2.3. MTT Cell Viability Assay

The MTT-based colorimetric assay for the nonradioactive quantification of cell viability was performed as previously described [27]. This assay is especially suitable as a sensitive and reliable indicator of the cellular metabolic activity, and it is therefore useful to assess the effects of cell treatments in short time intervals (i.e., 1–3 days). Specifically, it relies on the reduction of the water-soluble tetrazolium dye MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) into an insoluble colored formazan product primarily by the mitochondrial dehydrogenases of viable cells, thus evaluating mainly the metabolic state/viability of all cells in culture by virtue of a linear relationship between cell activity/viability and absorbance of the formazan product. Briefly, cells were seeded at the density of 2500 cells/well in 96 multiwell plates in RPMI-1640 medium supplemented with 10% serum. After adhesion, cells were treated with either Avn-A, Avn-C, YAvnI or YAvnII (50, 100, 200 μM) for 68 h. The medium was then removed, and cells were incubated for 4 h with fresh medium in the presence of 1.2 mM MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich, Milano, Italy), which is cleaved by viable cells to generate the corresponding pigmented formazan product, whose amount directly correlates to the number of metabolically active cells in the culture. Afterward, the medium was removed, 100 microliter of DMSO was added to each well to dissolve the blue formazan crystals, and the plates were incubated at 37 °C for 10 min. The absorbance of the formazan dye was then measured at 570 nm with a microplate reader (VersaMax Tunable Microplate Reader, Molecular Devices, San Jose, CA, USA), and used to calculate cell viability. Data were expressed as a percentage of the basal control.