To determine second order rate constants of reactions with O2•−, each reactant was individually mixed with 50 μM p-nitro blue tetrazolium (NBT), an established O2•− scavenger with a second order rate constant of kNBT = (5.94 ± 0.5) × 104 M−1 s−1 (Bielski and Richter, 1977 ). The reactions were initiated by adding xanthine/xanthine oxidase as a source of O2•−. The ultimate reactions conditions were as follows: 0.1 M Na4P2O7, 100 nM deferoxamine (Sigma), 100 μM xanthine (Sigma), 10 mU xanthine oxidase (Sigma) at pH 8.3. The reaction between O2•− and NBT was monitored spectrophotometrically at 560 nm. The second order rate constants of each reactant was determined as a steady-state approximation by evaluating the reaction when rNBT = rreactant ≡ kNBT[NBT][O2•−] = kreacant[reactant][O2•−]. Therefore, kreacant = kNBT[NBT]/[reactant].
Xanthine oxidase
Manufactured by Merck Group
Sourced in United States, Germany, Japan, Spain, Italy, Sao Tome and Principe, Macao, Chile, Poland, China
Xanthine oxidase is a lab equipment product used for the detection and quantification of xanthine and hypoxanthine levels. It catalyzes the oxidation of xanthine to uric acid, a key step in purine metabolism. The enzyme can be used in various biochemical and analytical applications to measure these purine metabolites.
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Lab products found in correlation
Xanthine, by Merck Group (106 mentions)
Hypoxanthine, by Merck Group (30 mentions)
Allopurinol, by Merck Group (26 mentions)
Cytochrome c, by Merck Group (24 mentions)
Catalase, by Merck Group (21 mentions)
Quercetin, by Merck Group (19 mentions)
Gallic acid, by Merck Group (19 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (19 mentions)
Nitroblue tetrazolium, by Merck Group (18 mentions)
Bovine serum albumin (bsa), by Merck Group (18 mentions)
Ascorbic acid, by Merck Group (15 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (14 mentions)
Hydrogen peroxide, by Merck Group (14 mentions)
Folin ciocalteu reagent, by Merck Group (13 mentions)
Superoxide dismutase, by Merck Group (11 mentions)
Glutathione reductase, by Merck Group (11 mentions)
Methanol, by Merck Group (11 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Reduced glutathione, by Merck Group (8 mentions)
5 5 dithiobis 2 nitrobenzoic acid dtnb, by Merck Group (8 mentions)
219 protocols using xanthine oxidase
1
Determining Second-Order Rate Constants for O2•− Reactions
2
Visualizing Guanine Deaminase Activity in Cells
3
Evaluating Oxidative Stress Sensitivity in P. brasiliensis
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For the phenotypic analysis, we tested the sensitivity of the PbSOD1 and PbSOD3 knockdown strains to conditions inducing oxidative stress. The sensitivity to exogenous ROS of SOD mutants was analyzed using hydrogen peroxide, xanthine oxidase and menadione. Hydrogen peroxide sensitivity was tested using H2O2-saturated filter disks. 1×105P. brasiliensis yeast cells were spread on BHI plates. After inoculation, sterile filter disks were loaded with 20 μl of PBS1X as a control, and 0.5, 1, 2, 4 and 8 M of H2O2 (02194057, MP Biomedicals). Plates were incubated at 36°C with 5% CO2 and after eight days the area lacking P. brasiliensis growth was measured. For xanthine oxidase induced-oxidative stress, cells were incubated in 50 mM Tris pH 8, 100 μM hypoxanthine (H9636, Sigma) and 5 mU/ml xanthine oxidase (X4500, Sigma). Yeasts were incubated for 4 h at 36°C with shaking [20 (link)]. After this time, we plated serial dilutions of the experiment on Kurita’s medium in order to determine viable CFUs. Finally, menadione (M5625, Sigma) sensitiveness was evaluated in a 96-well plate. 1×104P. brasiliensis yeast cells were inoculated in each well, containing 0.5, 5, 10, 20, 40, 80 and 160 μM menadione. Plates were incubated at 36°C during eight days, after incubation time, the plate results were recorded.
4
Multifunctional Nanomaterial Synthesis and Evaluation
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CTAC, TEA, xanthine, and xanthine oxidase (XO) were purchased from Sigma-Aldrich. TEOS and NaOH were obtained from Shanghai Lingfeng Chemical Reagent Co., Ltd. Fe(acac)3 was provided by J&K Scientific. Urea, RhB, iron chloride hexahydrate (FeCl3·6H2O) and 3,3′,5,5′-tetramethyl-benzidine (TMB) were bought from Sinopharm Chemical Reagents Co., Ltd. Methoxy PEG silane was purchased from Shanghai Yare Biotech, Co., Ltd. Gallic acid was acquired from Macklin. Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, and PBS were provided by Gibco. FITC, 4′, 6-diamidino-2-phenylindole (DAPI), DCFH-DA, CCK-8 assay, calcein-AM, PI, annexin V-FITC, H&E, paraformaldehyde (PFA) and TUNEL apoptosis assay kit were acquired from Beyotime. Primary antibody against GPX4 and a biotinylated secondary antibody (anti-rabbit IgG, HRP-linked antibody) were purchased from Cell Signaling Technology.
5
Investigating Cellular Mechanisms in Stress Response
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Pharmacological agents used: angiotensin II (Ang II), chloroquine (CQ), Xanthine oxidase (XO), and rapamycin (Rapa) were purchased from Sigma-Aldrich (St. Louis, MO, United States). Rhodamine phalloidin was acquired from Cytoskeleton (Denver, CO, United States). Mito-TEMPO and 4-PBA were obtained from Sigma-Aldrich (Sigma, St. Louis, MO, United States). Antibodies against LC3A/B, ATG5, p62, mTOR, p-mTOR, ATG5, ATG7, Beclin 1, PERK, XBP1, CHOP, β-actin, and GAPDH were purchased from Cell Signaling Technology (Boston, MA, United States). GRP78, GSK3β, p-GSK3β (Ser9), β-TrcP, and NRF2 were purchased from Abcam (Cambridge, MA, United States). NOX2, NOX4, and HO-1 were provided by Proteintech (Wuhan, China).
6
Oxidative Stress Induction in Placental Explants
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The explants were treated with 2.3 mM xanthine (X) and 0.015 U/mL xanthine oxidase (XO) (Sigma-Aldrich) to induce oxidative stress [8 (link),55 (link)]. Explants were incubated in X/XO in the presence or absence of 1 μg/mL hydroxychloroquine for 48 h at 37 °C in 20% oxygen, 5% CO2. Untreated cultures served as controls. Conditioned media were collected and stored at -80 °C in the presence of 0.005% butylated hydroxytoluene (BHT) (Sigma-Aldrich, St. Louis, Missouri, USA) to prevent autoxidation for activin A and 8-isoprostane assay measurements. Elevated levels of 8-isoprostane are a marker for lipid peroxidation caused by oxidative stress [56 (link)] and, in addition, high levels of activin A have been implicated in the pathway of placental oxidative stress [12 (link)].
7
Lipid Peroxidation Inhibition Assay
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Methanol-d4, tetramethylsilane, quercetin, linoleic acid, soybean 15-lipoxygenase (15-LO), hypoxanthine, xanthine oxidase (XO) from bovine milk, acarbose, 22-S-hydroxycholesterol (22-SHC), 4-nitrophenyl α-d -glucopyranoside (PNP-G), 2-chloro-4-nitrophenyl-α-d -maltotrioside (CNPG3), quercetin and gallic acid were from Sigma-Aldrich (St. Louis, MO, USA), Folin Ciocalteu reagent was from Merck (Darmstadt, Germany). Dulbecco‘s modified Eagle’s medium (DMEM-Glutamax™, 5.5 mM), DMEM, fetal bovine serum, Ultroser G, penicillin–streptomycin–amphotericin B and trypsin-EDTA were obtained from Gibco Life Technologies (Paisley, UK). d -[14C(U)]glucose (1 μCi/mL, 100 μM) was purchased from ARC (American Radiolabeled Chemicals, St. Louis, MO, USA). Corning CellBIND tissue culture plates were obtained from Corning Life-Sciences (Amsterdam, The Netherlands). The protein assay reagent was obtained from BioRad (Copenhagen, Denmark). All other reagents were of the highest purity available. All solvents were of analytical grade and water was purified by a MilliQ system (Millipore, Bedford, MA, USA).
8
Glutathione-Mediated Enzymatic Assay
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NADPH, dimethyl sulfoxide (DMSO), 1-chloro-2,4-dinitrobenzene (CDNB), 5,5′-dithiobis(2-nitrobenzoic) acid (DTNB), reduced glutathione (GSH), oxidized glutathione (GSSG), 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1), xanthine oxidase (XO), and all other chemicals of the highest analytical grade were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
9
Antioxidant Enzyme Evaluation Protocol
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1,1,3,3-Tetramethoxypropane (TMP), L-glutathione reduced, glutathione peroxidase (GPx), glutathione reductase (GR), xanthine, xanthine oxidase (XO), β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium (β-NADPH) salt, APAP, and NAC were purchased from the Sigma Chemical Company, St. Louis, MO, USA. Other chemical reagents were analytical grade and milli-Q water was used all through the experiments.
10
Xanthine Oxidase Activity Assay
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xanthine oxidase (XO) [grade III: from buttermilk, chromatographically purified suspension in 2.3 M (NH4)2SO4, 10 mM sodium phosphate buffer (pH 7.8), containing 1 mM EDTA and 1 mM sodium salicylate], xanthine, and superoxide dismutase were obtained from Sigma (St. Louis, MO). Hydrogen peroxide (H2O2) and FeSO4 were obtained from Wako Chemical (Osaka, Japan). 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was purchased from Labotec (Tokyo, Japan).
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