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

Detection of hydrogen peroxide in Jurkat cells using Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit (Cat#: 11505). Jurkat cells were stained with OxiVision™ Blue peroxide sensor for 30 minutes and treated with 100 µM hydrogen peroxide at 37 °C for 90 minutes. Cells stained with OxiVision™ Blue 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#: 11505). Jurkat cells were stained with OxiVision™ Blue peroxide sensor for 30 minutes and treated with 100 µM hydrogen peroxide at 37 °C for 90 minutes. Cells stained with OxiVision™ Blue peroxide sensor but without hydrogen peroxide treatment were used as control.
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Catalog Number11505
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Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
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H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501
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OverviewpdfSDSpdfProtocol


Hydrogen peroxide 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 many biological events 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™ Blue peroxide sensor to quantify hydrogen peroxide in live cells. OxiVision™ Blue peroxide sensor is cell-permeable, and generates blue 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.

Platform


Flow cytometer

Excitation405 nm laser
Emission450/40 nm filter
Instrument specification(s)Pacific Blue channel

Components


Component A: OxiVision™ Blue peroxide sensor1 vial
Component B: DMSO1 vial (200 µL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells in growth medium
  2. Stain cells with OxiVision™ Blue Peroxide Sensor
  3. Treat cells with test compounds
  4. Monitor fluorescence intensity with flow cytometer Pacific Blue Channel (Ex/Em = 405/450 nm)

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

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™ Blue Peroxide Sensor stock solution:
Add 100 µL of DMSO (Component B) into the vial of OxiVision™ Blue peroxide sensor (Component A), and mix them well. Note: 1 µL of reconstituted OxiVision™ Blue peroxide sensor stock solution is enough for 0.5 mL cells. The stock solution should be used promptly. Keep 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™ Blue Peroxide Sensor stock solution in full medium or in your desired buffer at 37°C for 20 - 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’s 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. Monitor the fluorescence intensity at Pacific Blue channel (Ex/Em=405/450 nm) using a flow cytometer. Gate on the cells of interest, excluding debris.

Citations


View all 1 citations: Citation Explorer
Plasma-activated medium selectively eliminates undifferentiated human induced pluripotent stem cells
Authors: Matsumoto, Ryo and Shimizu, Kazunori and Nagashima, Takunori and Tanaka, Hiromasa and Mizuno, Masaaki and Kikkawa, Fumitaka and Hori, Masaru and Honda, Hiroyuki
Journal: Regenerative Therapy (2016): 55--63

References


View all 152 references: Citation Explorer
Genetically encoded fluorescent indicator for intracellular hydrogen peroxide
Authors: Belousov VV, Fradkov AF, Lukyanov KA, Staroverov DB, Shakhbazov KS, Terskikh AV, Lukyanov S.
Journal: Nat Methods (2006): 281
Effects of hydrogen peroxide (H(2)O(2)) on alkaline phosphatase activity and matrix mineralization of odontoblast and osteoblast cell lines
Authors: Lee DH, Lim BS, Lee YK, Yang HC.
Journal: Cell Biol Toxicol (2006): 39
Fluorescent quenching method for determination of trace hydrogen peroxide in rain water
Authors: Chen H, Yu H, Zhou Y, Wang L.
Journal: Spectrochim Acta A Mol Biomol Spectrosc. (2006)
A parallel proteomic and metabolomic analysis of the hydrogen peroxide- and Sty1p-dependent stress response in Schizosaccharomyces pombe
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
Comparative effects of alpha-tocopherol and gamma-tocotrienol against hydrogen peroxide induced apoptosis on primary-cultured astrocytes
Authors: Mazlan M, Sue Mian T, Mat Top G, Zurinah Wan Ngah W.
Journal: J Neurol Sci (2006): 5
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
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)
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
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
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