Oxidized and reduced glutathione were measured using the Quantification kit for oxidized and reduced glutathione (38185, Sigma-Aldrich, Darmstadt, Germany) according to the manufacturer’s protocol. Briefly, 50 mg of tissue was homogenized in 0.2 mL of 5% SSA and centrifuged at 8000× g, 10 min, 4 °C. The supernatant was transferred to a new tube, and SSA concentration was reduced to 0.5% by the addition of ddH2O. Samples (40 µL) and GSH and GSSG standards were added into a microplate. For the determination of GSSG, only the “masking reagent” binding GSH was added to part of the samples. After 1 h incubation with buffer solution at 37 °C, the 20 µL of coenzyme solution and enzyme solution was added to each well. GSSG and total glutathione (GSH + GSSG) were detected spectrophotometrically at 415 nm. The GSH was quantified using the following equation: GSH = total glutathione − GSSG × 2.
Quantification kit for oxidized and reduced glutathione
Manufactured by Merck Group
Sourced in Germany, United States
The Quantification kit for oxidized and reduced glutathione is a laboratory product designed to measure the levels of reduced and oxidized glutathione in biological samples. It provides a reliable and efficient method for quantifying these important antioxidant molecules.
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12 protocols using quantification kit for oxidized and reduced glutathione
1
Spectrophotometric Quantification of Glutathione
2
Quantification of Oxidized and Reduced Glutathione
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Oxidized and reduced glutathione were measured using Quantification kit for oxidized and reduced glutathione (Sigma-Aldrich, Germany) according to the manufacturer’s protocol. Briefly, 100 mg of tissue were homogenized in 0.75 ml of 5 % SSA and centrifuged at 8000×g, 10 min, 4 °C. The supernatant was transferred to a new tube and SSA concentration was reduced to 0.5 % by addition of ddH2O. 40 μl of samples and GSH and GSSG standards were added into microplate. For determination of GSSG only the “masking reagent” binding GSH was added to part of the samples. After incubation with buffer solution 1 h, 37 °C the 20 μl of coenzyme solution and enzyme solution was added to each well. GSSG and total glutathione (GSH + GSSG) were determined detected spectrophotometrically at 415 nm. The GSH was quantified using following equation: GSH = total glutathione − GSSG × 2.
3
Glutathione Quantification in Mouse Liver
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Glutathione levels were measured from mouse hepatocytes in culture and irradiated liver tissue using the Quantification kit for oxidized and reduced glutathione (Sigma).
4
Mitochondrial Glutathione Quantification
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Mitochondrial glutathione levels were determined using the Quantification kit for oxidized and reduced glutathione (Sigma-Aldrich) based on the 5,5′-dithio-bis(2-nitro-benzoic acid; DTNB) assay. Briefly, Mitochondria were isolated as described previously. Mitochondrial pellets were resuspended in 10 mM HCl, and lysed by freezing and thawingtwice. 5-Sulfosalicylic acid was added to a final concentration of 0.5%. GSSG and GSH were determined following manufacture instructions. Color changes were monitored by absorbance at 405 nm. GSH and GSSG levels were quantified using standard curves.
5
Glutathione Quantification in Mice
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Plasma samples collected from mice were analyzed using the Quantification Kit for Oxidized and Reduced Glutathione (Sigma-Aldrich, St. Louis, MO, USA). Specifically, we assessed the levels of oxidized glutathione (GSSG) and reduced glutathione (GSH) according to the manufacturer’s guidelines. Fluorescence intensity was measured via a microplate reader (TECAN-Spark Cyto) at the wavelength of 490 nm for excitation and 520 nm for emission.
6
Quantification of Glutathione Levels
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For measurement of glutathione, cells were treated with 1.0 μM b-AP15 for 6 h. Cells were collected and concentrations of GSSG and total glutathione (GSH + GSSG) were analyzed using the quantification kit for oxidized and reduced glutathione (#38185, Sigma) as described. The final concentration of GSH was determined by equation of GSH = total glutathione (GSH + GSSG)–GSSG × 2. The data was analyzed using GraphPad Prism 7.
7
Quantification of Antioxidant Enzymes
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GSH was determined using a quantification kit for oxidized and reduced glutathione (Sigma-Aldrich, Darmstadt, Germany) according to manufacturer´s instructions. Activity of enzymes involved in the pentose phosphate pathway and oxidative defense: G6PD, GLD, and GPx was determined according to the methods recommended by the International Committee for Standardization in Haematology [60 (link)], as we previously described [61 (link),62 (link)]. Leukocyte- and platelet-free erythrocyte lysates were used, and the absorbance was measured by spectrophotometer (Infinite 200 Nanoquant; Tecan, Männedorf, Switzerland). All chemicals and purified enzymes were purchased from Sigma-Aldrich (Darmstadt, Germany).
8
Quantification of Glutathione Redox Status
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Tissue lysate was analyzed by Quantification Kit for Oxidized and Reduced Glutathione (Sigma Aldrich, St. Louis, MO, USA) according to the manufacturer’s guidelines. For the quantitative determination, samples were prepared by lysis of total cell protein in T-PER lysis buffer followed by dilution of 1:50 for glutathione (GSH) analysis. In brief, a serial dilution of reduced (GSH) and oxidized glutathione (GSSG) stock standards were prepared along with assay mixtures for detection of GSH and total GSH using 100 X Thiol green stock solutions, assay buffer, and GSSG probe. A one-step fluorometric reaction of the sample with the respective assay buffer was incubated for 30 min. Fluorescence intensity was assessed at Ex/Em of 490/520 nm. GSSG was determined by subtracting GSH from total GSH. Finally, GSH was plotted against GSSG to obtain the GSH/GSSG ratio.
9
Determination of Liver Glutathione Levels
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GSH levels were determined in liver tissue after homogenization in the TissueLyser II QIAGEN® equipment, Hilden, Germany. The ratio of oxidized glutathione, glutathione disulfide (GSSG), to GSH was determined using a commercial kit (Quantification kit for oxidized and reduced glutathione, Sigma Aldrich. St. Louis, MO, USA). Determinations were performed on a 96-well plate in an iMarkTM Microplate Absorbance Reader BIO-RAD. Hercules, CA, USA at 415 nm.
10
Glutathione Redox Status Quantification
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The analysis of cell lysate was performed using Quantification Kit for Oxidized and Reduced Glutathione (Sigma‐Aldrich). Specifically, we assessed the oxidized glutathione (GSSG) levels and reduced glutathione (GSH) according to the manufacturer's instruction. Fluorescence intensity was measured via a Bio‐Rad plate reader at specific excitation (Ex: 490 nm) and emission wavelengths (Em: 520 nm).
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