Pure Safranal (Safr), Cands (Candesartan), 3-NP (3-nitropropionic acid), paraffin oil, 0.5% carboxy methyl cellulose (CMC), thiobarbituric acid (TBA), vanadium chloride, sulfanilamide, N-(1-naphthyl) ethylenediamine, Folin-Ciocalteau reagent and bovine serum albumin (BSA) were obtained from Sigma (St. Louis, MO, USA). All other chemicals were of the highest analytical grades commercially available.
Vanadium chloride
Manufactured by Merck Group
Sourced in United States, Germany
Vanadium chloride is an inorganic chemical compound with the formula VCl3. It is a dark green crystalline solid that is soluble in water and organic solvents. Vanadium chloride is used as a precursor in various chemical processes and as a component in specialized laboratory equipment.
Automatically generated - may contain errors
Lab products found in correlation
Sulfanilamide, by Merck Group (7 mentions)
Sodium nitrite, by Merck Group (5 mentions)
N 1 naphthyl ethylenediamine, by Merck Group (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Naphthylethylenediamine dihydrochloride, by Merck Group (3 mentions)
Thiobarbituric acid, by Merck Group (3 mentions)
Folin ciocalteau reagent, by Merck Group (2 mentions)
Sulphanilamide, by Merck Group (2 mentions)
Ellman s reagent, by Merck Group (2 mentions)
Trichloroacetic acid, by Merck Group (2 mentions)
Tris base, by Merck Group (2 mentions)
N 1 naphthyl ethylenediaminedihydrochloride nedd, by Merck Group (1 mentions)
Griess reagent, by Merck Group (1 mentions)
Mercuric chloride, by Merck Group (1 mentions)
Sulfuric acid, by Thermo Fisher Scientific (1 mentions)
Potassium permanganate, by Thermo Fisher Scientific (1 mentions)
Potassium iodide, by Thermo Fisher Scientific (1 mentions)
Iodine, by Thermo Fisher Scientific (1 mentions)
Sodium iodide, by Thermo Fisher Scientific (1 mentions)
Hydrogen peroxide, by Thermo Fisher Scientific (1 mentions)
19 protocols using vanadium chloride
1
Safranal-Based Neuroprotective Assay
2
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)).
3
Nitric Oxide Metabolite Quantification by Griess Assay
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Griess method was used to measure plasma concentration of NO metabolites as described previously [49 (link)]. Briefly, plasma samples and standards (sodium nitrite, Sigma) were mixed with N-(1-Naphthyl) ethylenediamine dihydrochloride (NEDD, Sigma), sulfanilamide (SULF, Sigma), and vanadium chloride (Sigma) and incubated at 37 °C for 30 min. Then, 100 μl of prepared solutions was added to each well, and the absorbance of well-plate was detected at 540 nm.
4
Quantifying Nitric Oxide Levels
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The nitric oxide (NO) concentration in the medium (N = Table S1) was measured as described by Miranda et al.31 Briefly, vanadium chloride (Sigma) and Griess reagent (Sigma) were added to the medium, followed by incubation at 37°C for 30 min. Nitrite and nitrate (both Sigma) standards were read on the same plate.
5
Quantification of Nitrite and Nitrate
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Nitrite and nitrate concentrations were measured in the culture medium from aorta segment incubations by a modified method of the Griess assay [34 (link)]. Briefly, 100 μL of vanadium chloride (Sigma-Aldrich, St. Louis, MO, USA) were added to 100 μL of culture medium on a 96-well plate. Immediately after, 100 μL of the Griess reagent (1:1 mixture of 1% sulfanilamide (Merck Millipore, Darmstadt, Germany), and 0.1% naphthylethylenediamine dihydrochloride (Merck Millipore, Darmstadt, Germany)) were added to each well and incubated at 37 °C for 30 min. After incubation, absorbance was measured at 540 nm. Nitrite and nitrate concentrations were calculated using a NaNO2 standard curve and was expressed in µM.
6
Nitrate-Based Oxidative Assays
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Sodium nitrate (NaNO3), sulfuric acid (H2SO4 – 95.0–98.0%), potassium permanganate (KMnO4), hydrogen peroxide (H2O2 – 30 wt%), hydrochloric acid (HCl – 36 wt%), sodium iodide, potassium iodide, iodine, acetic acid (99.5%), sulfanilamide, and MTT assay were purchased from Thermo Scientific, Fisher Scientific, Acros, Nacalai Tesque and Alfa Aesar, respectively. Graphite flakes, N-(3-dimethylaminopropyl-N′-ethylcarbodiimide) hydrochloride (EDC), N-hydroxysuccinimide (NHS), phosphate buffered saline (PBS), potassium bromide (KBr), sodium nitrite (NaNO2 – 97%), potassium iodide, vanadium chloride, TEM copper grid, cysteamine, S-nitroso-N-acetyl-dl -penicillamine (SNAP), diethyldithiocarbamic acid sodium salt (DETC), iron (II) sulfate heptahydrate (FeSO4·7H2O, ≥99%), dichloromethane (CH2Cl2), mercuric chloride (HgCl2), 1 M hydrochloric acid (HCl) and the LDH assay kit (coulorimetric, MAK066) were purchased from Sigma Merck. ATCC medium containing 4 mM l -glutamine, 4500 mg/L glucose, 1 mM sodium pyruvate, and 1500 mg/L sodium bicarbonate was purchased from LGC Limited, UK. cGMP ELISA kits were purchased from Enzo Life Science, UK. BrdU cell proliferation assay kits were purchased from Cell Signalling Technology, UK. DAF-FM DA was purchased from Abcam, UK. All materials were used as received without any further purification unless stated otherwise.
7
Safranal-Based Neuroprotective Assay
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Pure Safranal (Safr), Cands (Candesartan), 3-NP (3-nitropropionic acid), paraffin oil, 0.5% carboxy methyl cellulose (CMC), thiobarbituric acid (TBA), vanadium chloride, sulfanilamide, N-(1-naphthyl) ethylenediamine, Folin-Ciocalteau reagent and bovine serum albumin (BSA) were obtained from Sigma (St. Louis, MO, USA). All other chemicals were of the highest analytical grades commercially available.
8
Pharmacological Effects in NMRI Mice
9
Quantifying Nitrite and Nitrate Levels in Aorta Segments
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As in González-Hedström et al.67 , using a modified method of the Griess assay75 (link), nitrite and nitrate concentrations were measured in the culture medium from aorta segment incubations. Briefly, to 100 μL of culture medium on a 96-well plate, 100 μL of vanadium chloride (Sigma‐Aldrich, St. Louis, MO, USA) were added. Immediately after, 100 μL of the Griess reagent (1:1 mixture of 1% sulfanilamide (Merck Millipore, Darmstadt, Germany) and 0.1% naphthylethylenediamine dihydrochloride (Merck Millipore, Darmstadt, Germany)) were added to each well and incubated at 37 °C for 30 min. Absorbance was measured at 540 nm after incubation. A NaNO2 standard curve was used to calculate nitrite and nitrate concentrations. Results were expressed in μM.
10
Antioxidant Assay Protocol
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Sulphanilamide, vanadium chloride (VCl3), zinc sulphate (ZnSO4), pyrogallol, tris base, Ellman’s reagent, trichloroacetic acid (TCA), sodium dodecyl sulphate, thiobarbituric acid (TBA), 1, 1′, 3, 3′-tetramethoxypropane were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA). Reduced glutathione was purchased from Alpha Chemika (Mumbai, India), and methanol and absolute ethanol from El-Nasr for Pharmaceutical Chemicals (Egypt). All other chemicals used in the study were of high analytical grade.
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