Cell Meter™ Fluorimetric Intracellular Nitric Oxide (NO) Activity Assay Kit *Orange Fluorescence Optimized for Microplate Reader*

Additional fluorescent color(s): 
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Fluorescence images of endogenous nitric oxide (NO) detection in RAW 264.7 macrophage. Cells were incubated with AAT’s Nitrixyte™ Orange (Left) or DAF-2 diacetate (Right) at the same concentration,  then treated with or without 20 µg/mL of lipopolysaccharide (LPS) and 1 mM L-arginine (L-Arg) at 37°C for 16 hours. The fluorescence signals were measured using a fluorescence microscope equipped with a TRITC (Left) or FITC (Right) filter set, respectively.
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Unit Size: Cat No: Price (USD): Qty:
200 Tests 16350 $345


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Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
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Overview

Ex/Em (nm)540/590
Storage F/D/L
InstrumentsFluorescence microplate reader, Fluorescence microscope
Category Neurobiology
Reactive Oxygen Species
Related Cell Signaling
Secondary Reagents
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. Cell Meter™ Fluorimetric Intracellular Nitric Oxide Assay Kit provides a sensitive tool to monitor intracellular NO level in live cells. Nitrixyte™ probes are developed and used in our kit as an excellent replacement for DAF-2 for the detection and imaging of free NO in cells. Compared to the commonly used DAF-2 probe, Nitrixyte™ probes have better photostability and enhanced cell permeability. This particular kit uses Nitrixyte™ Orange that can react with NO to generate a bright orange fluorescent product that has spectral properties similar to 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. This kit is optimized for fluorescence imaging and microplate reader applications.




Spectrum Advanced Spectrum Viewer

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Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.
At a glance

Protocol summary

  1. Prepare cells in growth medium
  2. Incubate cells with test compounds and Nitrixyte™ Orange working solution
  3. Add Assay Buffer II
  4. Monitor fluorescence intensity (bottom read mode ) at Ex/Em = 540/590 nm (Cutoff = 570 nm) or fluorescence microscope using TRITC filter

Important notes
Thaw all the kit components at room temperature before starting the experiment.

Key parameters
Instrument:Fluorescence microplate reader
Excitation:540 nm
Emission:590 nm
Cutoff:570 nm
Recommended plate:Solid black
Instrument specification(s):Bottom read mode
  
Instrument:Fluorescence microscope
Excitation:TRITC filter
Emission:TRITC filter
Recommended plate:Black wall/clear bottom
Preparation of working solution

Add 20 µL of 500X Nitrixyte™ Orange stock solution (Component A) into 10 mL of Assay Buffer I (Component B) and mix well to make Nitrixyte™ Orange. This Nitrixyte™ Orange working solution is stable for at least 2 hours at room temperature. Protect from light. Note: 20 µL of 500X Nitrixyte™ Orange stock solution is enough for one plate.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

Sample experimental protocol
  1. To stimulate endogenous NO, treat cells with 10 µL of 10X test compounds (96-well plate) or 5 µL of 5X test compounds (384-well plate) in cell culture medium or your desired buffer (such as PBS or HHBS). For control wells (untreated cells), add the corresponding amount of medium or compound buffer. Note: It is not necessary to wash cells before adding compound. However, if tested compounds are serum sensitive, growth medium and serum factors can be aspirated away before adding compounds. Add 90 µL/well (96-well plate) and 20 µL/well (384-well plate) of 1X Hank’s salt solution and 20 mM Hepes buffer (HHBS) or the buffer of your choice after aspiration. Alternatively, cells can be grown in serum-free media.

  2. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Nitrixyte™ Orange working solution in the cell plate. Co-incubate cells with test compound and Nitrixyte™ Orange working solution at 37°C for desired period of time, protected from light. Note: DO NOT remove the test compounds. For a NONOate positive control treatment: Cells were incubated with Nitrixyte™ Orange working solution at 37°C for 30 minutes. The working solution was removed and cells were further incubated with 1 mM DEA/NONOate at 37°C for 30 minutes to generate nitric oxide. Note: We have used Raw 264.7 cells incubated with 0.5X Nitrixyte™ Orange, 20 µg/mL of lipopolysaccharide (LPS) and 1 mM L-Arginine (L-Arg) in cell culture medium at 37°C for 16 hours. See Figure 1 for details.

  3. Remove solution in each well.

  4. Add Assay Buffer II (Component C) 100 µL/well for a 96-well plate or 25 µL/well for a 384-well plate. Note: DO NOT wash cells before adding Assay Buffer II.

  5. Monitor the fluorescence increase using microplate reader at Ex/Em = 540/590 nm (Cutoff = 570 nm) with bottom read mode, or take images using fluorescence microscope with a TRITC filter.
Example data analysis and figures

Figure 1. Fluorescence images of endogenous nitric oxide (NO) detection in RAW 264.7 macrophage. Cells were incubated with AAT’s Nitrixyte™ Orange (Left) or DAF-2 diacetate (Right) at the same concentration,  then treated with or without 20 µg/mL of lipopolysaccharide (LPS) and 1 mM L-arginine (L-Arg) at 37°C for 16 hours. The fluorescence signals were measured using a fluorescence microscope equipped with a TRITC (Left) or FITC (Right) filter set, respectively.
Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





References & Citations

Fluorescent real-time quantitative measurements of intracellular peroxynitrite generation and inhibition
Authors: Zhen Luo, Qin Zhao, Jixiang Liu, Jinfang Liao, Ruogu Peng, Yunting Xi, Zhenjun Diwu
Journal: Analytical biochemistry (2017): 44--48

Inducible Nitric Oxide Synthase (iNOS) Is a Novel Negative Regulator of Hematopoietic Stem/Progenitor Cell Trafficking
Authors: Mateusz Adamiak, Ahmed Abdelbaset-Ismail, Joseph B Moore, J Zhao, Ahmed Abdel-Latif, Marcin Wysoczynski, Mariusz Z Ratajczak
Journal: Stem Cell Reviews and Reports (2016): 1--12






Additional Documents

 
Safety Data Sheet (SDS)


Catalogs
1. Reactive Oxygen Species (ROS) Detection

Certificate of Analysis