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Cell Meter™ Fluorimetric Intracellular Peroxynitrite Assay Kit *Optimized for Flow Cytometry*
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 damage 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 broad 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. In order to address this unmet need, AAT Bioquest's Cell Meter™ Fluorimetric Intracellular Peroxynitrite (ONOO-) Assay Kit provides a sensitive tool to monitor ONOO- level in living cells. AAT Bioquest's DAX-J2™ PON Green is developed as an excellent fluorescent probe, which can specifically react with intercellular ONOO' to generate a bright green fluorescent product. This kit is optimized for flow cytometry.
Example protocol
AT A GLANCE
Protocol Summary
- Prepare cells in growth medium
- Co-incubate cells with test compounds (to stimulate ONOO¯) and DAX-J2™ PON Green working solution
- Monitor fluorescence intensity at Ex/Em = 490/530 nm
CELL PREPARATION
For guidelines on cell sample preparation, please visit https://www.aatbio.com/resources/guides/cell-sample-preparation.html
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.
DAX-J2™ PON Green stock solution (400X)
Add 25 µL of DMSO (Component C) into the vial of DAX-J2™ PON Green (Component A), and mix well. Note: 1 µL of reconstituted DAX-J2™ PON Green stock solution is enough for 0.4 mL of cells.SAMPLE EXPERIMENTAL PROTOCOL
- 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 is 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. Resuspend cells in 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 Jurkat cells with 200 µM SIN-1 and DAX-J2™ PON Green in full medium at 37°C for 1 hour to induce peroxynitrite. See Figure 1(A) for details.
- 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. See Figure 1(B) for details.
- Monitor the fluorescence intensity at FITC channel (Ex/Em=490/530 nm) using a flow cytometer. Gate on the cells of interest, excluding debris.
Citations
View all 9 citations: Citation Explorer
Copper nitroprusside: An innovative approach for targeted cancer therapy via ROS modulation
Authors: Asif, Kanwal and Adeel, Muhammad and Rahman, Md Mahbubur and Bartoletti, Michele and Brezar, Simona Kranjc and Cemazar, Maja and Canzonieri, Vincenzo and Rizzolio, Flavio and Caligiuri, Isabella
Journal: Biomedicine \& Pharmacotherapy (2024): 116017
Authors: Asif, Kanwal and Adeel, Muhammad and Rahman, Md Mahbubur and Bartoletti, Michele and Brezar, Simona Kranjc and Cemazar, Maja and Canzonieri, Vincenzo and Rizzolio, Flavio and Caligiuri, Isabella
Journal: Biomedicine \& Pharmacotherapy (2024): 116017
Cold atmospheric plasma sensitizes head and neck cancer to chemotherapy and immune checkpoint blockade therapy
Authors: Wang, Yanhong and Mang, Xinyu and Li, Danni and Wang, Zhao and Chen, Yiliang and Cai, Zhenyu and Tan, Fei
Journal: Redox Biology (2023): 102991
Authors: Wang, Yanhong and Mang, Xinyu and Li, Danni and Wang, Zhao and Chen, Yiliang and Cai, Zhenyu and Tan, Fei
Journal: Redox Biology (2023): 102991
Quantum Molecular Resonance Inhibits NLRP3 Inflammasome/Nitrosative Stress and Promotes M1 to M2 Macrophage Polarization: Potential Therapeutic Effect in Osteoarthritis Model In Vitro
Authors: Paolucci, Teresa and Pino, Vanessa and Elsallabi, Osama and Gallorini, Marialucia and Pozzato, Gianantonio and Pozzato, Alessandro and Lanuti, Paola and Reis, Victor Machado and Pesce, Mirko and Pantalone, Andrea and others,
Journal: Antioxidants (2023): 1358
Authors: Paolucci, Teresa and Pino, Vanessa and Elsallabi, Osama and Gallorini, Marialucia and Pozzato, Gianantonio and Pozzato, Alessandro and Lanuti, Paola and Reis, Victor Machado and Pesce, Mirko and Pantalone, Andrea and others,
Journal: Antioxidants (2023): 1358
Low level of antioxidant capacity biomarkers but not target overexpression predicts vulnerability to ROS-inducing drugs
Authors: Samarin, Jana and Fabrowski, Piotr and Kurilov, Roman and Nuskova, Hana and Hummel-Eisenbeiss, Johanna and Pink, Hannelore and Li, Nan and Weru, Vivienn and Alborzinia, Hamed and Yildiz, Umut and others,
Journal: bioRxiv (2023): 2023--01
Authors: Samarin, Jana and Fabrowski, Piotr and Kurilov, Roman and Nuskova, Hana and Hummel-Eisenbeiss, Johanna and Pink, Hannelore and Li, Nan and Weru, Vivienn and Alborzinia, Hamed and Yildiz, Umut and others,
Journal: bioRxiv (2023): 2023--01
Characterization and Valorization of ‘Sulmona Red Garlic’Peels and Small Bulbs
Authors: Lasalvia, Alba and Cairone, Francesco and Cesa, Stefania and Maccelli, Alessandro and Crestoni, Maria Elisa and Menghini, Luigi and Carradori, Simone and Marinacci, Beatrice and Gallorini, Marialucia and Elsallabi, Osama and others,
Journal: Antioxidants (2022): 2088
Authors: Lasalvia, Alba and Cairone, Francesco and Cesa, Stefania and Maccelli, Alessandro and Crestoni, Maria Elisa and Menghini, Luigi and Carradori, Simone and Marinacci, Beatrice and Gallorini, Marialucia and Elsallabi, Osama and others,
Journal: Antioxidants (2022): 2088
References
View all 10 references: Citation Explorer
Imaging of nucleolar RNA in living cells using a highly photostable deep-red fluorescent probe
Authors: Zhou B, Liu W, Zhang H, Wu J, Liu S, Xu H, Wang P.
Journal: Biosens Bioelectron (2015): 189
Authors: Zhou B, Liu W, Zhang H, Wu J, Liu S, Xu H, Wang P.
Journal: Biosens Bioelectron (2015): 189
RNA and DNA binding of inert oligonuclear ruthenium(II) complexes in live eukaryotic cells
Authors: Li X, Gorle AK, Ainsworth TD, Heimann K, Woodward CE, Collins JG, Keene FR.
Journal: Dalton Trans (2015): 3594
Authors: Li X, Gorle AK, Ainsworth TD, Heimann K, Woodward CE, Collins JG, Keene FR.
Journal: Dalton Trans (2015): 3594
Low molecular weight fluorescent probes with good photostability for imaging RNA-rich nucleolus and RNA in cytoplasm in living cells
Authors: Song G, Sun Y, Liu Y, Wang X, Chen M, Miao F, Zhang W, Yu X, Jin J.
Journal: Biomaterials (2014): 2103
Authors: Song G, Sun Y, Liu Y, Wang X, Chen M, Miao F, Zhang W, Yu X, Jin J.
Journal: Biomaterials (2014): 2103
Luminescence of [Ru(bpy)2(dppz)]2+ bound to RNA mismatches
Authors: McConnell AJ, Song H, Barton JK.
Journal: Inorg Chem (2013): 10131
Authors: McConnell AJ, Song H, Barton JK.
Journal: Inorg Chem (2013): 10131
Co-aggregation of RNA binding proteins in ALS spinal motor neurons: evidence of a common pathogenic mechanism
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Authors: Keller BA, Volkening K, Droppelmann CA, Ang LC, Rademakers R, Strong MJ.
Journal: Acta Neuropathol (2012): 733
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