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

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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. Cells were further treated with (Red line) or without (Blue line) 1 mM DEA NONOate in Assay Buffer (Component C) at 37 °C for an additional 30 minutes. The fluorescence signal was monitored at FL2 channel using a flow cytometer (BD FACSCalibur).
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Unit Size: Cat No: Price (USD): Qty:
100 Tests 16351 $345


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

Ex/Em (nm)488/590
Storage F/D/L
InstrumentsFlow cytometer
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 flow cytometry applications.




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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 (0.5 - 1 × 106 cells/mL)
  2. Add 1 µL 500X Nitrixyte™ Orange into 0.5 mL cell suspension
  3. Incubate cells with test compounds and Nitrixyte™ Orange at 37°C
  4. Analyze with a flow cytometer

Important notes
Thaw all the components at room temperature before use.

Key parameters
Instrument:Flow cytometer
Excitation:488 nm
Emission:590 nm
Instrument specification(s):FL2 channel or PE channel
Preparation of stock solutions
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. NONOate Positive Control stock solution (50 mM):
Add 200 µL of ddH2O into the vial of NONOate Positive Control (Component B) to make 50 mM stock solution.

Preparation of working solution

1. NONOate Positive Control working solution
Dilute the NONOate Positive Control stock solution to a 1 - 2 mM working solution with Assay Buffer (Component C).

Preparation of cell samples

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

Sample experimental protocol
  1. Add 1 µL of 500X Nitrixyte™ Orange (Component A) into 0.5 mL cell suspension. Note: For adherent cells, gently lift the cells with 0.5 mM EDTA to keep the cells intact, and wash the cells once with serum-containing media prior to incubation with Nitrixyte™ Orange.

  2. Incubate cells with test compounds and Nitrixyte™ Orange at 37°C for a desired period of time to generate endogenous or exogenous NO. Note: The appropriate incubation time depends on the individual cell type and test compound used. Optimize the incubation time for each experiment. Note: We have used Raw 264.7 cells incubated with 1X 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.

  3. Spin down cells that have pre-incubated with Nitrixyte™ Orange for 30 minutes. Resuspend cells with 1 mM DEA NONOate positive control working solution, and incubate at 37°C for another 30 minutes. See Figure 1 for details.

  4. Monitor the fluorescence intensity at the FL2 channel (Ex/Em = 488/590 nm) using a flow cytometer. Gate on the cells of interest, excluding debris.
Example data analysis and figures

Figure 1. 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. Cells were further treated with (Red line) or without (Blue line) 1 mM DEA NONOate in Assay Buffer (Component C) at 37 °C for an additional 30 minutes. The fluorescence signal was monitored at FL2 channel using a flow cytometer (BD FACSCalibur).
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