The level of NO secreted into the culture medium was determined by the Griess reaction as previously described [38 (link)]. Equal volumes of supernatant, Griess reagent A [0.1% N-(1-naphthyl)ethylenediamine dihydrochloride] and Griess reagent B (1% sulfanilamide in 5% phosphoric acid) (both from Sigma-Aldrich, St. Louis, MO, USA) were mixed, and the absorbance of the prepared solution was measured immediately at a wavelength of 540 nm in a Tecan Infinite 200 Pro spectrophotometer (Tecan, Mannedorf, Germany). The data were normalized to the level of NO released from the vehicle group in control OCCs (Experiment 1) or the vehicle group from the corresponding type of OCCs (Experiment 2) and are presented as the percentage of the control ± SEM.
Griess reagent a
Manufactured by Merck Group
Sourced in United States
Griess reagent A is a laboratory reagent used in the Griess test, a colorimetric method for the detection and quantification of nitrite ions in various samples. It is a key component in the two-step Griess reaction, which produces a purple azo dye that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Griess reagent b, by Merck Group (4 mentions)
Elx800, by Agilent Technologies (2 mentions)
Infinite 200 pro spectrophotometer, by Tecan (1 mentions)
Dmso d6, by Merck Group (1 mentions)
Cell proliferation kit 1 mtt, by Merck Group (1 mentions)
Caffeic acid, by Merck Group (1 mentions)
Vanillic acid, by Merck Group (1 mentions)
P coumaric acid, by Merck Group (1 mentions)
Benzoic acid, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Quercetin, by Merck Group (1 mentions)
Rutin, by Merck Group (1 mentions)
Kaempferol, by Merck Group (1 mentions)
Naringenin, by Merck Group (1 mentions)
4 hydroxybenzoic acid, by Merck Group (1 mentions)
Cd3od, by Merck Group (1 mentions)
4 hydroxybenzaldehyde, by Merck Group (1 mentions)
Hippuric acid, by Merck Group (1 mentions)
Quercetin 3 rhamnoside, by Merck Group (1 mentions)
7 protocols using griess reagent a
1
Quantification of Nitric Oxide Secretion
2
Fucoidan Modulates Macrophage Nitric Oxide
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Cultured RAW264.7 cells with 2 × 105 cells/well in a 96-well plate. After the cell adhesion, the fucoidan was added with different concentrations into the well plate, and added 1 μg/mL LPS separately to induce cells to produce nitrate for 24 h. Next, a portion of the supernatants was transferred to new 96 well plates. After mixing with Griess reagent A (Sigma Aldrich, USA) and Griess reagent B (Sigma Aldrich, USA) in dark, the absorption of samples was measured under 550 nm.
3
Griess Assay for Macrophage NO Production
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Griess reaction assay was used to study NO produced by treating macrophage cells with essential oil and its main compound, terpinolene. In this method, after exposing the macrophage cells (1 × 105/mL) with various concentration of essential oil (1/4 IC50, ½ IC50, and IC50) for 48 h, in a 96-well plate the supernatant of reaction (20 μL) was mixed with the nitrite assay buffer (80 μL), Griess reagent A (10 μL, Sigma-Aldrich) and B (10 μL) the amount of NO was recorded at 540 nm in an ELISA reader (BioTek-ELX800) (Albalawi et al., 2021b (link)). Standard curve (a serial dilution from 1 to 100 μM nitrite) was also prepared from 1 mM nitrite standard solution. The cells treated with the combination of lipopolysaccharide (LPS, 10 ng/mL) along with IFN-γ (10 U/mL) were considered as the positive control.
4
Polyphenolic Metabolite Profiling
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4-Hydroxybenzaldehyde, 4-hydroxybenzoic acid, caffeic acid, homovanillic acid, vanillic acid, protocatechuic acid, phloroglucinol aldehyde, hippuric acid, ferulic acid, p-coumaric acid, quercetin, chlorogenic acid, neochlorogenic acid, epicatechin, catechin, kaempferol, kaempferol-3-rutinoside, 3-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid, isorhamnetin-3-rutinoside, naringenin, isorhamnetin, rutin, quercetin, quercetin-3-rhamnoside, quercetin-3-glucoside, 4-methylcatechol, benzoic acid, DMSO-d6, CD3OD, Griess reagent A, Griess reagent B, cell proliferation kit I (MTT), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Proanthocyanidins B2, B5, and C1 were obtained from Plantchem (Klepp, Norway). Dulbecco‘s modified Eagle’s medium (DMEM-Glutamax™, 5.5 mM), fetal bovine serum, and penicillin–streptomycin–amphotericin B were obtained from Gibco Life Technologies (Paisley, UK). Corning CellBIND tissue culture plates were obtained from Corning Life-Sciences (Schiphol-Rijk, The Netherlands). All other reagents were of the highest purity available.
5
Quantifying Inflammatory Mediators in Macrophages
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The levels of NO, PGE2, and cytokines (TNF-α, IL-1β, IL-6) in the culture supernatant of RAW264.7 cells cocultured with KBL346 were measured. The amount of NO produced by macrophages was analyzed using the Griess reaction. The Griess reagent was prepared by mixing Griess reagent A (1% sulfanilamide in 5% phosphoric acid) (sulfanilamide, Sigma-Aldrich Co., St. Louis, MO, USA, 33626-100G) (phosphoric acid, Sigma-Aldrich Co., 49685-100ML) and Griess reagent B (0.1% N-(1-naphthyl) ethylenediamine dihydrochloride in DW) (Sigma-Aldrich Co., 33461-25G) at a 1:1 ratio. Absorbance was measured at 540 nm after mixing equal amounts of the sample and Griess reagent And incubating in the dark for 20 min. The concentration of NO was calculated from a standard calibration curve obtained using sodium nitrite (NaNO2) (Sigma-Aldrich Co., 7632-00-0). The levels of PGE2 and cytokines in the cocultured cell supernatant were measured using ELISA kits: PGE2 (Cayman CHEMICAL, Ann Arbor, Michigan, USA, 514010), TNF-α (558534), IL-6 (555240) (BD Biosciences, San Jose, CA, USA), and IL-1β/IL-1F2 (R&D SYSTEMS, Minneapolis, MN, USA, DY401).
6
Nitric Oxide Production in J774-A1 Macrophages
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J774-A1 macrophage cells (1×105/ml) were treated with various concentrations of AMCE (¼ IC50, ½ IC50, and IC50) for 48 h, in a 96-well plate. In the next step, 20 µl of the supernatant was mixed with the nitrite assay buffer (80 μl), Griess reagent A (10 μl, Sigma-Aldrich), and B (10 μl). Lastly the absorbance of the solution was read at 540 nm in an ELISA reader (BioTek-EL×800)18 . The cells treated with the combination of lipopolysaccharide (LPS, 10 ng/ml) along with IFN-γ (10 U/ml) were considered as the positive control20 (link).
7
Quantification of Nitric Oxide in THP-1 Cells
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In this method, after incubating the THP-1 cells 01׳1( 5 /mL) with various concentration of SLME (1/4 IC 50 , 1/3 IC 50 , and ½ IC 50 ) for 48 h, in a 96-well plate the supernatant of reaction (20 µl) was mixed with the nitrite assay buffer (80 µL), Griess reagent A (10 µL, Sigma-Aldrich) and B (10 µL) the amount of NO was recorded at 540 nm in an ELISA reader (BioTek-ELX800). The cells treated with the combination of lipopolysaccharide (LPS, 10 ng/mL) along with IFN-γ (10 U/mL) were considered as the positive control.
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