Actively helping customers, employees and the global community during the coronavirus SARS-CoV-2 outbreak.  Learn more >>

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.
Ordering information
Price ()
Catalog Number16360
Unit Size
Find Distributor
Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200
Related products
Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit *Green Fluorescence*
Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit *Blue Fluorescence*
Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit *Blue Fluorescence Optimized for Flow Cytometry*
Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit *Green Fluorescence Optimized for Flow Cytometry*
Cell Meter™ Mitochondrial Hydroxyl Radical Detection Kit *Red Fluorescence*
Cell Meter™ Fluorimetric Mitochondrial Superoxide Activity Assay Kit *Green Fluorescence*
Cell Meter™ Fluorimetric Intracellular Peroxynitrite Assay Kit *Green Fluorescence*
Cell Meter™ Fluorimetric Intracellular Peroxynitrite Assay Kit *Optimized for Flow Cytometry*
Cell Meter™ Live Cell Caspase 3/7 Binding Assay Kit *Green Fluorescence*
Cell Meter™ Live Cell Caspase 3/7 Binding Assay Kit *Red Fluorescence*
Cell Meter™ Live Cell Caspase 1 Binding Assay Kit *Green Fluorescence*
Cell Meter™ Live Cell Caspase 2 Binding Assay Kit *Green Fluorescence*
Cell Meter™ Live Cell Caspase 6 Binding Assay Kit *Green Fluorescence*
Cell Meter™ Live Cell Caspase 8 Binding Assay Kit *Green Fluorescence*
Cell Meter™ Live Cell Caspase 9 Binding Assay Kit *Green Fluorescence*
Cell Meter™ Live Cell Caspase 10 Binding Assay Kit *Green Fluorescence*
Cell Meter™ Live Cell Caspase 13 Binding Assay Kit *Green Fluorescence*
Cell Meter™ No-Wash Live Cell Caspase 3/7 Activity Assay Kit *Blue Fluorescence*
Cell Meter™ No-Wash Live Cell Caspase 3/7 Activity Assay Kit *Red Fluorescence*
Cell Meter™ Fluorimetric Intracellular pH Assay Kit
Cell Meter™ Fluorimetric Cell Cytotoxicity Assay Kit
Cell Meter™ Cell Viability Assay Kit *Red Fluorescence*
Cell Meter™ Cell Viability Assay Kit *Blue Fluorescence with 405 nm Excitation*
Cell Meter™ Cell Viability Assay Kit *Blue Fluorescence*
Cell Meter™ Cell Viability Assay Kit *Green Fluorescence*
Cell Meter™ Cell Viability Assay Kit *NIR Fluorescence Optimized for Fluorescence Microplate Reader*
Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Blue Fluorescence Optimized for Microplate Readers*
Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Green Fluorescence Optimized for Microplate Readers*
Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Red Fluorescence Optimized for Microplate Readers*
Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Deep Red Fluorescence Optimized for Microplate Readers*
Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Orange Fluorescence Optimized for Microplate Readers*
Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Blue Fluorescence*
Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Green Fluorescence*
Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Red Fluorescence*
Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Green Fluorescence*
Cell Meter™ Caspase 9 Activity Apoptosis Assay Kit *Green Fluorescence*
Cell Meter™ JC-10 Mitochondrion Membrane Potential Assay Kit *Optimized for Microplate Assays*
Cell Meter™ JC-10 Mitochondrion Membrane Potential Assay Kit *Optimized for Flow Cytometry Assays*
Cell Meter™ NIR Mitochondrion Membrane Potential Assay Kit *Optimized for Flow Cytometry*
Cell Meter™ NIR Mitochondrion Membrane Potential Assay Kit *Optimized for Microplate Reader*
Cell Meter™ Mitochondrion Membrane Potential Assay Kit *Orange Fluorescence Optimized for Flow Cytometry*
Cell Meter™ Mitochondrion Membrane Potential Assay Kit *Orange Fluorescence Optimized for Microplate Reader*
Cell Meter™ Intracellular GSH Assay Kit *Optimized for Flow Cytometry*
Cell Meter™ Nuclear Apoptosis Assay Kit *Green Fluorescence Optimized for Flow Cytometry*
Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Blue Fluorescence*
Cell Meter™ Caspase 9 Activity Apoptosis Assay Kit *Blue Fluorescence*
Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Red Fluorescence*
Cell Meter™ Caspase 9 Activity Apoptosis Assay Kit *Red Fluorescence*
Cell Meter™ Multiplexing Caspase 3/7, 8 and 9 Activity Assay Kit *Triple Fluorescence Colors*
Cell Meter™ Generic Fluorimetric Caspase Activity Assay Kit *Green Fluorescence Optimized for Flow Cytometry*
Cell Meter™ Generic Fluorimetric Caspase Binding Assay Kit *Red Fluorescence Optimized for Flow Cytometry*
Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Green Fluorescence Optimized for Flow Cytometry*
Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Green Fluorescence Optimized for Flow Cytometry*
Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Orange Fluorescence Optimized for Flow Cytometry*
Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Red Fluorescence Optimized for Flow Cytometry*
Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Deep Red Fluorescence Optimized for Flow Cytometry*
Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Blue Fluorescence Excited at 405 nm*
Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Green Fluorescence Excited at 405 nm*
Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Orange Fluorescence Excited at 405 nm*
Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Green Fluorescence Optimized for Flow Cytometry*
Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Deep Red Fluorescence Optimized for Flow Cytometry*
Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Blue Fluorescence Excited at 405 nm*
Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Green Fluorescence Excited at 405 nm*
Cell Meter™ APC-Annexin V Binding Apoptosis Assay Kit *Optimized for Flow Cytometry*
Cell Meter™ PE-Annexin V Binding Apoptosis Assay Kit *Optimized for Flow Cytometry*
Cell Meter™ Apoptotic and Necrotic Multiplexing Detection Kit I *Triple Fluorescence Colors*
Cell Meter™ Fluorimetric Live Cell Cycle Assay Kit *Green Fluorescence Optimized for Flow Cytometry*
Cell Meter™ Fluorimetric Fixed Cell Cycle Assay Kit *Red Fluorescence Optimized for Flow Cytometry*
Cell Meter™ Apoptotic and Necrotic Multiplexing Detection Kit II *Triple Fluorescence Colors*
Cell Meter™ Live Cell TUNEL Apoptosis Assay Kit *Red Fluorescence*
Cell Meter™ Fluorimetric Live Cell Cycle Assay Kit *Optimized for 405 nm Violet Laser Excitation*
Cell Meter™ Live Cell Caspase 3/7 and Phosphatidylserine Detection Kit *Triple Fluorescence Colors*
Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit*Green Fluorescence*
Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit*Red Fluorescence*
Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit*Orange Fluorescence*
Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit*Deep Red Fluorescence*
Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit*Optimized for Flow Cytometry*
Cell Meter™ Fluorimetric Mitochondrial Superoxide Activity Assay Kit*Optimized for Flow Cytometry*
Cell Meter™ Fluorimetric Mitochondrial Superoxide Activity Assay Kit*Optimized for Microplate Reader*
Cell Meter™ Autophagy Assay Kit *Blue Fluorescence*
Cell Meter™ Autophagy Fluorescence Imaging Kit
Cell Meter™ Autophagy Assay Kit *Green Fluorescence*
Cell Meter™ FITC-Annexin V Binding Apoptosis Assay Kit *Optimized for Flow Cytometry*
Cell Meter™ Colorimetric WST-8 Cell Quantification Kit
Cell Meter™ Multiplexing Live, Apoptotic and Necrotic Cell Detection Kit III *Triple Fluorescence Colors*
Cell Meter™ 2-NBDG Glucose Uptake Assay Kit
Cell Meter™ Intracellular NADH/NADPH Fluorescence Imaging Kit *Red Fluorescence*
Cell Meter™ Intracellular NADH/NADPH Flow Cytometric Analysis Kit *Red Fluorescence*
Cell Meter™ Live Cell TUNEL Apoptosis Assay Kit *Green Fluorescence*
Cell Meter™ No Wash and Probenecid-Free Endpoint Calcium Assay Kit *Optimized for microplate reader*
Cell Meter™ Mitochondrion Membrane Potential Assay Kit *Red Fluorescence Optimized for Flow Cytometry*
Cell Meter™ Mitochondrion Membrane Potential Assay Kit *Red Fluorescence Optimized for Microplate Reader*
Cell Meter™ Intracellular Colorimetric Lipid Peroxidation (MDA) Assay Kit
Cell Meter™ Flow Cytometric Calcium Assay Kit
Cell Meter™ Fluorimetric Live Cell Cycle Assay Kit *Red Fluorescence Optimized for Flow Cytometry*
Cell Meter™ Fluorimetric Cellular Lipid Peroxidation Assay Kit
Cell Meter™ Intracellular NADH/NADPH Fluorescence Imaging Kit *Deep Red Fluorescence*
Cell Meter™ Intracellular NADH/NADPH Flow Cytometric Analysis Kit *Deep Red Fluorescence*
Cell Meter™ Intracellular GSH Assay Kit *Optimized for Flow Cytometry with 405 nm excitation*
Cell Meter™ Mitochondrial Autophagy Imaging Kit *Red Fluorescence*
Cell Meter™ BX520 fixable viability dye
Cell Meter™ BX590 fixable viability dye
Cell Meter™ BX650 fixable viability dye
Cell Meter™ IX830 fixable viability dye
Cell Meter™ RX660 fixable viability dye
Cell Meter™ RX700 fixable viability dye
Cell Meter™ RX780 fixable viability dye
Cell Meter™ VX450 fixable viability dye
Cell Meter™ VX500 fixable viability dye
Cell Meter™ Live Cell Caspase 8 Binding Assay Kit *Red Fluorescence*
Cell Meter™ Fixed Cell and Tissue TUNEL Apoptosis Assay Kit *Green Fluorescence*
Cell Meter™ Fixed Cell and Tissue TUNEL Apoptosis Assay Kit *Red Fluorescence*
Cell Meter™ Fluorimetric Cellular Voltage Assay Kit
Cell Meter™ Cell Adhesion Assay Kit
Cell Meter™ Fixed Cell and Tissue TUNEL Apoptosis Assay Kit *Deep Red Fluorescence*
Cell Meter™ Fixed Cell and Tissue TUNEL Apoptosis Assay Kit *Blue Fluorescence*
Cell Meter™ Fluorescence Gap Junction Tracing Kit
Cell Meter™ Colorimetric MTT Cell Proliferation Kit
Cell Meter™ Live Cell Caspase 3/7 Imaging Kit *Green Fluorescence*
Cell Meter™ Beta-Arrestin Translocation GPCR Signaling Kit
Cell Meter™ Live Cell ATP Assay Kit
Cell Meter™ Colorimetric Cell Cytotoxicity Assay Kit
Cell Meter™ Fluorimetric Phagocytosis Assay Kit *Red Fluorescence*
Cell Meter™ Cellular Senescence Activity Assay Kit *Green Fluorescence*
Cell Meter™ Cellular Senescence Activity Assay Kit *Red Fluorescence*
Cell Meter™ Cell Proliferation Assay Kit
Show More (126)

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