Amplite® Fluorimetric Hydrogen Peroxide Assay Kit *Near Infrared Fluorescence*
Price | |
Catalog Number | |
Unit Size | |
Quantity |
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 |
Excitation (nm) | 648 |
Emission (nm) | 668 |
Certificate of Origin | Download PDF |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12171501 |
Amplite® Fluorimetric Hydrogen Peroxide Assay Kit *Red Fluorescence* |
Overview | SDSProtocol |
Excitation (nm) 648 | Emission (nm) 668 |
Platform
Fluorescence microplate reader
Excitation | 640 nm |
Emission | 680 nm |
Cutoff | 665 nm |
Recommended plate | Solid black |
Components
Example protocol
AT A GLANCE
Protocol summary
- Prepare H2O2 working solution (50 µL)
- Add H2O2 standards or test samples (50 µL)
- Incubate at room temperature for 0 - 30 minutes
- Monitor fluorescence intensity at Ex/Em = 640/680 nm (Cutoff = 665 nm)
Important notes
Thaw all the kit components at room temperature before starting the experiment. The Amplite™ Fluorimetric Hydrogen Peroxide Assay Kit can be used to measure the release of H2O2 from cells. The following is a suggested protocol that can be modified to meet the specific research needs. NADH and glutathione (reduced form of GSH) may interfere with the assay.
PREPARATION OF STOCK SOLUTION
1. Amplite™ IR Peroxidase Substrate stock solution (100X):
Add 250 µL of DMSO (Component E) into the vial of Amplite™ IR Peroxidase Substrate (Component A) to make 100X AmpliteTM IR Peroxidase Substrate stock solution. Note: Amplite™ IR Peroxidase Substrate (Component A) is unstable in the presence of thiols such as DTT and β mercaptoethanol. If the final concentration of the thiols is higher than 10 µM, it would significantly decrease the assay dynamic range.
2. Peroxidase stock solution (20 U/mL):
Add 1 mL of Assay Buffer (Component C) into the vial of Horseradish Peroxidase (Component D) to make 20 U/mL Peroxidase stock solution.
3. H2O2 standard solution (20 mM):
Add 22.7 µL of 3% H2O2 (0.88 M, Component B) into 977 µL of Assay Buffer (Component C) to make 20 mM H2O2 standard solution. Note: The diluted H2O2 stock solution is not stable. The unused portion should be discarded.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/11502
Add 1 µL of 20 mM H2O2 standard solution into 1999 µL of Assay Buffer (Component C) to get 10 µM H2O2 standard (HS7). Take 10 µM H2O2 standard (HS7) and perform 1:3 serial dilutions to get serially diluted H2O2 standard (HS6 - HS1) with Assay Buffer (Component C).
PREPARATION OF WORKING SOLUTION
Add 50 μL of 100X Amplite™ IR Peroxidase Substrate stock solution and 200 μL of 20 U/mL Peroxidase stock solution into 4.75 mL of Assay Buffer (Component C) to make H2O2 working solution. Keep from light.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of H2O2 standards and test samples in a solid black 96-well microplate. HS= H2O2 Standards (HS1 - HS7, 0.01 to 10 µM); BL=Blank Control; TS=Test Samples
BL | BL | TS | TS |
HS1 | HS1 | ... | ... |
HS2 | HS2 | ... | ... |
HS3 | HS3 | ||
HS4 | HS4 | ||
HS5 | HS5 | ||
HS6 | HS6 | ||
HS7 | HS7 |
Table 2. Reagent composition for each well.
Well | Volume | Reagent |
HS1 - HS7 | 50 µL | Serial Dilutions (0.01 to 10 µM) |
BL | 50 µL | Assay Buffer (Component C) |
TS | 50 µL | test sample |
Run H2O2 assay in supernatants reaction:
- Prepare H2O2 standards (HS), 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 H2O2 working solution to each well of H2O2 standard, blank control, and test samples to make the total H2O2 assay volume of 100 µL/well. For a 384-well plate, add 25 µL of H2O2 working solution into each well instead, for a total volume of 50 µL/well.
- Incubate the reaction at room temperature for 0 to 30 minutes, protected from light.
- Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 640/680 nm (Cutoff =665nm). Note: Amplite™ IR Peroxidase Substrate is easy to be self-oxidized, so read the fluorescence as soon as the H2O2 working solution was added to increase the signal to noise ratio. The contents of the plate can also be transferred to a white clear bottom plate and read by an absorbance microplate reader at the wavelength of 650 nm. The absorption detection has lower sensitivity compared to the fluorescence reading.
Run H2O2 assay for cells:
- The H2O2 working solution should be prepared as above except that the Assay Buffer (Component C) should be replaced with the media used in your cell culture system. Suggested media including (a) Krebs Ringers Phosphate Buffer (KRPB); (b) Hanks Balanced Salt Solution (HBSS); or (c) Serum-free media.
- Prepare cells in a 96-well plate (50 - 100 µL/well), and activate the cells as desired. Note: The negative controls (media alone and non-activated cells) are included for measuring the background fluorescence.
- Add 50 µL of H2O2 working solution into each well of cells and H2O2 standards. For a 384-well plate, add 25 µL of cells and 25 µL of H2O2 working solution into each well.
- Incubate the reaction at room temperature for 0 to 30 minutes, protected from light.
- Monitor the fluorescence intensity with a fluorescence plate reader at Ex/Em = 640/680 nm (Cutoff = 665nm).
Product Family
Name | Excitation (nm) | Emission (nm) |
Amplite® Fluorimetric Hydrogen Peroxide Assay Kit *Red Fluorescence* | 571 | 584 |
Images
Citations
Authors: Li, Chao and Wu, Yuxing and Chen, Qinjun and Luo, Yifan and Liu, Peixin and Zhou, Zheng and Zhao, Zhenhao and Zhang, Tongyu and Su, Boyu and Sun, Tao and others,
Journal: ACS nano (2023)
Authors: Wang, Yifei and Wang, Jia and Jiao, Yunke and Chen, Kangli and Chen, Tianhao and Wu, Xin-Ping and Jiang, Xingwu and Bu, Wenbo and Liu, Changsheng and Qu, Xue
Journal: Acta Biomaterialia (2023)
Authors: Li, Qinghua and Song, Huijuan and Li, Shuangyang and Hu, Pengbo and Zhang, Chuangnian and Zhang, Ju and Feng, Zujian and Kong, Deling and Wang, Weiwei and Huang, Pingsheng
Journal: Bioactive Materials (2023): 251--264
Authors: Hu, Can and Qiu, Yan and Guo, Jiajun and Cao, Yuchao and Li, Dairong and Du, Yonghong
Journal: International Journal of Nanomedicine (2023): 6257--6274
Authors: Liu, Shanshan and Xu, Jianpei and Liu, Yipu and You, Yang and Xie, Laozhi and Tong, Shiqiang and Chen, Yu and Liang, Kaifan and Zhou, Songlei and Li, Fengan and others,
Journal: ACS Applied Materials \& Interfaces (2022): 27743--27761
Authors: Song, Jialin and Liu, Huan and Lei, Miao and Tan, Haoqi and Chen, Zhanyi and Antoshin, Artem and Payne, Gregory F and Qu, Xue and Liu, Changsheng
Journal: ACS applied materials \& interfaces (2020): 8915--8928
Authors: Sakano, Daisuke and Uefune, Fumiya and Tokuma, Hiraku and Sonoda, Yuki and Matsuura, Kumi and Takeda, Naoki and Nakagata, Naomi and Kume, Kazuhiko and Shiraki, Nobuaki and Kume, Shoen
Journal: Diabetes (2020): 2377--2391
Authors: Li, Huaiguang and M{\"u}nchberg, Ute and Oughli, Alaa A and Buesen, Darren and Lubitz, Wolfgang and Freier, Erik and Plumer{\'e}, Nicolas
Journal: Nature communications (2020): 1--7
Authors: Yi, Zhengjun and Wang, Shuhui and Meng, Xiangying and Wu, Anqi and Li, Qian and Song, Yongjie and Zhao, Ronglan and Qiao, Jinjuan
Journal: Analytical and Bioanalytical Chemistry (2019): 1--9
Authors: Quan, Ying-Yao and Xia, Qiang and Liu, Yu-Hong and Lin, Chun-Mei and Wu, Sheng-Nan and Wang, Xiao-Ping and Chen, Tong-Sheng
Journal: Journal of Nanoscience and Nanotechnology (2017): 1740--1746
References
Authors: Belousov VV, Fradkov AF, Lukyanov KA, Staroverov DB, Shakhbazov KS, Terskikh AV, Lukyanov S.
Journal: Nat Methods (2006): 281
Authors: Lee DH, Lim BS, Lee YK, Yang HC.
Journal: Cell Biol Toxicol (2006): 39
Authors: Chen H, Yu H, Zhou Y, Wang L.
Journal: Spectrochim Acta A Mol Biomol Spectrosc. (2006)
Authors: Weeks ME, Sinclair J, Butt A, Chung YL, Worthington JL, Wilkinson CR, Griffiths J, Jones N, Waterfield MD, Timms JF.
Journal: Proteomics (2006): 2772
Authors: Mazlan M, Sue Mian T, Mat Top G, Zurinah Wan Ngah W.
Journal: J Neurol Sci (2006): 5
Authors: Almeida LE, Imasato H, Tabak M.
Journal: Biochim Biophys Acta (2006): 216
Authors: Bienert GP, Moller AL, Kristiansen KA, Schulz A, Moller IM, Schjoerring JK, Jahn TP.
Journal: J Biol Chem. (2006)
Authors: Onoda M, Uchiyama T, Mawatari K, Kaneko K, Nakagomi K.
Journal: Anal Sci (2006): 815
Authors: Yada T, Shimokawa H, Hiramatsu O, Haruna Y, Morita Y, Kashihara N, Shinozaki Y, Mori H, Goto M, Ogasawara Y, Kajiya F.
Journal: Am J Physiol Heart Circ Physiol (2006): H1138
Authors: Liang PF, Huang XY, Long JH, Xiao MZ, Yang XH, Zhang PH.
Journal: Zhonghua Shao Shang Za Zhi (2006): 175
Application notes
Design of potent inhibitors of acetylcholinesterase using morin as the starting compound
Acetylcholinesterase Inhibitory Activity of Pigment Echinochrome A
Induction of Neurite Outgrowth in PC12 Cells
Induction of Neuritogenesis in PC12 Cells by a Pulsed Electromagnetic Field
FAQ
How should I reconstitute an NADPH standard?
Will Amplite® Fluorimetric NAD/NADH Ratio Assay Kit *Red Fluorescence* work with NADP/NADPH? Can this kit measure NADP+ and NADPH?
What is the concentration of calcium inside cells?
What assay kits measure NADP/NADPH from cell samples?