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13547
Griess Reagent Nitrite Measurement Kit
Cellular Assay Kits
Assay Kit

Griess Reagent Nitrite Measurement Kit #13547

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Griess Reagent Nitrite Measurement Kit: Image 1

Figure 2: Detection of nitrite in supernatants of RAW 264.7 cells, untreated or treated with Mouse Interferon-γ (mIFN-γ) #5222 (100 ng/ml), Lipopolysaccharides (LPS) #14011 (10 ng/ml), or both. After 48 hours, cell culture supernatants were harvested and assayed for nitrite using the Griess Reagent Nitrite Measurement Kit.

Griess Reagent Nitrite Measurement Kit: Image 2

Figure 1: Representative standard curves in different assay matrices. Nitrite standard curves were generated in water, RPMI 10% FBS, serum, or urine.

To Purchase # 13547S
Product # Size Price
13547S
1 Kit  (500 assays) $ 122

Product Includes Quantity (with Count)
0.1 M Nitrite Standard 1 x 1 ml
Reagent A: Sulfanilamide Solution 1 x 25 ml
Reagent B (NED Solution) 1 x 25 ml

Protocol

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#13547 Griess Reagent Nitrite Measurement Kit Protocol

A. Solutions and Reagents

  1. Reagent A: Sulfanilamide Solution: Bring to room temperature before use.
  2. Reagent B: NED Solution: Bring to room temperature before use.
  3. Prepare Griess Reagent working solution by mixing equal volumes of Reagent A: Sulfanilamide Solution and Reagent B: NED Solution.
  4. 0.1 M Nitrite Standard: Bring to room temperature before use. Prepare a 100 µM working nitrite standard by diluting the 0.1 M Nitrite Standard 1:1000 in the same matrix/buffer used for unknown samples. Perform serial two-fold dilutions of the 100 µM working nitrite standard solution. The final concentrations of nitrite in the standard curve should be as follows: 100, 50, 25,12.5, 6.25, 3.13, and 1.56 µM. The blank should also be prepared in the same matrix/buffer.

B. Test Procedure

  1. Add 100 µl of sample or nitrite standard to each well of a microtiter plate.
  2. Add 100 µl of Griess Reagent working solution per well. The solution will turn a pinkish color in the presence of nitrite.
  3. Measure absorbance at 550 nM.
  4. To quantify the amount of nitrite in unknown samples, plot the average absorbance against each respective concentration in the standard curve in an X vs. Y plot. Extrapolate the concentration of unknown samples from a linear curve.

posted June 2020

Protocol Id: 2052

Product Description

The Griess Reagent Nitrite Measurement Kit can be used to indirectly detect nitric oxide (NO) through the measurement one of its stable oxidation products, nitrite. Nitrite reacts with sulfanilamide and N-(1-naphthyl)ethylenediamine dihydrochloride (NED) to yield a pink azo dye. The azo dye produced in this assay can be measured spectrophotometrically using its absorbance at 550 nM.

Specificity / Sensitivity

The Griess Reagent Nitrite Measurement Kit detects nitrite levels within a given sample. Detection of micromolar amounts of nitrite is possible using the Griess assay, with the detection range dependent upon the matrix or buffer used in the assay. An estimate of total NO produced requires determination of both nitrite and nitrate levels. As the kit measures only nitrite levels, nitrate should initially be reduced using a nitrate reductase.

Background

Nitric Oxide (NO) is a pleiotropic signaling molecule that plays key roles in multiple cells types and tissues. NO is generated by three distinct nitric oxide synthase (NOS) enzymes that catalyze the oxidation of L-arginine into L-citrulline and NO (1). Two of these enzymes, endothelial NOS (eNOS) and neuronal NOS (nNOS), are constitutively expressed in many cell types (1). Inducible NOS (iNOS) is transcriptionally regulated and is expressed in response to inflammatory stimuli, such as pro-inflammatory cytokines (1). NO functions as a potent vasodilator and neurotransmitter (1). In the context of cancer, NO may function as either a tumor suppressor or tumor promoter (2). High levels of NO produced by macrophages are cytotoxic (2). In contrast, research studies have shown that lower levels of NO may contribute to tumorigenesis through promoting angiogenesis (2).

  1. Hill, B.G. et al. (2010) J Biol Chem 285, 19699-704.
  2. Rahat, M.A. and Hemmerlein, B. (2013) Front Physiol 4, 144.

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