Amplite® Fluorimetric Peroxynitrite Quantification 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 damage a wide array of biomolecules including proteins, enzymes, lipids and nucleic acids, eventually contributing to cell death. Due to its extremely short half-life and low steady-state concentration, it has been challenging to detect and quantify peroxynitrite in solution. In order to address this need, AAT Bioquest's Amplite® Fluorimetric Peroxynitrite Quantification Kit provides a sensitive tool to measure ONOO- in solution. DAX-J2™ PON Green 99 reacts with ONOO- to generate a bright green fluorescent product. It specifically reacts with ONOO- with high selectivity over other reactive oxygen species (ROS) and reactive nitrogen species (RNS). This kit can be used with a fluorescence microplate reader and spectrometer.

Example protocol

AT A GLANCE

Important notes
Thaw all the kit components to room temperature before use.

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 99 stock solution (500X):
Add 20 µL of DMSO (Component C) into the vial of DAX-J2™ PON Green 99 (Component A) to make 500X stock solution. Note: 20 µL of reconstituted DAX-J2™ PON Green 99 stock solution is enough for 1 plate. Keep from light.

2. Peroxynitrite (ONOO¯ ) stock solution (not provided):
Peroxynitrite stock solution was synthesized according to literature report. Briefly, a mixture of sodium nitrite (0.6 M) and hydrogen peroxide (0.7 M) was acidified with hydrochloric acid (0.6 M), and sodium hydroxide (1.5 M) was added within 1 - 2 seconds to make the solution alkaline. The excess hydrogen peroxide was removed by passing the solution through a short column of manganese dioxide. The extinction coefficient of peroxynitrite solution in 0.1 M NaOH is 1670 M^-1cm^-1 at 302 nm. The ONOO¯ stock solution is not stable; we highly recommend make it fresh to use.

PREPARATION OF STANDARD SOLUTION

Peroxynitrite standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/16316

Dilute Peroxynitrite (ONOO¯) stock solution in Assay buffer (Component B) to have 20 µM ONOO¯ standard solution, and then perform 1:2 serial dilutions to get serially diluted ONOO¯ standard solution (O7 - O1).

PREPARATION OF WORKING SOLUTION

Add 20 μL of 500X DAX-J2™ PON Green 99 stock solution into 10 mL of Assay Buffer (Component B) and mix well. Note: This assay mixture is enough for one 96-well plate. Protect from light.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of ONOO¯ standards and test samples in a solid black 96-well microplate. O = ONOO¯ Standards (O1 - O7, 0.313 to 20 µM); BL = Blank Control; TS = Test Samples.

BL	BL	TS	TS
O1	O1	...	...
O2	O2	...	...
O3	O3
O4	O4
O5	O5
O6	O6
O7	O7

Table 2. Reagent composition for each well.

Well	Volume	Reagent
O1 - O7	50 µL	Serial Dilutions (0.313 to 20 µM)
BL	50 µL	assay buffer
TS	50 µL	test sample

Prepare ONOO¯ standards (O), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
Add 50 µL of working solution to each well of ONOO¯ standard, blank control, and test samples to make the total ONOO¯ assay volume of 100 µL/well. For a 384-well plate, add 25 µL of working solution into each well instead, for a total volume of 50 µL/well.
Incubate the reaction at room temperature for 5 to 10 minutes, protected from light.
Monitor the fluorescence increase at Ex/Em = 490/530 nm (cutoff at 515 nm) with a fluorescence plate reader.

Citations

View all 8 citations: Citation Explorer

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

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

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

Nanoparticles Encapsulating Nitrosylated Maytansine To Enhance Radiation Therapy
Authors: Gao, Shi and Zhang, Weizhong and Wang, Renjie and Hopkins, Sean P and Spagnoli, Jonathan C and Racin, Mohammed and Bai, Lin and Li, Lu and Jiang, Wen and Yang, Xueyuan and others,
Journal: ACS nano (2020): 1468--1481

Peroxynitrite Generation and Increased Heterotrophic Capacity Are Linked to the Disruption of the Coral--Dinoflagellate Symbiosis in a Scleractinian and Hydrocoral Species
Authors: Marangoni, Laura Fern and es de Barros , undefined and Mies, Miguel and Güth, Arthur Z and Banha, Thomás NS and Inague, Alex and Fonseca, Juliana da Silva and Dalmolin, Camila and Faria, Samuel Coelho and Ferrier-Pagès, Christine and Bianchini, Adalto
Journal: Microorganisms (2019): 426

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

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

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

Luminescence of [Ru(bpy)2(dppz)]2+ bound to RNA mismatches
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
Authors: Keller BA, Volkening K, Droppelmann CA, Ang LC, Rademakers R, Strong MJ.
Journal: Acta Neuropathol (2012): 733

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