Spectramax m5e microplate reader

The GSH levels in the mouse brain homogenate were measured using a GSH assay kit (Cayman Chemical, #703002, Ann Arbor, MI). Briefly, the mouse brain was homogenized per the manufacturer’s protocol followed by deproteinization with metaphosphoric acid. After neutralization, the samples were diluted with 50 mM MES buffer (pH = 6.0, containing 1 mM EDTA) before being analyzed in this enzymatic GSH recycling assay using DTNB (5,5′-dithiobis-2-nitrobenzoic acid). The yellow-colored 5-thio-2-nitrobenzoic acid (TNB) product formed was measured colorimetrically at 412 nm using a Molecular Devices SpectraMax M5e Microplate reader.

Cell viability was determined to examine the effect of drug treatments on U87, U87-R, U138, LN229, and SW1088 cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Cells were seeded in 200 μL medium at 5000 cells/well in 96-well plate and incubated at 37 °C and 5% CO₂ overnight. Next, the cells were incubated with drugs at designated concentrations for 48 hr. The concentration at which cellular viability decreased by half was noted as the CC₅₀ concentration for the rest of the study. At the end of the treatment, 20 μL of MTT solution (5 mg/mL) was added to each well and the plates were further incubated at 37 °C and 5% CO₂ for 3 hr and then media was aspirated and DMSO (100 μL/well) was added to dissolve the formazan crystals. The absorbance of the solution was measured at 570 nm using SpectraMax M5e microplate reader (Molecular Devices, USA). CC₅₀ values were calculated using GraphPad Prism 9 software by running log (inhibitor) vs normalized response on designated concentrations of drugs. Graphs with % viability vs log of concentration were plotted on GraphPad Prism 9.