4.10. Cell Proliferation Assay (MTT)

Cell proliferation was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT, M6494; Sigma-Aldrich). Cells were seeded at a density of 5 × 10³ cells per well in 96-well plates in supplemented DMEM medium. After culturing for 6, 24, 48, and 72 h, respectively, 10 µL of MTT in PBS solution (5 mg/mL) was added to each well and cells were incubated at +37 °C for 2 h in the dark. One hundred microliters of lysis buffer (20% SDS, 10 mM HCl) were then added to each well and cells were incubated overnight. The absorbance of the MTT reduction product formazan was measured at 540 nm using VICTOR^TM plate reader (Perkin Elmer, Waltham, MA, USA).