Cell viability (MTT) and cell proliferation (BrdU) assays

MTT: The cell viability assay was performed using a MTT reagent kit [3-(4, 5-dimethyl thiazolyl-2)-2, 5-diphenyltetrazolium bromide] according to the manufacturer’s instructions. Briefly, 10 μl MTT reagent (0.5 mg/ml) was added to 100 μl of medium bathing the cells in wells of tissue culture plates and the plates incubated for 4 h until a purple precipitate was visible. The medium was then removed and 100 μl of the detergent reagent was added into each well and incubation carried out in the dark for 2 h. Absorbance at 570 nm was spectrophotometrically measured using a microplate ELISA reader (μQuant, BioTek and Winooski, VT, USA) with a reference wavelength of 650 nm. BrdU: The cell proliferation assay was performed using a BrdU kit according to the manufacturer’s instructions (Cell Signaling, MA, USA). Briefly, the cells were incubated for 1–24 h with the BrdU solution followed by removal of the medium and incubation for 30 min with the fixing/denaturing solution. Subsequently, the cells were incubated for 1 h with BrdU detection antibody solution followed by three washes with the wash buffer. The HRP-conjugate solution was then added and incubated for 30 min followed by similar washing steps and incubation with the substrate solution for 30 min. The enzymatic reaction was finally stopped and the products quantified by measuring absorbance at 450 nm using a microplate ELISA reader.