Cell Meter™ Fluorimetric Intracellular Peroxynitrite Assay Kit *Green Fluorescence*
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.
Example protocol
AT A GLANCE
Protocol summary
- Prepare cells in growth medium
- Co-incubate cells with test compounds and DAX-J2™ PON Green working solution at 37oC for desired period of time
- 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
- 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.
- 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.
- 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.
- 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 9 citations: Citation Explorer
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
Silver Nitroprusside as an Efficient Chemodynamic Therapeutic Agent and a Peroxynitrite nanogenerator for Targeted Cancer Therapy
Authors: Asif, Kanwal and Adeel, Muhammad and Rahman, Md Mahbubur and Sfriso, Andrea Augusto and Bartoletti, Michele and Canzonieri, Vincenzo and Rizzolio, Flavio and Caligiuri, Isabella
Journal: Journal of Advanced Research (2023)
Authors: Asif, Kanwal and Adeel, Muhammad and Rahman, Md Mahbubur and Sfriso, Andrea Augusto and Bartoletti, Michele and Canzonieri, Vincenzo and Rizzolio, Flavio and Caligiuri, Isabella
Journal: Journal of Advanced Research (2023)
Triterpenoids and ultrasound dual-catalytic nanoreactor ignites long-lived hypertoxic reactive species storm for deep tumor treatment
Authors: Li, Ziying and Xie, Huanzhang and Shi, Huifang and Li, Dongmiao and Zhang, Zizhong and Chen, Haijun and Gao, Yu
Journal: Chemical Engineering Journal (2023): 139938
Authors: Li, Ziying and Xie, Huanzhang and Shi, Huifang and Li, Dongmiao and Zhang, Zizhong and Chen, Haijun and Gao, Yu
Journal: Chemical Engineering Journal (2023): 139938
Mechanisms of oxidative removal of 1, 4-dioxane via free chlorine rapidly mixing into monochloramine: Implications on water treatment and reuse
Authors: Wu, Liang and Patton, Samuel D and Liu, Haizhou
Journal: Journal of Hazardous Materials (2022): 129760
Authors: Wu, Liang and Patton, Samuel D and Liu, Haizhou
Journal: Journal of Hazardous Materials (2022): 129760
Interactions between pH, reactive species, and cells in plasma-activated water can remove algae
Authors: Mizoi, Ken and Rodr{\'\i}guez-Gonz{\'a}lez, Vicente and Sasaki, Mao and Suzuki, Shoki and Honda, Kaede and Ishida, Naoya and Suzuki, Norihiro and Kuchitsu, Kazuyuki and Kondo, Takeshi and Yuasa, Makoto and others,
Journal: RSC advances (2022): 7626--7634
Authors: Mizoi, Ken and Rodr{\'\i}guez-Gonz{\'a}lez, Vicente and Sasaki, Mao and Suzuki, Shoki and Honda, Kaede and Ishida, Naoya and Suzuki, Norihiro and Kuchitsu, Kazuyuki and Kondo, Takeshi and Yuasa, Makoto and others,
Journal: RSC advances (2022): 7626--7634
References
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