Product nameNitric Oxide Assay Kit (Fluorometric)
See all Nitric oxide kits
Sample typeUrine, Serum, Plasma, Other biological fluids, Tissue Extracts, Cell Lysate
Assay time3h 00m
Nitric Oxide Assay Kit (Fluorometric) ab65327 provides an accurate, convenient measure of total nitrate/nitrite concentration in a simple two-step process.
In the nitric oxide assay protocol:
- the first step converts nitrate to nitrite by nitrate reductase
- in the second step, nitrite reacts with the fluorescent probe DAN (2, 3 diaminonaphthalene)
The fluorescence can be measured at Ex/Em = 360/450 nm. NaOH enhances the fluorescent yield. The fluorescent intensity is proportional to the total nitric oxide production. The kit has been tested with culture media, plasma, and tissue homogenates.
Nitric oxide assay protocol summary:
- add samples and standards to wells
- add enzyme cofactor, to measure nitrite only add assay buffer or to measure total nitrate + nitrite add nitrate reductase
- incubate for 1-4 hr at room temp to convert nitrate to nitrite
- add enhancer and incubate for 30 min at room temp
- add DAN probe and incubate for 10 min at room temp
- add NaOH and incubate for 10 min at room temp
- analyze with microplate reader
If you are interested in a colorimetric detection kit, please check Nitric Oxide Assay Kit (Colorimetric) (ab65328).
For an assay kit to detect nitrite alone see Griess Reagent Kit ab234044.
Nitric oxide (NO) plays an important role in neurotransmission, vascular regulation, immune response and apoptosis. Since NO is rapidly converted to nitrite (NO2-) and nitrate (NO3-), the total concentration of nitrite and nitrate is used as a quantitative measure of NO production.
Storage instructionsStore at -20°C. Please refer to protocols.
Components Identifier 200 tests Assay Buffer WM 1 x 40ml DAN Probe Amber Red 1 x 1ml Enhancer (Lyophilized) Purple 1 vial Enzyme Cofactor (Lyophilized) Blue 1 vial Microtiter plate 2 units Nitrate Reductase (Lyophilized) Green 1 vial Nitrate Standard (Lyophilized) Yellow 1 vial Nitrite Standard (Lyophilized) Orange 1 vial Plate Cover 2 units Sodium Hydroxide Clear 1 x 1ml
RelevanceNitric oxide (NO) is a key vertebrate biological messenger, playing an important role in neurotransmission, vascular regulation, immune responses and apoptosis. NO , also known as "endothelium-derived relaxing factor" or "EDRF", is synthesized from L-arginine, oxygen and NADPH by various NO synthases. Most of the NO in the cell is oxidized to nitrite (NO2-) and nitrate (NO3-), and therefore the concentrations of these anions are generally as a quantitative measure of NO production.
Effect of arginase inhibitor and L-arginine on eATP-induced reduction of nitric oxide production in HSV endothelial cells.
HSVEC were either untreated (Ctrl), treated with ATP (2 mM), L-NAME, (100 µM), ATP with L-arginine (L-Arg, 2 mM), or ATP with NOHA (10 µM), for 2 hours. The cells were then stimulated with carbachol (CCH, 1 µM) for 10 minutes and the nitric oxide generated was measured as nitrite using the NO assay kit (ab65327) and relative percent of NO generated was calculated. NO generated with CCH was set as 100%, n = 6 passages, ∗p < 0.05, (One way ANOVA).
Nitric oxide measured in cell lysates showing quantity (micromol) per 106 cells tested
Nitric oxide measured in biologicals (average of 1:6 and 1:12 dilutions) showing concentration (micromolar)
Nitrite, nitrate assay in the presence and absence of nitrate reductase. Assays were performed according to the kit protocol with 1 hour conversion of nitrate to nitrite at Step 5.
ab65327 has been referenced in 9 publications.
- Krzywonos-Zawadzka A et al. Mixture of MMP-2, MLC, and NOS Inhibitors Affects NO Metabolism and Protects Heart from Cardiac I/R Injury. Cardiol Res Pract 2020:1561478 (2020). PubMed: 32322413
- Fodelianaki G et al. Nerve Growth Factor modulates LPS - induced microglial glycolysis and inflammatory responses. Exp Cell Res 377:10-16 (2019). PubMed: 30817930
- Devarakonda CV et al. CD13 deficiency leads to increased oxidative stress and larger atherosclerotic lesions. Atherosclerosis 287:70-80 (2019). PubMed: 31229835
- Coulombe P et al. Endothelial Sash1 Is Required for Lung Maturation through Nitric Oxide Signaling. Cell Rep 27:1769-1780.e4 (2019). PubMed: 31067462
- Huang Y et al. TRPM7 channels play a role in high glucose-induced endoplasmic reticulum stress and neuronal cell apoptosis. J Biol Chem 293:14393-14406 (2018). PubMed: 30076216
- Murugavel S et al. Valproic Acid Induces Endothelial-to-Mesenchymal Transition-Like Phenotypic Switching. Front Pharmacol 9:737 (2018). PubMed: 30050438
- Gupta A et al. PARK2 Depletion Connects Energy and Oxidative Stress to PI3K/Akt Activation via PTEN S-Nitrosylation. Mol Cell 65:999-1013.e7 (2017). PubMed: 28306514
- Komalavilas P et al. Vascular surgical stretch injury leads to activation of P2X7 receptors and impaired endothelial function. PLoS One 12:e0188069 (2017). PubMed: 29136654
- Huang Z et al. Stimulation of Unprimed Macrophages with Immune Complexes Triggers a Low Output of Nitric Oxide by Calcium-dependent Neuronal Nitric-oxide Synthase. J Biol Chem 287:4492-502 (2012). PubMed: 22205701