MitoROS™ 580 *Optimized for Detecting Reactive Oxygen Species (ROS) in Mitochondria*

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Fluorescence response of MitoROS™ 580 (10 µM)to different reactive oxygen species (ROS) and reactive nitrogen species (RNS). The fluorescence intensities were monitored at Ex/Em = 540/590 nm.
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Unit Size: Cat No: Price (USD): Qty:
500 Tests 16052 $195


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Additional Ordering Information
Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
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Overview

MWN/A
SolventDMSO
Storage Freeze (<-15 °C)
Minimize light exposure
Category Neurobiology
Reactive Oxygen Species
Related Cell Signaling
Reactive oxygen species (ROS) are chemically reactive molecules containing oxygen. Examples include superoxide, hydroxyl radical, singlet oxygen and peroxides. ROS is highly reactive due to the presence of unpaired valence shell electrons. ROS forms as a natural byproduct of the normal metabolism of oxygen and have important roles in cell signaling and homeostasis. However, during times of environmental stress (e.g., UV or heat exposure), ROS levels can increase dramatically. This may result in significant damage to cell structures. Cumulatively, this is known as oxidative stress. MitoROS™ 580 is a superoxide-sensitive dye that is localized in mitochondria upon loading into live cells. Oxidation of MitoROS™ 580 by superoxide generates red fluorescence. MitoROS™ 580 can be used for monitoring superoxide in mitochondria either with a fluorescence microscope or a fluorescence flow cytometer. MitoROS™ 580 reagent permeates live cells where it selectively targets mitochondria. It is rapidly oxidized by superoxide. It is less likely to be oxidized by other reactive oxygen species (ROS) and reactive nitrogen species (RNS). The oxidized product is highly fluorescent in cells. MitoROS™ 580 provides a valuable tool for investigating oxidative stress in various pathologies.




Protocol


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

Important notes
This protocol only provides a guideline, and should be modified according to your specific needs. Treat cells as desired before making the MitoROS™ 580 working solution.

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. MitoROS™ 580 Stock Solution (1000X)
Add 13 µL of DMSO to the MitoROS™ 580 vial and mix well. Note: The unused stock solution can be stored at -20 oC. Protect from light.

Preparation of working solution

MitoROS™ 580 working solution(2X):
Dilute the DMSO stock solution into Hanks solution with 20 mM Hepes buffer (HHBS) to make 2X working solution. Note: The 2X MitoROS™ 580 working solution is not stable, use it promptly.

Sample experimental protocol
  1. Treat cells as desired.

  2. Incubate the cells (such as 100 µL/well in 96-well plate) with equal volume of 2X MitoROS™ 580 working solution for 10-30 minutes at 37 °C, protected from light. Note: The final in cell concentration of the MitoROS™ 580 should not exceed 1 X. Concentrations exceeding 1 X can produce cytotoxic effects, including altered mitochondrial morphology and redistribution of fluorescence to nuclei and the cytosol. Note: Different cells react to MitoROS™ 580 differently, adjust the working concentration accordingly.

  3. Wash cells gently three times and replace it with HHBS buffer.

  4. Analyze the cells with a proper fluorescence instrument (e.g., a fluorescence microscope, flow cytometer) with Ex/Em = 510/580 nm.
Example data analysis and figures

Figure 1. Fluorescence response of MitoROS™ 580 (10 µM)to different reactive oxygen species (ROS) and reactive nitrogen species (RNS). The fluorescence intensities were monitored at Ex/Em = 540/590 nm.
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

Intracellular Fenton Reaction based on Mitochondria-Targeted Copper (Ⅱ)-Peptide Complex for Induced Apoptosis
Authors: Xia Li, Sijia Hao, Ailing Han, Yayu Yang, Guozhen Fang, Jifeng Liu, Shuo Wang
Journal: Journal of Materials Chemistry B (2019)

The contribution of oxidative stress to platelet senescence during storage
Authors: Li Wang, Rufeng Xie, Zhijia Fan, Jie Yang, Wei Liang, Qiang Wu, Mei X Wu, Zhicheng Wang, Yuan Lu
Journal: Transfusion (2019)

Carbon monoxide poisoning--induced delayed encephalopathy accompanies decreased in microglial cell numbers: distinctive pathophysiological features from hypoxemia--induced brain damage
Authors: Keisuke Sekiya, Tasuku Nishihara, Naoki Abe, Amane Konishi, Hideyuki Nandate, Taisuke Hamada, Keizo Ikemune, Yasushi Takasaki, Junya Tanaka, Migiwa Asano
Journal: Brain research (2018)






Documents

 
Safety Data Sheet (SDS)


Catalogs
1. Reactive Oxygen Species (ROS) Detection

Application Notes
1. AssayWise Letters 2013, Vol 2(1)

Certificate of Analysis