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MitoROS Brite™ 570 *Optimized for Detecting Reactive Oxygen Species (ROS) in Mitochondria*

Mitochondrial ROS (mtROS or mROS) are reactive oxygen species (ROS) produced in mitochondria. Selective detection of mtROS is a critical task to investigate the cellular functions of mitochondria. The cell-permeant MitoROS Brite™ 570 reagent is cell-permeable and selectively located in mitochondria. It is nonfluorescent and produces bright orange fluorescence upon ROS oxidation in mitochondria. The resulting fluorescence can be measured using fluorescence imaging, high-content imaging, microplate fluorometry, or flow cytometry. mtROS was considered to be the by-products of cellular metabolism. However, they are now recognized as important signaling molecules. mtROS is primarily formed during oxidative phosphorylation at the electron transport chain (ETC) on the inner mitochondrial membrane. Electrons leak from complexes I and III, partially reducing oxygen to form superoxide. Superoxide is rapidly converted to hydrogen peroxide by two dismutases: SOD2 in the mitochondrial matrix and SOD1 in intermembrane space. At low levels, mtROS are essential for metabolic adaptation (e.g., in hypoxia). They regulate inflammatory responses triggered by danger signals. High mtROS levels activate apoptosis and autophagy pathways, potentially inducing cell death. Mitochondrial dysfunction leads to increased ROS levels, contributing to aging. mtROS induces cellular senescence, a stress response. Recently it has been reported that monocytes/macrophages in the lungs produce mtROS in COVID-19 patients, affecting disease pathogenicity, thus targeting mtROS could be a therapeutic strategy for novel drugs against coronavirus.

Example protocol

AT A GLANCE

Important Note

Before use, thaw MitoROS Brite™ 570 at room temperature. Once thawed, briefly centrifuge to collect the dried pellet.

Subsection title
  1. Prepare the cells in a growth medium.

  2. Stain the cells using the MitoROS Brite™ 570 working solution.

  3. Treat the cells with your desired test compounds.

  4. Monitor fluorescence intensity with a Cy3 filter set or Ex/Em = 540/590 nm.

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™ 570 Stock Solution
  1. Prepare a 5 to 10 mM MitoROS Brite™ 570 stock solution in DMSO.

    Note: Prepare single-use aliquots of the MitoROS Brite™ 570 stock solution and store at ≤ -20°C, protected from light. Avoid freeze-thaw cycles.

PREPARATION OF WORKING SOLUTION

MitoROS Brite™ 570 Working Solution
  1. Prepare a 5 to 10 μM MitoROS Brite™ 570 working solution by diluting the MitoROS Brite™ 570 stock solution into Hanks solution with 20 mM Hepes buffer (HHBS).

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

    Note: Protect the working solution from light by covering it with foil or storing it in a dark place.

SAMPLE EXPERIMENTAL PROTOCOL

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

  2. Add 100 µL of the MitoROS Brite™ 570 working solution to the cells.

  3. Incubate the cells at 37°C for 30 to 60 minutes, protected from light.

    Note: The optimal concentration and incubation time for MitoROS Brite™ 570 may vary between different cell lines. You may need to test different concentrations to determine the best conditions.

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

  5. Treat cells as desired.

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

  7. Add HHBS buffer and examine the cells using a fluorescence microscope with a Cy3 filter set.

Spectrum

References

View all 50 references: Citation Explorer
Dual stimuli-responsive and sustained drug delivery NanoSensoGel formulation for prevention of cisplatin-induced ototoxicity.
Authors: Thakur, Neeraj S and Rus, Iulia and Sparks, Ethan and Agrahari, Vibhuti
Journal: Journal of controlled release : official journal of the Controlled Release Society (2024): 66-83
Exposure to low dose of Bisphenol A (BPA) intensifies kidney oxidative stress, inflammatory factors expression and modulates Angiotensin II signaling under hypertensive milieu.
Authors: Nagarajan, Manigandan and Maadurshni, Gobichettipalayam Balasubramaniam and Manivannan, Jeganathan
Journal: Journal of biochemical and molecular toxicology (2024): e23533
Menstrual blood-derived stem cells exosomal miR-let-7 to ameliorate pulmonary fibrosis through inhibiting ferroptosis by Sp3/HDAC2/Nrf2 signaling pathway.
Authors: Sun, Lifang and He, Xinxin and Kong, Jiao and Yu, Huan and Wang, Yunguang
Journal: International immunopharmacology (2024): 111316
Capsaicin attenuates Porphyromonas gingivalis-suppressed osteogenesis of periodontal ligament stem cells via regulating mitochondrial function and activating PI3K/AKT/mTOR pathway.
Authors: Wang, Weijia and Zhou, Zhiyan and Ding, Tian and Feng, Susu and Liu, Hongrui and Liu, Mengmeng and Ge, Shaohua
Journal: Journal of periodontal research (2024)
Molecular mechanism of high glucose-induced mitochondrial DNA damage in retinal ganglion cells.
Authors: Zhou, Jinzi and Chen, Fenghua and Yan, Aimin and Xia, Xiaobo
Journal: Cellular and molecular biology (Noisy-le-Grand, France) (2024): 219-224
Page updated on December 6, 2024

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Catalog Number15998
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Physical properties

Molecular weight

732.83

Solvent

DMSO

Spectral properties

Excitation (nm)

555

Emission (nm)

568

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

Platform

Fluorescence microscope

ExcitationCy3 Filter Set
EmissionCy3 Filter Set
Recommended plateBlack wall, clear bottom
The fluorescence response of MitoROS™ Brite 570 (5 µM) to varying concentrations of H2O2 in HeLa cells was investigated. Fluorescence intensities were measured using a fluorescence microscope equipped with a Cy3 filter.
The fluorescence response of MitoROS™ Brite 570 (5 µM) to varying concentrations of H2O2 in HeLa cells was investigated. Fluorescence intensities were measured using a fluorescence microscope equipped with a Cy3 filter.
The fluorescence response of MitoROS™ Brite 570 (5 µM) to varying concentrations of H2O2 in HeLa cells was investigated. Fluorescence intensities were measured using a fluorescence microscope equipped with a Cy3 filter.