Cs0260

Manufactured by Merck Group

Sourced in United States

The CS0260 is a laboratory equipment product manufactured by Merck Group. It is designed for performing essential functions within a research or analytical laboratory setting. The core function of the CS0260 is to provide a reliable and versatile tool for laboratory professionals. Further details on the intended use or specific capabilities of this product are not available.

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25 protocols using cs0260

Quantifying Cotinine and Glutathione in Biological Samples

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Blood collected in EDTA-coated tubes (Fisher Scientific, Pittsburg, PA) was centrifuged at 6000 rpm (10 min, 4 °C) to obtain plasma supernatant. Cotinine levels were assayed in plasma using the Immunalyis Cotinine Direct ELISA Kit according to the manufacturer’s instructions (Immunalysis, Pomona, CA, USA).
Glutathione (GSH) levels were measured in tissue samples of the liver using a kit for colorimetric enzymatic recycling containing glutathione reductase from S. cerevisiae (CS0260, Sigma-Aldrich, St. Louis, MO, USA). The right liver lobe (superior part) was dissected on an ice-cold platform. Samples were deproteinized with 5% 5-sulfosalicylic acid (centrifugation at 10,000 rpm, 4 °C, 10 min). Total (reduced and oxidized) GSH was measured with a kinetic assay based on the reduction of 5,5′-dithiobis (2-nitrobenzoic acid) to 2-nitro-5-thiobenzoic acid and recycling of the oxidized glutathione (GSSG) generated in the reaction by glutathione reductase and NADPH. For measuring GSSG, lysates were incubated with 4-vinylpyridine to block free thiols of reduced GSH (1 h, room temperature). Absorbance was measured at 405 nm (1 min intervals, 10 min) with an EnSpire® Multimode Plate Reader 2300 (Perkin Elmer, Waltham, MA, USA), and net slopes of experimental samples were compared to net slopes of standards using GraphPad Prism 7 software.

Wickramasekara R.N., Morrill S., Farhat Y., Smith S.J, & Yilmazer-Hanke D. (2018). Glutathione and Inter-α-trypsin inhibitor heavy chain 3 (Itih3) mRNA levels in nicotine-treated Cd44 knockout mice. Toxicology Reports, 5, 759-764.

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Reduced Glutathione Quantification in Tissues

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Measurement of reduced glutathione in brain and liver utilized a kinetic assay kit (Sigma #CS0260) and followed manufacturer’s instructions (n = 7–8/group). In brief, several hundred milligrams of tissue was required for this ELISA: liver and whole brains (minus hippocampus used in qPCR) were dissected from mice following cervical dislocation. Samples were immediately placed in liquid nitrogen and then transferred to −80 °C for long-term storage. For preparation, samples were pulverized, weighed (10–300 mg required for assay) and then 3 volumes of 5-sulfosalicylic acid added and vortexed, and another seven volumes of 5% SSA solution added. Samples were then homogenized with a pestle in a glass tube until an even suspension was achieved. Samples were left on ice for 10 min prior to centrifuging at 10,000× g for 10 min. The volume of the supernatant was measured. Samples were further diluted 1:20 with 5% SSA to obtain the working solution. Blanks, standard curve and samples were plated (samples were counterbalanced across conditions and organ type). Following manufacturer instructions, the plate was read in a plate reader, measuring absorption at 412 nm at one-minute intervals for five minutes. All samples were run in triplicate.

Vander Velden J.W, & Osborne D.M. (2021). Obesity Prevents S-Adenosylmethionine-Mediated Improvements in Age-Related Peripheral and Hippocampal Outcomes. Nutrients, 13(4), 1201.

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Quantifying Myocardial Oxidative Stress

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Myocardial levels (n = 7 LV sample/group) of oxidative stress markers were tested with enzyme-linked immunosorbent assay (ELISA). Hydrogen peroxide (H₂O₂) was assessed in LV tissue homogenates (n = 4–10/group). Samples containing equal amounts of total protein were analyzed for H₂O₂ formation. Total reduced glutathione in heart samples was determined in duplicate with a colorimetric glutathione assay kit (CS0260, Sigma Aldrich).

Herwig M., Kolijn D., Lódi M., Hölper S., Kovács Á., Papp Z., Jaquet K., Haldenwang P., Dos Remedios C., Reusch P.H., Mügge A., Krüger M., Fielitz J., Linke W.A, & Hamdani N. (2020). Modulation of Titin-Based Stiffness in Hypertrophic Cardiomyopathy via Protein Kinase D. Frontiers in Physiology, 11, 240.

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Enzymatic Assay for Skeletal Muscle Glutathione

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Glutathione measurement was performed using an enzymatic assay kit (Sigma #CS0260) per manufacture’s protocol. For glutathione extraction from skeletal muscle tissues, frozen tissue was pulverized with ~50 mg of samples collected and weighed in homogenizing tubes. 10 volumes of 5% 5-sulfosalicylic acid (SSA) solution were added to each vial. For glutathione extraction from cultured cells, ~1 million cells were collected and 50 ul 5% SSA was added to lyse the cells. After homogenization, samples were centrifuged at 10,000 g for 10 min and the supernatants were collected. Samples were diluted 1–5 times before glutathione assay as needed. Standard curve was generated using the provided glutathione standard. Samples were run in duplicates. Total glutathione was measured by this kit and normalized by tissue weight or total protein amount extracted from same number of cultured cells.

Li J., Lu M., Ahn Y., Cao K., Pinkus C.A., Stansfield J.C., Wu Z, & Zhang B.B. (2023). CHAC1 inactivation is effective to preserve muscle glutathione but is insufficient to protect against muscle wasting in cachexia. PLOS ONE, 18(4), e0283806.

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Quantifying Glutathione Levels in Listeria

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Reduced glutathione (GSH) concentrations were measured by using a commercial kit supplied by Sigma-Aldrich (CS0260) according to the manufacturer’s specifications. Briefly, overnight L. monocytogenes cultures were diluted to an OD₆₀₀ of 0.1 in 35 mL of fresh defined media and grown at 37°C shaking. After two hours, 10 OD₆₀₀ were transferred to 15 mL Falcon tube and 0.4 or 1.2 mM of MG was added to the indicated cultures. The media was supplemented with cysteine doubling the amount of MG added (0.8 mM or 2.2 mM) 15 minutes after the addition of MG. For the untreated cultures a total of 0.8 mM cysteine was added. Bacteria in the Falcon tube were washed twice in sterile PBS and resuspended in 200 μL of 5% 5-Sulfosalicylic Acid. Bacteria were lysed 0.1 mm-diameter silica-zirconium beads and 10 μL were used for the kit’s working reaction. Samples were taken at 15, 30 and 60 minutes post-challenge with MG. Absorbance at 412 nm was measured using a plate reader (Infinite M1000 PRO, TECAN). GSH concentrations as a percentage of wild-type without MG challenge are an average of two technical replicates from three independent experiments.

Anaya-Sanchez A., Feng Y., Berude J.C, & Portnoy D.A. (2021). Detoxification of methylglyoxal by the glyoxalase system is required for glutathione availability and virulence activation in Listeria monocytogenes. PLoS Pathogens, 17(8), e1009819.

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Oxidant-Antioxidant Defense Mechanisms Analysis

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A 5 ml of venous blood was drawn from an antecubital vein after a 12-h fast and 24 h after the last yoga exercise for the determination of selected biochemical parameters. For the determination of oxidant-antioxidant defence mechanism, lipid peroxide as malondialdehyde (MDA) (ELISA; MAK085, Sigma, USA),[20 (link)] SOD (ELISA; 19160, Sigma, USA),[21 ] CAT (ELISA; CAT100, Sigma, USA),[22 (link)] reduced GSH (ELISA; CS0260, Sigma, USA)[23 (link)] and ascorbic acid (ELISA; MAK074, Sigma, USA)[24 ] were estimated following standard methods.

, & Manna I. (2018). Effects of Yoga Training on Body Composition and Oxidant-Antioxidant Status among Healthy Male. International Journal of Yoga, 11(2), 105-110.

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Oxidative Stress Biomarker Assays

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Lipid peroxidation was determined by the assay of thiobarbituric acid reactive substances as described by Buege and Aust.31 (link) The material was centrifuged for 10 minutes at 13,000 rpm, and the supernatant was read in a spectrophotometer at 535 nm. The carbonyl protein assay was performed by the method described by Levine et al.32 (link) The CAT activity was measured by the decrease rate of H₂O₂ to an absorbance of 240 nm represented by U/mg of protein.33 (link) The SOD activity was measured according to the method described by Marklund and Marklund;34 (link) based on the enzyme ability to inhibit the autoxidation of pyrogallol; the absorbances were read in the ELISA reader at a wavelength of 570 nm. Glutathione is present in the cells mainly in its reduced form (GSH) and its remainder in oxidized form (GSSG). The glutathione assay was adapted from Sigma^® commercial kit # CS0260 and kinetic method was used to measure the levels of glutathione (GSH + GSSH) by the reduction of 5,5′-dithio-bis-2-nitrobenzoic acid in thionitrobenzoic acid. Protein content was analyzed on the samples of tissue homogenate by the Bradford method, and its concentration was represented in mg/mL.35 (link)

Pena K.B., Ramos C.D., Soares N.P., da Silva P.F., Bandeira A.C., Costa G.D., Cangussú S.D., Talvani A, & Bezerra F.S. (2016). The administration of a high refined carbohydrate diet promoted an increase in pulmonary inflammation and oxidative stress in mice exposed to cigarette smoke. International Journal of Chronic Obstructive Pulmonary Disease, 11, 3207-3217.

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Glutathione Concentration Determination

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Glutathione (GSH) concentration was determined using a glutathione assay kit (Sigma, CS0260) and according to manufacturer’s instructions [6 (link)].

Queiroz M., Leandro A., Azul L., Figueirinha A., Seiça R, & Sena C.M. (2021). Luteolin Improves Perivascular Adipose Tissue Profile and Vascular Dysfunction in Goto-Kakizaki Rats. International Journal of Molecular Sciences, 22(24), 13671.

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Myocardial Glutathione and Peroxide

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Total glutathione (GSH) in myocardial homogenates were determined in triplicate with a colorimetric glutathione assay kit (CS0260, Sigma Aldrich) to assess antioxidant levels. Stable hydrogen peroxide (H₂O₂) accumulation was also assessed in myocardial homogenates and subsequently in myocardial cytosol and mitochondria fractions using a colorimetric assay (Sigma Aldrich) (average of two runs with duplicates n=8) using a subcellular protein fractionation kit (78840; Thermo Fisher Scientific) according to manufacturer’s instructions.

Reil J.C., Reil G.H., Kovács Á., Sequeira V., Waddingham M.T., Lodi M., Herwig M., Ghaderi S., Kreusser M.M., Papp Z., Voigt N., Dobrev D., Meyhöfer S., Langer H.F., Maier L.S., Linz D., Mügge A., Hohl M., Steendijk P, & Hamdani N. (2020). CaMKII activity contributes to Homeometric Autoregulation of the heart: a novel mechanism for the Anrep effect. The Journal of physiology, 598(15), 3129-3153.

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Colorimetric Glutathione Quantification

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Total glutathione (GSH) in myocardial homogenates (n = 6 LV sample/group) was determined in triplicate with a colorimetric glutathione assay kit (CS0260, Sigma-Aldrich) according to manufacturer's instructions and as previously described (23 (link)).

Zhazykbayeva S., Hassoun R., Herwig M., Budde H., Kovács Á., Mannherz H.G., El-Battrawy I., Tóth A., Schmidt W.E., Mügge A, & Hamdani N. (2023). Oxidative stress and inflammation distinctly drive molecular mechanisms of diastolic dysfunction and remodeling in female and male heart failure with preserved ejection fraction rats. Frontiers in Cardiovascular Medicine, 10, 1157398.

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