Modification of an enzymatic glutathione assay for the microtiter plate and the determination of glutathione in rat primary cortical cells

A rapid and convenient microtiter-based method to determine total GSH is described and was used to assess the effect of human amylin on rat primary cortical cells in vitro. The method is a kinetic enzymatic recycling assay based on the oxidation of GSH by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). The assay was modified for a 96-well microtiter plate to pool addition of components in the reaction mixture and examine the impact of variations in the final concentration of the extractant sulfosalisylic acid. With this method GSH could be detected in freshly isolated and cultured rat primary cortical cells and could be determined in as few as 37.5 x 10(4) cells. Coincubation of the cultured cells with the amyloidogenic peptide human amylin resulted in a marked depletion of total GSH to 61% of controls. This microtiter method is rapid, reproducible, and sensitive where GSH is a desired endpoint in in vitro studies wherein hundreds of samples may be generated in a 96-well plate format.