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Intracellular Nitric Oxide (NO) Assays


Detection of exogenous nitric oxide (NO) in cells upon DEA NONOate treatment (NO donor) using Cell Meter™ Fluorimetric Intracellular Nitric Oxide Assay Kit (Cat#16350). CHO-K1 and HeLa cells at 50,000 cells/well/100 µL were seeded overnight in a 96-well black wall/clear bottom plate. Cells were incubated with Nitrixyte™ Orange working solution at 37 °C for 30 minutes. The cells were treated with or without 1mM DEA NONOate at 37 °C for 30 minutes. The fluorescence signal was monitored at Ex/Em = 540/590 nm (cut off = 570 nm) with bottom read mode.
Altered NO production is implicated in various immunological, cardiovascular, neurodegenerative and inflammatory diseases. As a free radical, NO is rapidly oxidized and exists in relatively low concentration. It has been challenging to detect and understand the role of NO in biological systems. Cell Meter™ Fluorimetric Intracellular Nitric Oxide Assay Kits provide a robust tool to monitor intracellular NO level in live cells.

Nitrixyte™ Orange and Nitrixyte™ Red are developed as excellent replacements for DAF-2 for the detection and imaging of free NO in cells. Compared to the widely used DAF-2 probes, Nitrixyte™ Orange and Nitrixyte™ Red have better photostability and enhanced cell permeability. Cell Meter™ Fluorimetric Intracellular Nitric Oxide Assay Kits (Cat# 16350 & 16351) use Nitrixyte™ Orange that reacts with NO to generate a bright orange fluorescent product. The NO-generated product of Nitrixyte™ Orange has spectral properties similar to those of Cy3® and TRITC. Nitrixyte™ Orange can be readily loaded into live cells, and its fluorescence signal can be conveniently monitored using the filter set of Cy3® or TRITC. Kit 16350 is optimized for fluorescence imaging and microplate reader applications. Kit 16351 is optimized for flow cytometry applications.


Fluorescence images of endogenous nitric oxide (NO) measurement in RAW 264.7 macrophage cells using Cell Meter™ Fluorimetric Intracellular Nitric Oxide Assay Kit (Cat#16350). Raw 264.7 cells at 100,000 cells/well/100 µL were seeded overnight in a 96-well black wall/clear bottom plate. Cells were incubated with Nitrixyte™ Orange, and treated with (Right) or without (Left) 20 µg/mL of lipopolysaccharide (LPS) and 1 mM L-Arginine (L-Arg) at 37 °C for 16 hours.



Detection of exogenous nitric oxide (NO) in Jurkat cells upon DEA NONOate treatment (NO donor) using Cell Meter™ Fluorimetric Intracellular Nitric Oxide Assay Kit (Cat#16351). Cells were incubated with Nitrixyte™ Orange at 37 °C for 30 minutes and washed with assay buffer twice. The cells were treated with (Red) or without (Blue) 1mM DEA NONOate at 37 °C for 30 minutes.
Cell Meter™ Fluorimetric Intracellular Nitric Oxide Assay Kit 16356 uses Nitrixyte™ Red that reacts with NO to generate a bright red fluorescent product. The NO-generated fluorescent product of Nitrixyte™ Red has spectral properties similar to those of Texas Red®. Nitrixyte™ Red can be readily loaded into live cells, and its fluorescence signal can be conveniently monitored using the filter set of Texas Red®. This kit is optimized for flow cytometry applications.