Total glutathione (GSH) is a cellular antioxidant, which is depleted when excessive reactive oxygen species (ROS) are produced. Therefore, GSH depletion was measured as an indicator of oxidative stress [32 (link)] using a GSH detection kit (Enzo life sciences, Belgium). After 24 h exposure, cell cultures were washed and harvested using trypsin 0.1% (Gibco, Belgium). Then, cells were resuspended in metaphosphoric acid 5% and homogenized using an ultra turrax t25 tissue homogenizer (Janke & kunkel, Germany). GSH was quantified according to manufacturer’s protocol and the protein content of cell cultures was assessed by the bicinchoninic acid (BCA) protein assay kit (Thermo Scientific Pierce, Belgium). GSH was normalized to the total protein content and the results were expressed as percentage of control (untreated) cells.
Gsh detection kit
Manufactured by Enzo Life Sciences
Sourced in United States, Belgium
The GSH detection kit is a laboratory tool used to quantify the levels of glutathione, a critical antioxidant, in various biological samples. The kit provides a simple, reliable, and sensitive method to measure glutathione concentrations.
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Lab products found in correlation
Mda assay kit, by Merck Group (7 mentions)
8 ohdg assay kit, by Abcam (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Trypsin 0.1, by Thermo Fisher Scientific (2 mentions)
Biochemical autoanalyzer, by Hitachi (2 mentions)
Colorimetric activity kit, by Thermo Fisher Scientific (2 mentions)
Mpo activity assay kit, by Abcam (2 mentions)
Triglyceride assay kit, by Abcam (2 mentions)
Automatic analyzer, by Hitachi (2 mentions)
8 ohdg elisa kit, by Abcam (1 mentions)
Elisa reader, by Thermo Fisher Scientific (1 mentions)
14 protocols using gsh detection kit
1
Measuring Cellular Oxidative Stress via GSH
2
Oxidative Stress Biomarker Analysis
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Malondialdehyde (MDA) and 8-hydroxy-2’-deoxyguanosine (8-OHdG) levels were analyzed using the MDA assay kit (Sigma-Aldrich, St. Louis, MO, USA) and the 8-OHdG assay kit (Abcam, Cambridge, MA, USA), respectively. Reduced glutathione (GSH) and oxidized glutathione (GSSG) levels were determined using the GSH detection kit (Enzo Life Sciences, Farmingdale, NY, USA). Activities of catalase and superoxide dismutase (SOD) were measured using commercial kits (Invitrogen, Carlsbad, CA, USA). All analyses were performed following the manufacturers’ instructions.
3
Assay of Cellular Oxidative Stress
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MCF-10A cells were plated in 90-mm dishes, allowed to grow for 24 hours then treated with 20 μM Z-ajoene for 24 hours. The pellets were suspended with cold 5% metaphosphoric acid and sonicated, and incubated on ice for 5 minutes. The suspension was transferred to a new tube and centrifuged for 5 minutes at 4°C. The oxidized GSH concentration was determined using GSH detection kit (Enzo Life Sciences, Plymouth, PA, USA). The assay was carried out according to the instructions supplied by the manufacturer. The levels of reduced GSH and its oxidized form (GSH disulfide, GSSG) were determined according to the manufacturer’s instructions. The rates of the reaction were compared to similarly prepared GSH and GSSG standard curves.
4
Biomarkers of Renal Oxidative Stress
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Serum creatinine and BUN levels were determined using a biochemical autoanalyzer (Hitachi, Osaka, Japan). Renal MDA and 8-OHdG levels were analyzed using the MDA assay kit (Sigma-Aldrich, St. Louis, MO, USA) and the 8-OHdG ELISA kit (Abcam, Cambridge, MA, USA), respectively. Renal GSH and GSSG levels were analyzed using the GSH detection kit (Enzo Life Sciences, Farmingdale, NY, USA). Catalase and SOD activities were determined using colorimetric activity kits (Invitrogen, Carlsbad, CA, USA). Serum TNF-α and IL-6 levels were determined using ELISA kits (R&D Systems, Minneapolis, MN, USA). All assays were performed according to the manufacturers’ protocols.
5
Biochemical Markers of Liver Injury
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An automatic analyzer (Hitachi, Osaka, Japan) was used for analyzing serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. Hepatic malondialdehyde (MDA) concentrations were assessed using the MDA assay kit (Sigma-Aldrich, St. Louis, MO, USA). Serum 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels were analyzed using the 8-OHdG assay kit (Abcam, Cambridge, MA, USA). Hepatic levels of GSH and oxidized GSH (GSSG) were assessed using the GSH detection kit (Enzo Life Sciences, Farmingdale, NY, USA). Serum levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β were determined using ELISA kits (R&D Systems, Minneapolis, MN, USA). Hepatic myeloperoxidase (MPO) activities were measured using the MPO activity assay kit (Abcam, Cambridge, MA, USA).
6
Oxidative Stress Biomarkers in Serum
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The serum concentrations of creatinine and BUN were measured using a biochemical autoanalyzer (Hitachi, Osaka, Japan). Catalase and SOD activities were analyzed using colorimetric activity kits (Invitrogen, Carlsbad, CA, USA). The serum concentrations of TNF-α and IL-6 were determined using ELISA kits (R&D Systems, Minneapolis, MN, USA). MDA and 8-OHdG levels were analyzed using an MDA assay kit (Sigma-Aldrich, St. Louis, MO, USA) and an 8-OHdG assay kit (Abcam, Cambridge, MA, USA), respectively. GSH levels were determined using a GSH detection kit (Enzo Life Sciences, Farmingdale, NY, USA). All analyses were carried out following the manufacturers’ protocols.
7
Measuring Oxidative Stress in HCFs
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HCFs were pretreated without or with respective inhibitors for 1 h and then incubated with 10 μM DDF for the indicated time intervals. The supernatants were used to measure the ratio of reduced GSH/oxidized GSH (GSSG) as the marker of oxidative stress, determined using a GSH detection kit, according to the manufacturer's instructions (Enzo Life Sciences, Farmingdale, NY, USA).
8
Oxidative Stress Biomarkers Quantification
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Malondialdehyde (MDA) and 8-OHdG levels were measured using the MDA assay kit (Sigma-Aldrich) and the 8-OHdG assay kit (Abcam), respectively. Reduced glutathione (GSH) and oxidized glutathione (GSSG) levels were determined using the GSH detection kit (Enzo Life Sciences, Farmingdale, NY, USA). Activities of catalase and SOD were measured using commercial kits (Invitrogen, Carlsbad, CA, USA). MPO activity was determined using the MPO activity assay kit (Abcam). All analyses were performed following the manufacturers’ instructions.
9
Glutathione Quantification by ELISA
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A GSH detection kit was purchased from Enzo Life Sciences (Farmingdale, NY, USA). The principle of the GSH quantitation kit is based on the production of yellow-colored 5-thio-2-nitrobenzoic acid (TNB) from GSH and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). Absorbance was measured by measurement by ELISA reader (Thermo Fisher Scientific, Waltham, MA, USA) at 405 nm for 10 min at 1-min intervals. The total amount of GSH was determined using a calibration curve and normalized to the protein concentration.
10
Measurement of Myocardial Glutathione Levels
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The levels of reduced glutathione (GSH) in the myocardium were determined 6 h after LPS initiation using a GSH detection kit (Enzo Life Sciences, Farmingdale, NY, USA) according to the manufacturer’s instructions.
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