Gsh gssg glo assay

Manufactured by Promega

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The GSH/GSSG-Glo Assay is a luminescent-based assay that measures the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) in biological samples.

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Lab products found in correlation

Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions) Ros glo h2o2 assay, by Promega (5 mentions) Enspire, by PerkinElmer (3 mentions) Glomax discover microplate reader, by Promega (3 mentions) Infinite 200 pro, by Tecan (3 mentions) Protein carbonyl content assay kit, by Merck Group (3 mentions) Mitotracker red, by Thermo Fisher Scientific (3 mentions) White walled clear bottom 96 well plate, by Corning (2 mentions) Glutamine glutamate glo assay, by Promega (2 mentions) Six well ultra low attachment culture plates, by Corning (2 mentions) Nadp nadph glo assay, by Promega (2 mentions) Gsh glo glutathione assay, by Promega (2 mentions) Dmem f12 50 50 medium, by Corning (2 mentions) Basic fibroblast growth factor (bfgf), by Merck Group (2 mentions) Epidermal growth factor (egf), by Merck Group (2 mentions) Celltiter glo luminescent cell viability assay, by Promega (2 mentions) Spectramax m3, by Molecular Devices (2 mentions) Fastprep 24 sample preparation instrument, by MP Biomedicals (2 mentions) Tcs sp5, by Leica (2 mentions) Synergy 2 plate reader, by Agilent Technologies (2 mentions)

Spelling variants of this product

GSH/GSSG-Glo (20 citations) GSH-Glo assay (20 citations) GSH/GSSG-GloTM Assay (19 citations) GSH/GSSG-GloTM Assay kit (12 citations)

146 protocols using gsh gssg glo assay

Evaluating Metabolic Changes in PGD-KO and 6AN-Treated Cells

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To study PGD-KO-induced metabolic changes, isogenic WM793 cells were treated with 1 μg/ml doxycycline. After 6 days, the cells were counted and re-suspended at a density of 1*10⁶/ml in PBS. 25 μl cells from each biological replicate were used for GSH/GSSG-Glo assay (Promega). This procedure was used in Figure 4L. To study 6AN-induced metabolic changes, WM793 cells were seeded in T75 flasks at a density of 1.5*10⁶/15 ml. 20 μM 6AN or vehicle control was added on the next day. After 2 days, the cells were counted and re-suspended at a density of 1*10⁶/ml in PBS. 25 μl cells from each biological replicate were used for GSH/GSSG-Glo assay (Promega) and 500 μl cells from each biological replicate were used for NADPH assay (AAT Bioquest). This procedure was used in Figures 4K and S4J.

Li H., Ericsson M., Rabasha B., Budnik B., Chan S.H., Freinkman E., Lewis C.A., Doench J.G., Wagner B.K., Garraway L.A, & Schreiber S.L. (2019). 6-Phosphogluconate Dehydrogenase Links Cytosolic Carbohydrate Metabolism to Protein Secretion via Modulation of Glutathione Levels. Cell chemical biology, 26(9), 1306-1314.e5.

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Quantifying Oxidative Stress with GSH/GSSG Assay

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Cellular ROS was measured through the detection glutathione in its reduced (GSH) and oxidized (GSSG) forms using the luminescence-based GSH/GSSG-Glo Assay (Promega, V6611). Briefly, the Promega GSH/GSSG-Glo Assay is a linked assay utilizing glutathione S-transferase and Luciferin-NT that generates a luminescent signal in response to levels of GSH present in the sample. The ratio of GSH to GSSG can then be calculated to give a read out of oxidative stress in the cells, where a decrease in the ratio indicates an increase in oxidative stress. All reactions and calculations were carried out as per the manufacturer’s instructions. The final ratio of GSH/GSSG was normalized to protein content to control for any changes in cell number. Protein was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23227) as per the manufacturer’s instructions.

Adams S.D., Csere J., D’angelo G., Carter E.P., Romao M., Arnandis T., Dodel M., Kocher H.M., Grose R., Raposo G., Mardakheh F, & Godinho S.A. (2021). Centrosome amplification mediates small extracellular vesicle secretion via lysosome disruption. Current Biology, 31(7), 1403-1416.e7.

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Quantifying Cellular Oxidative Stress

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Cellular ROS was measured through the detection glutathione in its reduced (GSH) and oxidized (GSSG) forms using the luminescence-based GSH/GSSG-Glo™ Assay (Promega, V6611). Briefly, the Promega GSH/GSSG-Glo™ Assay is a linked assay utilizing glutathione S-transferase and Luciferin-NT that generates a luminescent signal in response to levels of GSH present in the sample. The ratio of GSH to GSSG can then be calculated to give a read out of oxidative stress in the cells, where a decrease in the ratio indicates an increase in oxidative stress. All reactions and calculations were carried out as per the manufacturer's instructions. The final ratio of GSH/GSSG was normalized to protein content to control for any changes in cell number. Protein was quantified using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, 23227) as per the manufacturer's instructions.

Adams S.D., Csere J., D’angelo G., Carter E., Arnandis T., Dodel M., Kocher H.M., Grose R., Raposo G., Mardakheh F.K., & Godinho S.A. (2020). Centrosome amplification mediates small extracellular vesicles secretion via lysosome disruption.

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Measuring Cellular Redox Ratios

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Cells were seeded into the white-walled clear bottom 96-well plates (Corning). On day two, ratios of NADPH/NADP⁺ and GSH/GSSG were measured with NADP⁺/NADPH-Glo and GSH/GSSG-Glo Assays (Promega) according to the manufacturer’s instructions. Luminescence was measured using a Synergy H1 Multi-Mode reader.

Kiesel V.A., Sheeley M.P., Donkin S.S., Wendt M.K., Hursting S.D, & Teegarden D. (2022). Increased Ammonium Toxicity in Response to Exogenous Glutamine in Metastatic Breast Cancer Cells. Metabolites, 12(5), 469.

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Assessing Cellular Oxidative Stress and Apoptosis

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Materials purchased from Sigma Chemical Co. (St. Louis, MO, USA) were thymol, DMSO (dimethyl sulfoxide), DCFH-DA (2′,7′- dichlorofluorescein-diacetate), AO/EB (acridine orange/ethidium bromide) stain, SDS polyacrylamide gels, coomassie brilliant-blue-dye, powdered skim milk, trypan blue, comet assays, low-melting agarose, normal melting agarose, and lysis solution. Ham’s F-12 culture medium, fetal bovine serum (FBS), and antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin) were obtained from Gibco Invitrogen Corporation (Carlsbad, CA, USA). CellTiter-Glo Luminescent Cell Viability and GSH/GSSG-Glo assays were provided by Promega (Madison, MI, USA). Bax (N-20), Bcl-2 (C-21), Caspase-3 (H-277), Caspase-9 (H-170), and β-actin (AC-15) antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). Amersham ECL Plus Western Blotting Detection reagents were obtained from GE Healthcare (Piscataway, NJ, USA). Radioimmunoprecipitation assay (RIPA) buffer and a proteinase inhibitor cocktail were from Roche (Mannheim, Germany). Molecular weight marker BenchMark Pre-Stained Protein Ladder was from Invitrogen (Grand Island, New York, USA).

Günes-Bayir A., Kocyigit A., Guler E.M, & Dadak A. (2020). In Vitro Hormetic Effect Investigation of Thymol on Human Fibroblast and Gastric Adenocarcinoma Cells. Molecules, 25(14), 3270.

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Glutathione Redox Analysis in Neurons

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We measured both reduced (GSH) and oxidized (GSSG) glutathione for ratiometric analysis of glutathione redox metabolism using a luminescent-based GSH/GSSG-Glo assay (Promega). Primary hippocampal neurons were seeded in white 96-well plates with clear bottom and cultured for 15 days and subjected to the assay in accordance with the manufacturer’s instructions. Briefly, total and GSSG-converted GSH levels were separately measured from two identical configurations. Based on these measures, the levels of GSH and GSSG were calculated and normalized to DNA content per well quantified by using the CyQUANT assay (Thermo Fisher Scientific).

Chung K.M., Kim H., Roque C.G., McCurdy E.P., Nguyen T.T., Siegelin M.D., Hwang J.Y, & Hengst U. (2022). A systemic cell stress signal confers neuronal resilience toward oxidative stress in a Hedgehog-dependent manner. Cell reports, 41(3), 111488.

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Glutathione Redox Quantification

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Reduced and oxidized forms of glutathione were measured in three biological replicates with the GSH/GSSG-Glo™ assay (Promega) according to manufacturer’s instructions. A total of 5 × 10² fibroblasts were seeded in duplicates in 96-well plates. The next day, cells were lysed with either total or oxidized glutathione lysis reagent for 5 min on a plate shaker at room temperature (RT). Luciferin generation and detection reagents were subsequently added and incubated at RT for 30 and 15 min, respectively, before recording luminescence.

Cardon I., Grobecker S., Kücükoktay S., Bader S., Jahner T., Nothdurfter C., Koschitzki K., Berneburg M., Weber B.H., Stöhr H., Höring M., Liebisch G., Braun F., Rothammer-Hampl T., Riemenschneider M.J., Rupprecht R., Milenkovic V.M, & Wetzel C.H. (2024). Mitochondrial and Cellular Function in Fibroblasts, Induced Neurons, and Astrocytes Derived from Case Study Patients: Insights into Major Depression as a Mitochondria-Associated Disease. International Journal of Molecular Sciences, 25(2), 963.

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Glutamine/Glutamate and GSH/GSSG Assays

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Commercially available kits Glutamine/Glutamate-Glo™ Assay (Promega) and GSH/GSSG-Glo™ Assay (Promega) were used in accordance with the manufacturer’s instructions.

Merlin J., Ivanov S., Dumont A., Sergushichev A., Gall J., Stunault M., Ayrault M., Vaillant N., Castiglione A., Swain A., Orange F., Gallerand A., Berton T., Martin J.C., Carobbio S., Masson J., Gaisler-Salomon I., Maechler P., Rayport S., Sluimer J.C., Biessen E.A., Guinamard R.R., Gautier E.L., Thorp E.B., Artyomov M.N, & Yvan-Charvet L. (2021). Non-canonical glutamine transamination sustains efferocytosis by coupling redox buffering to oxidative phosphorylation. Nature metabolism, 3(10), 1313-1326.

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Glutathione Redox Balance Assay

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After nsPEFs exposure with or without calcium ions, cells were seeded into white 96-well microculture plates at the concentration of 5 × 10³ cells/well. The level of reduced and oxidized glutathione was determined after 24 h by a luminescence-based assay (GSH/GSSG-Glo™ Assay, Promega, Walldorf, Germany) using a multiwell scanning spectrophotometer at 570 nm (EnSpire Perkin Elmer, Warsaw, Poland). The experimental procedure was performed according to our previous study [40 (link)]. Experiments were repeated in triplicate. The results were expressed as a ratio of the reduced and oxidized glutathione (GSH/GSSG).

Kulbacka J., Rembiałkowska N., Szewczyk A., Rossowska J., Drąg-Zalesińska M., Kulbacki M, & Choromańska A. (2022). Nanosecond PEF Induces Oxidative Stress and Apoptosis via Proteasomal Activity Inhibition in Gastric Adenocarcinoma Cells with Drug Resistance. International Journal of Molecular Sciences, 23(21), 12943.

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Glutathione Measurement in Cells

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GSH (reduced glutathione) and GSSG (oxidized glutathione) were measured by use of GSH/GSSG Glo assay (Promega, Fitchburg, WI, US). The cells were seeded in white, 96-well plates at 4,000 cells per well. After adhering overnight, the cells were either left untreated or exposed to 5 mM DHA for 24 and 48 h. At the end of treatment time, cells were counted on a Celigo to normalize for variations in cell density, then assayed according to the manufacturer’s protocol. Following assay incubation, luminescence was recorded on Tecan M1000. Luminescence values were normalized to cell count and then expressed as a percentage of the untreated control. Results presented as the mean ± SEM of three replicates.

Smith K.R., Hayat F., Andrews J.F., Migaud M.E, & Gassman N.R. (2019). Dihydroxyacetone exposure alters NAD(P)H, induces mitochondrial stress and autophagy in HEK293T cells. Chemical research in toxicology, 32(8), 1722-1731.

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