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

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.
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.
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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Physical properties
Molecular weightN/A
Spectral properties
Excitation (nm)500
Emission (nm)582
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure


Molecular weight
Excitation (nm)
Emission (nm)
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.

Example protocol


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.


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.


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.


  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.


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Spectral properties

Excitation (nm)500
Emission (nm)582



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