Mtt reagent assay

Cell viability was assessed in 96-well plates (BD Falcon) by using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT reagent) assay (Promega, Madison, WI, USA) [39 (link),40 (link)]. Yellow MTT reagent enters the cells and passes into the mitochondria where mitochondrial dehydrogenases of viable cells cleave the tetrazolium ring, yielding reduced purple MTT formazan crystals, which are insoluble in aqueous solutions. This reduction occurs only when mitochondrial enzymes are active, and therefore conversion can be directly related to the number of viable cells. The formazan crystals can be dissolved in acidified isopropanol. The resulting purple solution is spectrophotometrically measured at 570 nm. An increase in cell number results in a large amount of MTT formazan formed and an increase in absorbance at 570 nm. After 24 h of RES treatment, 20 µL of MTT solution (2 mg/mL) in medium M199 were added to the cells and incubated at 37 °C in a cell culture incubator for 2 h. At the end of the incubation period, the solution was removed, and the purple formazan product was solubilized with acidic isopropanol (0.04 N HCl in absolute isopropanol). Then, plates were analyzed at 570 nm using a GENios plus micro-plate reader (Tecan). Results were calculated as the means ± SD of five measurements and expressed as a percentage of untreated control cells.

Cell viability was assessed in 96-well plates (BD Falcon) by using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT reagent) assay (Promega, Madison, WI, USA) [51 (link),62 (link)]. Yellow MTT reagent enters the cells and passes into the mitochondria where mitochondrial dehydrogenases of viable cells cleave the tetrazolium ring, yielding a reduced purple MTT formazan crystals which are insoluble in aqueous solutions. This reduction occurs only when mitochondrial enzymes are active, and therefore conversion can be directly related to the number of viable cells. The formazan crystals can be dissolved in acidified isopropanol. The resulting purple solution is spectrophotometrically measured at 570 nm. An increase in cell number results in a large amount of MTT formazan formed and an increase in absorbance at 570 nm. After 24 h of RES treatment, 20 µl of MTT solution (2 mg/mL) in medium M199 were added to the cells and incubated at 37 °C in a cell culture incubator for 2 h. At the end of the incubation period, the solution was removed, and the purple formazan product was solubilized with acidic isopropanol (0.04 N HCl in absolute isopropanol). Then plates were analyzed at 570 nm using a GENios plus micro-plate reader (Tecan). Results are expressed as a percent of untreated control cells.