Gsh gssg detection assay kit

For measurement of the amount of GSH and GSSG in wild-type and Wobbler spinal cord (p40) the GSH/GSSG detection assay kit (#ab138881, Abcam, Cambridge, UK) was used according to manufacturer’s protocol. In brief, spinal cord tissue was lysed and homogenized in extraction buffer (#ab65349, Abcam, Cambridge, UK), centrifuged for 15 min at 4°C at 12,700 rpm and the supernatant was collected. For measurement of reduced glutathione (GSH) and total glutathione (GSH+GSSG), 50 μl of the samples were plated in duplicates in a 96-well plate suitable for fluorescence measurements. For GSH detection, 50 μl of GSH assay mixture were added to samples, for total glutathione (GSH+GSSG), 50 μl of total glutathione assay mixture were added to samples. After incubation at room temperature for 15 min protected from light, the fluorescence was measured at 490 nm excitation and 520 nm emission with a TriStar2 Multimode Reader LB 942 (Berthold Technologies GmbH and Co. KG, Bad Wildbad, Germany). Twelve animals per genotype were used.