Reactive oxygen species (ROS) were determined in black 96-well microplates by labeling approximately 7×103 cells/well with 25 μM of 2’,7’-dichoroflurescein diacetate (DCFDA) for 30 min at 37°C in the dark; then, co-cultures were treated with ethanol for 48 hours followed by LPS for 3 h. Fluorescence intensity was measured at 535 nm (excitation at 485 nm). Glutathione (GSH) content was determined after alcohol and/or LPS treatments according to the GSH assay kit instructions (CS0260, Sigma, St. Louis, MO) [25 (link)].
Gsh assay kit
Manufactured by Merck Group
Sourced in United States, Germany
The GSH assay kit is a laboratory product designed to measure the concentration of glutathione (GSH) in biological samples. It provides a quantitative assessment of GSH levels, which is a key indicator of cellular oxidative status and antioxidant capacity.
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Lab products found in correlation
Mda assay kit, by Merck Group (3 mentions)
Colorimetry kits, by Cayman Chemical (2 mentions)
Tbars assay kit, by Cayman Chemical (2 mentions)
Iron assay kit, by Merck Group (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Infinite 200 pro, by Tecan (1 mentions)
Acetylcholinesterase activity colorimetric assay kit, by Abcam (1 mentions)
Multiskan fc microplate photometer, by Thermo Fisher Scientific (1 mentions)
Sod determination kit, by Merck Group (1 mentions)
Metaphosphoric acid, by Merck Group (1 mentions)
Sod assay kit, by Merck Group (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Mes buffer, by Merck Group (1 mentions)
Epoch 2, by Agilent Technologies (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Elisa plate reader, by Agilent Technologies (1 mentions)
Sod activity assay kit, by Beyotime (1 mentions)
Mda assay kit, by Beyotime (1 mentions)
Bovine erythrocyte sod, by Merck Group (1 mentions)
Lipid peroxidation colorimetric fluorometric assay kit, by Abcam (1 mentions)
28 protocols using gsh assay kit
1
Measuring ROS and Glutathione Levels
2
Measuring Glutathione in Xenopus Oocytes
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The total intracellular GSH concentration from Xenopus oocytes was determined by using the GSH assay kit from Sigma-Aldrich. For intracellular measurements, selected oocytes were kept in ND96 solution with low Ca2+ (0.25 mM) containing or not 20 mM BSO. After 48 and 72 h, the collected oocytes first were deproteinized with 5% 5-sulfosalicylic acid solution, followed by centrifugation to remove the precipitated protein, and then assayed for GSH as per the manufacturer’s instructions. For measuring extracellular GSH release, 30 oocytes per condition were incubated in 100 µl of low Ca2+ (0.25 mM) solution 1 d after injection or not with hCx26 mRNA. The extracellular medium was collected 6 and 24 h after incubation. The measurement of GSH was achieved by using a kinetic assay in which catalytic amounts (nmol) of GSH cause a continuous reduction of 5,5′-dithiobis(2-nitrobenzoic acid) to TNB, and the oxidized form of GSH (GSSG) formed is recycled by GSH reductase and NADPH. The GSSG present will also react to give a positive value in this reaction. The assay uses a standard curve of rGSH to determine the amount of GSH in the biological sample. A plate reader was set to 412 nm with kinetic read at 1-min intervals for 5 min to calculate the nanomoles of GSH in the sample, as suggested by the manufacturer.
3
Quantifying Glutathione in Zebrafish Embryos
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The embryo extracts were prepared in 5% 5-sulfosalicylic acid and subjected to GSH quantification using GSH assay kit according to manufacturers’ instruction (Sigma-Aldrich). Control and BMAA170 treated extracts (250 μL) from 96 hpf embryos were mixed with 250 μL of working solution which consist of reaction buffer, diluted GSH reductase (enzyme) and 5,5′-dithiobis-2-nitrobenzoic acid (DTNB). This reaction produces GSH disulfide (GSSH) and 5-thio-2-nitrobenzoic acid (TNB) which is a yellow product measured photometrically at 412 nm. Fifty microliters of this solution were added to the 96-well plates, and incubated at 37 °C for 10 minutes. After this step, 50 μL of diluted NADPH stock solution in reaction buffer were added to each well plate, and incubated again for 20 minutes at 37 °C. In the presence of NADPH, GSH reductase converts GSSG to GSH which is utilized in the previous reaction and produces even more TNB. Therefore, this recycling reaction improves the sensitivity of total GSH detection. The absorbance of each well was measured using a multiplate fluorimeter (Tecan Infinite 200 Pro, Männerdorf, Switzerland).
4
Evaluation of Oxidative Stress Biomarkers
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The lipid peroxidation assay kit for malondialdehyde evaluation (MDA, Lot no: MDA-2409, Product code: NWK-MDA01) was obtained from Northwest Life Science (Vancouver, WA, USA). The reduced glutathione assay kit (GSH Assay Kit, Lot no: 095M4114V, Product code: 1002170877) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The assay kit for superoxide dismutase (SOD, Batch no: 0538703, Item: 706002) was obtained from Cayman Chemical (Ann Arbor, MI, USA). The assay kit for catalase (CAT, Batch no: 0539007, Item: 707002) was purchased from Cayman Chemical (Ann Arbor, MI, USA). The acetylcholinesterase activity colorimetric assay kit (Lot no: GR 3295454-2, Product: ab65345) was purchased from BioVision (Milpitas, CA, USA) to assess AChE activity. All kits were utilized following the manufacturers’ instructions.
5
Glutathione Quantification Assay
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Total GSH was determined by using the GSH assay kit (Sigma). Briefly, larval pellets (50 mg) were homogenized in 1 volume of PBS. An aliquot was used for protein determination, and another aliquot was added to 3 volumes of 5% 5-sulfosalicylic acid and mixed. Samples were then frozen (−80°C) and thawed at 37°C twice, left for 5 min at 4°C and finally centrifuged at 10 000g for 10 min. The supernatant was used for glutathione determination by performing a kinetic assay in which catalytic amounts (nmoles) of GSH caused a continuous reduction of 5,5′-dithiobis(2-nitrobenzoic acid) to 5-thio-2-nitrobenzoic acid measured spectrophotometrically at 405 nm with a Thermo Scientific™ Multiskan™ FC Microplate Photometer.
6
Oxidative Stress Markers in Lung Tissue
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Lung tissues were homogenized, and malondialdehyde (MDA) activity was assayed using the MDA assay kit (Sigma) [16 (link)]. Furthermore, glutathione (GSH) levels and SOD activity were examined using a GSH assay kit and SOD determination kit (Sigma), respectively.
7
Quantifying Glutathione in CLL Cells
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GSH was quantified using a GSH assay kit, based on enzymatic recycling reactions. Following CLL cell culture under various experimental conditions, the cells were collected, sonicated at speed four (five times) at 4°C, and de-proteinated by precipitation with an equal volume of 10% metaphosphoric acid (Sigma-Aldrich). The precipitated proteins were removed by centrifugation at 3,000 × g for 5 min at 4°C. The supernatant was collected, neutralized with triethanolamine (cat. no. T58300; Sigma-Aldrich) and assayed for GSH (reduced) and GSSG (oxidized) using a Cayman GSH assay kit, according to the manufacturer's instructions. The data were obtained from triplicate measurements.
8
Quantification of Glutathione Levels
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For the quantification of GSH, a commercially available GSH assay kit, acquired from Sigma-Aldrich Chemie GmbH, Steinheim, Germany, was used in the present study. The samples were deproteinized with 5% 5-sulfosalicylic acid. The sample (10 μL) or standard and 150 μL of the working mixture were added to the assay buffer, 5,5′-dithiobis (2-nitrobenzoic acid), and GSH reductase. Then, 50 µL of NADPH in a 96-well plate was added to the reaction mixture for optimal color development, and five kinetic readings were recorded in 6 min at 412 nm. The GSH content was analyzed and expressed as μM of GSH.
9
Quantifying GSH and GSSG in Tumor Tissues
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For the detection of reduced glutathione and GSSG levels in tumor tissues and cell lysate, the GSH Assay Kit (Sigma-Aldrich) and GSSG assay kit (Sigma-Aldrich) were used following the manufacturer’s instructions. Details are provided in Supplementary Info, “Materials and Methods” section.
10
Quantification of Cellular Glutathione
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GSH content was analyzed using a commercially available GSH assay kit (Sigma-Aldrich Chemie GmbH, Stein Heim, Germany) in all experimental groups. Briefly, the samples were deproteinized using 5% 5-sulfosalicylic acid, and 10 μL of sample or standard was dispensed in a 96-well plate, followed by 150 μL of a working mixture consisting of assay buffer, 5,5′-dithiobis (2-nitrobenzoic acid), and GSH reductase. NADPH (50 µL) was added to the reaction mixture for optimal color development, and kinetic readings were recorded for 6 min at 412 nm. GSH content was calculated and expressed as micromolar GSH.
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