Cell Meter™ Mitochondrial Hydroxyl Radical Detection Kit *Red Fluorescence*

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Fluorescence images of hydroxyl radical measurement in HeLa cells using MitoROS™ OH580 (Cat#16055). HeLa cells were incubated with MitoROS™ OH580 working solution at 37 °C for 1 hour, then washed once with HHBS. Fenton Reaction: Cells were then treated with 10 µM CuCl2 and 100 µM H<sub>2</sub>O<sub>2</sub> in 1X HBSS buffer at 37 °C for 1 hour. Control: HeLa cells were kept in 1X HBSS buffer without treatment. After washing 3 times with HHBS, HeLa cells were measured using a fluorescence microscope with a TRITC filter set (Red). Cell nuclei were stained with Hoechst 33342 (Cat#17530, Blue).
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Unit Size: Cat No: Price (USD): Qty:
200 Tests 16055 $295


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Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
International: See distributors





Overview

Ex/Em (nm)576/598
Storage Freeze (<-15 °C)
Minimize light exposure
InstrumentsFluorescence microplate reader, Fluorescence microscope
Category Neurobiology
Reactive Oxygen Species
Related Cell Signaling
Secondary Reagents
The detection of intracellular hydroxyl radical is of central importance to understanding proper cellular redox regulation and the impact of its dysregulation on various pathologies. The hydroxyl radical ('OH) is one of the reactive oxygen species (ROS) highly reactive with other molecules to achieve stability. In general, hydroxyl radical is considered to be a harmful by-product of oxidative metabolism, which can cause molecular damage in living system. It shows an average lifetime of 10-9 nano seconds and can react with nearly every biomolecule such as nuclear DNA, mitochondrial DNA, proteins and membrane lipids. AAT Bioquest's Cell Meter™ Mitochondrial Hydroxyl Radical Detection Kit is optimized for detecting hydroxyl radical in mitochondria. MitoROS™ OH580 is live-cell permeant probe and can rapidly and selectively target hydroxyl radical in live cells. It generates red fluorescence when it reacts with 'OH, and can be easily read at Ex/Em= 540/590 nm. Cell Meter™ Mitochondrial Hydroxyl Radical Detection Kit provides a sensitive fluorimetric probe to detect OH' in live cells with one hour incubation. This kit can be used for fluorescence microplate readers and fluorescence microscopy applications.




Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.
At a glance

Protocol summary

  1. Prepare cells
  2. Incubate cells with MitoROS™ OH580 working solution at 37°C for 60 minutes
  3. Incubate cells with test compounds (to induce OH-)
  4. Monitor the fluorescence increase at Ex/Em= 540/590 nm

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

Key parameters
Instrument:Fluorescence microplate reader
Excitation:540 nm
Emission:590 nm
Cutoff:570 nm
Instrument specification(s):Bottom read mode
Recommended plate:Solid black
  
Instrument:Fluorescence microscope
Excitation:545 nm
Emission:570 nm
Recommended plate:Black wall/clear bottom
Instrument specification(s):TRITC Filter Set
Preparation of stock solutions
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. MitoROS™ OH580 stock solution (500X):
Add 50 µL of DMSO (Component C) into the vial of MitoROS™ OH580 (Component A), and mix them well. Note: 25 uL of stock solution is enough for 1 plate.

Preparation of working solution

Add 25 μL of 500X DMSO reconstituted MitoROS™ OH580 stock solution into 10 mL of Assay Buffer (Component B). Mix well. This working solution is stable for at least 2 hours at room temperature.

Preparation of cell samples

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

Sample experimental protocol
  1. Remove medium, and add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of MitoROS™ OH580 working solution into the cell plate. Incubate cells at 37°C for 1 hour.

  2. To induce hydroxyl radical, treat cells with test compounds in your desired buffer (such as PBS or HHBS) at 37°C for a desired period of time, protected from light. Note: We treated HeLa cells with Fenton reaction (10 µM CuCl2 and 100 µM H2O2) at 37°C for 1 hour to induce exogenous hydroxyl radical. See Figure 1 for details. We treated RAW 264.7 cells with PMA (phorbol 12-myristate 13-acetate) in growth medium at 37°C for 4 hours to stimulate endogenous hydroxyl radical. See Figure 2 for details.

  3. Wash cells 2 - 3 times with HHBS or DPBS, and add 100 µL Assay Buffer (Component B) to each well.

  4. Monitor the fluorescence signal in cells using fluorescence microscope with a TRITC filter set, or measure fluorescence increase using fluorescence microplate reader at Ex/Em = 540/590 nm (cut off = 570 nm) with bottom read mode.
Example data analysis and figures

Figure 1. Fluorescence images of hydroxyl radical measurement in HeLa cells using MitoROS™ OH580 (Cat#16055). HeLa cells were incubated with MitoROS™ OH580 working solution at 37 °C for 1 hour, then washed once with HHBS. Fenton Reaction: Cells were then treated with 10 µM CuCl2 and 100 µM H2O2 in 1X HBSS buffer at 37 °C for 1 hour. Control: HeLa cells were kept in 1X HBSS buffer without treatment. After washing 3 times with HHBS, HeLa cells were measured using a fluorescence microscope with a TRITC filter set (Red). Cell nuclei were stained with Hoechst 33342 (Cat#17530, Blue).
Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





References & Citations

Oxyl and hydroxyl radical transfer in mitochondrial amidoxime reducing component-catalyzed nitrite reduction
Authors: Yang J, Giles LJ, Ruppelt C, Mendel RR, Bittner F, Kirk ML.
Journal: J Am Chem Soc (2015): 5276

Arbutin, an intracellular hydroxyl radical scavenger, protects radiation-induced apoptosis in human lymphoma U937 cells
Authors: Wu LH, Li P, Zhao QL, Piao JL, Jiao YF, Kadowaki M, Kondo T.
Journal: Apoptosis (2014): 1654

Chloroplast-located BjFer1 together with anti-oxidative genes alleviate hydrogen peroxide and hydroxyl radical injury in cytoplasmic male-sterile Brassica juncea
Authors: Yang J, Liu S, Yang X, Zhang M.
Journal: Mol Biol Rep (2012): 4169

Hydroxyl radical (.OH) played a pivotal role in oridonin-induced apoptosis and autophagy in human epidermoid carcinoma A431 cells
Authors: Yu Y, Fan SM, Song JK, Tashiro S, Onodera S, Ikejima T.
Journal: Biol Pharm Bull (2012): 2148

Excess no predisposes mitochondrial succinate-cytochrome c reductase to produce hydroxyl radical
Authors: Chen J, Chen CL, Alevriadou BR, Zweier JL, Chen YR.
Journal: Biochim Biophys Acta (2011): 491

Postresuscitation syndrome: potential role of hydroxyl radical-induced endothelial cell damage
Authors: Huet O, Dupic L, Batteux F, Matar C, Conti M, Chereau C, Lemiale V, Harrois A, Mira JP, Vicaut E, Cariou A, Duranteau J.
Journal: Crit Care Med (2011): 1712

Endogenous 3,4-dihydroxyphenylalanine and dopaquinone modifications on protein tyrosine: links to mitochondrially derived oxidative stress via hydroxyl radical
Authors: Zhang X, Monroe ME, Chen B, Chin MH, Heibeck TH, Schepmoes AA, Yang F, Petritis BO, Camp DG, 2nd, Pounds JG, Jacobs JM, Smith DJ, Bigelow DJ, Smith RD, Qian WJ.
Journal: Mol Cell Proteomics (2010): 1199

Hyperglycemia induces apoptosis in rat liver through the increase of hydroxyl radical: new insights into the insulin effect
Authors: Frances DE, Ronco MT, Monti JA, Ingaramo PI, Pisani GB, Parody JP, Pellegrino JM, Sanz PM, Carrillo MC, Carnovale CE.
Journal: J Endocrinol (2010): 187

Acarbose reduces myocardial infarct size by preventing postprandial hyperglycemia and hydroxyl radical production and opening mitochondrial KATP channels in rabbits
Authors: Minatoguchi S, Zhang Z, Bao N, Kobayashi H, Yasuda S, Iwasa M, Sumi S, Kawamura I, Yamada Y, Nishigaki K, Takemura G, Fujiwara T, Fujiwara H.
Journal: J Cardiovasc Pharmacol (2009): 25

Endogenous activation of mitochondrial KATP channels protects human failing myocardium from hydroxyl radical-induced stunning
Authors: Maack C, Dabew ER, Hohl M, Schafers HJ, Bohm M.
Journal: Circ Res (2009): 811


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