DHA and Brusatol (Nrf2 inhibitor) were purchased from Tauto. GSH assay kits and antibodies against β‐actin were purchased from Beyotime. The hydroxyproline kit was purchased from the Jiancheng Bioengineering Institute. The TNF‐α and TGF‐β ELISA kits were purchased from Elabscience. Antibodies against HO‐1 were purchased from Abcam. Antibodies against tumor necrosis factor α (TNF‐α), transforming growth factor‐β (TGF‐β), GPX4, Nrf2, electron microscope fixative, hydrogen peroxide (H2O2), hematoxylin, eosin, osmium acid, lead citrate, uranium acetate and the immunohistochemistry (IHC) special goat anti‐rabbit secondary antibody were purchased from Servicebio. The goat anti‐rabbit IgG (H + L) fluorescent secondary antibody was purchased from Cell Signaling Technology; excitation is 777 nm and peak fluorescence emission is 794 nm. The diaminobenzidine (DAB), sodium dodecyl sulfate (SDS), radioimmunoprecipitation assay (RIPA) buffer and Tris Buffered Saline Tween (TBST) were purchased from Solarbio. The polyvinylidene fluoride membrane was purchased from Millipore. The acetone and ethanol were purchased from Chron Chemicals.
Gsh assay kit
Manufactured by Beyotime
Sourced in China
The GSH assay kit is a laboratory tool designed to quantify the levels of glutathione (GSH) in various biological samples. It provides a simple and reliable method to measure the total GSH content, which is an important antioxidant molecule in living cells.
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Lab products found in correlation
Mda assay kit, by Beyotime (15 mentions)
Sod assay kit, by Beyotime (6 mentions)
Iron assay kit, by Merck Group (5 mentions)
Lipid peroxidation mda assay kit, by Beyotime (5 mentions)
Atp assay kit, by Beyotime (4 mentions)
Acetonitrile, by Thermo Fisher Scientific (2 mentions)
Methanol, by Thermo Fisher Scientific (2 mentions)
Cell culture reagents, by Thermo Fisher Scientific (2 mentions)
Mda assay kit, by Nanjing Jiancheng (2 mentions)
Cell ferrous iron colorimetric assay kit, by Elabscience (2 mentions)
Total superoxide dismutase assay kit, by Beyotime (2 mentions)
Lipid peroxidation assay kit, by Beyotime (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Hoechst 33342, by Solarbio (2 mentions)
Ros assay kit, by Beyotime (2 mentions)
Cat assay kit, by Beyotime (2 mentions)
Bca assay kit, by Beyotime (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Reactive oxygen species assay kit, by Beyotime (2 mentions)
Anti β actin, by Abcam (2 mentions)
95 protocols using gsh assay kit
1
Investigating Nrf2 Inhibition and Oxidative Stress
2
Chebula Fruit Extract for Ulcerative Colitis
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T. chebula fruits were purchased from the Hehua Chi Medicine Market in Chengdu, Sichuan Province, and the sample was preserved and identified by Dr. Gao Zhou from Hubei University of Technology (sample number: 20211213). Dextran Sulfate Sodium Salt (DSS) (MW: 36000∼50000) was purchased from MP Biomedicals company (Santa Ana, California, United States). 5-Aminosalicylic acid was purchased from Shanghai Yi’en Chemical Technology Co., Ltd. (Shanghai, China). CAT, GSH, MDA, GSH assay kits, and staining reagents were purchased from Beyotime Biotechnology (Shanghai, China). All analytical standards were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). TLR4, β-actin antibody, and horseradish peroxidase-conjugated anti-rabbit IgG purchased from Servicebio Biotechnology Co., Ltd. (Wuhan, China). MyD88, NF-κBp65 antibody purchased from Proteintech Group (Wuhan, China). Methanol and acetonitrile were chromatographically pure from Thermo Fisher Scientific Inc. (Shanghai, China).
3
Neuronal Injury Protection Protocol
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PTE, EX527, and DCFH-DA and DAPI fluorescent probes were obtained from Sigma-Aldrich (St. Louis, MO, USA). Lactate dehydrogenase (LDH) cytotoxicity, Cell counting kit-8 (CCK-8), MDA, SOD, and GSH assay kits were obtained from Beyotime Biotechnology (Shanghai, China). TUNEL kit was obtained from Roche (Mannheim, Germany). TNF-α and IL-6 ELISA kits were obtained from Sangon Biotech (Shanghai, China). Antibodies against SIRT-1, p65, and acetylated p65 at Lys310 were obtained from Cell Signalling Technology (Danvers, MA, USA). Antibodies against β-actin, caspase 3, cleaved-caspase 3, Bax, Bcl-2, and secondary antibodies labelled by HRP were obtained from Wanleibio (Shenyang, China). Transwell Chamber was purchased from Corning (NY, USA). Cell culture reagents were obtained from Gibco (Grand Island, NY, USA). SH-SY5Y human neuroblastoma cell line and BV-2 mouse microglia cell line were obtained from the Neurological Lab of Tangdu Hospital.
4
Oxidative Stress Assay Protocol
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The cells were treated as indicated in LIP protocol, and cellular MDA and GSH levels were assessed using MDA and GSH assay kits, respectively (Beyotime Biotechnology), according to the manufacturer's instructions.
5
Endoplasmic Reticulum Stress Pathway Analysis
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γ-GC with a purity of over 95% (CAS no. 636-58-8) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Antibodies against 78 kDa glucose-regulated protein (GRP78), protein kinase R-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1α (IRE1α), TNF receptor-associated factor 2 (TRAF2), eukaryotic initiation factor 2 (eIF2α), phosphorylated eIF2α (p-eIF2α), C/EBP homologous protein (CHOP), c-Jun NH 2-terminal kinase (JNK), and phosphorylated JNK (p-JNK) were obtained from Cell Signaling Technology (Massachusetts, USA). Antibodies against Bax, Bcl-2, and GAPDH were purchased from Bioworld Technology (St. Louis, Missouri, USA). Antibodies against caspase-3 were purchased from Proteintech Group (Wuhan, China). An antibody against p-PERK was obtained from Signalway Antibody LLC (Maryland, USA), and an antibody against p-IRE1α was obtained from Affinity Biosciences (Jiangsu, China). Cell Counting Kit-8 (CCK-8) and calcein-AM/propidium iodide (PI) were purchased from Dojindo Molecular Technologies (Tokyo, Japan). An Annexin V-PE/7-AAD apoptosis detection kit and TUNEL kit were obtained from Vazyme Biotech Co. (Nanjing, China). ROS, malondialdehyde (MDA), and GSH assay kits were obtained from Beyotime Biotechnology (Shanghai, China).
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γ-GC with a purity of over 95% (CAS no. 636-58-8) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Antibodies against 78 kDa glucose-regulated protein (GRP78), protein kinase R-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1α (IRE1α), TNF receptor-associated factor 2 (TRAF2), eukaryotic initiation factor 2 (eIF2α), phosphorylated eIF2α (p-eIF2α), C/EBP homologous protein (CHOP), c-Jun NH 2-terminal kinase (JNK), and phosphorylated JNK (p-JNK) were obtained from Cell Signaling Technology (Massachusetts, USA). Antibodies against Bax, Bcl-2, and GAPDH were purchased from Bioworld Technology (St. Louis, Missouri, USA). Antibodies against caspase-3 were purchased from Proteintech Group (Wuhan, China). An antibody against p-PERK was obtained from Signalway Antibody LLC (Maryland, USA), and an antibody against p-IRE1α was obtained from Affinity Biosciences (Jiangsu, China). Cell Counting Kit-8 (CCK-8) and calcein-AM/propidium iodide (PI) were purchased from Dojindo Molecular Technologies (Tokyo, Japan). An Annexin V-PE/7-AAD apoptosis detection kit and TUNEL kit were obtained from Vazyme Biotech Co. (Nanjing, China). ROS, malondialdehyde (MDA), and GSH assay kits were obtained from Beyotime Biotechnology (Shanghai, China).
6
Quantification of Cellular Glutathione
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The content of GSH was measured using GSH Assay Kit (Beyotime, Shanghai,China) according to the instruction. The cells were treated with protein removal reagents, freezed-thawed twice, and then placed in ice bath for 5 min. Followed centrifugatng, the supernatant was used for the determination of GSH. The reagents and samples were added into 96-well plate in turn according to the instructions, mixed and incubated for 5 min at room temperature. After adding NADPH solution, the absorbance at 412 nm was detected by a microplate reader. The standard curve was drawn according to the different absorbance measured by different concentrations of standard materials. The total glutathione or GSSG content was calculated by comparing the sample to the standard curve. GSH = Total Glutathione-GSSG × 2.
7
Antioxidant Levels in Tissues
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The MDA, SOD, and GSH levels in tissues were detected using a lipid peroxidation MDA assay kit, GSH assay kit, and SOD assay kit (Beyotime, Shanghai, China), respectively, following the instructions of the manufacturer.
8
Investigating ROS Modulation by 2-DG and CsA
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The effect of 2-DG on the production of ROS induced by CsA was evaluated using flow cytometry. Briefly, 5x10 5 cells/well were seeded into 6-well plates with DMEM, containing 10% fetal bovine serum, and cultured at 37˚C for 24 h. The medium was replaced with DMEM without serum and the cells were cultured at 37˚C for a further 12 h. The medium was changed and the cells were treated with 2-DG (25 mM) for 30 min prior to the addition of 10 µM CsA. Rosup was added 30 min prior to cell digestion. The cells were incubated at 37˚C for 12 h, and changes in the levels of ROS were detected using flow cytometry.
Determination of glutathione (GSH). GSH was detected using a GSH assay kit (Beyotime Institute of Biotechnology Co. Ltd.), according to the manufacturer's instructions. Briefly, 5x10 5 cells/ well were seeded into 6-well plates with DMEM, containing 10% fetal bovine serum, and cultured at 37˚C for 24 h. The medium was replaced with DMEM without serum and the cells were cultured at 37˚C for a further 12 h. The medium was replaced again, and the cells were treated with 2-DG (25 mM) for 30 min prior to the addition of 10 µM CsA and culture for 24 h. The cells were then washed with cold PBS and lysed. The supernatant was assessed using an ELISA reader to determine the absorbance at the wavelength of 405 nm.
Determination of glutathione (GSH). GSH was detected using a GSH assay kit (Beyotime Institute of Biotechnology Co. Ltd.), according to the manufacturer's instructions. Briefly, 5x10 5 cells/ well were seeded into 6-well plates with DMEM, containing 10% fetal bovine serum, and cultured at 37˚C for 24 h. The medium was replaced with DMEM without serum and the cells were cultured at 37˚C for a further 12 h. The medium was replaced again, and the cells were treated with 2-DG (25 mM) for 30 min prior to the addition of 10 µM CsA and culture for 24 h. The cells were then washed with cold PBS and lysed. The supernatant was assessed using an ELISA reader to determine the absorbance at the wavelength of 405 nm.
9
Measuring Glutathione Content in Oocytes
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The measurement of GSH content has been reported in our previous study (Huan et al., 2015b (link)). Briefly, for GSH staining, matured oocytes were incubated with 5 μM 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin (CMF2HC, Invitrogen) for 30 min and manipulation medium for another 30 min. Then, oocytes were washed and examined to analyze the fluorescence intensity in each oocyte. For quantitative measurement, GSH content in matured oocytes was determined using a GSH assay kit (Beyotime). After oocytes were frozen and thawed three times using liquid nitrogen and 37°C water, GSH content was measured by the 5,5′-dithiobis (2-nitrobenzoic acid) oxidized glutathione reductase recycling assay. According to the standard curve, total GSH amounts in samples were calculated and divided by the number of oocytes to get GSH content per oocyte.
10
Oxidative Stress Biomarker Quantification
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The levels of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) in cells were determined using a commercial MDA assay kit (Beyotime), SOD assay kit (Nanjing Jiancheng, China), and GSH assay kit (Beyotime), respectively, according to the respective manufacturer’s protocols.
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