The rate of superoxide anion release was measured using a standard protocol [31 (link)] and determined as a superoxide dismutase-inhibitable reduction of cytochrome C. In both treated and untreated cells, 100 μl of cytochrome C were added, and in another sample, 100 μL of superoxide dismutase were also added for 30 min in incubator (all substances from Sigma-Aldrich). The absorbance was measured at 550 nm in a spectrometer (VICTORX3 Multilabel Plate Reader) and the O2 was expressed as means ± SD% of nanomoles per reduced cytochrome C per microgram of protein compared to control [35 (link)].
Superoxide dismutase
Manufactured by Merck Group
Sourced in United States, Germany, Italy, United Kingdom
Superoxide dismutase is an enzyme that catalyzes the dismutation of superoxide radicals into oxygen and hydrogen peroxide. It is an important antioxidant defense in nearly all cells exposed to oxygen.
Automatically generated - may contain errors
Lab products found in correlation
Catalase, by Merck Group (39 mentions)
Cytochrome c, by Merck Group (22 mentions)
Xanthine oxidase, by Merck Group (11 mentions)
Nadph, by Merck Group (9 mentions)
Amplex red, by Thermo Fisher Scientific (9 mentions)
Horseradish peroxidase, by Merck Group (8 mentions)
Folin ciocalteu reagent, by Merck Group (7 mentions)
Bovine serum albumin (bsa), by Merck Group (7 mentions)
Isocitrate dehydrogenase, by Merck Group (6 mentions)
Xanthine, by Merck Group (6 mentions)
Nitroblue tetrazolium, by Merck Group (5 mentions)
Gallic acid, by Merck Group (5 mentions)
Nadp na2, by Avantor (4 mentions)
Ammonium acetate, by Merck Group (4 mentions)
Formic acid, by Merck Group (4 mentions)
Ammonium formate, by Merck Group (4 mentions)
Malondialdehyde, by Merck Group (4 mentions)
L name, by Merck Group (4 mentions)
Isocitrate, by Merck Group (4 mentions)
Ascorbic acid, by Merck Group (4 mentions)
125 protocols using superoxide dismutase
1
Superoxide Anion Release Quantification
2
Quantifying Superoxide Anion Release
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Superoxide anion release was quantified using a standard methodology based on cytochrome C reduction [5 (link),32 (link)]. A Tecan spectrophotometer was used to detect the absorbance in culture supernatants at 550 nm after adding 100 μL of cytochrome C (Merck, Milan, Italy) to each well. Comparatively, empty wells were filled with 100 μL of superoxide dismutase (Merck, Milan, Italy) and 100 μL of cytochrome C, and the plate was incubated for 30 min. The O2 rate was quantified as the average standard deviation (%) of nanomoles per decreased cytochrome C per microgram of protein relative to the control (0 line).
3
Quantification of Superoxide Anion Release
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The quantification of superoxide anion release was obtained following a standard protocol based on the reduction in cytochrome C [38 (link)], and the absorbance in culture supernatants was measured at 550 nm using the spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland). Specifically, 100 μL of cytochrome C (Merck, Milan, Italy) was added to all the wells, while 100 μL of superoxide dismutase (Merck, Milan, Italy) and 100 μL of cytochrome C were added to empty wells and the plate was then incubated for 30 min. After that, 100 μL was taken from each well and the absorbance was measured with a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland) at 550 nm. The O2 rate was expressed as the mean ± SD (%) of nanomoles per reduced cytochrome C per microgram of protein compared to the control (0 line).
4
Quantification of Superoxide Anion Release
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The quantification of superoxide anion release was obtained following a standard protocol based on the reduction in cytochrome C [51 (link)], and the absorbance in culture supernatants was measured at 550 nm using the spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland). Specifically, 100 μL of cytochrome C (Merck Life Science, Rome, Italy) was added to all the wells, while 100 μL of superoxide dismutase (Merck Life Science, Rome, Italy) and 100 μL of cytochrome C were added to empty wells and the plate was then incubated for 30 min. After that, 100 μL was taken from each well and the absorbance was measured with a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland) at 550 nm. The O2 rate was expressed as the mean ± SD (%) of nanomoles per reduced cytochrome C per microgram of protein compared to the control (0 line).
5
Synthesis and Characterization of Novel 2-Oxo-PCX Hydrochlorides
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2-Oxo-PCcP hydrochloride, 2-oxo-PCE hydrochloride, 2-oxo-PCiP hydrochloride, 2-oxo-PCMe hydrochloride, and 2-oxo-PCPr were synthesized by the standard methods and analytically characterized by 1H and 13C nuclear magnetic resonance (NMR), high-performance liquid chromatography (HPLC) (>95% pure on HPLC and NMR), and high-resolution mass spectrometry (HRMS.) Acetonitrile (ACN), ammonium formate, dichloromethane (DCM), ethyl acetate, hydrochloric acid (HCl), isopropanol, methanol (MeOH), sodium hydroxide solution (NaOH), and all other chemicals were obtained from VWR International GmbH (Darmstadt, Germany). Diazepam-d5 was obtained from LGC Standards GmbH (Wesel, Germany). NADP+ was obtained from Biomol (Hamburg, Germany), superoxide dismutase, magnesium chloride (MgCl2), isocitrate, and isocitrate dehydrogenase were from Merck (Darmstadt, Germany). pHLMs (20 mg microsomal protein/mL, 330 pmol total CYP/mg protein) were obtained from Corning (Amsterdam, the Netherlands). All enzyme-containing preparations were thawed at 37 °C after delivery, aliquoted, snap frozen in liquid nitrogen and stored at −80 °C until use. Water was purified with a Millipore (Merck, Darmstadt, Germany) filtration unit, which purifies water to a resistance of 18.2 Ω × cm. Isolute HCX solid-phase extraction (SPE) cartridges (130 mg, 3 mL) were obtained from Biotage (Uppsala, Sweden).
6
Quantitative PCYP Metabolic Analysis
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PCYP hydrochloride was provided by the State Bureau of Criminal Investigation Schleswig-Holstein (E.U. project ADEBAR plus, Kiel, Germany) for research purposes. The chemical purity of >93% and the identity of the compound was verified by MS and nuclear magnetic resonance analysis. Ammonium formate, ammonium acetate, creatinine-d3, dipotassium phosphate, formic acid, D-glucose-1,2,3,4,5,6,6-d7, isocitrate dehydrogenase, isocitrate, magnesium chloride, palmitic acid-d31, superoxide dismutase, and tripotassium phosphate were obtained from Merck (Darmstadt, Germany). Acetonitrile, ethanol, methanol (all LC-MS grade), and NADP-Na2 were from VWR (Darmstadt, Germany). L-Tryptophan-d5 was obtained from Alsachim (Illkirch-Graffenstaden, France). 1-Palmitoyl-d9-2-palmitoyl-sn-glycero-3-PC and prostaglandin-E3-d9 were from Cayman Chemical (Ann Arbor, MI, USA). Water was purified with a millipore filtration unit (18.2 W × cm water resistance). pHLMs (20 mg microsomal protein × mL−1, 360 pmol total CYP/mg, 26 donors) were obtained from Corning (Amsterdam, The Netherlands). After delivery, pHLMs were thawed at 37 °C, aliquoted, snap-frozen in liquid nitrogen, and stored at −80 °C until use.
7
Neurochemical Markers of Oxidative Stress
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Trimethyltin chloride, dimethyl sulfoxide (DMSO), paraformaldehyde (PFA), malondialdehyde (MDA), hydrogen peroxide, superoxide dismutase (SOD), sodium dodecyl sulfate, trichloroacetic acid, epinephrine, ethylenediamine tetraacetic acid (EDTA), ethanol, xylene, cresyl violet and luxol fast blue were purchased from Merck, Millipore.
8
In Vitro Metabolism of Benzylguanine and Zaleplon
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O 6 -benzylguanine (O6BG), its metabolite, 8-oxo-benzylguanine, and zaleplon were purchased from Toronto Research Chemicals (North York, ON). N-(2-dimethylamino)ethyl)acridine-4-carboxamide (DACA) and its metabolite (DACA-acridone) were synthesized as described previously 23 . Phenacetin as the internal standard, catalase, phthalazine, 1-phthalazinone, chlorzoxazone (CZN), and 3,4-Methylenedioxymethamphetamine (MDMA) were purchased from Sigma-Aldrich (St. Louis, MO). 5-oxozaleplon was biosynthesized and puri ed as previously described 23 . Dihydroxymethamphetamine (DHMA) was purchased from Cayman Chemical (Ann Arbor, MI). CYP2D6 BACULOSOMES® Plus Reagents and tris(2-carboxyethyl) phosphine were purchased from Thermo Fisher (Waltham, MA). Formic acid was purchased from Fischer Scienti c (Pittsburgh, PA). Acetic acid, acetonitrile, superoxide dismutase, and NADPH were purchased from EMD Millipore Corp. (Billerica, MA) and EMD Chemicals (San Diego, CA), respectively. The monobasic potassium phosphate, dibasic potassium phosphate, and EDTA used for the potassium phosphate buffer were purchased from JT Baker (Center Valley, PA).
Human CYP 2E1 Supersomes TM was purchased from BD Gentest (Franklin Lakes, NJ). Expression and puri cation of recombinant human aldehyde oxidase (hAO) was performed following the method previously described 23, 24 .
Human CYP 2E1 Supersomes TM was purchased from BD Gentest (Franklin Lakes, NJ). Expression and puri cation of recombinant human aldehyde oxidase (hAO) was performed following the method previously described 23, 24 .
9
Evaluating Neutrophil Activation Pathways
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Bisphenol A (BPA), Superoxide Dismutase from bovine erythrocytes, Cytochrome C from equine heart, Griess reagent, nitroblue tetrazolium (NBT), N-Formyl-Met-Leu-Phe (fMLP) and Latex were from Merck Millipore, Burlington, USA. May-Grunwald and Giemsa were from Aqua-med; Polimorphoprep™ (Axis Shield), PBS with or without ions of CaCl
10
Antioxidant Enzyme Activity Assay
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All enzymes and all other reagents were purchased from Merck KGaA. Other chemicals were prepared analytically. Superoxide dismutase (from bovine erythrocytes, 300 KU, lyophilized powder, ≥3.000units/mg protein), catalase (from bovine liver, lyophilized powder, ≥10.000 units/mg protein), Glutation peroksidaz (from bovine erythrocytes, 300 KU, lyophilized powder, White, ≥100 units/mg), Glutathion reductase (from baker's yeast (S. cerevisiae), 2.5 KU, ammonium sulphate suspension, 100-300 units/mg protein (biuret)), Malathion
Pralidoxime [2-pyridine aldoxime methyl chloride] were purchased form Merck KGaA. All pesticides were prepared in ethanol. The chemical structures of the pesticides and Pralidoxime (PAM) were shown at Table 1. circulating water bath (core IC 302, Turkey), magnetic stirrer (Biosan magnetic stirrer MSH 300) was used. Glass and disposable polypropylene or polystyrene plastics tend to absorb light in the UV range so that 96-well quartz plates were used in the UV range. For obtaining good results, using the appropriate microplate for UV region is more important (Held 2001) .
Pralidoxime [2-pyridine aldoxime methyl chloride] were purchased form Merck KGaA. All pesticides were prepared in ethanol. The chemical structures of the pesticides and Pralidoxime (PAM) were shown at Table 1. circulating water bath (core IC 302, Turkey), magnetic stirrer (Biosan magnetic stirrer MSH 300) was used. Glass and disposable polypropylene or polystyrene plastics tend to absorb light in the UV range so that 96-well quartz plates were used in the UV range. For obtaining good results, using the appropriate microplate for UV region is more important (Held 2001) .
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