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Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit *Green Fluorescence*

Fluorescence images of intracellular hydrogen peroxide in HeLa cells using Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit (Cat#11503). HeLa cells were treated with (left) or without (right) 100 µM hydrogen peroxide at 37<sup>o</sup>C for 90 minutes.
Fluorescence images of intracellular hydrogen peroxide in HeLa cells using Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit (Cat#11503). HeLa cells were treated with (left) or without (right) 100 µM hydrogen peroxide at 37<sup>o</sup>C for 90 minutes.
Fluorescence responses of OxiVision Green™ to various ROS species.
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Catalog Number11503
Unit Size
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Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Spectral properties
Excitation (nm)498
Emission (nm)517
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501
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OverviewpdfSDSpdfProtocol


Excitation (nm)
498
Emission (nm)
517
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 hydrogen peroxide 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. This Cell Meter™ Hydrogen Peroxide Assay Kit uses our unique OxiVision™ Green hydrogen peroxide sensor to quantify hydrogen peroxide in live cells. OxiVision™ Green is cell-permeable, and generates the green fluorescence when it reacts with hydrogen peroxide. The kit is an optimized 'mix and read' assay format that is compatible with HTS liquid handling instruments.

Platform


Fluorescence microscope

ExcitationFITC channel
EmissionFITC channel
Recommended plateBlack wall/clear bottom

Fluorescence microplate reader

Excitation490 nm
Emission525 nm
Cutoff515 nm
Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode

Components


Component A: OxiVision Green™ hydrogen peroxide sensor1 vial
Component B: H2O21 vial (3% stabilized solution, 200 µL)
Component C: Assay Buffer1 bottle (20 mL)
Component D: DMSO1 vial (200 µL)

Example protocol


AT A GLANCE

Protocol summary for Solution Assay

  1. Prepare and add Amplite™ Green Peroxide Sensor working solution (50 µL)
  2. Add H2O2 standards or test samples (50 µL)
  3. Incubate at room temperature for 15 - 60 minutes
  4. Read fluorescence intensity at Ex/Em = 490/525 nm (Cutoff = 515 nm)

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

Protocol summary for Live Cell Assay

  1. Prepare cells in growth medium
  2. Stain cells with Amplite™ Green Peroxide Sensor working solution and incubate for your desired period of time
  3. Treat cells with test compounds
  4. Monitor fluorescence intensity at Ex/Em = 490/525 nm (Cutoff = 515 nm) with bottom read mode

Important notes
Amplite™ Green Peroxide Sensor can be loaded passively into living cells and report the micromolar changes in intracellular H2O2 concentrations. The following is a suggested microscope imaging protocol that can be modified to meet specific research needs.

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™ Green Peroxide Sensor stock solution (250X):
Add 50 µL of DMSO (Component D) into the vial of Amplite™ Green Peroxide Sensor (Component A) to make 250X Amplite™ Green Peroxide Sensor stock solution. Protect from light.

2. 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 H2Ostandard solution. Note: The diluted H2O2 standard solution is not stable. The unused portion should be discarded.

PREPARATION OF STANDARD SOLUTION

H2O2 standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/11503

Add 50 µL of 20 mM H2O2 standard solution into 950 µL of Assay Buffer (Component C) to get 1000 µM H2O2 standard solution. Take 1000 µM H2O2 standard solution and perform 1:3 serial dilutions to get serially diluted H2O2 standards (HS1 - HS7) with Assay Buffer (Component C).

PREPARATION OF WORKING SOLUTION

Add 20 μL of 250X Amplite™ Green Peroxide Sensor stock solution into 5 mL of Assay Buffer (Component C) to make Amplite™ Green Peroxide Sensor working solution.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

SAMPLE EXPERIMENTAL PROTOCOL

Run H2O2 assay in supernatants reaction:

Table 1. Layout of H2O2 standards and test samples in a solid black 96-well microplate. HS= H2O2 Standards (HS1 - HS7, 300 to 0.3 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 (300 to 0.3 µM)
BL 50 µL Assay Buffer (Component C)
TS 50 µL test sample
  1. 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.

  2. Add 50 µL of Amplite™ Green Peroxide Sensor 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 Amplite™ Green Peroxide Sensor working solution into each well 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 = 490 ± 10, Emission = 520 ± 10 nm (optimal Ex/Em = 490/525 nm, Cutoff = 515 nm).

Run H2O2 assay in live cells:

  1. Activate the cells as desired.

  2. Wash the cells with PBS buffer, incubated the cells with 100 µL/well Amplite™ Green Peroxide Sensor working solution for 5 to 60 minutes or your desired time. For a 384-well plate, add 25 µL/well of Amplite™ Green Peroxide Sensor working solution. Note: For a kinetic measurement, cells can be stained before adding the treatment.

  3. Monitor the fluorescence increase with a fluorescence plate reader (bottom read mode) at Ex/Em = 490/525 nm (Cutoff = 515 nm) Or image the fluorescence change with a fluorescence microscope using FITC channel.

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)498
Emission (nm)517

Citations


View all 7 citations: Citation Explorer
Homogeneously catalytic oxidation of phenanthrene by the reaction of extracellular secretions of pyocyanin and Nicotinamide Adenine Dinucleotide
Authors: Nie, Hongyun and Nie, Maiqian and Diwu, Zhenjun and Wang, Lei and Qiao, Qi and Zhang, Bo and Yang, Xuefu
Journal: Environmental Research (2020): 110159
Semaphorin 4D inhibits neutrophil activation and is involved in the pathogenesis of neutrophil-mediated autoimmune vasculitis
Authors: Nishide, Masayuki and Nojima, Satoshi and Ito, Daisuke and Takamatsu, Hyota and Koyama, Shohei and Kang, Sujin and Kimura, Tetsuya and Morimoto, Keiko and Hosokawa, Takashi and Hayama, Yoshitomo and others, undefined
Journal: Annals of the Rheumatic Diseases (2017): annrheumdis--2016
Hydrogen peroxide detection with high specificity in living cells and inflamed tissues
Authors: Rong, Lei and Zhang, Chi and Lei, Qi and Hu, Ming-Ming and Feng, Jun and Shu, Hong-Bing and Liu, Yi and Zhang, Xian-Zheng
Journal: Regenerative Biomaterials (2016): rbw022
Aggravation of brain infarction through an increase in acrolein production and a decrease in glutathione with aging
Authors: Uemura, Takeshi and Watanabe, Kenta and Ishibashi, Misaki and Saiki, Ryotaro and Kuni, Kyoshiro and Nishimura, Kazuhiro and Toida, Toshihiko and Kashiwagi, Keiko and Igarashi, Kazuei
Journal: Biochemical and biophysical research communications (2016): 630--635
Modification of lignin in sugarcane bagasse by a monocopper hydrogen peroxide-generating oxidase from Thermobifida fusca
Authors: Chen, Cheng-Yu and Lee, Cheng-Cheng and Chen, Hung-Shuan and Yang, Chao-Hsun and Wang, Shu-Ping and Wu, Jyh-Horng and Meng, Menghsiao
Journal: Process Biochemistry (2016): 1486--1495
Dopamine-mediated oxidation of methionine 127 in α-synuclein causes cytotoxicity and oligomerization of α-synuclein
Authors: Nakaso, Kazuhiro and Tajima, Naoko and Ito, Satoru and Teraoka, Mari and Yamashita, Atsushi and Horikoshi, Yosuke and Kikuchi, Daisuke and Mochida, Shinsuke and Nakashima, Kenji and Matsura, Tatsuya
Journal: PLoS One (2013): e55068
Hydrogen peroxide stimulates the epithelial sodium channel through a phosphatidylinositide 3-kinase-dependent pathway
Authors: Ma, He-Ping
Journal: Journal of Biological Chemistry (2011): 32444--32453