Gsh gssg ratio detection assay kit fluorometric green

The Glutathione/Glutathione disulfide (GSH/GSSG) ratio was determined using the GSH/GSSG ratio detection assay kit (fluorometric - green) (#ab205811 from Abcam) and the transcriptional activity of Nrf2 was determined using the Nrf2 transcription factor assay kit (colorimetric) (#ab207223 from Abcam). Methylglyoxal (MG) formation was determined using the methylglyoxal assay kit (#ab241006 from Abcam) and 3-nitrotyrosine formation was measured using the 3-nitrotyrosine ELISA kit (#ab116691 from Abcam) according to the manufacturer’s instructions.

Intracellular GSH levels were quantified using the GSH/GSSG Ratio Detection Assay Kit (Fluorometric - Green) (abcam), following the manufacturer’s instructions. Briefly, 1 × 10⁶ cells were plated in a 60 mm plate and treated with the compound of interest. After 24 h, cells were collected and washed with cold PBS 1× and PBS 0.5% NP-40. After, samples were centrifugated and the supernatant was collected. We removed the enzymes from the samples by following the deproteinization protocol. Next, we prepared the Thiol Green Solution (1:50) and mixed it with the samples, after 30 min we measured the fluorescence (490/520 nm).

Reduced glutathione (GSH) levels and oxidised glutathione (GSSG) contents in experimental and normal lymphocytes were analysed using the GSH/GSSG Ratio Detection Assay Kit (Fluorometric—Green) (Abcam, UK) according to the manufacturer protocol. Briefly, isolated lymphocytes were harvested overnight in 6-well plate then treated with chemicals for 1 h. Cells were washed with cold PBS and re-suspended in 100 µl of cold lysis buffer supplemented with 10 µl of protease inhibitors then thoroughly mixed by pipetting and centrifuged at 400g for 5 min to remove insoluble. The supernatant (sample) was collected and kept on ice for further use. 50 µl of each GSH and GSSG standards (kit components) were added to 96-well plates in duplicates. For GSH detection, 50 µl of the GSH assay mixture (GAM) (provided with the kit) was added to each GSH standard and sample. For GSSG detection, total GSH assay mixture (TGAM) was added to each GSSG standard and sample. Incubated for 60 min at room temperature in the dark and then fluorescence was measured at 490/520 nm using Promega Glumax explorer version 2.4. Data were analysed using Graph Pad Prism 7.02.