Celltiter 96 non radioactive cell proliferation assay mtt reagent

In order to assess cell proliferation and viability, the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was performed as previously described (33 (link)–39 ). Briefly, subconfluent HCT116 cells were infected with AdBMP2 or AdGFP (MOI=20) for 16 h and seeded in 96-well plates (1,000 cells/well). The plated cells were incubated in DMEM supplied with 1% FBS. At the indicated time points, the cells were incubated with 10 μl of the CellTiter 96^® Non-Radioactive Cell Proliferation Assay (MTT) reagent (Promega, Madison, WI, USA) at 37°C for 4 h, followed by addition of 100 μl DMSO to dissolve the formazan products for 10 min at room temperature with gentle agitation. The absorbance was measured at 492 nm using a microtiter plate reader. Each assay condition was carried out in five replicates. The overall experiments were repeated at least in three batches of independent experiments.

The reference compound staurosporine was purchased from Sigma-Aldrich (Saint Louis, MO, USA). Compounds 2k and 2n were dissolved in DMSO as 50 mM stock. CellTiter 96^® Non-Radioactive Cell Proliferation Assay (MTT) reagent was purchased from Promega (Cat# G4000). The normal Vero cell line was purchased from ATCC. Normal Vero cells were cultured with EMEM medium plus 10% FBS. In total, 100 µg/mL penicillin and 100 µg/mL streptomycin were added to the culture media. Cultures were maintained at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air.