4.7. MTT Assay

The U87, U87-TxR and primary GBM cells were seeded into flat-bottomed 96-well cell culture plates (4000 cells per well) and incubated overnight in 100 µL of the appropriate medium. U87 and U87-TxR cells were treated for 24 h with increasing concentrations of Si306 and pro-Si306 (2.5, 5, and 10 µM) to assess cellular metabolic activity. Primary GBM cells were treated for 72 h with increasing concentrations of Si306, pro-Si306 and dasatinib (1, 2.5, 5, 10, and 25 µM). This colorimetric assay is based on the 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) enzymatic reduction into formazan dye by active mitochondria in viable cells, indicative of their metabolic activity. The insoluble formazan crystals are dissolved with a solubilization solution and the resulting purple-colored solution is quantified by measuring absorbance. MTT was purchased from Sigma-Aldrich Chemie GmbH, Germany. After treatment, 100 μL of MTT solution (2 mg/mL) was added to each well and the plates were incubated at 37 °C for 4 h. Subsequently, formazan product formed in cells with intact/viable mitochondria was dissolved in 200 µL of DMSO and absorbance was measured at 540 nm using an automated microplate reader (LKB 5060–006 Micro Plate Reader, LKB, Vienna, Austria). The IC₅₀ values for Si306, pro-Si306, and dasatinib were calculated by non-linear regression analysis using GraphPad Prism 6.0 software (GraphPad Software, La Jolla, CA, USA).