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Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit *Green Fluorescence Optimized for Flow Cytometry*

Hydrogen peroxide is a reactive oxygen metabolic by-product that serves as a key regulator for a number of oxidative stress-related events. It is involved in many biological processes that are linked to asthma, atherosclerosis, diabetic vasculopathy, osteoporosis, a number of neurodegenerative diseases and Down's syndrome. The measurement of this reactive species is helpful for determining how oxidative stress modulates various intracellular pathways. This Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit uses our unique OxiVision™ Green peroxide sensor to quantify hydrogen peroxide in live cells. OxiVision™ Green peroxide sensor is cell-permeable, and generates green fluorescence when it reacts with hydrogen peroxide. This kit provides a sensitive tool to monitor hydrogen peroxide level in living cells, and it is optimized to be used in flow cytometry.

Example protocol

AT A GLANCE

Protocol summary

  1. Prepare cells in growth medium
  2. Stain cells with OxiVision™ Green peroxide sensor
  3. Treat cells with test compounds
  4. Monitor fluorescence intensity with flow cytometer FITC channel (Ex/Em = 490/530 nm)

Important notes
Thaw all the four components at room temperature before use.

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. OxiVision™ Green Peroxide Sensor stock solution:
Add 100 µL of DMSO (Component B) into the vial of OxiVision™ Green Peroxide Sensor (Component A), and mix well. Note: 1 µL of reconstituted OxiVision™ Green Peroxide Sensor stock solution is for 0.5 mL cells. The stock solution should be used promptly. Protect from light.

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

SAMPLE EXPERIMENTAL PROTOCOL

  1. Stain cells with OxiVision™ Green Peroxide Sensor stock solution in full medium or in your desired buffer at 37°C for 30 minutes, protected from light.

  2. Treat cells with test compounds in full medium or in your desired buffer at 37°C for desired period of time. For control samples (untreated cells), add the corresponding amount of compound buffer. Note: It is recommended to treat cells in full medium. However, if tested compounds are serum sensitive, growth medium and serum factors can be aspirated away before treatment. Resuspend cells in 1X Hank’s salt solution and 20 mM Hepes buffer (HHBS) or the buffer of your choice after aspiration. Alternatively, cells can be treated in serum-free media. Note: We treated Jurkat cells with 100 µM hydrogen peroxide in full medium at 37°C for 90 minutes to induce hydrogen peroxide. See Figure 1 for details.

  3. Alternatively, treat cells with tested compounds at 37°C for desired period of time. Remove the treatment solution, then stain cells with OxiVision™ Green Peroxide Sensor stock solution in full medium or in your desired buffer at 37°C for desired period of time.

  4. Monitor the fluorescence intensity at FITC channel (Ex/Em = 490/530 nm) using a flow cytometer. Gate on the cells of interest, excluding debris.

Spectrum

Citations

View all 7 citations: Citation Explorer
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)
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)
Antiviral Effects of Pyrroloquinoline Quinone through Redox Catalysis To Prevent Coronavirus Infection
Authors: Ishak, Nur Syafiqah Mohamad and Numaguchi, Tomoe and Ikemoto, Kazuto
Journal: ACS Omega (2023)
An Oxygen Supply Strategy for Sonodynamic Therapy in Tuberculous Granuloma Lesions Using a Catalase-Loaded Nanoplatform
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
Low level of antioxidant capacity biomarkers but not target overexpression predicts vulnerability to ROS-inducing drugs
Authors: Samarin, Jana and Fabrowski, Piotr and Kurilov, Roman and Nuskova, Hana and Hummel-Eisenbeiss, Johanna and Pink, Hannelore and Li, Nan and Weru, Vivienn and Alborzinia, Hamed and Yildiz, Umut and others,
Journal: bioRxiv (2023): 2023--01

References

View all 152 references: Citation Explorer
Effect of antisense oligonucleotide against Smac/DIABLO on inhibition of hydrogen peroxide induced myocardial apoptosis of neonatal rats
Authors: Liang PF, Huang XY, Long JH, Xiao MZ, Yang XH, Zhang PH.
Journal: Zhonghua Shao Shang Za Zhi (2006): 175
Cardioprotective role of endogenous hydrogen peroxide during ischemia-reperfusion injury in canine coronary microcirculation in vivo
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
Simple and rapid determination of hydrogen peroxide using phosphine-based fluorescent reagents with sodium tungstate dihydrate
Authors: Onoda M, Uchiyama T, Mawatari K, Kaneko K, Nakagomi K.
Journal: Anal Sci (2006): 815
Specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes
Authors: Bienert GP, Moller AL, Kristiansen KA, Schulz A, Moller IM, Schjoerring JK, Jahn TP.
Journal: J Biol Chem. (2006)
Enzymatic oxidation of dipyridamole in homogeneous and micellar solutions in the horseradish peroxidase-hydrogen peroxide system
Authors: Almeida LE, Imasato H, Tabak M.
Journal: Biochim Biophys Acta (2006): 216
Page updated on October 6, 2024

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Catalog Number11506
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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

Flow cytometer

Excitation488 nm laser
Emission530, 30 nm filter
Instrument specification(s)FITC channel

Components

Detection of hydrogen peroxide in Jurkat cells using Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit (Cat#: 11506). Jurkat cells were stained with OxiVision™ Green peroxide sensor for 30 minutes and treated with 100 µM hydrogen peroxide at 37 °C for 90 minutes. Cells stained with OxiVision™ Green peroxide sensor but without hydrogen peroxide treatment were used as control.
Detection of hydrogen peroxide in Jurkat cells using Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit (Cat#: 11506). Jurkat cells were stained with OxiVision™ Green peroxide sensor for 30 minutes and treated with 100 µM hydrogen peroxide at 37 °C for 90 minutes. Cells stained with OxiVision™ Green peroxide sensor but without hydrogen peroxide treatment were used as control.
Detection of hydrogen peroxide in Jurkat cells using Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit (Cat#: 11506). Jurkat cells were stained with OxiVision™ Green peroxide sensor for 30 minutes and treated with 100 µM hydrogen peroxide at 37 °C for 90 minutes. Cells stained with OxiVision™ Green peroxide sensor but without hydrogen peroxide treatment were used as control.