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Cell Meter™ Fluorimetric Intracellular Nitric Oxide (NO) Activity Assay Kit *NIR 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 (NO) Activity Assay Kit. Cells were incubated with Nitrixyte™ NIR for 30 minutes, and further treated with or without 1 mM DEA NONOate in Assay Buffer for 60 minutes. Fluorescence intensity was measured using ACEA NovoCyte flow cytometer in APC channel.
Detection of exogenous nitric oxide (NO) in Jurkat cells upon DEA NONOate treatment (NO donor) using Cell Meter™ Fluorimetric Intracellular Nitric Oxide (NO) Activity Assay Kit. Cells were incubated with Nitrixyte™ NIR for 30 minutes, and further treated with or without 1 mM DEA NONOate in Assay Buffer for 60 minutes. Fluorescence intensity was measured using ACEA NovoCyte flow cytometer in APC channel.
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Catalog Number16360
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Telephone1-408-733-1055
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H-phraseH303, H313, H333
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Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


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 Kits provide sensitive tools to monitor intracellular NO level in live cells. Nitrixyte™ probes are developed and used in our assay kits 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™ NIR that can react with NO to generate strong near-infrared (NIR) fluorescence signal. Nitrixyte™ NIR can be readily loaded into live cells, and its fluorescence signal can be conveniently monitored using the filter set of Cy5® or APC. 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: Nitrixyte™ NIR1 vial (100 uL, 500X)
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™ NIR
  3. Incubate cells with test compounds and Nitrixyte™ NIR at 37 ºC for desired period of time
  4. Analyze cells with a flow cytometer using APC 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™ NIR (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™ NIR.

  3. Incubate cells with test compounds and Nitrixyte™ NIR 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™ NIR 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™ NIR 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 APC channel (Ex/Em = 640/675 nm) using a flow cytometer. Gate on the cells of interest, excluding debris.

Citations


View all 3 citations: Citation Explorer
The Selective Acetamidine-Based iNOS Inhibitor CM544 Reduces Glioma Cell Proliferation by Enhancing PARP-1 Cleavage In Vitro
Authors: Gallorini, Marialucia and Maccallini, Cristina and Ammazzalorso, Aless and ra , undefined and Amoia, Pasquale and De Filippis, Barbara and Fantacuzzi, Marialuigia and Giampietro, Letizia and Cataldi, Amelia and Amoroso, Rosa
Journal: International Journal of Molecular Sciences (2019): 495
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
Authors: Novensa L, Novella S, Medina P, Segarra G, Castillo N, Heras M, Hermenegildo C, Dantas AP.
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
Authors: Zhou X, He P.
Journal: Am J Physiol Heart Circ Physiol (2011): H1788
Polyamines, polyamine oxidases and nitric oxide in development, abiotic and biotic stresses
Authors: Wimalasekera R, Tebartz F, Scherer GF.
Journal: Plant Sci (2011): 593
Rapid upregulation of cytoprotective nitric oxide in breast tumor cells subjected to a photodynamic therapy-like oxidative challenge
Authors: Bhowmick R, Girotti AW.
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
Authors: Ye X, Xie F, Romanova EV, Rubakhin SS, Sweedler JV.
Journal: ACS Chem Neurosci (2010): 182