One hundred thousand RAW 264.7 cells were seeded into each well of a 24-well plate and incubated under cell culture conditions of 95% relative humidity (RH), 5% CO2 and 37 °C for 24 h. Next, the cells were treated with 1 µg/ml lipopolysaccharide (LPS) plus 250 µg/ml AE extracts or 125 µg/ml VB or 62.50 µg/ml VB depending on the tested cytokines. Five micrograms per milliliter of hydrocortisone were the positive control. The treated cells were incubated for 24 h and the supernatants were collected for determination of the amount of IL-1β and TNF-α using an ELISA Kit (Invitrogen, Vienna, Austria). The amount of nitric oxide was determined using the chemical reaction of Griess’s reagent (Sigma-Aldrich, Steinheim, Germany).
Griess s reagent
Manufactured by Merck Group
Sourced in Germany
Griess's reagent is a chemical compound used in analytical chemistry for the detection and quantification of nitrite ions (NO2−) in aqueous solutions. It is a colorimetric reagent that reacts with nitrite to produce a characteristic pink or purple azo dye, which can be measured spectrophotometrically.
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Lab products found in correlation
Ethylene glycol tetraacetic acid (egta), by Merck Group (1 mentions)
Sb366791, by Merck Group (1 mentions)
Hc 030031 hc, by Merck Group (1 mentions)
14 15 eet, by Cayman Chemical (1 mentions)
1 2 bis 2 aminophenoxy ethane n n n n tetra acetic acid acetoxymethyl ester bapta am, by Merck Group (1 mentions)
Drabkin s reagent kit 525, by Merck Group (1 mentions)
14 15 ee5ze, by Cayman Chemical (1 mentions)
Turbofect, by Thermo Fisher Scientific (1 mentions)
Capsaicin, by Merck Group (1 mentions)
Capsazepine, by Merck Group (1 mentions)
Matrigel, by BD (1 mentions)
5 protocols using griess s reagent
1
Cytokine and Nitric Oxide Modulation
2
Quantification of Nitric Oxide Levels
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Nitric oxide (NO) level was determined as in Shaheen et al. (2016 ). Nitrate and nitrite are final products of NO metabolism and these levels used for the quantification of NO. Sum of nitrate and nitrite accounts for the total NO concentration. Griess's reaction was used for the determination of NO level in serum based on the nitrite concentration. In the serum, nitrate was reduced nitrite in the presence of cadmium (Sigma‐Aldrich Inc., Shanghai, China). Then, nitrite was converted to nitric acid that reacts with Griess's reagent (Sigma‐Aldrich Inc., Shanghai, China) to produce color. Serum level of nitrite was determined by measuring absorbance at 540 nm in a spectrophotometer.
3
Calcium Signaling in Cell Lines
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14,15-EET and 14,15-EE5ZE were from Cayman (Ann Arbor, MI). Quest Fluo-8 NW Calcium Assay Kit was from AAT Bioquest (AAT Bioquest Inc., Sunnyvale, CA, USA). Ca2+ free medium, capsaicin, capsazepine (CPZ), SB366791, HC030031 (HC), 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl ester (BAPTA/AM), ethylene glycol tetraacetic acid (EGTA), Griess's reagent, and Drabkin's reagent kit 525 were from Sigma-Aldrich (St. Louis, MO, USA). TurboFect was from Fermentas (Glen Burnie, MD, USA). Matrigel was from BD Biosciences (San Jose, CA, USA).
4
Indirect Measurement of Nitric Oxide
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Nitrite (NO2−) and nitrate (NO3−) are stable final products of NO metabolism and may be used as indirect markers of NO. Total NO concentration was determined as sum of NO2− and NO3− concentrations in indirect method based on measurement of NO2− concentration in serum according to Griess reaction. In the presence of cadmium (Sigma-Aldrich, Steinheim, Germany), NO3− was reduced to NO2−, which is able to react in a colorful reaction with Griess’s reagent (Sigma-Aldrich, Steinheim, Germany). Nitrite concentrations were determined by spectrophotometric analysis at 540 nm (UVN-340 ASYS Hitech GmbH microplate reader; Biogenet, Eugendorf, Austria) with reference to a standard curve. NO products were expressed as μM.
5
Spectrophotometric Nitric Oxide Assay
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Nitrite (NO2-) and nitrate (NO3-) are stable final products of NO metabolism and may be used as indirect markers of NO presence. Total NO concentration is commonly determined as a sum of nitrite and nitrate concentrations. NO concentration was determined in serum using an indirect method based on measurement of nitrite concentration in serum according to Griess's reaction (Jablonska et al., 2007) .
In the samples analyzed, nitrate was reduced to nitrite in the presence of cadmium (Sigma-Aldrich, Steinheim, Germany), and then converted to nitric acid that gave a color reaction with Griess's reagent (Sigma-Aldrich, Steinheim, Germany). Nitrite concentrations were determined by spectrophotometric analysis at 540 nm (UVN-340 ASYS Hitech GmbH microplate reader; Biogenet, Eugendorf, Austria) regarding a standard curve. NO products were expressed as (nmol/ml)
In the samples analyzed, nitrate was reduced to nitrite in the presence of cadmium (Sigma-Aldrich, Steinheim, Germany), and then converted to nitric acid that gave a color reaction with Griess's reagent (Sigma-Aldrich, Steinheim, Germany). Nitrite concentrations were determined by spectrophotometric analysis at 540 nm (UVN-340 ASYS Hitech GmbH microplate reader; Biogenet, Eugendorf, Austria) regarding a standard curve. NO products were expressed as (nmol/ml)
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