GSH was measured in serum using the GSH reductase enzyme method [22 (link)]. Briefly, the assay is based on the reaction of GSH (Sigma-Aldrich, St. Louis, MO, USA) with 5,5′-dithio-bis (2-nitrobenzoic acid) (DNTB, Sigma-Aldrich) to form 5-thio-2-nitrobenzoic acid (TNB), in the presence of β-NADPH (Sigma-Aldrich) and GSH reductase (Sigma-Aldrich). The absorbance at 412 nm was immediately measured at 30 s intervals for 2 min to compare with the GSH standard. The test is specific to GSH based on the specificity of the GSH reductase enzyme (Sigma-Aldrich) to GSH: the rate of accumulation of TNB is directly proportional to the concentration of GSH in the sample.
Glutathione (gsh)
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, China, Italy, Sao Tome and Principe, India, France, Spain, Japan, Macao, Poland, Canada, Australia, Morocco, Belgium, Thailand
GSH is a high-performance laboratory equipment designed for a variety of applications in research and development. It serves as a versatile tool for general laboratory tasks.
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Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (49 mentions)
Nadph, by Merck Group (47 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (42 mentions)
N acetyl cysteine (nac), by Merck Group (37 mentions)
Cysteine, by Merck Group (36 mentions)
Thiobarbituric acid, by Merck Group (36 mentions)
Glutathione reductase, by Merck Group (35 mentions)
Dithiothreitol (dtt), by Merck Group (35 mentions)
Ascorbic acid, by Merck Group (30 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (26 mentions)
Sodium hydroxide (naoh), by Merck Group (24 mentions)
Hydrogen peroxide, by Merck Group (24 mentions)
5 5 dithiobis 2 nitrobenzoic acid, by Merck Group (24 mentions)
Formic acid, by Merck Group (20 mentions)
Trichloroacetic acid, by Merck Group (19 mentions)
L cysteine, by Merck Group (19 mentions)
Sodium chloride (nacl), by Merck Group (18 mentions)
Hydrogen peroxide h2o2, by Merck Group (18 mentions)
1 chloro 2 4 dinitrobenzene, by Merck Group (16 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (16 mentions)
877 protocols using glutathione (gsh)
1
Glutathione Quantification in Serum
2
Natural Compounds Screening Protocol
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Eugenol, β-escin, curcumin, berberine hydrochloride, sclareol, capsaicin, parthenolide, ellagic acid, glutathione, L -ascorbic acid were from Sigma–Aldrich (United Kingdom); osthole and pterostilbene were from Stratech (United Kingdom) and mitoquinol from Cayman Chemical Company (United Kingdom); all other NPs were components of the Puretitre natural compound library from Caithness Biotechnologies (United Kingdom). All of the above except Eugenol (70% ethanol), glutathione, and L -ascorbic acid (dH2O) were dissolved in dimethyl sulfoxide (DMSO, Sigma United Kingdom) and added to growth media from the following stock solutions prepared in those solvents: Eugenol, 500 mM; glutathione, 375 mM, L -ascorbic acid, 500 mM; osthole, 200 mM; pterostilbene, 200 mM; β-escin, 50 mM; curcumin, 50 mM; berberine hydrochloride, 200 mM; sclareol, 130 mM; capsaicin, 200 mM; parthenolide, 130 mM; ellagic acid, 33.3 mM, mitoquinol, 2.94 mM.
3
Studying Oxidative Stress in Nematodes
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For GSH treatment, NGM plates containing 5 mM GSH (Sigma) were poured and allowed to dry. The respective E.coli food source was added to each plate and once dry, animals (young adult) were added to the plate and allowed to lay eggs. Nematodes grew for one generation on these plates prior to ROS analysis or GFP scoring. Inhibition of BLI-3 by DPI (Diphenyleneiodonium chloride, Sigma) was performed by treating animals with 80 µM of the compound in M9 solution for 15 min prior to analysis. Removal of mitoROS by the mitochondrial-targeted superoxide scavenger Mito-Tempo (Sigma) occurred by exposing animals to 250 µM of the antioxidant for 2 hr in M9 solution prior to ROS determination.
4
Chardonnay Bottle Aging with Glutathione
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Chardonnay wines from Burgundy were produced from grapes originating from the same vineyard during 2008 and 2009 vintages, following the same winemaking process at the same winery. GSH (Sigma Aldrich, France) spiking was done after alcoholic fermentation at 50 mg/L (50AF thereafter) or prior bottling at 20 and 50 mg/L (20B and 50B, respectively thereafter) and compared to the control wines with no GSH spiking. Wines were conditioned in standard commercial 75 cl green glass bottles and stored at 16°C until analysis. In order to enhance differences in oxygen exposure, samples were sealed either with synthetic coextruded stoppers (S) or screw caps (C) allowing high and low oxygen ingress, respectively during aging. Wines were analyzed in April 2016 (i.e., after 7 and 6 years of bottle aging) in biological duplicates.
5
Glutathione Quantification Protocol
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The treated cells were washed and harvested in extraction buffer (0.1 Trition-X 100 and 0.6% sulfosalicylic acid in 0.1 M potassium phosphate buffer, pH 7.5), homogenized, and centrifuged at 3000 g for 10 min at 4 °C. The supernatant of the samples was incubated with Ellman's reagent (Sigma Aldrich, India) and glutathione reductase (Sigma Aldrich, India) for 10 min to allow for the conversion of glutathione disulfide to glutathione (GSSG to GSH). The sample was later incubated with β-NADPH2 (Sigma Aldrich, India) for 1 min. The absorbances of the samples were read at 412 nm in a microplate reader using a kinetic program which monitored the reaction at 1 min interval for 10 min [23 ]. The concentration of total glutathione (tGSH) was determined from the samples using the standard curve of GSH (Sigma Aldrich, India). The values were expressed as tGSH μM/mg of protein.
6
Glutathione Quantification in Tissue
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Briefly, 100 µl of tissue homogenate was mixed with 100 µl of 10% trichloro acetic acid and vortexed. The contents were then centrifuged at 5000 rpm for 10 min. Subsequently 0.05 ml of supernatant was mixed with a reaction mixture containing 3.0 ml 0.3 M phosphate buffer (pH 8.4) and 0.5 ml DTNB [5, 5′ dithiobis-(2-nitrobenzoic acid)]. Within 10 min, the absorbance was measured at 412 nm using a spectrophotometer. GSH was determined from a standard curve produced using commercially available standard GSH (Sigma Chemicals, USA). The amount was expressed as µg/mg of protein30 (link).
7
Investigating GFPT2 Expression in MDA-MB-231 Cells
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The MDA-MB-231 cells were seeded in 24-well plates at 300,000 cells/well and cultured for 48 h followed by treatment with 2 μM hydrogen peroxide (H2O2, Honeywell, 18304H) for 2 h. GFPT2 gene expression was tested by RT-qPCR.
The MDA-MB-231 cells were seeded in 24-well plates at 200,000 cells/well and cultured for 24 h, followed by treatment with 50 mg/l reduced glutathione (GSH, Sigma, G4251) for 48 h. Cells were changed with fresh GSH medium 2 h before the RNA extraction. GFPT2 gene expression was tested by RT-qPCR.
The MDA-MB-231 cells were cultured in the H14 medium as described for the D492 cell lines, then seeded in 24-well plates at 200,000 cells/well and cultured for 24 h followed by treatments with medium deprived of insulin or EGF for 48 h. Fresh medium was changed for the cells 2 h before the RNA extraction. GFPT2 gene expression was tested by RT-qPCR.
The MDA-MB-231 cells were seeded in 24-well plates at 200,000 cells/well and cultured for 24 h, followed by treatment with 50 mg/l reduced glutathione (GSH, Sigma, G4251) for 48 h. Cells were changed with fresh GSH medium 2 h before the RNA extraction. GFPT2 gene expression was tested by RT-qPCR.
The MDA-MB-231 cells were cultured in the H14 medium as described for the D492 cell lines, then seeded in 24-well plates at 200,000 cells/well and cultured for 24 h followed by treatments with medium deprived of insulin or EGF for 48 h. Fresh medium was changed for the cells 2 h before the RNA extraction. GFPT2 gene expression was tested by RT-qPCR.
8
Comprehensive Blood Oxidative Analysis
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Four hundred microliters of whole blood was taken for evaluating GSH. The residual plasma was separated by centrifugation at 6,000 rpm for 10 minutes and immediately assayed for TAC. Residual plasma was separated to determine MDA, PrOOH, Vit C, Vit A, and Vit E. GSH was determined by following the previous spectrophotometry protocol of Leelarungrayub et al.21 The GSH concentration was calculated by comparing with the absorbance of standard GSH (Sigma-Aldrich Co., St Louis, MO, USA), and finally presented as milligrams in 1 g of hemoglobin (mg/g Hb) from CBC analysis.
TAC of fresh plasma was assayed following the 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid decolorization method,22 (link) by spectrophotometry. TAC was represented as millimoles of standard Trolox per liter (mmol Trolox/L).
Plasma MDA from lipid oxidation was evaluated by following a previous method with the protocol of high performance liquid chromatography (HPLC)23 (link) under the thiobarbituric acid reactive substances test. The plasma MDA (µmol/L) was calculated by comparing with the standard tetramethoxypropane (Sigma-Aldrich Co.).
Plasma PrOOH was evaluated by following the previous ferrous oxidation xylenol-orange protocol.24 (link) The PrOOH (µmol/L) was determined by spectrophotometry at 560 nm, and its yield was compared to the standard tert-butylhydroperoxide (Sigma-Aldrich Co.).
TAC of fresh plasma was assayed following the 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid decolorization method,22 (link) by spectrophotometry. TAC was represented as millimoles of standard Trolox per liter (mmol Trolox/L).
Plasma MDA from lipid oxidation was evaluated by following a previous method with the protocol of high performance liquid chromatography (HPLC)23 (link) under the thiobarbituric acid reactive substances test. The plasma MDA (µmol/L) was calculated by comparing with the standard tetramethoxypropane (Sigma-Aldrich Co.).
Plasma PrOOH was evaluated by following the previous ferrous oxidation xylenol-orange protocol.24 (link) The PrOOH (µmol/L) was determined by spectrophotometry at 560 nm, and its yield was compared to the standard tert-butylhydroperoxide (Sigma-Aldrich Co.).
9
Stability of GSH in DMSO and Water
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Two 1 mM GSH (Sigma-Aldrich, Darmstadt, Germany) solutions were prepared: (A) in 10% (v/v) DMSO; (B) in pure water. The samples were left at room temperature for 48 h. The first measurement for both solutions was performed immediately, at time t = 0 min (the controls). Further measurements were recorded after 1, 3, 5, 7, 24, and 48 h. The determination of the GSH level is based on Ellman’s method [55 (link)], in which 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB, Sigma-Aldrich, Darmstadt, Germany) is reduced by -SH groups present in the sample, resulting in a product with an intense yellow color. The reaction mixture consisted of 850 µL of 0.2 M phosphate buffer, pH 8.2, 100 µL of 6 mM DTNB, and 50 µL of GSH solutions (A or B), respectively. Absorbance was measured spectrophotometrically against a blank containing DTNB and buffer at 412 nm. The total content of GSH was calculated from a standard curve prepared for 1 mM GSH. The results obtained for the DMSO-containing group were normalized to the GSH concentration determined in the non-DMSO-treated group and expressed as a percentage relative to the control (t = 0, 100%).
10
Intracellular GSH Quantification Protocol
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Intracellular GSH changes were measured using Ellman’s reagent (DTNB; Sigma-Aldrich, St. Louis, MO, USA) employing the protocol proposed by Tan et al. [81 (link)]. The absorbance was measured at 405 nm using a microplate reader (BioSan, Riga, Latvia). The concentration of free thiols in the samples was calculated using a GSH (Sigma-Aldrich, St. Louis, MO, USA) standard curve.
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