The serum glutathione (GSH) content was assessed by an EZ-Glutathione Assay Kit (DoGen Bio Co., Ltd., Seoul, Korea). Glutathione peroxidase (GPx) and glutathione reductase (GRed) were determined using the glutathione peroxidase cellular activity assay kit and glutathione reductase assay kit, respectively (Sigma-Aldrich, St. Louis, MO, USA). The above assays were performed strictly following the manufacturer’s instructions.
Glutathione reductase assay kit
Manufactured by Merck Group
Sourced in United States, Germany
The Glutathione Reductase Assay Kit is a laboratory equipment designed to measure the activity of the enzyme glutathione reductase. Glutathione reductase plays a crucial role in maintaining the cellular redox balance by catalyzing the reduction of oxidized glutathione (GSSG) to its reduced form (GSH). The kit provides the necessary reagents and protocols to quantify the enzymatic activity of glutathione reductase in biological samples.
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Lab products found in correlation
Glutathione peroxidase cellular activity assay kit, by Merck Group (3 mentions)
Ez glutathione assay kit, by DoGenBio (1 mentions)
Glutathione s transferase assay kit, by Merck Group (1 mentions)
Sod determination kit, by Merck Group (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Versamax tunable microplate reader, by Molecular Devices (1 mentions)
Glutathione assay kit, by Merck Group (1 mentions)
Synergy h4, by Agilent Technologies (1 mentions)
Microplate reader, by Molecular Devices (1 mentions)
11 protocols using glutathione reductase assay kit
1
Assessing Glutathione and Antioxidant Enzymes
2
Evaluating Antioxidant Enzyme Activity
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Both floating and adherent A549 cells were harvested at 24, 48, and 72 h after treatment with compound 49 (20 µM). SOD determination kit, Glutathione Peroxidase Cellular Activity Assay kit, Glutathione Reductase Assay Kit and Glutathione-S-transferase (GST) Assay Kit (all Sigma Aldrich, Germany) were used for the detection of relevant enzyme activity. Reduced glutathione (GSH) content was measured by the method originally described by Floreani et al. [53 (link)]. Assays were performed on an M 501 single beam UV/VIS spectrophotometer (Spectronic Camspec Ltd., Leeds, United Kingdom). All measured parameters were calculated per milligram or gram of protein determined using the bicinchoninic acid assay.
3
Antioxidant Enzyme Activity Assays
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Glutathione reductase was measured by monitoring the oxidation of NADPH at 340 nm in presence of oxidized glutathione. The glutathione reductase activity is expressed as nmol/min/g lung according to the glutathione reductase assay kit (Sigma-Aldrich, St. Louis, MO, USA) instructions.
Glutathione peroxidase was measured by monitoring the oxidation of NADPH at 340 nm according to the method of Paglia and Valentine [17 (link)]. The glutathione peroxidase activity is expressed as nmol/min/g lung.
Catalase (CAT) activity was determined spectrophotometrically according to the method of Higgins et al. [18 (link)], namely, via the assay of hydrogen peroxide (H2O2). Catalase activity is expressed as μmol/min/g lung using a molar absorbance of 43.6 for H2O2.
The superoxide dismutase assay was a slight modification of the indirect inhibition assay developed by McCord and Fridovich [19 (link)]. Superoxide dismutase was measured by monitoring the decrease of the rate of detector 2(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride (INT) reaction with superoxide anion. The superoxide dismutase activity was measured at 505 nm and is expressed as U/g tissue.
Glutathione peroxidase was measured by monitoring the oxidation of NADPH at 340 nm according to the method of Paglia and Valentine [17 (link)]. The glutathione peroxidase activity is expressed as nmol/min/g lung.
Catalase (CAT) activity was determined spectrophotometrically according to the method of Higgins et al. [18 (link)], namely, via the assay of hydrogen peroxide (H2O2). Catalase activity is expressed as μmol/min/g lung using a molar absorbance of 43.6 for H2O2.
The superoxide dismutase assay was a slight modification of the indirect inhibition assay developed by McCord and Fridovich [19 (link)]. Superoxide dismutase was measured by monitoring the decrease of the rate of detector 2(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride (INT) reaction with superoxide anion. The superoxide dismutase activity was measured at 505 nm and is expressed as U/g tissue.
4
Glutathione Reductase Activity Assay
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GR activity was measured using the glutathione reductase assay kit from Sigma-Aldrich, which is a colorimetric assay monitoring the reduction of DTNB. 100 μg of CL1-0 and CL1-0ΔGR cell extracts was used directly in this assay. One unit of enzyme activity caused the reduction of 1 μmol of DTNB to TNB at 25°Cat pH 7.5.
5
Hepatic Glutathione Quantification Protocol
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The hepatic glutathione (GSH) content was evaluated according to previous method [28 (link)]. Briefly, 50 μL of each liver tissue sample was mixed with 80 μL of a mixture solution of DTNB/NADPH (1:7 (v/v); 4 mM DTNB and 0.3 mM NADPH). After 0.06 U of GSH reductase solution was added, the absorbance was measured at 405 nm. Glutathione peroxidase (GSH-px) and glutathione reductase (GSH-red) were determined using the glutathione peroxidase cellular activity assay kit and glutathione reductase assay kit, respectively (Sigma, St. Louis, MO, USA). The above assays were performed strictly following the manufacturer’s instructions.
6
Measuring Glutathione Reductase Activity
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Glutathione reductase activity was measured with a Glutathione Reductase Assay Kit (Sigma–Aldrich, Darmstadt, Germany, Cat. No. GRSA). The activity was measured by the increase in absorbance caused by the reduction of DTNB [5,5”-dithiobis(2-nitrobenoic acid)] at 412 nm.
7
Enzymatic Activities in Inner Ear
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Mitochondrial and cytosolic fractions from two inner ears (cochlea and vestibuli) per experimental group were isolated as described [25 (link)]. Glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (PGD) activity were measured in cytosolic fractions [37 (link)], and glutathione reductase (GSR) activity was measured in both cytosolic and mitochondrial fractions using the Glutathione Reductase Assay Kit (Sigma-Aldrich, Madrid, Spain). All spectrophotometric measurements were performed at least in triplicate in a 96-well format in a VERSA max Tunable Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). Protein concentrations were determined using the DC Protein Assay kit (Bio-Rad Laboratories, Hercules, CA, USA ).
8
Quantification of Glutathione Metabolism
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All parameters were measured in hippocampal homogenates using commercial kits purchased from Sigma-Aldrich (Munich, Germany)—Glutathione Assay Kit (S0260), Glutathione Reductase Assay Kit (GRSA), and Glutathione Peroxidase Assay Kit (CGP1) according to the manufacturer’s instructions. Changes in absorbance were measured using a BioTek Synergy H4 hybrid microplate reader (Agilent Technologies, Santa Clara, CA, USA).
9
Glutathione Reductase Activity Assay
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Glutathione reductase activity was measured using the Glutathione Reductase Assay kit (Sigma-Aldrich) according to the manufacturer’s instructions. In brief, 20 μl of cytosolic lysate was added to a well in the 96 well plate and then 180 μl of mixture containing 50 μl of 1 mM GSSG, 20 μl of assay buffer, 50 μl of 0.75 mM DTNB, and 60 μl of 0.1 mM NADPH was added to the well. The Absorbance was read at 405 nm every 10 s for 2 min in a spectrometer (Bio-Tek) to calculate the activity. All samples were run in duplicate.
10
Glutathione Reductase Activity Assay
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The activity of GSR was measured in cytosolic or mitochondrial fractions using the Glutathione Reductase Assay Kit (Sigma-Aldrich, St. Louis, MO), according to the manufacturer’s instructions. The absorbance was read at 405 nm every 10 s for 2 min in a microplate reader (Molecular Devices, Sunnyvale, CA) to calculate the activity. All samples were run in duplicate.
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