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

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.

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

 

BLBLTSTS
HS1HS1......
HS2HS2......
HS3HS3  
HS4HS4  
HS5HS5  
HS6HS6  
HS7HS7  

Table 2. Reagent composition for each well.

WellVolumeReagent
HS1 - HS750 µLSerial Dilutions (300 to 0.3 µM)
BL50 µLAssay Buffer (Component C)
TS50 µLtest 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

Citations

View all 14 citations: Citation Explorer
Exploring the Cellular Impact of Size-Segregated Cigarette Aerosols: Insights into Indoor Particulate Matter Toxicity and Potential Therapeutic Interventions
Authors: Shen, Yu-Xin and Lee, Pe-Shuen and Wang, Chia C and Teng, Ming-Chu and Huang, Jhih-Hong and Fan, Hsiu-Fang
Journal: Chemical Research in Toxicology (2024)
Application of Synephrine to Grape Increases Anthocyanin via Production of Hydrogen Peroxide, Not Phytohormones
Authors: Suzuki, Masaya and Kimura, Aoi and Suzuki, Shunji and Enok, Shinichi
Journal: (2024)
Ultrasound-Triggered Azo Free Radicals for Cervical Cancer Immunotherapy
Authors: Wang, Yumeng and Lv, Bin and Wang, Han and Ren, Tingting and Jiang, Qian and Qu, Xinyu and Ni, Dalong and Qiu, Junjun and Hua, Keqin
Journal: ACS nano (2024)
Influence of Cigarette Aerosol in Alpha-Synuclein Oligomerization and Cell Viability in SH-SY5Y: Implications for Parkinson’s Disease
Authors: Shen, Yu-Xin and Lee, Pe-Shuen and Teng, Ming-Chu and Huang, Jhih-Hong and Wang, Chia C and Fan, Hsiu-Fang
Journal: ACS Chemical Neuroscience (2024)
Hyaluronan-decorated copper-doxorubicin-anlotinib nanoconjugate for targeted synergistic chemo/chemodynamic/antiangiogenic tritherapy against hepatocellular carcinoma
Authors: Tan, Gang and Hou, Guanghui and Qian, Junmin and Wang, Yaping and Xu, Weijun and Luo, Wenjuan and Chen, Xiaobing and Suo, Aili
Journal: Journal of Colloid and Interface Science (2024)
Page updated on December 11, 2024

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Catalog Number11503
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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

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