Cell Meter™ Fluorimetric Intracellular Peroxynitrite Assay Kit Optimized for Flow Cytometry

Detection of peroxynitrite in Jurkat cells upon SIN-1 treatment using AAT Cell Meter™ Fluorimetric Intracellular Peroxynitrite Assay Kit (Cat#16317). (A) Jurkat cells were co-incubated with DAX-J2™ PON Green and 200 µM SIN-1 in full medium at 37 ºC for 1 hour. (B) Cells were stained with DAX-J2™ PON Green for 1 hour, washed with PBS and then incubated with 200 µM SIN-1 in full medium at 37 ºC for 16 hours. Cells stained with DAX-J2™ PON Green but without SIN-1 treatment were used as a control. Fluorescence intensity was measured using ACEA NovoCyte flow cytometer in FITC channel.

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Alternative formats

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

Overview SDS Protocol

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.

Platform

Flow cytometer

Excitation	488 nm laser
Emission	530/30 nm filter
Instrument specification(s)	FITC channel

Components

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

Important Allow all the components to warm to room temperature before begining protocol.

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.

Images

Figure 1. Detection of peroxynitrite in Jurkat cells upon SIN-1 treatment using AAT Cell Meter™ Fluorimetric Intracellular Peroxynitrite Assay Kit (Cat#16317). (A) Jurkat cells were co-incubated with DAX-J2™ PON Green and 200 µM SIN-1 in full medium at 37 ºC for 1 hour. (B) Cells were stained with DAX-J2™ PON Green for 1 hour, washed with PBS and then incubated with 200 µM SIN-1 in full medium at 37 ºC for 16 hours. Cells stained with DAX-J2™ PON Green but without SIN-1 treatment were used as a control. Fluorescence intensity was measured using ACEA NovoCyte flow cytometer in FITC channel.

Citations

View all 6 citations: Citation Explorer

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

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

Peroxynitrite Decomposition Catalyst Enhances Respiratory Function in Isolated Brain Mitochondria
Authors: Albuck, Aaron L and Sakamuri, Siva Sankara Vara Prasad and Sperling, Jared A and Evans, Wesley R and Kolli, Lahari and Sure, Venkata NLR and Katakam, Prasad VG
Journal: American Journal of Physiology-Heart and Circulatory Physiology (2020)

Concept and connotation of oxidative stress in preeclampsia
Authors: Al-Kuraishy, Hayder M and Al-Gareeb, Ali I and Al-Maiahy, Thabat J and others, undefined
Journal: Journal of Laboratory Physicians (2018): 276

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)

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