for 30 min, stained for DNA with 1 μg/mL DAPI (Sigma-Aldrich) for 20 min and transferred to flow cytometry tubes for cell cycle analysis in a BD LSR-Fortessa flow cytometer (BD Biosciences, San Jose, CA, USA), using the ModFit LT software (Verity Software House, Topsham, ME). For proliferation assays, cells were harvested and plated in triplicate onto 12-well microplates at (Corning, Costar), at a density of <sup>1</sup> <sup>×</sup> <sup>10</sup><sup>3</sup> cells/mL. Cell proliferation was assessed for 10 days using the MTT [3-(4,5-dimethylthiazole)- 2-5-diphenyl tetrazolium bromide] commercial kit (Sigma-Aldrich) and following the manufacturer's instructions. Absorbance was measured at 570 nm on a Molecular Devices Spectra Max Plus384 microplate reader (Molecular Devices, Sunnyvale, CA, USA).
