Glutathione gssg gsh detection kit

H₂O₂ was measured using OxiSelect Hydrogen Peroxide/Peroxidase Assay Kit (Cell Biolabs), following manufacturer’s instructions. Protein oxidation was measured by flow cytometry using the FlowCellect oxidative stress kit (Sigma/Merck), following manufacturer’s instructions. For glutathione quantification, cells were FACS-sorted from spleen and LN of C57Bl/6J or B6.TCRα^−/− tumor-free mice and lysed in 5% metaphosphoric acid at a concentration of 2 × 10⁶ cells per mL (for smaller cell numbers, the volume was adjusted accordingly). Glutathione (GSSG/GSH) detection kit (Enzo Life Sciences) was used to quantify total glutathione according to manufacturer’s instructions.

Measurement of oxidized glutathione (GSSG) and total glutathione (tGSH) are based on enzymatic recycling method for quantification of glutathione; GR reduces oxidized glutathione (GSSG) to reduced glutathione (GSH). The sulfhydryl group of GSH reacts with DTNB (5,5′-dithiobis-2-nitrobenzoic acid) to produce a yellow-colored 5-thio-2-nitrobenzoic acid (TNB) that absorbs at 405 nm. The rate of TNB production is directly proportional to the concentration of glutathione in the sample. The ratio of GSSG to tGSH was measured using the glutathione (GSSG/GSH) detection kit (Enzo Life Sciences, Farmingdale, New York, USA) according to the manufacturer’s instruction. In brief, freshly isolated mitochondrial fractions were suspended in ice-cold 5% metaphosphoric acid (20 μL/mg tissue) and centrifuged at 12,000 × g for 15 min at 4 °C. Supernatants reacted to the freshly prepared reaction mix. The absorbances were detected at 405 nm every minute for 10 min. Determination of GSSG was the same protocol with GSH assay with an exception in which mitochondrial fraction was suspended in 5% metaphosphoric acid containing 2 M 4-vinylpyridine.

Total GSH (tGSH) and GSSG levels were measured as described previously (Akerboom & Sies, 1981 (link)). Measurement of tGSH and GSSG is based on the enzymatic recycling method for quantification of glutathione; GR reduces oxidized glutathione (GSSG) to reduced glutathione (GSH). The sulfhydryl group of GSH reacts with DTNB (5,5′‐dithiobis‐2‐nitrobenzoic acid) to produce a yellow‐colored 5‐thio‐2‐nitrobenzoic acid (TNB) that absorbs at 405 nm. The rate of TNB production is directly proportional to the concentration of glutathione in the sample. The ratio of GSSG to tGSH was measured using the glutathione (GSSG/GSH) detection kit (Enzo Life Sciences, Farmingdale, New York, USA) according to the manufacturer's instructions. In brief, freshly isolated mitochondrial fractions were suspended in ice‐cold 5% metaphosphoric acid (20 μl/mg tissue) and centrifuged at 12,000 × g for 15 min at 4 °C. Supernatants reacted to the freshly prepared reaction mix. The absorbances were detected at 414 nm every minute for 10 min. Determination of GSSG was the same protocol with GSH assay with an exception in which mitochondrial fraction was suspended in 5% metaphosphoric acid containing 2 M 4‐vinyl pyridine.