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

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#16356). Cells were incubated with Nitrixyte™ Red at 37 °C for 30 minutes. Cells were further treated with (Red line) or without (Blue line) 1 mM DEA NONOate at 37 °C for another 30 minutes. The fluorescence signal was monitored at FL4 channel using a flow cytometer (BD FACSCalibur).
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#16356). Cells were incubated with Nitrixyte™ Red at 37 °C for 30 minutes. Cells were further treated with (Red line) or without (Blue line) 1 mM DEA NONOate at 37 °C for another 30 minutes. The fluorescence signal was monitored at FL4 channel using a flow cytometer (BD FACSCalibur).
Ordering information
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Catalog Number16356
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Spectral properties
Excitation (nm)586
Emission (nm)607
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Excitation (nm)
586
Emission (nm)
607
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™ Red that can react with NO to generate a bright red fluorescent product that has spectral properties similar to Texas Red®. Nitrixyte™ Red can be readily loaded into live cells, and its fluorescence signal can be conveniently monitored using the filter set of Red. This kit is optimized for flow cytometry applications.

Platform


Flow cytometer

Excitation640 nm laser
Emission660/20 nm filter
Instrument specification(s)APC channel

Components


Component A: 500X Nitrixyte™ Red1 vial (100 µL)
Component B: NONOate Positive Control1 vial (lyophilized powder)
Component C: Assay Buffer1 bottle (10 mL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells (0.5 - 1×106 cells/mL)
  2. Add 1 µL 500X Nitrixyte™ Red
  3. Incubate cells with test compounds and Nitrixyte™ Red at 37 ºC for desired period of time
  4. Analyze cells with a flow cytometer using FL4 channel

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

PREPARATION OF STOCK SOLUTION

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

PREPARATION OF WORKING SOLUTION

Dilute 50 mM NONOate Positive Control treatment stock solution with Assay Buffer (Component C) to make 1-2 mM NONOate positive control working solution.

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

SAMPLE EXPERIMENTAL PROTOCOL

  1. For each sample, prepare cells in 0.5 mL warm medium or buffer of your choice at a density of 5×105 to 1×106 cells/mL. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density for NO induction.                                                                                                                                                                                         
  2. Add 1 µL of 500X Nitrixyte™ Red (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™ Red.

  3. Incubate cells with test compounds and Nitrixyte™ Red at 37 ºC 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 Nitrixyte™ Red working solution, 20 µg/mL of lipopolysaccharide (LPS) and 1 mM L-Arginine (L-Arg) in cell culture medium at 37 ºC for 16 hours.

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

  5. Monitor the fluorescence intensity at the FL4 channel (Ex/Em = 630/660 nm) using a flow cytometer. Gate on the cells of interest, excluding debris.

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)586
Emission (nm)607

Citations


View all 5 citations: Citation Explorer
HEMA-induced oxidative stress inhibits NF-$\kappa$B nuclear translocation and TNF release from LTA-and LPS-stimulated immunocompetent cells
Authors: Schweikl, Helmut and Birke, Margaritha and Gallorini, Marialucia and Petzel, Christine and Bolay, Carola and Waha, Claudia and Hiller, Karl-Anton and Buchalla, Wolfgang
Journal: Dental Materials (2020)
MAGI1 Mediates eNOS Activation and NO Production in Endothelial Cells in Response to Fluid Shear Stress
Authors: Ghimire, Kedar and Zaric, Jelena and Alday-Parejo, Begona and Seebach, Jochen and Bousquenaud, Mélanie and Stalin, Jimmy and Bieler, Grégory and Schnittler, Hans-Joachim and Rüegg, Curzio
Journal: Cells (2019): 388
Functions of transcription factors NF-kB and Nrf2 in the inhibition of LPS-stimulated cytokine release by the resin monomer HEMA
Authors: Schweikl, Helmut and Gallorini, Marialucia and P{\"o}schl, Gerd and Urmann, Vera and Petzel, Christine and Bolay, Carola and Hiller, Karl-Anton and Cataldi, Amelia and Buchalla, Wolfgang
Journal: Dental Materials (2018): 1661--1678
Fluorescent real-time quantitative measurements of intracellular peroxynitrite generation and inhibition
Authors: Luo, Zhen and Zhao, Qin and Liu, Jixiang and Liao, Jinfang and Peng, Ruogu and Xi, Yunting and Diwu, Zhenjun
Journal: Analytical biochemistry (2017): 44--48
Inducible Nitric Oxide Synthase (iNOS) Is a Novel Negative Regulator of Hematopoietic Stem/Progenitor Cell Trafficking
Authors: Adamiak, Mateusz and Abdelbaset-Ismail, Ahmed and Moore, Joseph B and Zhao, J and Abdel-Latif, Ahmed and Wysoczynski, Marcin and Ratajczak, Mariusz Z
Journal: Stem Cell Reviews and Reports (2016): 1--12

References


View all 139 references: Citation Explorer
Pitfalls and limitations in using 4,5-diaminofluorescein for evaluating the influence of polyphenols on nitric oxide release from endothelial cells
Authors: Uhlenhut K, Hogger P.
Journal: Free Radic Biol Med (2012): 2266
Effects of moderate electrical stimulation on reactive species production by primary rat skeletal muscle cells: cross talk between superoxide and nitric oxide production
Authors: Lambertucci RH, Silveira Ldos R, Hirabara SM, Curi R, Sweeney G, Pithon-Curi TC.
Journal: J Cell Physiol (2012): 2511
Improved measurements of intracellular nitric oxide in intact microvessels using 4,5-diaminofluorescein diacetate
Authors: Zhou X, He P.
Journal: Am J Physiol Heart Circ Physiol (2011): H108
Aging negatively affects estrogens-mediated effects on nitric oxide bioavailability by shifting ERalpha/ERbeta balance in female mice
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Journal: PLoS One (2011): e25335
Temporal and spatial correlation of platelet-activating factor-induced increases in endothelial [Ca(2)(+)]i, nitric oxide, and gap formation in intact venules
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Journal: Am J Physiol Heart Circ Physiol (2011): H1788
Polyamines, polyamine oxidases and nitric oxide in development, abiotic and biotic stresses
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Journal: Plant Sci (2011): 593
Rapid upregulation of cytoprotective nitric oxide in breast tumor cells subjected to a photodynamic therapy-like oxidative challenge
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Journal: Photochem Photobiol (2011): 378
Sleep deprivation triggers inducible nitric oxide-dependent nitric oxide production in wake-active basal forebrain neurons
Authors: Kalinchuk AV, McCarley RW, Porkka-Heiskanen T, Basheer R.
Journal: J Neurosci (2010): 13254
Production and scavenging of nitric oxide by barley root mitochondria
Authors: Gupta KJ, Kaiser WM.
Journal: Plant Cell Physiol (2010): 576
Production of Nitric Oxide within the Aplysia Californica Nervous System
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Journal: ACS Chem Neurosci (2010): 182