Gsh gssg glo assay kit

Manufactured by Promega

Sourced in United States, United Kingdom, Australia

The GSH/GSSG-Glo Assay kit is a laboratory tool designed to measure the levels of reduced glutathione (GSH) and oxidized glutathione (GSSG) in biological samples. The kit provides a luminescent-based assay that enables the quantification of these important cellular redox markers.

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Close spelling variants of this product

GSH/GSSG-Glo (20 citations) GSH-Glo assay (20 citations) GSH-Glo kit (18 citations) GSH/GSSG-GloTM Assay kit (12 citations) GSH/GSSG Assay kit (4 citations) GSH assay kit (4 citations)

98 protocols using gsh gssg glo assay kit

Glutamine Modulates Cellular Redox State

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Cells were plated into 96 well plates (5000/well) and the next day treated with or without stabilized glutamine (Gemini Bio-Products, Sacramento, CA) for 24 hours. The GSH and GSSG concentrations were measured in each well of cells using the GSH/GSSG-Glo Assay kit (Promega, Madison, WI) following the manufacturer’s instructions..
Whole frozen tissues which were leftover from the metabolomics analysis were utilized to measure absolute GSH and GSSG levels using the GSH/GSSG-Glo Assay kit (Promega, Madison, WI) following the manufacturer’s instructions.

Wettersten H.I., Hakimi A.A., Morin D., Bianchi C., Johnstone M.E., Donohoe D.R., Trott J.F., Aboud O.A., Stirdivant S., Neri B., Wolfert R., Stewart B., Perego R., Hsieh J.J, & Weiss R.H. (2015). Grade-dependent metabolic reprogramming in kidney cancer revealed by combined proteomics and metabolomics analysis. Cancer research, 75(12), 2541-2552.

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Glucose Deprivation Induces Oxidative Stress and Apoptosis

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First, cells were cultured in glucose-deprived medium and were incubated with 10 mmol/L 2, 7-dichlorodihydrofluorescein diacetate (H2-DCFDA, Thermo Fisher Scientific, cat. no. D399) at 37°C for 30 min. Then, we collected the cells and washed them twice with 4°C PBS; PBS was used to resuspend the cells; FACScan Flow Cytometer (Beckman-Coulter) was immediately used to measure the fluorescence. The NADP/NADPH-Glo Kit (Promega, cat. no. G9081) was used to measure the intracellular levels of NADPH and for the total NADP measurement. The intracellular levels of GSH/GSSG was measured with a GSH/GSSG-Glo Assay kit (Promega, WI, USA). Annexin V-FITC and PI (4A Biotech Co. cat. no. FXP018) were used for cell apoptosis analysis with a flow cytometer. The cell cycle was analyzed by PI/RNase staining buffer (BD Pharmingen™) with a flow cytometer.

Li H., Fu X., Yao F., Tian T., Wang C, & Yang A. (2019). MTHFD1L-Mediated Redox Homeostasis Promotes Tumor Progression in Tongue Squamous Cell Carcinoma. Frontiers in Oncology, 9, 1278.

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Measuring Glutathione Levels in HaCaT Cells

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GSH/GSSG-Glo assay kit (Promega, Cat.V6611) was used for glutathione measurements. After 48h 800μM H₂O or BSO pretreatment, 10,000 H₂O-treated HaCaTs cells/per well or 15,000 BSO-treated cells/per well were transferred into white 96 well plates. Then, another 24h H₂O or 800μM BSO treatment was followed. To measure total GSH levels, 50μl per well total glutathione lysis reagent (containing Luciferin-NT, passive lysis buffer) was added to the cells. To measure oxidized GSSG, 50ul per well oxidized lysis reagent (containing Luciferin-NT, passive lysis buffer, and 25mM NEM) was added. Plates were incubated at room temperature and shaken for 5 minutes, then 50μl per well luciferin generation reagent (containing 100mM DTT, glutathione S-transferase and GSH reaction buffer) was added. The plate was shaken and incubated at room temperature for another 30 minutes. Finally, 100μl/well luciferin detection reagent was added and the plate was shaken and incubated at room temperature for another 15 minutes. The luminescence was measured using a DTX-800 multimode plate reader (Beckman Coulter). Standard curves were generated using serial dilutions of standard GSH (provided by the kit) ranging from 16μM to 0.25μM.

Li S., Bronnimann M.P., Williams S.J, & Campos S.K. (2019). Glutathione contributes to efficient post-Golgi trafficking of incoming HPV16 genome. PLoS ONE, 14(11), e0225496.

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Quantifying Oxidative Stress and Mitochondrial Function

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To determine oxidative stress, cells were washed once with PBS and incubated with PBS containing 2 μM CM-H₂DCFDA (Life Technologies) for the determination of reactive oxygen species for 30 min at 37 °C. Live cells were imaged using an inverted fluorescence microscope. For quantitative comparison of cellular ROS between WT and TKO MEFs, DCF-labelled cells were analyzed by flow cytometry. Levels of NAD(H), NADP(H), and GSH were quantified using a NAD/NADH Quantification Kit Sigma-Aldrich, St. Louis, MO, NADP/NADPH Quantification Kit (Sigma-Aldrich), and GSH/GSSG-Glo assay kit (Promega, Madison, WI; V6611) according to the manufacturer's protocols. Oxygen consumption rates were measured using a XF-24 extracellular flux analyzer (SeaHorse Bioscience Inc., Chicopee, MA). To assess mitochondrial function, the XF Cell Mito Stress Test Kit (101706-100) was employed.

Song J.H., An N., Chatterjee S., Kistner-Griffin E., Mahajan S., Mehrotra S, & Kraft A.S. (2014). Deletion of Pim Kinases Elevates the Cellular Levels of Reactive Oxygen Species and Sensitizes to K-Ras-Induced Cell Killing. Oncogene, 34(28), 3728-3736.

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Quantifying Cellular Glutathione Levels

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Reduced glutathione (GSH) and oxidized glutathione (GSSG) plays key roles in cellular redox systems. It is essential to examine intracellular glutathione levels in experiments, because fluctuations in the GSH/GSSG ratio are related to human disease therapy, aging and other cell signaling activities. The level of GSSG reflects cell health and oxidative stress. Briefly, 24 h after plasma treatment, cells were washed with PBS and total cell extract was prepared separately for GSH and GSSG quantification in the lysis reagent provided with the kit. GSH, GSSG and GSH/GSSG were determined by a luminescence based biochemical method using the GSH/GSSG-Glo Assay Kit (Promega, Korea) following the manufacturer’s instructions. Luminescence was detected using a microplate reader (Biotek, VT, USA).

Kaushik N.K., Kaushik N., Park D, & Choi E.H. (2014). Altered Antioxidant System Stimulates Dielectric Barrier Discharge Plasma-Induced Cell Death for Solid Tumor Cell Treatment. PLoS ONE, 9(7), e103349.

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Quantifying Cellular Redox Status

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Reduced glutathione (GSH) and oxidised glutathione (GSSG) were measured with GSH/GSSG-Glo™ Assay Kit (Promega,UK) according to the manufacturer’s instructions. Cells were treated for 4 h before 5 × 10⁴ cells were transferred into 96-well luminescence plates and luminescence recorded using a luminometer (Victor X3, Perkin Elmer Inc., UK).

Jiang Y., Southam A.D., Trova S., Beke F., Alhazmi B., Francis T., Radotra A., di Maio A., Drayson M.T., Bunce C.M, & Khanim F.L. (2021). Valproic acid disables the Nrf2 anti-oxidant response in acute myeloid leukaemia cells enhancing reactive oxygen species-mediated killing. British Journal of Cancer, 126(2), 275-286.

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Usnic Acid Impact on Cellular Glutathione

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Total intracellular glutathione (GSH) levels of HepG2 cells were measured after usnic acid treatment for 24 h using HT glutathione assay kit (Trevigen). The data were normalized against total protein content as determined by a Bio-Rad protein assay (Bio-Rad, Hercules, CA). The GSH/oxidized glutathione (GSSG) ratios in usnic acid-treated cells were determined using GSH/GSSG-Glo assay kit (Promega) as described by the manufacturer.

Chen S., Zhang Z., Qing T., Ren Z., Yu D., Couch L., Ning B., Mei N., Shi L., Tolleson W.H, & Guo L. (2016). Activation of the Nrf2 signaling pathway in usnic acid-induced toxicity in HepG2 cells. Archives of toxicology, 91(3), 1293-1307.

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Cellular Glutathione Homeostasis Assay

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Reduced (GSH), oxidized (GSSG) and total cellular GSH/GSSG ratio levels [51 (link)] were determined by a GSH/GSSG-Glo assay kit (V6612, Promega, USA) following the manufacturer's instructions. Briefly, cells were seeded at a concentration of 15,000 cells/cm² in white opaque-bottom, 96-well plates, with a final volume of 100 μl per well and allowed to proliferate for 24 h. Then, 25 μM of AntiOxCIN₄ was added to cells for 48 h. In the last 24 h of the experiment, the cell culture medium was changed to OXPHOSm. Afterward, cell culture medium was removed, and 50 μL of total glutathione or oxidized glutathione lysis reagents were added and were mixed for 5 min on an orbital shaker to induce cell lysis. After, 50 μL of luciferin generation reagent was added and incubated at 22 °C for 30 min. Lastly, 100 μL of luciferin detection reagent was added, and after 15 min of incubation at 22 °C, the luminescence signal was monitored in a Biotek Cytation 3 spectrophotometer (BioTek Instruments Inc., USA). A glutathione standard curve was also generated following the manufacturer's instructions.

Deus C.M., Pereira S.P., Cunha-Oliveira T., Teixeira J., Simões R.F., Cagide F., Benfeito S., Borges F., Raimundo N, & Oliveira P.J. (2021). A mitochondria-targeted caffeic acid derivative reverts cellular and mitochondrial defects in human skin fibroblasts from male sporadic Parkinson's disease patients. Redox Biology, 45, 102037.

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Glutathione Redox Status Assay

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GSH/GSSH ratio was determined using the GSH/GSSG-Glo assay kit (Promega, WI).
Briefly, 5000 cells were seeded in triplicates into 96-well plates and incubated for 24–48 h. At the time of the assay, growth media was aspirated and replaced with either total glutathione lysis buffer or oxidized glutathione lysis buffer. To achieve efficient lysis, the plate was placed on a plate shaker for 5 min at room temperature. Next, Luciferin generation reagent was added to each well, and the plate was incubated for 30 minutes at room temperature. Finally, Luciferin detection reagent was added to each well and the resulting luminescence was quantified using a Cytation 1 Imager (BioTek, VT). Graphs were plotted using Microsoft Excel (Microsoft, WA).

Oyelakin A., Nayak K.B., Glathar A.R., Gluck C., Wrynn T., Tugores A., Romano R.A, & Sinha S. (2022). EHF is a novel regulator of cellular redox metabolism and predicts patient prognosis in HNSCC. NAR Cancer, 4(2), zcac017.

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Leelamine Modulates CXCR7/CXCR4 Signaling

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Leelamine (LEE, Figure 1A) was purchased from Cayman Chemical (Ann Arbor, MI, USA). LEE stock solution (10 mM) was prepared in EtOH, storage at −20 °C and finally diluted in cell culture medium for use. Anti-CXCR7 and anti-CXCR4 antibodies were purchased from abcam (Cambridge, UK). Anti-MnSOD, anti-fibronectin, anti-vimentin, anti-MMP-9, anti-MMP-2, anti-N-cadherin, anti-E-cadherin, anti-twist, anti-snail, anti-occludin, and anti-b-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-STAT3(Tyr705), anti-STAT3, anti-phospho-JAK1(Tyr1022/1023), anti-JAK1, anti-phospho-JAK2(Tyr1007/1008), and anti-JAK2 were purchased from Cell Signaling Technology (Beverly, MA, USA). Alexa Fluor^® 488 donkey anti-rabbit IgG (H+L) antibody and Alexa Fluor^® 594 donkey anti-mouse IgG (H+L) antibody was obtained from Life Technologies (Grand Island, NY, USA). The GSH/GSSG-Glo™ Assay kit was purchased from Promega (Madison, WI, USA).

Jung Y.Y., Um J.Y., Sethi G, & Ahn K.S. (2022). Potential Application of Leelamine as a Novel Regulator of Chemokine-Induced Epithelial-to-Mesenchymal Transition in Breast Cancer Cells. International Journal of Molecular Sciences, 23(17), 9848.

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