Amplite® Fluorimetric Peroxynitrite Quantification Kit *Green Fluorescence*
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
Overview | ![]() ![]() |
Platform
Fluorescence microplate reader
Excitation | 490 nm |
Emission | 530 nm |
Cutoff | 515 nm |
Recommended plate | Solid black |
Components
Example protocol
AT A GLANCE
Important notes
Thaw all the kit components to room temperature before use.
PREPARATION OF STOCK SOLUTION
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
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.
Images
Citations
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: Wu, Liang and Patton, Samuel D and Liu, Haizhou
Journal: Journal of Hazardous Materials (2022): 129760
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: 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
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
Authors: Zhenyukh, Olha and González-Amor, Maria and Rodrigues-Diez, Raul R and Esteban, Vanesa and Ruiz-Ortega, Marta and Salaices, Mercedes and Mas, Sebastian and Briones, Ana M and Egido, Jesus
Journal: Journal of Cellular and Molecular Medicine (2018)
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
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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Application notes
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