Overview

  • Product name

    Nitric Oxide Assay Kit (Fluorometric - Orange)
    See all Nitric oxide kits
  • Detection method

    Fluorescent
  • Sample type

    Adherent cells, Suspension cells
  • Assay type

    Cell-based
  • Species reactivity

    Reacts with: Other species, Mammals
  • Product overview

    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.

  • Notes

    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.

  • Platform

    Microplate reader, Fluor. microscope, Flow cyt.

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components 200 tests
    500X NO Orange Dye 1 x 50µl
    Assay Buffer I 1 x 20ml
    Assay Buffer II 1 x 20ml
  • Research areas

  • Relevance

    Nitric 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.

Images

  • 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 37ºC 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 37ºC 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).

  • Endogenous nitric oxide (NO) production in RAW 264.7 macrophage cells. Raw 264.7 cells were seeded overnight (10cells/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 37ºC 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 37ºC 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.

Protocols

References

ab219932 has not yet been referenced specifically in any publications.

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