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Cell Meter™ Fluorimetric Intracellular Peroxynitrite Assay Kit *Green Fluorescence*

Fluorescence images of intracellular peroxynitrite in RAW 264.7 macrophage cells using Cell Meter™ Fluorimetric Intracellular Peroxynitrite Assay Kit (Cat#16315). Raw 264.7 cells at 100,000 cells/well/100 µL were seeded overnight in a Costar black wall/clear bottom 96-well plate. SIN-1 Treatment: Cells were co-incubated with DAX-J2™ PON Green and 100 µM SIN-1 at 37 °C for 1 hour. Untreated control: The RAW 264.7 cells were incubated with DAX-J2™ PON Green without SIN-1 treatment. The fluorescence signals were measured using a fluorescence microscope with a FITC filter
Fluorescence images of intracellular peroxynitrite in RAW 264.7 macrophage cells using Cell Meter™ Fluorimetric Intracellular Peroxynitrite Assay Kit (Cat#16315). Raw 264.7 cells at 100,000 cells/well/100 µL were seeded overnight in a Costar black wall/clear bottom 96-well plate. SIN-1 Treatment: Cells were co-incubated with DAX-J2™ PON Green and 100 µM SIN-1 at 37 °C for 1 hour. Untreated control: The RAW 264.7 cells were incubated with DAX-J2™ PON Green without SIN-1 treatment. The fluorescence signals were measured using a fluorescence microscope with a FITC filter
Detection of peroxynitrite in living cells upon SIN-1 treatment using Cell Meter™ Fluorimetric Intracellular Peroxynitrite Assay Kit (Cat#16315). RAW 264.7 cells at 100,000 cells/well/100 µL were seeded overnight in a Costar black wall/clear bottom 96-well plate. Cells were co-incubated with DAX-J2™ PON Green working solution and SIN-1 at the concentration from 50 to 200 µM at 37 ºC for 1 hour. Cells incubated with DAX-J2™ PON Green without SIN-1 treatment were used as control. The fluorescence signal were monitored at Ex/Em = 490/530 nm (cut off = 515 nm) with bottom read mode using a FlexStation microplate reader (Molecular Devices).
Microplate reader measurement of RAW 264.7 cells labeled with (A) DAX-J2 PON Green or (B) DHR 123. RAW 264.7 cells were treated with different concentrations of SIN-1. Ebselen at the concentration of 20 µM was used as an ONOO- scavenger. Compared to DHR 123, the fluorescence increase of DAX-J2 PON Green labeled cells upon SIN-1 treatment was more fully inhibited by ebselen. As in previous findings, DHR 123 oxidation in any given cell type may involve not only ONOO- but also other related ROS/RNS. These results further highlight the high selectivity of DAX-J2 PON Green for intracellular ONOO- detection.
Effects of gene silencing of endothelial vWF during NOX activation. (A) Representative images of DHE (dihydroethidium) staining of wild type and vWF-knockdown cell in resting conditions and after the exposure to PMA. vWF downregulation prevented the increase of anion superoxide generation; in panel B, the results are shown as number of DHE positive cells per sample; (C) vWF downregulation prevented the increase in peroxynitrite levels observed in control cells after exposure to PMA; in panel D, results are shown as arbitrary units of fluorescence intensity. (E) vWF downregulation prevented the increase in NOX-4 subunit expression observed in control cells after exposure to PMA. In panel F, levels of NOX-4 are expressed as arbitrary units of NOX-4 (70 kDa, MW)/GAPDH (37 kDa, MW) ratio. (G) vWF downregulation prevented the increase in NOX-2 subunit expression observed in control cells after exposure to PMA. In panel H, levels of NOX-2 are expressed as arbitrary units of NOX-2 (67 kDa, MW)/GAPDH (37 kDa, MW) ratio. (I) vWF silencing downregulates ET-1 expression after exposure to PMA. ET-1 levels do not increase in response to PMA in vWF silenced cells. In panel L, levels of ET-1 are expressed as arbitrary units of ET-1 (24 kDa, MW)/alpha-Tubulin (50 kDa, MW) ratio. experiments performed in triplicate. *p < 0.05 vs. siRNA-NT at rest; #p < 0.05 vs. siRNA-vWF at rest; §p < 0.01 vs. siRNA-NT under PMA. Source: <strong>Gene silencing of endothelial von Willebrand Factor attenuates angiotensin II-induced endothelin-1 expression in porcine aortic endothelial cells </strong>by Dushpanova et al., <em>Scientific Reports</em>, July 2016.
Effects of gene silencing of endothelial vWF on Ang II-induced O2− production, peroxynitrite levels and NADPH oxidase activity. (A) Representative images of DHE staining of wild type and vWF-knockdown cell in resting conditions and after the exposure to AngII. (B) vWF downregulation prevented the increase of anion superoxide generation; the results are shown as number of DHE positive cells per sample; (C) vWF downregulation prevented the increase in peroxynitrite levels observed in control cells after exposure to AngII. In panel D, results are shown as arbitrary units of fluorescence intensity; (E) vWF downregulation prevented the increase in NADPH activity levels observed in control cells after exposure to AngII. Results are shown as arbitrary units of luminescence intensity. siRNA NT: non-targeting siRNA; siRNA vWF: anti-vWF siRNA; AngII: angiotensin II. All measurements are mean ± SD, n = 3 independent experiments performed in triplicate. *p < 0.05 vs. control at rest; §p < 0.05 vs. siRNA-NT under AngII. Source: <strong>Gene silencing of endothelial von Willebrand Factor attenuates angiotensin II-induced endothelin-1 expression in porcine aortic endothelial cells </strong>by Dushpanova et al., <em>Scientific Reports</em>, July 2016.
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Catalog Number16315
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Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
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H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Peroxynitrite (ONOO-) is a strong oxidizing species and a highly active nitrating agent. Peroxynitrite is formed from the reaction between superoxide radicals and nitric oxide generated in cells. It can cause damages to a wide array of biomolecules including proteins, enzymes, lipids and nucleic acids, eventually contributing to cell death. Meanwhile, peroxynitrite can also have protective activities in vivo by contributing to host-defense responses against invading pathogens. Therefore, peroxynitrite is an essential biological oxidant involved in a board range of physiological and pathological processes. Due to its extremely short half-life and low steady-state concentration, it has been challenging to detect and understand the role of peroxynitrite in biological systems. AAT Bioquest's DAX-J2™ PON Green has been developed to address this unmet need. It provides a sensitive tool to monitor ONOO- level in living cells. AAT Bioquest's DAX-J2™ PON Green specifically reacts with intercellular ONOO- to generate a bright green fluorescent product. It can be used in fluorescence imaging, flow cytometry and fluorescence microplate reader-based assays.

Platform


Fluorescence microscope

Excitation490 nm
Emission530 nm
Recommended plateBlack wall/clear bottom
Instrument specification(s)FITC Filter Set

Fluorescence microplate reader

Excitation490 nm
Emission530 nm
Cutoff515 nm
Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode

Components


Component A: DAX-J2™ PON Green1 vial
Component B: Assay Buffer1 vial (1 mL/vial)
Component C: DMSO1 vial (100 µL/vial)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells in growth medium
  2. Co-incubate cells with test compounds and DAX-J2™ PON Green working solution at 37oC for desired period of time
  3. Monitor fluorescence intensity at Ex/Em = 490/530 nm (Cutoff=515 nm)

Important notes
Bring all the kit 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. DAX-J2™ PON Green stock solution (500X):
Add 20 µL of DMSO (Component C) into the vial of DAX-J2™ PON Green (Component A), and mix well. Note: 20 µL of reconstituted DAX-J2™ PON Green stock solution is enough for 1 plate.

PREPARATION OF WORKING SOLUTION

Add 10 μL of 500X DMSO reconstituted DAX-J2™ Peroxynitrite Sensor stock solution into 500 μL of Assay Buffer (Component B) and mix well. Note: The working solution is not stable; prepare it as needed before use.

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

SAMPLE EXPERIMENTAL PROTOCOL

  1. Add 10 µL/well (96-well plate), or 2.5 µL/well (384-well plate) of DAX-J2™ PON Green working solution in 90 µL (96-well plate) or 22.5 µL (384-well plate) cell culture per well in the cell plate. Note: It is not necessary to wash cells before staining. It’s recommended to stain the cells in full medium.

  2. Co-incubate cells with DAX-J2™ PON Green with test compounds in full medium or in your desired buffer at 37°C for desired period of time, protected from light. For control wells (untreated cells), add the corresponding amount of compound buffer. Note: It’s recommended to stain the cells in full medium. However, if tested compounds are serum sensitive, growth medium and serum factors can be aspirated away before staining. Add 90 µL/well (96-well plate) and 22.5 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 stained in serum-free media. We co-incubated RAW 264.7 macrophage cells with 50 - 200 µM SIN-1 and DAX-J2™ PON Green in full medium at 37°C for 1 hour to induce peroxynitrite. See Figure 1 for details.

  3. Alternatively, stain cells with DAX-J2™ PON Green at 37°C for 1 hour, protected from light. Remove the working solution, then treat cells with test compounds in full medium or in your desired buffer at 37°C for desired period of time.

  4. Monitor the fluorescence increase using microplate reader at Ex/Em = 490/530 nm (cut off = 515 nm) with bottom read mode, or take images using fluorescence microscope with a FITC filter.

Citations


View all 4 citations: Citation Explorer
Peroxynitrite (ONOO-) generation from the HA-TPP@ NORM nanoparticles based on synergistic interactions between nitric oxide and photodynamic therapies for elevating anticancer efficiency
Authors: Jiang, Dawei and Yue, Tao and Wang, Guichen and Wang, Chaochao and Chen, Chao and Cao, Hongliang and Gao, Yun
Journal: New Journal of Chemistry (2020): 162--170
Nitric oxide and reactive oxygen species-releasing polylactic acid monolith for enhanced photothermal therapy of osteosarcoma
Authors: Lee, Ji-Hye and Uyama, Hiroshi and Kwon, Oh-Kyoung and Kim, Young-Jin
Journal: Journal of Industrial and Engineering Chemistry (2020)
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
Gene silencing of endothelial von Willebrand Factor attenuates angiotensin II-induced endothelin-1 expression in porcine aortic endothelial cells
Authors: Dushpanova, Anar and Agostini, Silvia and Ciofini, Enrica and Cabiati, Manuela and Casieri, Valentina and Matteucci, Marco and Del Ry, Silvia and Clerico, Aldo and Berti, Sergio and Lionetti, Vincenzo
Journal: Scientific Reports (2016)

References


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