Nitric Oxide Assay Kit (Fluorometric - Orange)
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(2 Publications)
- Fluorescence Microscopy
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Fluorescence Microscopy - Nitric Oxide Assay Kit (Fluorometric - Orange) (AB219932)
Endogenous nitric oxide (NO) production in RAW 264.7 macrophage cells. Raw 264.7 cells were seeded overnight (105 cells/well/100 μL) in a black wall/clear bottom 96-well plate. Left : cells were treated with 20 μg/mL of lipopolysaccharide (LPS) and 1 mM L-Arginine (L-Arg) and co-incubated with 0.5X NO stain working solution at at 37°C for 16 hours. Right : control RAW 264.7 cells were left untreated in cell culture medium and co-incubted with 0.5X NO stain working solution at 37°C for 16 hours. The solution in each well was removed and Assay Buffer II was added before fluorescence measurement. The fluorescence signal was measured using fluorescence microscope with a TRITC filter.
- FuncS
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Functional Studies - Nitric Oxide Assay Kit (Fluorometric - Orange) (AB219932)
Exogenous nitric oxide (NO) production upon DEA/NONOate treatment (NO donor). CHO-K1 (blue) and HeLa (red) cells were incubated with NO stain working solution at 37°C for 30 minutes, and then removed to stop the staining. Cells were further treated with or without 1mM DEA/NONOate in HBSS buffer (with 10 mM HEPES (pH=6.2)) at 37°C for 30 minutes. The solution in each well was removed and Assay Buffer II was added before measuring fluorescence. The fluorescence signal was monitored at Ex/Em = 540/590 nm (cut off = 570 nm) with bottom read mode using a FlexStation microplate reader (Molecular Devices).
Product details
Nitric Oxide Assay Kit (Fluorometric - Orange) (ab219932) is a sensitive fluorometric assay to monitor intracellular nitric oxide (NO) levels in live cells. The assay uses an orange dye that can react with NO to generate a bright orange fluorescent product that can be easily detected at Ex/Em = 540/590 nm, using the same filter set as Cy3® or TRITC.
The assay can be detected by fluorescence microscopy, microplate fluorometry or high-content imaging. It can be easily adapted to use in 384-well microplate format.
Nitric oxide (NO) is an important biological regulator involved in numbers of physiological and pathological processes. Altered NO production is implicated in various immunological, cardiovascular, neurodegenerative and inflammatory diseases. As a free radical, NO is rapidly oxidized and there is relatively low concentrations of NO existing in vivo. It has been challenging to detect and understand the role of NO in biological systems.
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Biological function summary
Nitric oxide mediates vasodilation neurotransmission and immune response modulation. It is important for maintaining vascular homeostasis where it relaxes smooth muscle tissue promoting blood flow. In the central nervous system nitric oxide acts as a neurotransmitter and neuromodulator influencing synaptic plasticity. Within the immune system it functions to regulate leukocyte adhesion and can mediate microbial destruction. Nitric oxide often works within complexes associating with proteins like guanylate cyclase enhancing its signaling efficacy through nitrosylation.
Pathways
Nitric oxide integrates into key biological processes such as the nitric oxide signaling pathway and the endothelial nitric oxide pathway. It activates soluble guanylate cyclase increasing cyclic GMP levels leading to smooth muscle relaxation and vasodilation. In the endothelial pathway nitric oxide links with other proteins like endothelium-derived relaxing factor and interacts with oxidative stress pathways showing its role in cellular defense mechanisms. These pathways manage critical physiological responses and connect nitric oxide to other cardiovascular regulators.
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Publications (2)
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Communications biology 5:379 PubMed35440683
2022
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Nature communications 13:646 PubMed35115501
2022
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