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

Fluorescence images of intracellular hydrogen peroxide in HeLa cells using Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit (Cat# 11504). HeLa cells at 10,000 cells/well/100 µL were seeded overnight in a Costar black wall/clear bottom 96-well plate. 100 µM H<sub>2</sub>O<sub>2</sub>: HeLa cells were stained with OxiVision™ Blue peroxide sensor for 30 minutes and treated with 100 µM hydrogen peroxide at 37 °C for 90 minutes. Control: Cells were stained with OxiVision™ Blue peroxide sensor but without hydrogen peroxide treatment. The fluorescence signals were measured using fluorescence microscope with a DAPI filter.
Fluorescence images of intracellular hydrogen peroxide in HeLa cells using Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit (Cat# 11504). HeLa cells at 10,000 cells/well/100 µL were seeded overnight in a Costar black wall/clear bottom 96-well plate. 100 µM H<sub>2</sub>O<sub>2</sub>: HeLa cells were stained with OxiVision™ Blue peroxide sensor for 30 minutes and treated with 100 µM hydrogen peroxide at 37 °C for 90 minutes. Control: Cells were stained with OxiVision™ Blue peroxide sensor but without hydrogen peroxide treatment. The fluorescence signals were measured using fluorescence microscope with a DAPI filter.
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Catalog Number11504
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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

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 for fluorescence microscopy.

Platform


Fluorescence microscope

ExcitationDAPI filter
EmissionDAPI filter
Recommended plateBlack wall/clear bottom

Components


Component A: OxiVision™ Blue Peroxide Sensor1 vial
Component B: Assay Buffer1 bottle (20 mL)
Component C: DMSO1 vial (100 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 DAPI filter or Ex/Em = 405/450 nm

Important notes
Thaw all the 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 (500X):
Add 40 µL of DMSO (Component C) into the vial of OxiVision™ Blue Peroxide Sensor (Component A) and mix well to make 500X OxiVision™ Blue Peroxide Sensor stock solution.  Note: 20 µL of reconstituted OxiVision™ Blue peroxide sensor stock solution is enough for 1 plate. The stock solution should be used promptly. Protect from light.

PREPARATION OF WORKING SOLUTION

Add 10 μL of 500X OxiVision™ Blue Peroxide Sensor stock solution into 500 μL of Assay Buffer (Component B) and mix well to make OxiVision™ Blue Peroxide Sensor working solution. Note: This OxiVision™ Blue Peroxide Sensor working solution is not stable; prepare it as needed before use.

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

SAMPLE EXPERIMENTAL PROTOCOL

  1. Add 10 µL/well (96-well plate) or 2.5 µL/well (384-well plate) of OxiVision™ Blue Peroxide Sensor working solution in 90 µL cell culture per well in a 96-well cell plate or 22.5 µL cell culture per well in a 384-well cell plate. Note: It is not necessary to wash cells before staining. It’s recommended to stain the cells in full medium.

  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. Add 1X Hank’s salt solution and 20 mM Hepes buffer (HHBS) or the buffer of your choice into the cells after aspiration. Alternatively, cells can be treated in serum-free media. 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. Wash cells with DPBS 1 - 2 times, and replace with 100 uL/well (for 96-well plate) or 25 uL/well (for 384- well plate) Assay Buffer (Component C).

  4. Take images using fluorescence microscope with a DAPI filter.

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