The concentration of NO in the culture medium was determined using the Griess reagent. A volume of 80 μL of the culture media collected after the exposure was pipetted into 96-well plates. A stoichiometric solution (v/v) of 1% sulphanilamide (Sigma-Aldrich, St. Louis, MO, USA) and 0.1% dihydrochloride N-(1-naphthyl) ethylenediamine (Sigma-Aldrich) was prepared just before and it was added to wells in a ratio of 1:1 (v/v). The absorbance of the colored product formed was measured spectrophotometrically at 550 nm at the Flex Station plate reader (Molecular Devices, San Jose, CA, USA), and the NO concentration in the samples was determined by extrapolation on a standard sodium nitrite (Sigma-Aldrich) curve with concentrations between 3125 and 100 μM, which was carried out under the same conditions.
Sulphanilamide
Manufactured by Merck Group
Sourced in United States, Germany, Canada, Italy
Sulphanilamide is a chemical compound that functions as a laboratory reagent. It is a white, crystalline solid used in various analytical and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Phosphoric acid, by Merck Group (26 mentions)
Sodium nitrite, by Merck Group (13 mentions)
N 1 naphthyl ethylenediamine dihydrochloride, by Merck Group (12 mentions)
Ascorbic acid, by Merck Group (10 mentions)
Sodium nitroprusside, by Merck Group (9 mentions)
Naphthylethylenediamine dihydrochloride, by Merck Group (9 mentions)
Quercetin, by Merck Group (9 mentions)
Methanol, by Merck Group (9 mentions)
Hydrogen peroxide, by Merck Group (8 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (8 mentions)
Thiobarbituric acid, by Merck Group (7 mentions)
Bovine serum albumin (bsa), by Merck Group (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
N 1 naphthyl ethylenediamine, by Merck Group (6 mentions)
Trichloroacetic acid, by Merck Group (6 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (6 mentions)
Hydrochloric acid (hcl), by Merck Group (5 mentions)
Ferric chloride, by Merck Group (5 mentions)
Disodium hydrogen phosphate, by Merck Group (5 mentions)
Linoleic acid, by Merck Group (4 mentions)
81 protocols using sulphanilamide
1
Nitric Oxide Quantification in Cell Culture
2
Synthesis and Characterization of NQC Compound
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N′-(Pyridine-3-carbonyl)quinoxaline-2-carbohydrazide (NQC) was synthesised and purified to 99.2% purity in International Medical University (IMU). Structure of the quinoxaline-2-carbohydrazide derivative was confirmed by spectroscopic methods. All solvents used in this research were of high performance column chromatography (HPLC) grade. Chemical reagents including formic acid and dipotassium phosphate (K2HPO4) from Fisher Scientific, dimethyl sulfoxide (DMSO) and monopotassium phosphate (KH2PO4) from Merck, and acetonitrile (ACN) from Friedemann Schmidt were used. (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide), sulphanilamide, N-(1-naphthyl)ethylenediamine and biological grade DMSO were procured from Sigma-Aldrich Sdn. Bhd, Malaysia. β-NADPH was procured from Sigma Aldrich, USA. RAW 264.7 cells and LPS (Escherichia coli O111:b4) were procured from American Type Culture Collection, Manassas, USA. HLM, RLM and MLM (20 mg/mL, Catalog #HMMCPL, Gibco) was procured from Life Technologies, Singapore.
Synthesis of N′-(pyridine-3-carbonyl)quinoxaline-2-carbohydrazide is as shown in Fig.4 .![]()
Synthesis of N′-(pyridine-3-carbonyl)quinoxaline-2-carbohydrazide is as shown in Fig.
Synthetic route of N′-(pyridine-3-carbonyl) quinoxaline-2-carbohydrazide (NQC)
3
Cardiac Stress Biomarker Evaluation
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Isoproterenol (1-(3,4-dihydroxyphenyl)-2-isopropylaminoethanol hydrochloride, molecular weight: 247.72) and TMQ (2-isopropyl-5-methyl-1, 4-benzoquinone; TMQ) (CAS number 490-91-5, molecular formula C10H12O2, molecular weight 164.20, and purity 99%, Figure 1 ) were procured from Sigma Aldrich (St. Louis, MO, USA). The chemicals and reagents including bovine serum albumin (BSA), 5-sulfosalicylic acid (SSA), naphthylene diamine dihydrochloride, sulphanilamide, phosphoric acid, HEPES, sucrose, 1,4-dithiothreitol (DTT), CHAPS, sodium chloride, protease inhibitors, phenyl methyl sulfonyl fluoride (PMSF), Tween-20, sodium nitrate, 3,3,5,5′-tetramethyl benzidine (TMB), and reduced form of glutathione (GSH) assay kit were purchased from Sigma Aldrich. The enzyme linked immunosorbent assay (ELISA) kit was obtained from R&D Systems, USA. The aspartate transaminase (AST), alanine transaminase (ALT), creatine kinase (CK), and lactate dehydrogenase (LDH) kits were procured form Roche Diagnostics, USA.
4
Nitrite Quantification in Cell Supernatants
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NO was determined by measuring nitrites in cell supernatants. Supernatants were combined with an equal volume of sulphanilamide (10 mM; Sigma) and N-1 naphthyletliylenediamine dihydrochloride (10 mM; Sigma) and incubated at room temperature for 5–10 min, and the absorbance was measured at 490 nm in a microplate reader. Nitrite levels were determined based on a standard curve of known concentrations of sodium nitrite.
5
Nitrite Quantification in Macrophages
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NO production was determined as follows. Flounder HK macrophages in a 96-well microplate (~105 cells/well) were incubated with TXhfq, TX01, or TXhfqC (106 CFU/well) for 1 h or 2 h. The supernatants were removed to a separate 96-well plate (50 μL/well), followed by adding into each well 100 μL of 1% sulphanilamide and 100 μL of 0.1% N-naphthylethylene-diamine (Sigma, St. Louis, MO, USA). The plate was read at 540 nm, and the molar concentration of nitrite was determined from standard curve generated using known concentrations of sodium nitrate.
6
Antioxidant Enzyme Activity Assay
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Vitamin C, dimethylsulfoxide (DMSO), sulphanilamide, phosphoric acid (H3PO4), ethylene diamine tetraacetic Acid (EDTA), o-Phthalaldehyde (OPA), and n-ethylmaleimide (NEM) were purchased from Sigma, USA. Commercial kits for the analysis of superoxide dismutase (SOD), xanthine oxydase (XOD) and myeloperoxidase (MPO) were purchased from Nanjing Jiancheng Bioengineering Institute, China. QlAamp DNA Mini Kit was obtained from Qiagen, China. 2×Taq PCR MasterMix for PCR amplication was purchased from CWBIO, China. Primers for PCR amplification were synthesized by Sangon Biotech, Shanghai, China.
7
Plant Cell Culture Optimization
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Murashige and Skoog medium, 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin, 2-(N-morpholino)ethanesulfonic acid (MES), triphenil-tetrazolium chloride (TTC), xylenol orange, EDTA, succinate, 4-morpholinepropanesulfonic acid (MOPS), Polyvinylpyrrolidone (PVP-40), hydroxylamine, sulphanilamide, α-naphthylamine, ampicillin, NP40, safranin, Salicylhydroxamic acid (SHAM), kalium-cyanide (KCN), linoleic acid, fatty acid free bovine serum albumin (BSA), luminol, p-Coumaric acid, Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), were obtained from Sigma-Aldrich. ProBond Purification System was purchased from Invitrogen. Amicon Ultra 30K Centrifugal Filter Units were purchased from Merck. IPTG was obtained from Duchefa Biochemie, cytochrome c was purchased from Fluka. Primary and secondary antibodies were purchased from Agrisera Antibodies. All other chemicals were of analytical or HPLC grade, and were purchased from Reanal, Hungary. Pierce BCA Protein Assay Kit, GeneJET Plant RNA Purification Kit, and RevertAid First-Strand cDNA Synthesis Kit were obtained from Thermo Scientific; SensiFAST SYBR No-ROX Kit was purchased from Bioline.
8
Enzyme-Assisted Extraction and Analysis
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Alcalase and Flavourzyme were purchased from Brentag (Mülheim, Germany). Neutrase was purchased from Novozymes (Bagsværd, Denmark). Acetic acid, ethanol and phosphoric acid were purchased from Merck (Gibbstown, NJ, USA). Acetonitrile (ACN), l -α-amino-n-butyric acid, bovine serum albumin (BSA), budesonide, curcumin from Curcuma longa (turmeric), Coomassie brilliant blue G-250, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), disodium hydrogen phosphate, Dulbecco’s modified Eagle medium (DMEM), foetal bovine serum (FBS), phosphoric acid, formic acid, hydrochloric acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), methanol, monosodium dihydrogen orthophosphate, mouse interferon gamma (IFN-γ), lipopolysaccharides (LPS) from Escherichia coli, potassium persulfate, sodium nitrite, (1-naphthyl)ethylenediamine (NED), sodium nitroprusside (SNP), sodium pyruvate, streptomycin sulphate, sulphanilamide, and trifluoroAcetic acid (TFA) were purchased from Sigma-Aldrich, Merck (St. Louis, MO, USA).
9
Measuring Nitric Oxide Levels in Cell Cultures
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Nitric oxide (NO) was measured by the Griess staining
method. At 48 and 72 hours, we collected the supernatant
(cell culture media) of the 4T1 cells that had been exposed
to different doses of the exosomes (0.1, 0.5 and 1 mg/
ml). We deproteinized 400 μl of supernatant by adding 6
mg of zinc sulfphate (Sigma-Aldrich, Canada). The vials
were centrifuged at 4˚C and 12 000 rpm for 12 minutes.
Then, 100 µl of the supernatant of the deproteinized
samples were added to the wells of the 96-well plate and
100 µl of vanadium chloride (Sigma-Aldrich, Canada),
50 µl of sulphanilamide (Sigma-Aldrich, Canada) and 50
µl of N-(1-Naphthyl) ethylenediamine dihydrochloride
(NEDD, Sigma-Aldrich, Canada) were added to each well.
The plate was incubated for 30 minutes at 37˚C. Next,
100 µl standard sodium nitrate solution at concentrations
of 0, 6, 12.5, 25, 50, 100 and 200 µM, was prepared and
we added vanadium chloride, sulphanilamide and NEDD
to the standard wells, which was similar to the approach
used for the experimental samples. The standards and the
samples were read at 540 and 630 nm with an ELISA
reader (Stat Fax 2100, USA) (18 (link)).
method. At 48 and 72 hours, we collected the supernatant
(cell culture media) of the 4T1 cells that had been exposed
to different doses of the exosomes (0.1, 0.5 and 1 mg/
ml). We deproteinized 400 μl of supernatant by adding 6
mg of zinc sulfphate (Sigma-Aldrich, Canada). The vials
were centrifuged at 4˚C and 12 000 rpm for 12 minutes.
Then, 100 µl of the supernatant of the deproteinized
samples were added to the wells of the 96-well plate and
100 µl of vanadium chloride (Sigma-Aldrich, Canada),
50 µl of sulphanilamide (Sigma-Aldrich, Canada) and 50
µl of N-(1-Naphthyl) ethylenediamine dihydrochloride
(NEDD, Sigma-Aldrich, Canada) were added to each well.
The plate was incubated for 30 minutes at 37˚C. Next,
100 µl standard sodium nitrate solution at concentrations
of 0, 6, 12.5, 25, 50, 100 and 200 µM, was prepared and
we added vanadium chloride, sulphanilamide and NEDD
to the standard wells, which was similar to the approach
used for the experimental samples. The standards and the
samples were read at 540 and 630 nm with an ELISA
reader (Stat Fax 2100, USA) (18 (link)).
10
Nitrite Quantification by Griess Assay
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NO production by astrocyte and microglia cultures was assessed by indirect measurement of nitrite concentration by colorimetric Griess assay in a 96-well plate (Nunc). Initially, 100 μl of the cell supernatant were mixed with the same volume of 58 mM sulphanilamide (Sigma-Aldrich) in 2.5% phosphoric acid and incubated for 10 min. Subsequently, 100 μl of 12 mM N-(1-naphthyl)ethylenediamine (Sigma-Aldrich) in 2.5% phosphoric acid were added and the samples were incubated for 10 min in the dark. The absorbance was then measured by a microplate reader (Tecan Infinite M200) at 550 nm. Standards of sodium nitrite in range of 100–3.125 μM (two-fold serial dilution) were used for calibration of the assay.
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