Amplite™ Fluorimetric Hydrogen Peroxide Assay Kit *Red Fluorescence*

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Hydrogen Peroxide dose response was measured in a solid black 384-well plate with the Amplite™ Flourimetric Hydrogen Peroxide Assay Kit using a Gemini fluorescence microplate reader (Molecular Devices).

Unit Size: Cat No: Price (USD): Qty:
500 Tests 11501 $245

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Ex/Em (nm)575/590
Storage Freeze (<-15 °C)
Minimize light exposure
InstrumentsFluorescence microplate reader
Category Enzyme Detection
Horseradish Peroxidase (HRP)
Related Reactive Oxygen Species
Microplate Readers
Hydrogen peroxide (H2O2) is a reactive oxygen metabolic by-product that serves as a key regulator for a number of oxidative stress-related states. It is involved in a number of biological events that have been linked to asthma, atherosclerosis, diabetic vasculopathy, osteoporosis, a number of neurodegenerative diseases and Down's syndrome. Perhaps the most intriguing aspect of H2O2 biology is the recent report that antibodies have the capacity to convert molecular oxygen into hydrogen peroxide to contribute to the normal recognition and destruction processes of the immune system. Measurement of this reactive species will help to determine how oxidative stress modulates varied intracellular pathways. Amplite™ Hydrogen Peroxide Assay Kit uses our Amplite™ Red peroxidase substrate to quantify hydrogen peroxide in solutions and cell extracts. It can also be used to detect a variety of oxidase activities through enzyme-coupled reactions. The kit is an optimized 'mix and read' assay that is compatible with HTS liquid handling instruments.

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This protocol only provides a guideline, and should be modified according to your specific needs.
At a glance

Protocol summary

  1. Prepare H2O2 working solution (50 µL)
  2. Add H2O2 standards or test samples (50 µL)
  3. Incubate at room temperature for 10 - 30 minutes
  4. Monitor fluorescence intensity at Ex/Em = 540/590 nm

Important notes
Thaw all the kit components at room temperature before starting the experiment.

Important notes
The component A is unstable in the presence of thiols such as DTT and β-mercaptoethanol. Thiols higher than 10 uM (final concentration) would significantly decrease the assay dynamic range.

Important notes
NADH and glutathione (reduced form: GSH) may interfere with the assay.

Key parameters
Instrument:Fluorescence microplate reader
Excitation:540 nm
Emission:590 nm
Cutoff:570 nm
Recommended plate:Solid black
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. Amplite™ Red Peroxidase Substrate stock solution (100X):
Add 250 µL of DMSO (Component E) into the vial of Amplite™ Red Substrate (Component A). This stock solution should be used promptly.

2. Peroxidase stock solution (20 U/mL):
Add 1 mL of Assay Buffer (Component C) into the vial of Horseradish Peroxidase (Component D).

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). Note: Diluted H2O2 solution is not stable. Any unused portions should be discarded.

Preparation of standard solution
H2O2 standard

For convenience, use the Serial Dilution Planner:

Add 1 µL of 20 mM H2O2 stock solution into 1999 µL of Assay Buffer (Component C) to get a 10 µM H2O2 standard (HS7). Take 10 µM H2O2 standard and perform 1:3 serial dilutions to get serial dilutions of H2O2 standard (HS6 - HS1).

Preparation of working solution

Add 50 μL of Amplite™ Red Peroxidase Substrate stock solution (100X) and 200 μL of Peroxidase stock solution (20 U/mL) into 4.75 mL of Assay Buffer (Component C) to make a total volume of 5 mL. Note: Keep from light.

For guidelines on cell sample preparation, please visit

Sample experimental protocol

Table 1. Layout of H2O2 standards and test samples in a solid black 96-well microplate. HS = H2O2 standard (HS1-HS7, 0.01 to 10 µM); BL = blank control; TS = test sample.

HS1 HS1 ... ...
HS2 HS2 ... ...
HS3 HS3    
HS4 HS4    
HS5 HS5    
HS6 HS6    
HS7 HS7    

Table 2. Reagent composition for each well. Note that high concentrations of H2O2 (e.g., >100 µM, final concentration) may cause reduced fluorescence signals due to the overoxidation of Amplite™ Red (to non-fluorescent products).

Well Volume Reagent
HS1 - HS7 50 µL serial dilution (0.01 to 10 µM)
BL 50 µL Assay Buffer (Component C)
TS 50 µL sample

H2O2 assay in supernatants

  1. Prepare H2O2 standards (HS), blank controls (BL), and test samples (TS) into a solid black 96-well microplate according to the layout provided in Table 1 and Table 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.

  2. Add 50 µL of H2O2 working solution into 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 instead, for a total volume of 50 µL/well.

  3. Incubate the reaction at room temperature for 15 to 30 minutes, protected from light.

  4. Monitor the fluorescence increase with a fluorescence plate reader at Excitation = 540 ± 10 nm, Emission = 590 ± 10 nm (optimal Ex/Em = 540/590 nm). Note: The contents of the plate can also be transferred to a white clear bottom microplate and read by an absorbance microplate reader at the wavelength of 576 ± 5 nm. However, the absorption detection has a lower sensitivity compared to that of a fluorescence reading.

H2O2 assay for cells
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.

  1. The H2O2 cell working solution should be prepared as above, except that the Assay Buffer (Component C) should be replaced with the media that is used in your cell culture system. Suggested medias include (a) Krebs Ringers Phosphate Buffer (KRPB); (b) Hanks Balanced Salt Solution (HBSS); or (c) Serum-free media.

  2. 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 background fluorescence. For a 384-well plate, use 25 µL/well of cell media instead.

  3. Add 50 µL of H2O2 cell working solution into each well of cells and H2O2 standards. For a 384-well plate, add 25 µL of H2O2 cell working solution into each well instead.

  4. Incubate the reaction at room temperature for 15 to 30 minutes, protected from light.

  5. Monitor the fluorescence increase with a fluorescence plate reader at Excitation = 540 ± 10 nm, Emission = 590 ± 10 nm (optimal Ex/Em = 540/590 nm).
Example data analysis and figures

The reading (RFU) obtained from the blank standard well is used as a negative control. Subtract this value from the other standards' readings to obtain the base-line corrected values. Then, plot the standards' readings to obtain a standard curve and equation. This equation can be used to calculate Hydrogen Peroxide samples. We recommend using the Online Linear Regression Calculator which can be found at:

Figure 1. Hydrogen Peroxide dose response was measured in a solid black 384-well plate with the Amplite™ Flourimetric Hydrogen Peroxide Assay Kit using a Gemini fluorescence microplate reader (Molecular Devices).

AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email if you have any questions.

References & Citations

Bio-inspired redox-cycling antimicrobial film for sustained generation of reactive oxygen species
Authors: Huan Liu, Xue Qu, Eunkyoung Kim, Miao Lei, Kai Dai, Xiaoli Tan, Miao Xu, Jinyang Li, Yangping Liu, Xiaowen Shi
Journal: Biomaterials (2018)

PPARα-mediated peroxisome induction compensates PPARγ-deficiency in bronchiolar club cells
Authors: Srikanth Karnati, Gani Oruqaj, Harshavardhan Janga, Srinu Tumpara, Claudia Colasante, Paul P Van Veldhoven, Nancy Braverman, Adrian Pilatz, Thomas J Mariani, Eveline Baumgart-Vogt
Journal: PloS one (2018): e0203466

Semaphorin 4D inhibits neutrophil activation and is involved in the pathogenesis of neutrophil-mediated autoimmune vasculitis
Authors: Masayuki Nishide, Satoshi Nojima, Daisuke Ito, Hyota Takamatsu, Shohei Koyama, Sujin Kang, Tetsuya Kimura, Keiko Morimoto, Takashi Hosokawa, Yoshitomo Hayama
Journal: Annals of the Rheumatic Diseases (2017): annrheumdis--2016

Aggravation of brain infarction through an increase in acrolein production and a decrease in glutathione with aging
Authors: Takeshi Uemura, Kenta Watanabe, Misaki Ishibashi, Ryotaro Saiki, Kyoshiro Kuni, Kazuhiro Nishimura, Toshihiko Toida, Keiko Kashiwagi, Kazuei Igarashi
Journal: Biochemical and biophysical research communications (2016): 630--635

Hydrogen peroxide detection with high specificity in living cells and inflamed tissues
Authors: Lei Rong, Chi Zhang, Qi Lei, Ming-Ming Hu, Jun Feng, Hong-Bing Shu, Yi Liu, Xian-Zheng Zhang
Journal: Regenerative Biomaterials (2016): rbw022

Modification of lignin in sugarcane bagasse by a monocopper hydrogen peroxide-generating oxidase from Thermobifida fusca
Authors: Cheng-Yu Chen, Cheng-Cheng Lee, Hung-Shuan Chen, Chao-Hsun Yang, Shu-Ping Wang, Jyh-Horng Wu, Menghsiao Meng
Journal: Process Biochemistry (2016): 1486--1495

Dopamine-mediated oxidation of methionine 127 in α-synuclein causes cytotoxicity and oligomerization of α-synuclein
Authors: Kazuhiro Nakaso, Naoko Tajima, Satoru Ito, Mari Teraoka, Atsushi Yamashita, Yosuke Horikoshi, Daisuke Kikuchi, Shinsuke Mochida, Kenji Nakashima, Tatsuya Matsura
Journal: PLoS One (2013): e55068

Hydrogen peroxide stimulates the epithelial sodium channel through a phosphatidylinositide 3-kinase-dependent pathway
Authors: He-Ping Ma
Journal: Journal of Biological Chemistry (2011): 32444--32453

Additional Documents

Safety Data Sheet (SDS)

1. Enzyme Probes & Assay Kits
2. Reactive Oxygen Species (ROS) Detection

Certificate of Analysis