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mtSOX Deep Red – Mitochondrial Superoxide Detection

$120.00$270.00

Content: 100 nmol x 1; 100 nmol x 3

∼ Features ∼
– Absorption/emission maxima: ~550/690 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


*This offer is for the trial size kit of mtSOX Deep Red (MT14-10).


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DescriptionDataQ & AManualS.D.S

Sensitive detection of mitochondrial ROS via deep red fluorescent probe. Compatible with many other mitochondrial probes.

Limited Time Discount

A special discount ($40 off) will be offered for a limited time!
To obtain the promotion code, please answer the following short survey!
*This offer is for the trial size kit of mtSOX Deep Red (MT14-10).

>> Survey Link
(First time US users only)

 

Mitochondrial ROS 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 correlate with cancer, aging, and neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. More specifically, mitochondrial mass (MM), mitochondrial membrane potential (MMP), and mitochondrial ROS (mtROS) are all important and promising targets for mitochondria-related diseases. These mitochondrial attributes are dynamic and therefore require simultaneous analysis via multiple staining in a single sample.

MitoSOX™ Red is a product widely used to detect mitochondrial superoxide. However, the emission wavelength is the common red, which overlaps with other mitochondrial probes, such as the MMP detection probe TMRE. Because of this overlap, MitoSOX Red is therefore not applicable for simultaneous staining with these other mitochondrial markers in a single sample.

Dojindo’s mtSOX Deep Red addresses and overcomes these limitations. For example, this dye emits deep red fluorescence that does not overlap with emission wavelengths that other red fluorescent markers use. Furthermore, 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 it 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

A bar graph juxtaposes the selectivity of mtSOX Deep Red and MitoSOX Red towards various ROS. MitoSOX Red fluorescence is about 3 times more intense in the presence of superoxide compared to other ROS, but mtSOX Deep Red intensity increases by a factor of 6. Additionally, fluorescence images depict HeLa cells following mitochondrial superoxide induction by antimycin treatment. mtSOX Deep Red and MitoSOX each display minor fluorescence in the control and strong fluorescence after treatment.

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

Fluorescence images depict co-staining being performed with mtSOX Deep Red and MitoBright LT Green. The two dyes are highly co-localized.

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


Applications

The mtSOX Deep Red and antimycin experimental process: Wash the sample twice with HBSS before incubating for 30 minutes with mtSOX Deep Red and antimycin. The sample is then ready for mitochondrial superoxide measurement with a fluorescence microscope or microplate reader. For use with flow cytometry, proceed to wash the cells once again with HBSS. Then trypsinize the cells and wash twice with HBSS.

Fluorescence microscopes, flow cytometers, and microplate readers all detect significant fluorescent signal from mtSOX Deep Red.

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


Fluorescence Properties

mtSOX Deep Red's excitation maximum is at 550 nano meters. mtSOX Deep Red's emission maximum is at 690 nano meters.

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.

2 multiple stains involving a nuclear stain, a mitochondrial mass stain, a mitochondrial membrane potential stain, and a mitochondrial superoxide stain. In the control, we see the mitochondrial dyes colocalizing outside of the nuclei. After antimycin treatment, we see that mitochondrial membrane potential has disappeared, and mitochondrial superoxide signal has intensified.

(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 Mitochondrial ROS in HeLa Cells – Microplate Reader

The selectivities of mtSOX Deep Red and MitoSOX Red are compared, using a microplate reader. MitoSOX Red displays a more intense signal under hydrogen peroxide treatment compared to control conditions. And a slightly stronger response to antimycin treatment. mtSOX Deep Red displays a similar difference in signal under hydrogen peroxide treatment, but a much stronger response to antimycin treatment, indicating that mtSOX Deep Red is more superoxide specific than MitoSOX Red in HeLa cells.

<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

InstrumentBest 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 microscopeTexas Red filter
Ex: 560/40 nm, Em: 630/75 nm
Microplate readerEx/Em: 550/675 nm
(Bottom reading)
Flow cytometryAPC filter
Ex: 640 nm, Em: 670/30 nm

Detection of Apoptosis and Mitochondrial Superoxide due to Antimycin Treatment

 

Annexin 5 - FITC is used in conjunction with mtSOX Deep Red during an antimycin treatment experiment. The procedure is simple, allowing for incubation with the two reagents and antimycin all at the same time before trypsinization and centrifugation. Flow cytometry results using different filters indicate induction of both apoptosis and mitochondrial superoxide production due to antimycin.

 

[Protocol]
1. HeLa cells (3.0 × 105 cells/well) in MEM (10% FBS) were seeded in a 6-well microplate and cultured at 37°C overnight in a 5% CO2 incubator.
2. After removing the supernatant, the cells were washed twice with HBSS.
3. mtSOX Deep Red working solution containing Antimycin (10 μmol/l) and Annexin V-FITC/HBSS was added to the cells.
4. The cells were incubated at 37°C for 30 minutes.
5. After the supernatant was removed, the cells were washed once with HBSS.
6. The cells were harvested by trypsin and resuspended in MEM (10% FBS).
7. Centrifuged at 300 × g for 5 minutes and discarded the supernatant.
8. Added HBSS and suspend.
9. The cells were observed via Flow cytometry. (Annexin V-FITC: FITC filter, mtSOX Deep Red: APC filter)

 

Changes in Mitochondrial Membrane Potential and Superoxide due to Antimycin Treatment

Multiple stain results produced by using JC-1 and mtSOX Deep Red. Antimycin treatment causes the Red signal from JC-1 to disappear and the mtSOX signal to become more intense.

<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

Co-staining with mtSOX Deep Red and JC-1 was used to simultaneously detect the changes in mitochondrial superoxide and mitochondrial membrane potential after treatment with antimycin (an inhibitor of the mitochondrial electron transfer system). 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 indicates mitochondrial membrane potential.

 

<General Protocol of MT-1>

The procedure for using the MT-1 mitochondrial membrane potential kit with mtSOX Deep Red and antimycin. Excluding washing steps, the cells are incubated with MT-1 for 30 minutes before incubation for 30 minutes with mtSOX Deep Red and Antimycin.


Confocal fluorescent microscopy of a co-stain between MT-1 and mtSOX Deep Red. Antimycin treatment causes the MT-1 signal to almost disappear and the mtSOX Deep Red signal to become more intense.

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

 

Plate reader measurements of the MT-1 and mtSOX Deep Red co-stain corroborate the microscopy results; the MT-1 mitochondrial membrane signal is diminished while the mtSOX Deep Red signal indicates an increase in mitochondrial superoxide.

<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 Mitochondrial Hydrogen Peroxide and Mitochondrial Superoxide due to Antimycin Treatment

A co-stain between mtSOX Deep Red and HS DCFH-DA, a general ROS detection dye produced by Dojindo. Hydrogen peroxide treatment elicits strong signal from only the ROS detection dye, and antimycin treatment elicits strong signal only from mtSOX Deep Red.

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

mtSOX Deep Red
Purple: 561/640-700 nm

Co-staining was performed with mtSOX Deep Red and ROS Assay Kit – Highly Sensitive DCFH-DA- (HS DCFH-DA), which is a total ROS detection dye, in order to detect changes in mitochondrial superoxide and hydrogen peroxide, respectively. This experiment confirmed that antimycin treatment results in increased superoxide presence without affecting hydrogen peroxide. These results suggest that antimycin inhibits the mitochondrial electron transfer system.

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

 

Co-Staining with DHE (Total ROS Probe)

mtSOX Deep Red and DHE produce highly correlated images in distinct emission filters

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

Fluorescence images show MitoSOX Red fluorescence accumulating inside of nuclei as staining time increases. Bar graphs show that the presence of DNA has a large impact on mitoSOX Red signal intensity, whereas it has much smaller effect on mtSOX Deep 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 mitochondrial ROS stimulation decreases mitochondrial membrane potential, 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

Fluorescence images show MitoSOX Red fluorescence accumulating inside of nuclei as staining time increases.

 

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

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