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

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 has 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 to monitor superoxide in mitochondria either with a fluorescence microscope or 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. MitoROS™ 580 is equivalent to the MitoSOX™ Mitochondrial Superoxide Indicator (#M36008) that is often used for live-cell imaging (MitoSOX™ is the trademark of ThermoFisher).

Example protocol

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

MitoROS™ 580 Stock Solution (1000X)

  1. Add 13 µL of DMSO to the MitoROS™ 580 vial and mix well.

    Note: Any unused stock solution can be stored at -20 °C, protected from light.

PREPARATION OF WORKING SOLUTION

MitoROS™ 580 Working Solution(2X)

  1. Dilute the DMSO stock solution into Hanks solution with 20 mM Hepes buffer (HHBS) to make a 2X working solution.

    Note: The 2X MitoROS™ 580 working solution is not stable, use it promptly.

SAMPLE EXPERIMENTAL PROTOCOL

Important Note

This protocol is a mere guideline and should be optimized to suit your specific requirements. Prior to making the MitoROS™ 580 working solution, treat cells as desired.

  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 1X. Higher concentrations can lead to cytotoxic effects, such as altered mitochondrial morphology and fluorescence redistribution to nuclei and 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.

Spectrum

Citations

View all 31 citations: Citation Explorer
Sirtuin 3-activated superoxide dismutase 2 mediates fluoride-induced osteoblastic differentiation in vitro and in vivo by down-regulating reactive oxygen species
Authors: Yang, Liu and Li, Qiao and Wang, Sa and Ji, Yi and Ma, Xinbo and Qin, Ming and Gao, Yanhui and Yang, Yanmei
Journal: Archives of Toxicology (2024): 1--13
Tetrandrine Targeting SIRT5 Exerts Anti-melanoma Properties via Inducing ROS, ER Stress, and Blocked Autophagy
Authors: Ji, Yacong and Li, Chongyang and Wan, Sicheng and Dong, Zhen and Liu, Chaolong and Guo, Leiyang and Shi, Shaomin and Ci, Mingxin and Xu, Minghao and Li, Qian and others,
Journal: Journal of Pharmaceutical Analysis (2024): 101036
Exploring the Cellular Impact of Size-Segregated Cigarette Aerosols: Insights into Indoor Particulate Matter Toxicity and Potential Therapeutic Interventions
Authors: Shen, Yu-Xin and Lee, Pe-Shuen and Wang, Chia C and Teng, Ming-Chu and Huang, Jhih-Hong and Fan, Hsiu-Fang
Journal: Chemical Research in Toxicology (2024)
Quercetin Impairs the Growth of Uveal Melanoma Cells by Interfering with Glucose Uptake and Metabolism
Authors: Tura, Ayseg{\"u}l and Herfs, Viktoria and Maa{\ss}en, Tjorge and Zuo, Huaxin and Vardanyan, Siranush and Prasuhn, Michelle and Ranjbar, Mahdy and Kakkassery, Vinodh and Grisanti, Salvatore
Journal: International Journal of Molecular Sciences (2024): 4292

References

View all 46 references: Citation Explorer
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
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
Page updated on September 19, 2024

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Catalog Number16052
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Physical properties

Molecular weight

N/A

Solvent

DMSO

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

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12352200
Fluorescence response of MitoROS&trade; 580 (10 &micro;M)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&trade; 580 (10 &micro;M)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&trade; 580 (10 &micro;M)to different reactive oxygen species (ROS) and reactive nitrogen species (RNS). The fluorescence intensities were monitored at Ex/Em = 540/590 nm.
The detection of intracellular superoxide in Jurkat cells was performed using MitoROS™ 580. For the AMA treatment (Red), cells were treated with 50 µM Antimycin A (AMA) at 37°C for 30 minutes, followed by incubation with MitoROS™ 580 for 1 hour. For the control (Blue), cells were incubated with MitoROS™ 580 at 37°C for 1 hour without prior AMA treatment. The fluorescence signal was monitored using the FL2 channel of a BD FACSCalibur flow cytometer.
Fluorescence imaging of superoxide measurement in HeLa cells was performed using MitoROS™ 580. HeLa cells were seeded at a density of 100,000 cells per well in 100 µL and incubated overnight in a 96-well plate with black walls and a clear bottom. For the AMA treatment group, cells were treated with 50 µM Antimycin A (AMA) at 37°C for 30 minutes, followed by incubation with MitoROS™ 580 for 1 hour. In the untreated control group, cells were incubated with MitoROS™ 580 at 37°C for 1 hour without any prior AMA treatment. Fluorescence signals were measured using a fluorescence microscope with a TRITC filter.
Detection of intracellular superoxide in HeLa Cells using MitoROS™ 580. HeLa cells were seeded at a density of 100,000 cells per well in 100 µL of medium and incubated overnight in a 96-well plate with black walls and a clear bottom. The next day, the cells were treated with either 50 µM Pyocyanin (Pyo), 50 µM Antimycin A (AMA), or left untreated as a control. These treatments were applied at 37°C for 30 minutes. Following this, the cells were incubated with MitoROS™ 580 at 37°C for 1 hour. Fluorescence signals were measured using a CLARIOstar microplate reader (BMG Labtech) with an excitation/emission wavelength of 540/590 nm and a cutoff of 570 nm, using the bottom read mode.

Documents

pdfSafety data sheet
pdfProduct protocol
pdfCertificate of analysis
Reactive Oxygen Species (ROS)
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