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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.
Ordering information
Price ()
Catalog Number16052
Unit Size
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Additional ordering information
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
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


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


View all 8 citations: Citation Explorer
Linoleic Acid Upregulates Microrna-494 to Induce Quiescence in Colorectal Cancer
Authors: Ogata, Ruiko and Mori, Shiori and Kishi, Shingo and Sasaki, Rika and Iwata, Naoya and Ohmori, Hitoshi and Sasaki, Takamitsu and Nishiguchi, Yukiko and Nakashima, Chie and Goto, Kei and others,
Journal: International Journal of Molecular Sciences (2022): 225
Photoactivation of mitochondrial reactive oxygen species-mediated Src and protein kinase C pathway enhances MHC class II-restricted T cell immunity to tumours
Authors: Chang, Haocai and Zou, Zhengzhi and Li, Jie and Shen, Qi and Liu, Lei and An, Xiaorui and Yang, Sihua and Xing, Da
Journal: Cancer Letters (2021)
Graphene Oxide (GO)-based Nanosheets With Combined Chemo/photothermal/Photodynamic Therapy to Overcome Gastric Cancer (GC) Paclitaxel Resistance by Reducing Mitochondria-Derived Adenosine-Triphosphate (ATP)
Authors: Guo, Weihong and Chen, Zhian and Feng, Xiaoli and Shen, Guodong and Huang, Huilin and Liang, Yanrui and Zhao, Bingxia and Li, Guoxin and Hu, Yanfeng
Journal: (2021)
Icariside II overcomes BRAF inhibitor resistance in melanoma by inducing ROS production and inhibiting MITF
Authors: Liu, Xiao and Li, Zheng and Li, Ming and Chai, Jingxiu and He, Shan and Wu, Jinfeng and Xu, Jinhua
Journal: Oncology Reports (2020)
The contribution of oxidative stress to platelet senescence during storage
Authors: Wang, Li and Xie, Rufeng and Fan, Zhijia and Yang, Jie and Liang, Wei and Wu, Qiang and Wu, Mei X and Wang, Zhicheng and Lu, Yuan
Journal: Transfusion (2019)
Carbon monoxide poisoning--induced delayed encephalopathy accompanies decreased microglial cell numbers: Distinctive pathophysiological features from hypoxemia--induced brain damage
Authors: Sekiya, Keisuke and Nishihara, Tasuku and Abe, Naoki and Konishi, Amane and Nandate, Hideyuki and Hamada, Taisuke and Ikemune, Keizo and Takasaki, Yasushi and Tanaka, Junya and Asano, Migiwa and others,
Journal: Brain research (2019): 22--32
Carbon monoxide poisoning--induced delayed encephalopathy accompanies decreased in microglial cell numbers: distinctive pathophysiological features from hypoxemia--induced brain damage
Authors: Sekiya, Keisuke and Nishihara, Tasuku and Abe, Naoki and Konishi, Amane and N, undefined and ate, Hideyuki and Hamada, Taisuke and Ikemune, Keizo and Takasaki, Yasushi and Tanaka, Junya and Asano, Migiwa and others, undefined
Journal: Brain research (2018)
Comparison of the detrimental features of microglia and infiltrated macrophages in traumatic brain injury: A study using a hypnotic bromovalerylurea
Authors: Abe, Naoki and Choudhury, Mohammed E and Watanabe, Minori and Kawasaki, Shun and Nishihara, Tasuku and Yano, Hajime and Matsumoto, Shirabe and Kunieda, Takehiro and Kumon, Yoshiaki and Yorozuya, Toshihiro and others, undefined
Journal: Glia (2018)


View all 46 references: Citation Explorer
Nonthermal plasma induces head and neck cancer cell death: the potential involvement of mitogen-activated protein kinase-dependent mitochondrial reactive oxygen species
Authors: Kang SU, Cho JH, Chang JW, Shin YS, Kim KI, Park JK, Yang SS, Lee JS, Moon E, Lee K, Kim CH.
Journal: Cell Death Dis (2014): e1056
An oxidative stress mechanism of shikonin in human glioma cells
Authors: Yang JT, Li ZL, Wu JY, Lu FJ, Chen CH.
Journal: PLoS One (2014): e94180
Low Amounts of Mitochondrial Reactive Oxygen Species Define Human Sperm Quality
Authors: Marques M, Sousa AP, Paiva A, Almeida-Santos T, Ramalho-Santos J.
Journal: Reproduction. (2014)
Subneurotoxic copper(II)-induced NF-kappaB-dependent microglial activation is associated with mitochondrial ROS
Authors: Hu Z, Yu F, Gong P, Qiu Y, Zhou W, Cui Y, Li J, Chen H.
Journal: Toxicol Appl Pharmacol (2014): 95
HPLC-based monitoring of products formed from hydroethidine-based fluorogenic probes--the ultimate approach for intra- and extracellular superoxide detection
Authors: Kalyanaraman B, Dranka BP, Hardy M, Michalski R, Zielonka J.
Journal: Biochim Biophys Acta (2014): 739
The acute inhibitory effect of iodide excess on sodium/iodide symporter expression and activity involves the PI3K/Akt signaling pathway
Authors: Serrano-Nascimento C, da Silva Teixeira S, Nicola JP, Nachbar RT, Masini-Repiso AM, Nunes MT.
Journal: Endocrinology (2014): 1145
Association rule mining of cellular responses induced by metal and metal oxide nanoparticles
Authors: Liu R, France B, George S, Rallo R, Zhang H, Xia T, Nel AE, Bradley K, Cohen Y.
Journal: Analyst (2014): 943
Muscadine grape skin extract reverts snail-mediated epithelial mesenchymal transition via superoxide species in human prostate cancer cells
Authors: Burton LJ, Barnett P, Smith B, Arnold RS, Hudson T, Kundu K, Murthy N, Odero-Marah VA.
Journal: BMC Complement Altern Med (2014): 97
The omega-3 polyunsaturated fatty acid DHA induces simultaneous apoptosis and autophagy via mitochondrial ROS-mediated Akt-mTOR signaling in prostate cancer cells expressing mutant p53
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Journal: Biomed Res Int (2013): 568671
Moderate hypoxia followed by reoxygenation results in blood-brain barrier breakdown via oxidative stress-dependent tight-junction protein disruption
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