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Glutathione fluorometric assay kit

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The Glutathione Fluorometric Assay Kit is a laboratory instrument designed to measure the total glutathione content in various sample types. It utilizes a fluorometric method to quantify the level of glutathione, an important antioxidant molecule, in a given sample.

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Lab products found in correlation

Piercetm bca protein assay kit, by Thermo Fisher Scientific (3 mentions) Infinite m200 pro, by Tecan (2 mentions) Nadp nadph quantification colorimetric kit, by Abcam (2 mentions) Unicel dxh 900, by Beckman Coulter (1 mentions) Elisa kit, by Tecan (1 mentions) Elisa kit, by Cayman Chemical (1 mentions) Fluostar optima, by BMG Labtech (1 mentions) Deproteinizing sample preparation kit, by Abcam (1 mentions) Glucose 6 phosphate colorimetric assay kit, by Abcam (1 mentions) Glycogen assay kit, by Abcam (1 mentions) Ros detection assay kit, by Abcam (1 mentions) Nadph quantitation fluorometric assay kit, by Abcam (1 mentions) Glomax multi detection system, by Promega (1 mentions) Varioskan flash, by Thermo Fisher Scientific (1 mentions) Dxc800, by Beckman Coulter (1 mentions) Enzyme linked immunosorbent assay kit, by Cayman Chemical (1 mentions) Infinite f200, by Tecan (1 mentions) Tbars assay kit, by Cayman Chemical (1 mentions) Synergy h1 hybrid reader, by Agilent Technologies (1 mentions) Reduced glutathione gsh assay kit, by Abcam (1 mentions)

31 protocols using glutathione fluorometric assay kit

1

Quantification of NADP/NADPH and Glutathione

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Nicotinamide adenine dinucleotide phosphate (NADP+) and its reduced form (NADPH) levels were analyzed using an NADP/NADPH Quantification Colorimetric Kit (BioVision, Milpitas, CA, USA), and the total cellular glutathione (GSH) and oxidized glutathione (GSSG) were measured using a Glutathione Fluorometric Assay Kit (BioVision, Milpitas, CA, USA). NADP and NADPH levels were analyzed using NADP/NADPH Quantification Colorimetric Kit (Biovision, Milpitas, CA). A total of 4 × 106 cells were washed with cold PBS and lysed with NADP/NADPH extraction buffer. The supernatant was collected for measurement of NADPH and NADP+/NADPH by UV spectrophotometer at 450 nm. Total Cellular glutathione (GSH) and oxidized glutathione (GSSG) was measured using Glutathione Fluorometric Assay Kit (Biovision, Milpitas, CA, USA). Then, 0.4 × 106 cells were prepared by deproteination and subsequent reagents were added following the manufacturer’s instructions. The fluorescence intensity of samples and standards was measured at excitation/emission (Ex/Em) wavelengths = 340/420 nm. Cellular GSH and GSSG contents were calculated with the standard curve generated in parallel experiments and normalized to cell counts.
You X., Jiang W., Lu W., Zhang H., Yu T., Tian J., Wen S., Garcia-Manero G., Huang P, & Hu Y. (2019). Metabolic reprogramming and redox adaptation in sorafenib-resistant leukemia cells: detected by untargeted metabolomics and stable isotope tracing analysis. Cancer Communications, 39, 17.
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2

Biomarkers and Exercise Performance

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On the experiment day, participants finished their lunch 2 h before arrival. Blood samples were collected by registered phlebotomists before baseline and 5 min after finishing the exercise. Times to complete all race stages were recorded. The Borg Rating of Perceived Exertion (RPE, 1–10 scale) was used to determine the immediate RPE score during sprint triathlon.
To obtain plasma or serum, the blood samples with 5 ml collected into blood sampling tubes with or without ethylene diamine tetraacetic acid (EDTA), respectively, were centrifuged at 3000 rpm at 4°C for 10 min and then stored at −80°C until analysis. Full blood counts were obtained using automated haemocytometry (UniCel DxH900, Beckman Coulter®, America). Thiobarbituric acid reactive substances (TBARS), SOD, and CAT were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Cayman Chemical, Ann Arbor, MI, United States); cortisol and testosterone levels were also measured using an ELISA kit (IBL®, Minneapolis, MN, America). GSH and oxidized glutathione (GSSG) were estimated using Glutathione fluorometric assay kit (BioVision, Milpitas, CA, United States) and an ELISA plate reader (Infinite M200Pro, Tecan Group Ltd., Mannedorf, Switzerland).
Wang J.P., Wei C.C., Peng Y.D., Wang H.Y., Hung C.H., Hong Y.H., Liou Y.F, & Hou C.W. (2022). Dose caffeinated energy drink is a consideration issue for endurance performance. Frontiers in Physiology, 13, 999811.
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3

Quantitative Glutathione Analysis

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Glutathione assay was performed using the Glutathione Fluorometric Assay Kit (Biovision).
Cano M., Datta S., Wang L., Liu T., Flores‐Bellver M., Sachdeva M., Sinha D, & Handa J.T. (2021). Nrf2 deficiency decreases NADPH from impaired IDH shuttle and pentose phosphate pathway in retinal pigmented epithelial cells to magnify oxidative stress‐induced mitochondrial dysfunction. Aging Cell, 20(8), e13444.
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4

Quantification of Intracellular Glutathione

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Intracellular GSH was quantified using Glutathione Fluorometric Assay Kit (Biovision). 4 × 106 cells were collected, lysed in glutathione assay buffer and mixed with pre-chilled perchloric acid. For patient-derived xenograft tumor samples, 40 mg of tumors were homogenized in 100 μl of ice-cold glutathione assay buffer and mixed with pre-chilled perchloric acid. Supernatants were collected, OPA (o-phthalaldehyde) probe was added, the samples were incubated at room temperature for 40 min and fluorescence was measured at Ex/Em = 340/420 nm (Fluostar OPTIMA, BMG LABTECH). Quantification of GSH was done by comparing with GSH standard curve; GSH levels were normalized against protein concentration.
Wang H., Nicolay B.N., Chick J.M., Gao X., Geng Y., Ren H., Gao H., Yang G., Williams J.A., Suski J.M., Keibler M.A., Sicinska E., Gerdemann U., Haining W.N., Roberts T.M., Polyak K., Gygi S.P., Dyson N.J, & Sicinski P. (2017). The metabolic function of cyclin D3-CDK6 kinase in cancer cell survival. Nature, 546(7658), 426-430.
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5

Glycogen and ROS Assay in Astrocytes

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The presence of various enzymes and proteins could interfere with the detection of metabolites in cultured astrocytes and consequently be removed using the Deproteinizing Sample Preparation Kit (Biovision, USA). A glycogen assay kit (Biovision) was used to detect glycogen levels in cultured astrocytes. ROS levels were determined using the ROS Detection Assay Kit (Biovision). A glucose-6-phosphate colorimetric assay kit (Biovision) was used to detect the glucose-6-phosphate levels. NADPH levels were determined with the NADPH Quantitation Fluorometric Assay Kit (Biovision). Glutathione levels were analyzed with the Glutathione Fluorometric Assay Kit (Biovision).
To determine the glycogen levels in the brain, the mice were decapitated, and brain tissues in the penumbra were immersed in liquid nitrogen. Then, the brain tissues (10 mg) were homogenized with 200 μL 30% KOH on ice, and the homogenates were boiled for 10 min to inactivate enzymes. The boiled samples were centrifuged at 12,000 rpm at 4 °C for 10 min to remove insoluble sediments. Then, the supernatant was ready for the assay using a glycogen assay kit (Biovision), and the results were normalized to the protein levels in homologous samples.
Guo H., Fan Z., Wang S., Ma L., Wang J., Yu D., Zhang Z., Wu L., Peng Z., Liu W., Hou W, & Cai Y. (2021). Astrocytic A1/A2 paradigm participates in glycogen mobilization mediated neuroprotection on reperfusion injury after ischemic stroke. Journal of Neuroinflammation, 18, 230.
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6

Glutathione Redox Balance Assay

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Intracellular reduced glutathione (GSH) and oxidised glutathione (GSSG) levels were measured using a glutathione fluorometric assay kit (BioVision) according to the manufacturer’s protocol. In this assay, o-phthalaldehyde (OPA) reacts with GSH present in the sample emitting a fluorescent signal (ex. 340 nm, em. 420 nm) that is measured using a GLOMAX Multidetection System (Promega). To measure GSSG specifically, a GSH Quencher is added to remove GSH, preventing its reaction with OPA. GSSG is reduced to GSH, which is measured using the preceding method. The levels of intracellular GSH were quantified using a standard curve for known amounts of GSH. The GSH/GSSG ratio was calculated as an indicator of redox balance.
Miglionico R., Ostuni A., Armentano M.F., Milella L., Crescenzi E., Carmosino M, & Bisaccia F. (2017). ABCC6 knockdown in HepG2 cells induces a senescent-like cell phenotype. Cellular & Molecular Biology Letters, 22, 7.
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7

Glutathione Quantification Assay in Feeder-free Cells

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Feeder-free cells were cultured on Matrigel-coated tissue culture plates in MEF-conditioned ESC medium. On day 3, the cells were washed in PBS and scraped and pelleted by centrifugation. Subsequent steps were carried out using a glutathione fluorometric assay kit (catalogue no K264-100, BioVision) according to the manufacturer’s manual. Briefly, cell pellets were homogenized in ice cold glutathione assay buffer, preserved in perchloric acid and centrifuged. Supernatants were neutralized with potassium hydroxide. After centrifugation, either the supernatant was used to detect reduced glutathione (GSH), or total glutathione was measured by reducing oxidized glutathione (GSSG) to GSH before measurement. For measuring GSSG concentrations specifically, existing GSH was quenched before reducing agent was applied. o-phthalaldehyde probe, which reacts with GSH and emits fluorescence, was added to samples, and signal was acquired at Ex/Em = 340 nm/420 nm on a Varioskan Flash by Thermo Scientific. The oxidation capacity of glutathione was determined by the quantity of total glutathione (GSH+GSSG).
Skamagki M., Correia C., Yeung P., Baslan T., Beck S., Zhang C., Ross C.A., Dang L., Liu Z., Giunta S., Chang T.P., Wang J., Ananthanarayanan A., Bohndorf M., Bosbach B., Adjaye J., Funabiki H., Kim J., Lowe S., Collins J.J., Lu C.W., Li H., Zhao R, & Kim K. (2017). ZSCAN10 expression corrects the genomic instability of iPSCs from aged donors. Nature cell biology, 19(9), 1037-1048.
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8

ROS and Glutathione Quantification

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To measure ROS activity, a fluorometric assay involving 2′,7′-Dichlorodihydrofluorescein diacetate (H2DCFDA) was used as previously described [39 (link)]. To assess the total level of GSH and quantified GSH/GSSG enzyme levels, a glutathione fluorometric assay kit (BioVision, Milpitas Boulevard, Milpitas, CA, USA, Catalog #: K264-100) was used according to the manufacturer’s instructions.
Saeed K., Jo M.H., Park J.S., Alam S.I., Khan I., Ahmad R., Khan A., Ullah R, & Kim M.O. (2021). 17β-Estradiol Abrogates Oxidative Stress and Neuroinflammation after Cortical Stab Wound Injury. Antioxidants, 10(11), 1682.
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9

Exercise-Induced Metabolic Biomarkers

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Venous blood samples were drawn following an overnight fast using vacutainers at four time points: (a) At rest, (b) 5 min post exercise, (c) 60 min post exercise, and (d) 24 h post exercise. Plasma samples were separated by centrifugation at 3000 rpm for 10 min and stored at −80 °C for further assays.
Serum plasma GOT, GPT, CKMB, and myoglobin were analyzed by the enzymatic rate method based on their respective reagents using a chemistry analyzer (DxC 800, Beckman Coulter, Chaska, MN, USA). TBARS, SOD, and CAT were analyzed by the enzyme-linked immunosorbent assay kit (Cayman Chemical, Ann Arbor, MI, USA), and GSH and GSSG were analyzed by the Glutathione fluorometric assay kit (BioVision, K264-100, Milpitas, CA, USA), using an ELISA reader (Infinite M200 Pro, Tecan Group Ltd., Mannedorf, Switzerland).
Alkhatib A., Feng W.H., Huang Y.J., Kuo C.H, & Hou C.W. (2020). Anserine Reverses Exercise-Induced Oxidative Stress and Preserves Cellular Homeostasis in Healthy Men. Nutrients, 12(4), 1146.
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10

Glutathione Fluorometric Assay Protocol

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The intracellular GSH level was assessed by using Glutathione Fluorometric Assay Kit (BioVision, San Francisco). The treated cell (2*10^6 per sample) was homogenized on ice with 100 μL of ice cold Glutathione Assay Buffer. To 60 μL of the cell homogenate was added 20 μL perchloric acid (6N) and further homogenized by vortexing for several seconds. After incubation on ice (5 min) the samples were centrifuged for 2 min at 13,000g and 4°C. The collected supernatant (40 μL) was neutralized with 20 μL ice cold KOH and incubation for 5 min on ice. After centrifugation for 2 min at 13,000g, 4°C samples (10 μL) were added to 90 μL of assay buffer. The samples were read on a fluorescence plate reader (Infinite F200, Tecan, Switzerland) after incubation with o‐phthalaldehyde (10 μL) for 40 min at RT. The glutathione concentration was determined based on the standard curve. All samples were performed in triplicates.
Miran T., Vogg A.T., El Moussaoui L., Kaiser H., Drude N., von Felbert V., Mottaghy F.M, & Morgenroth A. (2017). Dual addressing of thymidine synthesis pathways for effective targeting of proliferating melanoma. Cancer Medicine, 6(7), 1639-1651.
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