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MitoROS Brite™ NIR 780 *Optimized for Detecting Reactive Oxygen Species (ROS) in Mitochnodria*

The fluorescence response of MitoROS Brite™ NIR 780 (0.5 µM) to varying concentrations of H2O2 in HeLa cells was assessed. Fluorescence intensities were monitored using a fluorescence microscope equipped with a Cy7 filter.
The fluorescence response of MitoROS Brite™ NIR 780 (0.5 µM) to varying concentrations of H2O2 in HeLa cells was assessed. Fluorescence intensities were monitored using a fluorescence microscope equipped with a Cy7 filter.
The fluorescence response of MitoROS Brite™ NIR 780 (0.5 µM) to varying concentrations of H2O2 in HeLa cells was assessed. Fluorescence intensities were monitored using a fluorescence microscope equipped with a Cy7 filter.
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Physical properties
Molecular weight746.35
SolventDMSO
Spectral properties
Excitation (nm)764
Emission (nm)778
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
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Molecular weight
746.35
Excitation (nm)
764
Emission (nm)
778
Mitochondrial reactive oxygen species (mtROS or mROS) are generated within mitochondria, primarily during oxidative phosphorylation at the electron transport chain (ETC) on the inner mitochondrial membrane. Electron leakage from complexes I and III partially reduces oxygen to superoxide, which is dismutated to hydrogen peroxide by SOD2 in the mitochondrial matrix and SOD1 in the intermembrane space. Low levels of mtROS are essential for metabolic adaptation and inflammatory response regulation, while elevated mtROS can induce apoptosis, autophagy, cellular senescence, and aging. Recent findings implicate mtROS production in lung monocytes and macrophages in COVID-19 pathogenesis, highlighting mtROS as a potential therapeutic target for novel coronavirus treatments. MitoROS Brite™ NIR 780 is a superoxide-sensitive fluorescent probe specifically designed for targeting mitochondria in live cells. Upon oxidation by superoxide anions, it emits near-infrared fluorescence, allowing precise quantification of mitochondrial superoxide levels via fluorescence microscopy or flow cytometry. Its high cell permeability and mitochondrial selectivity ensure specific detection of superoxide with minimal cross-reactivity to other ROS or reactive nitrogen species (RNS). The retention of the oxidized fluorophore within cells makes MitoROS Brite™ NIR 780 invaluable for investigating oxidative stress-related pathologies. Furthermore, its compatibility with multiplexing techniques facilitates concurrent use with other fluorescent mitochondrial probes, enabling comprehensive analysis of multiple mitochondrial parameters within the same cell population. For instance, it can be combined with MitoLite™ Green FM to assess ROS levels and mitochondrial mass or morphology, or with TMRE or TMRM to measure both mitochondrial membrane potential and ROS levels, advancing research into mitochondrial-associated diseases.

Platform


Fluorescence microscope

ExcitationCy7 filter set
EmissionCy7 filter set
Recommended plateBlack wall/clear bottom

Example protocol


AT A GLANCE

Important Note

Before using MitoROS Brite™ NIR 780 for the first time, allow it to thaw at room temperature. Then, briefly centrifuge it to collect the dried pellet. 

Protocol Summary
  1. Prepare the cells in a growth medium.

  2. Stain cells with MitoROS Brite™ NIR 780 working solution.

  3. Treat cells with test compound.

  4. Monitor the fluorescence intensity using a fluorescence microscope equipped with a Cy7 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

MitoROS Brite™ NIR 780 Stock Solution
  1. Prepare a 5 to 10 mM MitoROS Brite™ NIR 780 stock solution in DMSO. For example, to make a 10 mM stock solution, add 141 μL of DMSO to the MitoROS Brite™ NIR 780 vial.

    Note: Any unused stock solution can be stored at -20 °C, protected from light. Avoid repeated freeze-thaw cycles.

PREPARATION OF WORKING SOLUTION

MitoROS Brite™ NIR 780 Working Solution
  1. Prepare a working solution of 0.5 to 5 μM by diluting the MitoROS Brite™ NIR 780 stock solution in Hanks solution with 20 mM Hepes buffer (HHBS).

    Note: For optimal results, use this solution within a few hours of preparation.

    Note: Cover the working solution with foil or store it in a dark place to protect it from light.

SAMPLE EXPERIMENTAL PROTOCOL

Sample Protocol
  1. Plate the cells as desired in a 96-well black wall-clear bottom plate.

  2. Add 100 µL of the MitoROS Brite™ NIR 780 working solution directly to the cells.

  3. Incubate the cells at 37°C for 15-30 minutes, protected from light.

    Note: The concentration and incubation time of MitoROS Brite™ NIR 780 may vary depending on the cell line. Test different concentrations to determine the optimal dose.

  4. Remove the dye working solution and wash the cells twice with HHBS buffer.

  5. Treat cells as desired.

  6. Remove the treatment and was the cells twice with HHBS buffer.

  7. Add HHBS buffer and analyze the cells using a fluorescence microscope equipped with a Cy7 filter set.

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of MitoROS Brite™ NIR 780 *Optimized for Detecting Reactive Oxygen Species (ROS) in Mitochnodria* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM133.985 µL669.927 µL1.34 mL6.699 mL13.399 mL
5 mM26.797 µL133.985 µL267.971 µL1.34 mL2.68 mL
10 mM13.399 µL66.993 µL133.985 µL669.927 µL1.34 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Spectrum


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spectrum

Spectral properties

Excitation (nm)764
Emission (nm)778

Images


References


View all 50 references: Citation Explorer
Hydrophilic oxygen radical absorbance capacity values of low-molecular-weight phenolic compounds containing carbon, hydrogen, and oxygen.
Authors: Sakurai, Shuhei and Kikuchi, Akito and Gotoh, Hiroaki
Journal: RSC advances (2022): 4094-4100
EPR spectroscopy investigation of oxygen radical production by methylene blue and indocyanine green in aqueous solutions under laser irradiation in the context of antibacterial photodynamic therapy.
Authors: Tanev, Mihail and Tomov, Georgi and Karakirova, Yordanka
Journal: Folia medica (2021): 372-376
Metabolic Rate and Oxygen Radical Levels Increase But Radical Generation Rate Decreases with Male Age in Drosophila melanogaster Sperm.
Authors: Turnell, Biz R and Reinhardt, Klaus
Journal: The journals of gerontology. Series A, Biological sciences and medical sciences (2020): 2278-2285
Evaluation of oxidative stress levels in obesity and diabetes by the free oxygen radical test and free oxygen radical defence assays and correlations with anthropometric and laboratory parameters.
Authors: Găman, Mihnea-Alexandru and Epîngeac, Mirela Elena and Diaconu, Camelia Cristina and Găman, Amelia Maria
Journal: World journal of diabetes (2020): 193-201
Effect of free oxygen radical anions and free electrons in a Ca12Al14O33 cement structure on its optical, electronic and antibacterial properties.
Authors: Phrompet, Chaiwat and Sriwong, Chaval and Srepusharawoot, Pornjuk and Maensiri, Santi and Chindaprasirt, Prinya and Ruttanapun, Chesta
Journal: Heliyon (2019): e01808
Improving Microcirculatory Reperfusion Reduces Parenchymal Oxygen Radical Formation and Provides Neuroprotection.
Authors: Taskiran-Sag, Aslihan and Yemisci, Muge and Gursoy-Ozdemir, Yasemin and Erdener, Sefik Evren and Karatas, Hulya and Yuce, Deniz and Dalkara, Turgay
Journal: Stroke (2018): 1267-1275
The Transcription Factor Nrf2 Protects Angiogenic Capacity of Endothelial Colony-Forming Cells in High-Oxygen Radical Stress Conditions.
Authors: Gremmels, Hendrik and de Jong, Olivier G and Hazenbrink, Diënty H and Fledderus, Joost O and Verhaar, Marianne C
Journal: Stem cells international (2017): 4680612
Metabolomic Analyses of Brain Tissue in Sepsis Induced by Cecal Ligation Reveal Specific Redox Alterations--Protective Effects of the Oxygen Radical Scavenger Edaravone.
Authors: Hara, Naomi and Chijiiwa, Miyuki and Yara, Miki and Ishida, Yusuke and Ogiwara, Yukihiko and Inazu, Masato and Kuroda, Masahiko and Karlsson, Michael and Sjovall, Fredrik and Elmér, Eskil and Uchino, Hiroyuki
Journal: Shock (Augusta, Ga.) (2015): 578-84
Role of heat shock proteins in oxygen radical-induced gastric apoptosis.
Authors: Leung, Anna M and Redlak, Maria J and Miller, Thomas A
Journal: The Journal of surgical research (2015): 135-44
Oxygen radical formation in anoxic transgression and anoxia-reoxygenation: foe or phantom? Experiments with a hypoxia tolerant bivalve.
Authors: Rivera-Ingraham, Georgina A and Rocchetta, Iara and Meyer, Stefanie and Abele, Doris
Journal: Marine environmental research (2013): 110-9