NADPH oxidase activity was measured both in retina and RPE/choroid homogenates by lucigenin-enhanced chemiluminescence, following routine protocols in our laboratory [43 (link)]. To confirm the source(s) of superoxide anion (O2•−), homogenate samples were preincubated for 5 min at 37 °C with the following inhibitors at 0.1 mmol/L: diphenyleneiodonium, DPI (inhibitor of flavoproteins; Sigma-Aldrich, Madrid, Spain); oxypurinol (inhibitor of xanthine oxidase; Sigma-Aldrich, Madrid, Spain); and rotenone (mitochondrial chain inhibitor of electron transport; Sigma-Aldrich, Madrid, Spain). Following the same protocol, the inhibitor of NOX1/4 (0.1 µmol/L GKT136901; Sigma-Aldrich, Madrid, Spain, 492000), specific NOX1 inhibitor (0.5 µmol/L ML171; Sigma-Aldrich, Madrid, Spain, 175226) and the pan-NADPH oxidase inhibitor (10 µmol/L VAS2870; Sigma-Aldrich, Madrid, Spain, 5340320001) were used to explore the relative contribution of each NOX isoform in O2•− production [35 (link)]. Hydrogen peroxide (H2O2) levels were measured in retina homogenates by AmplexTM Red hydrogen peroxide/peroxidase assay kit (A22188, ThermoFisher Scientific, Invitrogen, Spain) following the manufacturer’s instructions. Absorbance readings were obtained in 96-well plates at 560 nm. All measurements referred to the samples’ protein content, and results were always expressed as relative to the control group.
Oxypurinol
Manufactured by Merck Group
Sourced in United States, Spain
Oxypurinol is a laboratory reagent manufactured by Merck Group. It is a white crystalline powder used for various analytical and research applications. Oxypurinol serves as a reference standard and is commonly employed in analytical techniques such as chromatography and spectroscopy. Its core function is to provide a reliable and consistent chemical compound for laboratory analyses and research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Rotenone, by Merck Group (4 mentions)
Vas2870, by Merck Group (3 mentions)
Ml171, by Merck Group (3 mentions)
Allopurinol, by Merck Group (3 mentions)
Gkt136901, by Merck Group (2 mentions)
Wst 1 cell proliferation and cytotoxicity assay kit, by Beyotime (1 mentions)
C57bl 6 mice, by CLEA Japan (1 mentions)
Syto13, by Thermo Fisher Scientific (1 mentions)
Secondary alexa fluor antibodies 488 and 546, by Thermo Fisher Scientific (1 mentions)
2 7 dichlorodihydrofluorescein diacetate h2dcfda, by Thermo Fisher Scientific (1 mentions)
Anti vegf, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Merck Group (1 mentions)
Anti enos antibody af950, by R&D Systems (1 mentions)
Noc 18, by Santa Cruz Biotechnology (1 mentions)
Sodium nitrite nano2, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Human thrombin, by Merck Group (1 mentions)
Gly pro arg pro, by Merck Group (1 mentions)
Ng nitro l arginine methyl ester l name, by Merck Group (1 mentions)
Adenosine 5 diphosphate adp, by Merck Group (1 mentions)
15 protocols using oxypurinol
1
Measuring NADPH Oxidase Activity in Retina and RPE/Choroid
2
WST-1 Assay for Oxypurinol-Mediated HCC Proliferation
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A WST-1 Cell Proliferation and Cytotoxicity Assay Kit (Beyotime Institute of Biotechnology, China) was used to detect HCC cell proliferation, as described in our previous report.52 (link) In brief, the abovementioned cells were seeded in 96-well culture plates at a density of 2000 cells/well. To evaluate the effects of oxypurinol (Sigma-Aldrich, Co. LLC., Shanghai, China, cat. no. O6881), a potent xanthine oxidase inhibitor,53 (link) on cell proliferation, we incubated the cells with or without 50 μmol/l (μM ) oxypurinol. Cell proliferation was monitored over a 72-h time period and measured according to the manufacturer’s instruction. All experiments were performed at least three times and in triplicate.
3
Febuxostat and Xanthine Oxidase Inhibition
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Six- to nine-week-old male C57BL/6 mice (CLEA Japan Inc., Tokyo), weighing 19–22 g, were allowed free access to food and water before the experiments. All experimental procedures in this study were approved by the institutional animal care committee of Nippon Medical School. Extreme care was taken throughout the study to minimize the pain and discomfort to the animals.
Febuxostat, 2-[3-cyano-4-(2-methylpropoxy) phenyl]-4-methyl-5-thiazolecarboxylic acid, was obtained from Carbosynth Ltd. (Berkshire, UK). Allopurinol [4-hydroxypyrazolo(3,4-d)pyrimidine] and oxypurinol [4,6-dihydroxypyrazolo(3,4-d) pyrimidine] were obtained from Sigma-Aldrich Co. LLC (St. Louis, Missouri, USA). All other chemicals and reagents were of reagent grade or equivalent and were purchased from Wako Pure Chemical Industries, Ltd., Osaka.
Febuxostat, 2-[3-cyano-4-(2-methylpropoxy) phenyl]-4-methyl-5-thiazolecarboxylic acid, was obtained from Carbosynth Ltd. (Berkshire, UK). Allopurinol [4-hydroxypyrazolo(3,4-d)pyrimidine] and oxypurinol [4,6-dihydroxypyrazolo(3,4-d) pyrimidine] were obtained from Sigma-Aldrich Co. LLC (St. Louis, Missouri, USA). All other chemicals and reagents were of reagent grade or equivalent and were purchased from Wako Pure Chemical Industries, Ltd., Osaka.
4
Immunohistochemical and Biochemical Assays
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Anti-eNOS (ab5589) and anti-iNOS (ab3523) used for immunohistochemistry were from Abcam (Paris, France). Anti-eNOS antibody (AF950) used for immunoprecipitation experiments was from R&D Systems (Bio-Techne, France). Anti-glutathione antibody recognizing GS-S-proteins was from Virogen (Watertown, MA, USA). Secondary antibodies anti-mouse and anti-rabbit HRP-conjugated were from Cell Signaling Technology (Ozyme, France). Anti-Von Willebrand Factor (VWF) (AB7356) was from Chemicon (Merck Millipore) and anti-VEGF was from Sigma. Secondary anti-goat HRP-conjugated was purchased from Southern Biotech (Clinisciences, France). Secondary Alexa Fluor antibodies (488 and 546) were from Life Technologies (Courtaboeuf, France). Dihydroethidine (DHE), DAF-FM diacetate (4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate), dithiotreitol (DTT), 4,6-Diamidino 2-phenylindole dihydrochloride (DAPI), oxypurinol, VAS2870, L-NAME (N-Nitro-L -arginine methyl ester hydrochloride), BH4 (tetrahydrobiopterin dihydrochloride) were from Sigma-Aldrich (Saint Quentin Fallavier, France). 2′,7′-Dichlorodihydrofluorescein diacetate (H2DCFDA) and SYTO-13 were from Thermofisher (Villebon sur Yvette, France), NOC-18 (diethylenetriamine/nitric oxide adduct; DETA NONOate), was from Santa Cruz Biotechnology (Clinisciences, France).
5
Platelet Activation Assay Protocol
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Sodium nitrite (NaNO2), adenosine 5′ diphosphate (ADP), collagen, human thrombin, NG-Nitro-L-Arginine Methyl Ester (L-NAME), Gly-Pro-Arg-Pro (GPRP), oxypurinol and paraformaldehyde were purchased from Sigma (St.Louis, MO). Diethylamine diazeniumdiolate (DEANONOate) was purchased from Cayman chemical (Ann Arbor, MI), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (C-PTIO) was purchased from Alexis (Lausen, Switzerland). FITC-labeled PAC1, PE-labeled anti-human CD62P, PE-Cy5 anti-human CD41a and FITC- or PE-conjugated isotypic MoAbs, and flow cytometry graded PBS were purchased from Becton Dickinson (BD; San Jose, CA).
6
Oxidative Stress Measurements Protocols
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Potassium phosphate monobasic (KH2PO4), dl -dithiothreitol (DTT), citric acid, diethylenetriaminepentaacetic
acid (DTPA), oxypurinol, menadione (vitamin K3), and N-acetyl-l -cysteine (NAC) were purchased from Sigma-Aldrich
(St. Louis, MO). Acetonitrile (ACN), ammonium acetate, methanol, an
iodine solution, and potassium iodide were purchased from Thermo-Fisher.
Octyl sulfate sodium salt (OSA) was purchased from Acros Organics
(Waltham, MA). 7,8-Dihydro-l -biopterin (BH2),
dihydroxanthopterin (XH2), pterin (P), isoxanthopterin
(IX), xanthopterin (XP), andl -biopterin (B) were purchased
from Schircks Laboratories (Jone, Switzerland). (6R)-l -Tetrahydrobiopterin (BH4), l -NG-nitroarginine methyl ester (l -NAME),
and Mn(III)TBAP were purchased from Cayman Chemical (Ann Arbor, MI).
Dihydroethidium (DHE) and 4′,6-diamidino-2-phenylindole dihydrochloride
(DAPI) were purchased from Invitrogen (Carlsbad, CA). Anti-NOS3 antibodies
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The
anti-glutathione antibody was purchased from Virogen (Boston, MA).
The superoxide probe tetrathiatriarylmethyl (TAM) radical with a single
aromatic hydrogen (CT02-H) was synthesized in house as reported previously.43 ,44 (link)
acid (DTPA), oxypurinol, menadione (vitamin K3), and N-acetyl-
(St. Louis, MO). Acetonitrile (ACN), ammonium acetate, methanol, an
iodine solution, and potassium iodide were purchased from Thermo-Fisher.
Octyl sulfate sodium salt (OSA) was purchased from Acros Organics
(Waltham, MA). 7,8-Dihydro-
dihydroxanthopterin (XH2), pterin (P), isoxanthopterin
(IX), xanthopterin (XP), and
from Schircks Laboratories (Jone, Switzerland). (6R)-
and Mn(III)TBAP were purchased from Cayman Chemical (Ann Arbor, MI).
Dihydroethidium (DHE) and 4′,6-diamidino-2-phenylindole dihydrochloride
(DAPI) were purchased from Invitrogen (Carlsbad, CA). Anti-NOS3 antibodies
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The
anti-glutathione antibody was purchased from Virogen (Boston, MA).
The superoxide probe tetrathiatriarylmethyl (TAM) radical with a single
aromatic hydrogen (CT02-H) was synthesized in house as reported previously.43 ,44 (link)
7
Corneal NADPH Oxidase Activity Assay
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NADPH oxidase activity was measured in cornea homogenates by lucigenin-enhanced chemiluminescence, following routine protocols in our laboratory [48 (link)]. Potential sources of superoxide anion (O2.−) production in corneal samples were discriminated at 37 °C after a 5-min preincubation with 0.1 mmol/L DPI, oxypurinol, or rotenone (respective inhibitors of flavoproteins, xanthine oxidase, and mitochondrial electron transport chain; Sigma-Aldrich, Madrid, Spain). Similar protocols were followed when using an inhibitor of NOX1/4 (0.1 µmol/L GKT136901; Sigma-Aldrich, 492,000), specific NOX1 inhibitor (0.5 µmol/L ML171; Sigma-Aldrich, 175,226), and the pan-NADPH oxidase inhibitor (10 µmol/L VAS2870; Sigma-Aldrich, 5,340,320,001) to determine the relative contribution of each NOX isoform in total O2.− production. All measurements were referred to the samples’ protein content, and results were always expressed as relative to those in the control group.
8
Genetic Manipulation and Pharmacological Assays in HepG2 Cells
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The E. coli DHB10 strain containing the Gateway ptREX-DEST30 vector with the cDNA encoding XO (BC166696) was from ImgaGenes (Berlin, Germany) and was propagated and enriched according to the manufacturer’s instructions. Plasmids were isolated with the S.N.A.P Plasmid DNA Midi kit (Life Technologies, Carlsbad, CA, USA).
Fetal calf serum (FCS), Lipofectamine 2000, and Opti-MEM were from Life Technologies. Pencillin-streptomycin, 6MP, AP, and oxypurinol were from Sigma Aldrich (St Louis, MO, USA).
HepG2 cells (ATCC® HB-8065, LGC standards, Teddington, UK) were maintained in Eagle’s minimum essential medium (LGC standards) supplemented with 10% FCS, and penicillin-streptomycin (100 U mL-1 resp. 100 μg mL-1) at 37°C in a humidified atmosphere with 5% CO2. Cells were grown in 6-well trays (0.2x106 cells per well) overnight in medium without antibiotics before experiments were started. Thereafter 2 μg plasmid was mixed with Optimem and Lipofectamine 2000 and transfection was performed according to the manufacturer´s instructions. Cells not transfected to express XO were not MOCK-transfected as comparisons were made within each condition (i.e. +/-XO). Drugs [6MP (6 μM), AP (100 μM) or the combination of 6MP+AP] were dissolved in 0.1 M NaOH, diluted in growth medium and added to the cell cultures grown overnight. Control cultures received the same concentration of solvent.
Fetal calf serum (FCS), Lipofectamine 2000, and Opti-MEM were from Life Technologies. Pencillin-streptomycin, 6MP, AP, and oxypurinol were from Sigma Aldrich (St Louis, MO, USA).
HepG2 cells (ATCC® HB-8065, LGC standards, Teddington, UK) were maintained in Eagle’s minimum essential medium (LGC standards) supplemented with 10% FCS, and penicillin-streptomycin (100 U mL-1 resp. 100 μg mL-1) at 37°C in a humidified atmosphere with 5% CO2. Cells were grown in 6-well trays (0.2x106 cells per well) overnight in medium without antibiotics before experiments were started. Thereafter 2 μg plasmid was mixed with Optimem and Lipofectamine 2000 and transfection was performed according to the manufacturer´s instructions. Cells not transfected to express XO were not MOCK-transfected as comparisons were made within each condition (i.e. +/-XO). Drugs [6MP (6 μM), AP (100 μM) or the combination of 6MP+AP] were dissolved in 0.1 M NaOH, diluted in growth medium and added to the cell cultures grown overnight. Control cultures received the same concentration of solvent.
9
Antioxidant Activity Assay Protocol
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xanthine oxidase from bovine milk, luminol sodium salt, xanthine, oxypurinol, PBS tabs, Na-EDTA salt, gelatin from bovine skin, penicillin/streptomycin, trypsin-EDTA, Trolox, and 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium perborate, boric acid, NaOH, and FeCl2 were from Carlo Erba (Milan, Italy). M200 medium, low serum growth supplements, and fetal bovine serum, RNaseOUT, were purchased from Thermo Fisher Scientific (Waltham, MA, USA). RNeasy Mini Kit was from QIAGEN (Hilden, Germany). Primers for RT-PCR were purchased from IDT (Coralville, IA, USA). Cell counting kit-8 (CCK8) and LDH assay kit were purchased from Dojindo Molecular Technologies (Rockville, MD, USA). SuperScript® III First-Strand Synthesis SuperMix and EXPRESS SYBR® GreenER™ qPCR SuperMix were purchased from Life Technologies (Carlsbad, CA, USA). All the other chemicals and solvents were of the highest analytical grade.
10
Synthetic Cyadox Compounds and Assays
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CYA, bisdesoxycyadox (Cy1), cyadox 1-monoxide (Cy2) and cyadox 4-monoxide (Cy10) (Fig 1 ) were synthesized by the Institute of Veterinary Pharmaceuticals (HZAU) (Wuhan, Hubei, China). OLA was obtained from the China Institute of Veterinary Drug Control (Beijing, China). MEQ was purchased from Beijing Zhongnongfa Pharmaceutical Co. Ltd. (Huanggang, Huibei, China). An ROS assay kit and dihydroethidium were purchased from Beyotime (Shenzhen, China). 3'-(p-hydroxyphenyl) fluorescein (HPF) and the Trizol Reagent were bought from Invitrogen (Carlsbad, CA, USA). Ciprofloxacin, carbenicillin, enrofloxacin, gentamycin, tirapazamine (TPZ), sodium 4,5-dihydroxybenzene-1,3-disulfonate (tiron), β-mercaptoethanol (β-ME), xanthine oxidase of E. coli K12, xanthine, oxypurinol, 4-methylpyrazole, and raloxifene were purchased from Sigma (St Louis, MO, USA). 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO), N-tert-butyl-alpha-phenylnitrone (PBN) and 2,2-dipyridyl were obtained from Fluka (Buchs, Switzerland). Auranofin and dicoumarol were purchased from J&K Chemical Company (Shanghai, China). Oxonic acid was obtained from Tokyo Chemical Industry (Shanghai, China). Rotenone and dimercaprol were purchased from Aladdin (Shanghai, China). All other chemicals and reagents commercially available were of the highest analytical grade.
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