Inducible nitric oxide enzymatic activity in RAW264.7 cells was determined as previously described (Babcock et al., 2013 ; Matsebatlela et al., 2015 ). In brief, cells were plated at 105 cells/well in 96-well plates. After treating with LPS or sediment extracts to a total of 200 ul, cells were incubated for 24 h. One hundred μl of supernatants from each well were then pipetted into a new 96-well plate along with 100 ul of Griess reagent added. Griess reagent is a 1:1 ratio of (N-(1-naphthyl) ethylenediamine dihydrochloride; (0.1 g NED (Sigma) in 100 of dH2O), and sulfanilamide (1.0 g sulfanilamide (Sigma) in 100 of 5% phosphoric acid) (Dietert, 1995 ). A NaNO2 solution in dH2O was used to establish a standard curve. Under these conditions LPS-stimulated RAW264.7 macrophages typically produce ≈ 30 ug/mL NO2 following 24 h stimulation. ELISA assays to monitor the effects of treatment on the secretion of cytokines in the remaining supernatants per well were performed using ELISA MAX™ Deluxe Sets for mouse IL-10 and IL-6 (BioLegend). Protocols followed exact instructions provided by the commercial kits.
Sulfanilamide
Manufactured by Merck Group
Sourced in United States, Germany, India, Sao Tome and Principe, United Kingdom, Japan, Portugal, Australia, Switzerland, Spain
Sulfanilamide is a chemical compound used in laboratory settings. It is a white, crystalline powder with a melting point of 165-167°C. Sulfanilamide is a sulfonamide drug that has been used as a bacteriostatic agent. Its core function is to inhibit the growth and reproduction of certain bacteria.
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Lab products found in correlation
N 1 naphthyl ethylenediamine dihydrochloride, by Merck Group (61 mentions)
Phosphoric acid, by Merck Group (50 mentions)
Sodium nitrite, by Merck Group (43 mentions)
Naphthylethylenediamine dihydrochloride, by Merck Group (31 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (31 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (24 mentions)
Thiobarbituric acid, by Merck Group (19 mentions)
N 1 naphthyl ethylenediamine, by Merck Group (19 mentions)
Trichloroacetic acid, by Merck Group (17 mentions)
Bovine serum albumin (bsa), by Merck Group (15 mentions)
H3po4, by Merck Group (13 mentions)
Sodium nitroprusside, by Merck Group (12 mentions)
Ascorbic acid, by Merck Group (12 mentions)
Gallic acid, by Merck Group (12 mentions)
N 1 napthylethylenediamine dihydrochloride, by Merck Group (11 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (10 mentions)
Quercetin, by Merck Group (10 mentions)
Griess reagent, by Merck Group (10 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (10 mentions)
Lipopolysaccharide, by Merck Group (9 mentions)
269 protocols using sulfanilamide
1
Nitric Oxide Induction and Cytokine Release in RAW264.7 Cells
2
Quantifying Nitrite Accumulation in Microglia
3
Colorimetric Assay for Nitrite Quantification
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Accumulation of nitrite in microglial
conditioned media was measured by a colorimetric assay based on the
Griess reaction. A nitrate standard curve was performed with NaNO2 at known concentrations. Sulfanilamide (5 mM; Sigma-Aldrich)
was added to the culture medium, and the standard curve was obtained.
Sulfanilamide reacts with nitrite under acidic conditions to form
a diazonium cation, which subsequently couples to N-naphthyl-ethylenediamine dihydrochloride (40 mM; Sigma-Aldrich)
to produce a colored azo dye. After 15 min of incubation at room temperature
in the dark, absorbance was read at 540 nm with a multiplate spectrophotometric
reader (BioRad Laboratories Srl, Segrate, Milano, Italy).
conditioned media was measured by a colorimetric assay based on the
Griess reaction. A nitrate standard curve was performed with NaNO2 at known concentrations. Sulfanilamide (5 mM; Sigma-Aldrich)
was added to the culture medium, and the standard curve was obtained.
Sulfanilamide reacts with nitrite under acidic conditions to form
a diazonium cation, which subsequently couples to N-naphthyl-ethylenediamine dihydrochloride (40 mM; Sigma-Aldrich)
to produce a colored azo dye. After 15 min of incubation at room temperature
in the dark, absorbance was read at 540 nm with a multiplate spectrophotometric
reader (BioRad Laboratories Srl, Segrate, Milano, Italy).
4
Anti-inflammatory Effects of IMP
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IMP was isolated from Notopterygium incisum. Lipopolysaccharides (LPS), Dimethyl sulfoxide (DMSO), Dexamethasone (Dexa), Sodium nitrite, Paraformaldehyde, Bovine serum albumin (BSA), sulfanilamide, Phosphoric acid, N-1-naphthyl ethylenediamine dihydrochloride, Sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent (MTT), N-1-naphthyl ethylenediamine dihydrochloride, sulfanilamide, Phosphoric acid, Fetal bovine serum (FBS), Penicillin, and streptomycin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Gibco (Grand Island, NY, USA). ELISA Kits of IL-1β, IL-6, TNF-α, TRIzol™ Reagent, SuperScript™ II Reverse Transcriptase, RNaseOUT™ Recombinant RNase Inhibitor, Hoechst 33258, Alexa Fluor 488, and Alexa Fluor 594 were purchased from Invitrogen (Carlsbad, CA, USA). Primary antibody anti-Akt, p-Akt, Nrf2, SOD1, SOD2, CAT, and HO-1 were purchased from Cell Signaling (Beverly, MA, USA). PGE2 ELISA kit was purchased from Cayman (Ann Arbor, MI, USA). Enhanced chemiluminescence (ECL) was purchased from Perkin Elmer (Waltham, MA, USA). IRdye-labeled NF-κB oligonucleotide was purchased from LI-COR Biosciences (Lincoln, NE, USA).
5
Optimizing Noncovalent Complex Detection
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All experiments were
performed under the optimum conditions found for the detection of
noncovalent complexes on a Bruker Solarix 12 T electrospray ionization
source Fourier transform mass spectrometer. Mass spectra were recorded
in the positive ion mode with a mass range from 50 to 6000 m/z for broadband low-resolution acquisition.
Each spectrum was an average of 32 transients (scans) composed of
1 or 2 megabyte data points. All aspects of pulse sequence control
and data acquisition were controlled by Solarix control software.
A bovine carbonic anhydrase II (bCAII) sulfanilamide complex was used
as a control for optimization.26 −28 (link) bCAII (29 089 Da, Sigma-Aldrich)
was dissolved in ammonium acetate (10 mM, pH 7) to generate a stock
solution (34 μM). sulfanilamide (172 Da, Sigma-Aldrich) was
dissolved in methanol to make a stock solution (5.8 mM). bCAII (100
μL, 3.4 μM) in ammonium acetate (10 mM) was mixed with
the sulfanilamide (10 μL, 581 μM) and incubated for 1
h at room temperature (20 °C). The inhibitor/protein ratio was
16:1. TRAP SBPs were buffer-exchanged to 10 mM ammonium acetate, pH
7, using size-exclusion chromatography prior to analysis. The Δm/z for the unbound protein and the protein–ligand
complex ions was multiplied by the charge state (z) to directly afford the MW of the bound ligand, using the following
equation: MWligand = Δm/z × z.
performed under the optimum conditions found for the detection of
noncovalent complexes on a Bruker Solarix 12 T electrospray ionization
source Fourier transform mass spectrometer. Mass spectra were recorded
in the positive ion mode with a mass range from 50 to 6000 m/z for broadband low-resolution acquisition.
Each spectrum was an average of 32 transients (scans) composed of
1 or 2 megabyte data points. All aspects of pulse sequence control
and data acquisition were controlled by Solarix control software.
A bovine carbonic anhydrase II (bCAII) sulfanilamide complex was used
as a control for optimization.26 −28 (link) bCAII (29 089 Da, Sigma-Aldrich)
was dissolved in ammonium acetate (10 mM, pH 7) to generate a stock
solution (34 μM). sulfanilamide (172 Da, Sigma-Aldrich) was
dissolved in methanol to make a stock solution (5.8 mM). bCAII (100
μL, 3.4 μM) in ammonium acetate (10 mM) was mixed with
the sulfanilamide (10 μL, 581 μM) and incubated for 1
h at room temperature (20 °C). The inhibitor/protein ratio was
16:1. TRAP SBPs were buffer-exchanged to 10 mM ammonium acetate, pH
7, using size-exclusion chromatography prior to analysis. The Δm/z for the unbound protein and the protein–ligand
complex ions was multiplied by the charge state (z) to directly afford the MW of the bound ligand, using the following
equation: MWligand = Δm/z × z.
6
Nitrite Quantification via Griess Assay
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For indirect measurement of NOS activity, nitrite concentration was measured using Griess protocol. Briefly, serum proteins were precipitated using ZnSO4 (Merck), and then the reduction of nitrate to nitrite was performed by saturated solutions of VCl3 (Vanadium Chloride) (0.8% w/v) in 1 M HCl (Merck) and then the color generating solution containing N-L(naphthyl) ethylenenndiamine dihydrochloride (NEDD; 0.1% w/v, Merck) in H2O and sulfanilamide (2% w/v, Merck) in 5% HCl (Griess reagents) were added to the reaction vessels. After 20 min of incubation at room temperature, the absorbance was read in 540 nm (21 (link)) using spectrophotometer.
7
Antioxidant and Cytotoxicity Assays
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2, 2-Diphenyl-1-picrylhydrazyl (DPPH), trichloroacetic acid (TCA), 2,4-dinitrophenyl hydrazine (DNPH), 2,4,6-Tripyridyl-s-triazine (TPTZ), ferric chloride (FeCl3), nitroblue tetrazolium (NBT), nicotinamide adenine dinucleotide (NADH), phenazine metho-sulfate (PMS), Dulbecco's modified Eagle medium (DMEM), (3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), sulfanilamide, N-(1-Naphthyl) ethylenediamine (NED), 2’,7’-dichlorofluorescein diacetate (DCFDA), lipopolysaccharide (LPS), and all other chemicals used were purchased from Merck, HiMedia, and Sigma-Aldrich. The high-performance-thin-layer chromatography (HP-TLC) plates Si 60F254 (20 * 10) were purchased from E. Merck (Darmstadt, Germany).
8
Electrochemical Characterization of MnO₂ Nanorods
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An X-ray diffractometer (Panalytical X’Pert Pro, Almelo, The Netherlands) using copper/Kα radiation (λ = 1.5418 Å) captured XRD patterns. A Tensor II spectrometer (Bruker, Denkendorf, Baden-Württemberg, Germany) was utilized to record the FT-IR spectra. A scanning electron microscope (MIRA3, Tescan, Brno, Czech Republic) provided the FE-SEM images and the EDX patterns. An autolab potentiostat/galvanostat (PGSTAT-302N, Eco Chemie, Utrecht, The Netherlands) recorded all electrochemical determinations. The General Purpose Electrochemical System (GPES) as selected software monitored all testing protocols. A conventional three-electrode system was used at 25 ± 1 °C. An Ag/AgCl/KCl (3.0 M) electrode, a platinum wire and MnO2NR-IL/CPE were used as the reference, auxiliary and working electrodes, respectively. A pH meter (Metrohm type 713) was utilized to determine all solution pH values. All solutions were prepared freshly by deionized water (DIW, Millipore Direct-Q® 8 UV water purification system, Darmstadt, Germany). Sulfanilamide, 1-butyl-3-methylimidazolium hexafluorophosphate (BMIM-PF6) ionic liquid, and all materials were purchased from Merck (Darmstadt, Germany) with analytical research purity. Orthophosphoric acid as well as relevant salts have been applied to achieve all phosphate buffer solutions (PBS), set in a pH range of 2.0–9.0.
9
Thermoplastic PU and PEG Hydrogel Synthesis
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Polymers including thermoplastic PU and PEG (2000 MW) were obtained from Desmopan (Leverkusen, Germany) and Sigma-Aldrich (Shanghai, China) respectively. However, gelatin was purchased from Sigma-Aldrich (St. Louis, MO, USA). The solvents of tetrahydrofuran (THF), ethanol, glutaraldehyde, dimethyl sulfoxide (DMSO), hexane, hydrochloric acid (HCL) and the cross-linkers of 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-Hydroxysuccinimide (NHS) were bought from Merck (Hohenbrunn, Germany). The other chemical materials such as sulfanilamide, naphtylethelenediamine-dihydrochloride, NaNO2 and H3PO4 were also purchased from Merck too. Aspirin, toluidine blue, CaCl2 and 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) were provided from Sigma-Aldrich (St. Louis, MO, USA). Heparin (Heparin sodium injection parenteral, 5000 U/mL) was provided by DarouPhakhsh (Tehran, Iran). The cell culture medium Dulbecco’s modified Eagle’s medium (DMEM) with high glucose content, fetal bovine serum (FBS), trypsin and phosphate buffered saline (PBS) were purchased from Gibco (Grand Island, NY, USA). Moreover, the HUVEC line was purchased from the Pasture Institute cell bank (Tehran, Iran).
10
Measurement of Oxidative Stress Biomarkers
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Trichloroacetic acid (TCA), 5,5-dithionitrobenzoic acid (DTNB), thiobarbituric acid (TBA), isopropanol, p-dimethylamino benzaldehyde, 2,4-dinitrophenylhydrazine (DNPH), meta-phosphoric acid, 2-amino-2-hydroxymethyl-propane-1,3-diol-hydrochloride (Tris—HCl), glacial acetic acid, guanidine hydrochloride, phenylmethylsulfonyl fluoride (PMSF), bovine serum albumin (BSA), diethyl pyrocarbonate (DEPC), chloroform, boric acid, and ammonium molybdate were obtained from Kalazist Co. (Tehran, Iran). Ascorbic acid, vanadium chloride, sodium nitrite, ethylenediamine tetra-acetic acid (EDTA), Tris base, tetra ethoxy propane (TEP), N-(l-naphthyl)-ethylendiamine-dihydrochloride (NEDD), sulfanilamide, sodium acetate, ethyl acetate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), dithiothreitol (DTT), Coomassie blue, hydrogen peroxide, and Triton X-100, were obtained from Merck (Darmstadt, Germany). Ethanol, formalin, and hydrochloric acid (HCl) were obtained from Mojallali Co. (Tehran, Iran). 2,4,6-Tripyridyl-S-triazine (TPTZ) was obtained from Sigma-Aldrich (Missouri, US).
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