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mtSOX Deep Red – Mitochondrial Superoxide Detection

Item # Unit Size
MT14-10
100 nmol x 1
MT14-12
100 nmol x 3

For Research Use Only Products

Content: 100 nmol x 1; 100 nmol x 3

∼ Features ∼
– Absorption/emission maxima: ~540/670 nm
– High selectivity towards superoxide and mitochondria
– Applicable for co-staining with other mitochondrial markers in a single sample
– Used for live cell imaging

The general number of usable assays per 100 nmol:
・35 mm dish x 5
・96 well plate x 1

Storage Condition: Store at 0-5oC
Shipping Condition: Ambient temperature




Detection Principle

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.

MitoSOX™ 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 this 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 MitoSOX ™ 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.


Comparison Between mtSOX Deep Red and MitoSOX™ Red



mtSOX Deep Red has high superoxide selectivity and detects mitochondrial ROS induced by Antimycin in HeLa cells, similarly to MitoSOX™ Red.

 
Co-Staining with Another Mitochondria-Staining Probe



mtSOX Deep Red permeates live cells and selectively stains mitochondria.
*MitoBright LT Green (MT10): Mitochondria-staining dye designed for long-term visualization.


Applications





mtSOX Deep Red allows for sensitive detection of superoxide via fluorescence imaging, flow cytometry, and microplate assay.


Fluorescence Properties



mtSOX Deep Red emits deep red fluorescence.

 
Experimental Example

Multiple Staining with Other Mitochondrial Markers

mtSOX Deep Red is applicable for multiple staining with other mitochondrial markers.



(Blue) Nuclear stain: Hoechst33342 (Ex: 405 nm, Em: 450–495 nm)
(Green) Mitochondrial mass stain: MitoTracker™ Green FM (Ex: 488 nm, Em: 500–550 nm)
(Red) Mitochondrial membrane potential stain: TMRE (Ex: 561 nm, Em: 560–620 nm)
(Purple) Mitochondrial superoxide stain: mtSOX Deep Red (Ex: 633 nm, Em 640–700 nm)

 
Selectivity of mtSOX Deep Red Toward ROS in Cells – Microplate Reader



<Detection Condition>
HeLa cells
mtSOX Deep Red + Antimycin or H2O2: Ex 550 / Em 675 nm
MitoSOX™ Red + Antimycin or H2O2: Ex 500 / Em 580 nm

mtSOX Deep Red has high superoxide selectivity and is compatible with quantitative assay by microplate reader.



Optimal Detection Conditions

Instrument Best detection condition
Confocal microscope - Single staining
Ex: 561, Em: 640-670 nm
- Co-staining with red fluorescent dye
Ex: 633, Em: 640-670 nm
Epifluorescence microscope Texas Red filter
Ex: 560/40 nm, Em: 630/75 nm
Microplate reader Ex/Em: 550/675 nm
(Bottom reading)
Flow cytometry APC filter
Ex: 640 nm, Em: 670/30 nm

Changes in Mitochondrial Membrane Potential and Superoxide due to Antimycin Treatment



<Detection Condition>
JC-1 (Ex/Em)
Green: 488/500-550 nm
Red:     561/560-610 nm

mtSOX Deep Red
Purple: 633/640-700 nm

 


<Detection Condition>
JC-1 (Ex/Em)
Green: 485/535 nm
Red:     535/595 nm

mtSOX Deep Red
550/675 nm

The changes in mitochondrial superoxide and mitochondrial membrane potential after treatment with antimycin (an inhibitor of the mitochondrial electron transfer system) were detected by co-staining with mtSOX Deep Red and JC-1. It was confirmed that superoxide presence increased and the membrane potential strongly decreased due to the antimycin treatment.

*JC-1 MitoMP Detection Kit (MT09): JC-1 is used to determine mitochondrial membrane potential.

 
<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

*MT-1 MitoMP Detection Kit (MT13): MT-1 is used to determine mitochondrial membrane potential.

 
Changes in Hydrogen Peroxide and Superoxide due to Antimycin Treatment



<Detection Condition>
ROS Assay Kit -Highly Sensitive DCFH-DA- (Ex/Em)
Green: 488/500-550 nm

mtSOX Deep Red
Purple: 561/640-700 nm

The Changes in mitochondrial superoxide and hydrogen peroxide were detected by co-staining with mtSOX Deep Red and ROS Assay Kit -Highly Sensitive DCFH-DA- (HS DCFH-DA), which is a total ROS detection dye. It was confirmed that superoxide presence increased and that hydrogen peroxide was unchanged by the antimycin treatment. These results suggest that antimycin inhibits the mitochondrial electron transfer system.

*ROS Assay Kit -Highly Sensitive DCFH-DA- (R252): HS DCFH-DA allows ROS detection with higher sensitivity than conventional DCFH-DA.

 
Co-Staining with DHE (Total ROS Probe)



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

 
Co-staining with MitoTracker™ Green – FCM Analysis



mtSOX Deep Red is compatible with APC filter (640 nm laser) in FCM, and its fluorescence does not overlap with emission wavelengths that other green/red fluorescent markers use.

 
Comparison Between mtSOX Deep Red and MitoSOX™ Red



MitoSOX Red tends to be localized at DNA in cells, but mtSOX Deep Red does not localize to DNA.
What is the detection principle of mtSOX Deep Red?

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.


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

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


What instruments are available, and what filters are appropriate?

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



Is it possible to stain after stimulation/superoxide induction?

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.



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

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

No. Sample Instrument Reference (Link)
1) Cell
(NRK52E Cell)
Fluorescent microscope D. Sun, S. Cui, H. Ma, P. Zhu, N. Li, X. Zhang, L. Zhang, L. Xuan, J. Li , "Salvianolate ameliorates renal tubular injury through the Keap1/Nrf2/ARE pathway in mouse kidney ischemia-reperfusion injury", 2022, J. Ethnopharmacol., doi:10.1016/j.jep.2022.115331.
2) Cell
(TIG-1 Cell)
Fluorescent Microscope Y. Fujita, M. Iketani, M. Ito, I. Ohsawa, "Temporal changes in mitochondrial function and reactive oxygen species generation during the development of replicative senescence in human fibroblasts", 2022, doi:10.1016/j.exger.2022.111866.