05 Cell Staining

mtSOX Deep Red - Mitochondrial Superoxide Detection

 mtSOX Deep Red - Mitochondrial Superoxide Detection

Mitochondrial Superoxide Detection

  • Allow to detecting mitochondrial superoxide with a long wavelength(Deep Red)
  • Allow to co-staining with a green or red fluorescent probe
  • High selectivity to superoxide
  • Product code
    MT14  mtSOX Deep Red - Mitochondrial Superoxide Detection
Unit size Price Item Code
100 nmol x 1 $120.00 MT14-10
100 nmol x 3 $270.00 MT14-12
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Component
100 nmol x 1
100 nmol x 3

Description

  The mitochondrion is an important organelle that uses oxygen to synthesize ATP, producing the necessary energy for living cells to thrive. Decreased mitochondrial activity and mitochondrial dysfunction are associated with cancer, aging, and neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. Mitochondrial mass (MM), mitochondrial membrane potential (MMP), and mitochondrial ROS (mtROS) have been widely studied as promising targets for mitochondria-related diseases. Since these mitochondrial attributes are dynamic, simultaneous analysis using multiple staining in a single sample is required.

  Company T' product Red has widely been used to detect mitochondrial superoxide. However, the emission wavelength is the common red, which overlaps with other MMP detection probes such as TMRE, and is therefore not applicable for simultaneous staining with these other mitochondrial markers in a single sample.

  Dojindo’s mtSOX Deep Red overcomes these limitations. This dye emits deep red fluorescence; its fluorescence does not overlap with emission wavelengths that other red fluorescent markers use. Furthermore, the mtSOX Deep Red is better able to selectively detect superoxide, compared to Company T' product Red.

  Altogether, mtSOX Deep Red is a powerful tool for researchers with a limited number of cells and can provide an understanding of how mitochondria are altered during different treatments and physiological or pathological states.

 

 

Manual

Technical info

Comparison with other products

mtSOX Deep Red has higher superoxide selectivity than Company T's existing product Red.

In addition, mtSOX Deep Red has a special fluorescence spectrum (λex: 540 nm, λem: 670 nm), making it possible to co-staining with mitochondrial membrane potential reagent (JC-1, Code: MT09, TMRE, MT-11, code: MT13), which is difficult for others mitochondrial superoxide reagent.

 

Company T's product Red tends to be localized at DNA in cells, but mtSOX Deep Red does not localize to DNA.

Application Data: Simultaneously evaluation of mitochondrial superoxide and membrane potential

After HeLa cells were washed with HBSS, co-stained with mtSOX Deep Red and mitochondrial membrane potential staining dye (JC-1: code MT09 or MT-1: code MT13), and the generated mitochondrial ROS and membrane potential were observed simultaneously. As a result, the decrease in mitochondrial membrane potential and the generation of mitochondrial ROS are simultaneously observed.

<General protocol of JC-1>

<Imaging Conditions>(Confocal microscopy)
JC-1: Green Ex = 488, Em = 490-520 nm, Red: Ex = 561, Em = 560-600 nm
mtSOX :Ex = 633 nm, Em = 640-700 nm
Scale bar: 10 μm

 

<Examination Conditions>(Plate Reader)Tecan, Infinite M200 Pro
JC-1: Green Ex=480-490 nm, Em=525-545 nm; Red: Ex= 530-540 nm, Em=585-605 nm
mtSOX: Ex=545-555 nm, Em = 665-685 nm

 

<General Protocol of MT-1>

<Imaging Conditions>(Confocal microscope)
MT-1: Ex=561, Em=560-600 nm
mtSOX: Ex=633 nm, Em=640-700 nm
Scale bar: 10 μm

 

<Examination Conditions>(Plate Reader)Tecan, Infinite M200 Pro
MT-1: Ex=540-550 nm, Em=590-610 nm (Gain=200)
mtSOX: Ex=545-555 nm, Em = 665-685 nm

Application Data: Co-Staining with Mitochondrial Fluorescent probe(Company T's Product Green)

Application Data: Simultaneously evaluation of mitochondrial superoxide and intracellular total ROS

HeLa cells were washed with HBSS and co-stained with mtSOX and intracellular total ROS reagent (ROS Assay Kit -Highly Sensitive DCFH-DA-: code R252), and separately treated with mitochondrial superoxide inducer Antimycin or hydrogen peroxide. 

As a result, intracellular total ROS and mitochondrial superoxide were observed separately.

General Protocol

Treated with H2O2

Treated with Antimycin

<Imaging Conditions>(Confocal microscopy)
Intracellular ROS: Ex = 488, Em = 490-520 nm
mtSOX: Ex = 633 nm, Em = 640-700 nm
Scale bar: 10 μm

Application Data: Co-staining with DHE(Total ROS Probe)

mtSOX Deep Red is applicable for multiple staining with DHE (total ROS probe).

Application Data: Simultaneously evaluate mitochondrial mass, membrane potential and mitochondrial superoxide

HeLa cells stained with nuclear staining reagent (Hoechst33342: code H342), mitochondrial mass detection dye (MitoTrackerTM Green FM), and mitochondrial membrane potential detection dye (Tetramethylrhodamine ethyl ester (TMRE)). The cells were washed with HBSS and stained with mtSOX Deep Red working solution containing Antimycin, and the generated mitochondrial ROS and membrane potential, mitochondrial mass, and nuclei were observed simultaneously.
 As a result, the decrease in mitochondrial membrane potential associated with the generation of mitochondrial ROS without any change in mitochondrial mass was simultaneously observed.

<Imaging Conditions>(Confocal microscopy)
(Blue) Nucleus: Hoechst33342 (Ex=405, Em=450-495 nm)

(Green) Mitochondrial Mass: MitoTrackerTM Green FM (Ex=488 nm, Em=500-550 nm)
(Red) Mitochondrial Membrane Protential: TMRE (Ex=561 nm, Em=560-620 nm)
(Deep Red) Mitochondrial Superoxide: mtSOX Deep Red (Ex=633 nm, Em=640-700 nm
Scale bar: 10 μm

The general number of usable assays per 100 nmol

・96-well plate X 1
・ibidi 8-well plate X 6
・35 mm dish X 5

Fluorescence spectra

Q & A

Q

What is the detection principle of mtSOX Deep Red?

A

mtSOX Deep Red is a fluorescent dye that is selectively oxidized by superoxide.
This dye localizes to intracellular mitochondria in a membrane-potential-dependent manner, allowing for the specific detection of mitochondrial superoxide.
When mitochondrial membrane potential disappears following superoxide induction, dye is dispersed throughout the nucleolus and cytoplasm.

Q

Is it possible to prepare the working solution with buffers other than culture medium?

A

Yes, the working solution can be produced using HBSS or PBS

Q

What instruments are available, and what filters are appropriate?

A

The signal can be detected via confocal microscopy, epi-fluorescence microcopy, microplate reader, and flow cytometry.

・Confocal laser microscopy
Ex: 561, Em: 640-670 nm (Single staining)
Ex: 633, Em: 640-670 nm (Co-staining with red fluorescent dye)

・Epi-fluorescence microscopy
Texas Red filter

・Microplate reader
Ex/Em: 550/675 nm (Bottom reading)

・Flow cytometry
APC filter
 

Q

Is it possible to stain after stimulation/superoxide induction?

A

This is possible under conditions where the mitochondrial membrane potential does not decrease.
This dye accumulates in mitochondria in a membrane-potential-dependent manner. Thus, if the membrane potential is decreased via stimulation, the dye cannot accumulate in the mitochondria. Therefore, the dye cannot detect accurate superoxide, as the stain will be inaccurate.
Thus, we recommend pre-stimulus staining.

Q

What is the difference in mitochondrial specificity between mtSOX Deep Red and MitoSOX™ Red?

A

MitoSOX™ Red tends to accumulate in the nucleus as the staining time increases, whereas mtSOX Deep Red does not accumulate in the nucleus.
However, depending on the type of drug treatment, localization may be seen in nucleolus and other non-mitochondrial organelles.
(Signal change similar to that of MitoSOX™ Red)

Comparison between mtSOX Deep Red and MitoSOX™ Red images at different staining times

Detection of superoxide in HeLa cells treated with antimycin by mtSOX Deep Red and MitoSOX™ Red

 

 

 

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