I have been recently trying to study mitochondrial structures in our cell line B16. Previously we used the Mitotracker Deep Red dye for this study and it worked fine. But in the last few experiments I have realised that staining with Mito deep red for even 20min @500nM concentration in both live and fixed cells led to a total degradation of the mitochondria. I have done parallel staining of experimental duplicates with Mito tracker Green dye and checked that mitochondria in these cells are in good condition. It is only when I add Mitotracker Deep Red do I see a degradation. Has anyone faced anything similar or could give some explanation for this problem as I am not able to solve it myself.
The regular dose of MitoTracker Deep Red is 25 nM (50 nM already gives near saturation fluorescence), with 30-min incubation at 37C.
Your mitochondria (live) may have been overloaded. In fact all mitotrackers (green, red) work around this dose range, if your microscope lasers are efficient enough.
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As for staining fixed mitochondria, it is not recommended based on chemical knowledge of the tracker structures. All commercial mitochondria trackers carry a positive charge nested in a lipophilic benzimidzole structure, meaning the targeting of these dyes is dependent on mitochondrial membrane potential. In fixed cells, there is no mitochondrial membrane potential to start with, and the staining results are confounded.
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Lastly, some dyes have phototoxicity (they could either induce it directly or sensitize other molecules to become photoreactive and damaging). The deep red core is different from that of the red and green trackers. Therefore, there's no sure way to say that one dye's behavior will predict that of another.
I recommend you reduce the deep red tracker dose and do the staining in live cells (followed by fixing if needed, and check if the the localization is retained).
Good luck
The regular dose of MitoTracker Deep Red is 25 nM (50 nM already gives near saturation fluorescence), with 30-min incubation at 37C.
Your mitochondria (live) may have been overloaded. In fact all mitotrackers (green, red) work around this dose range, if your microscope lasers are efficient enough.
**
As for staining fixed mitochondria, it is not recommended based on chemical knowledge of the tracker structures. All commercial mitochondria trackers carry a positive charge nested in a lipophilic benzimidzole structure, meaning the targeting of these dyes is dependent on mitochondrial membrane potential. In fixed cells, there is no mitochondrial membrane potential to start with, and the staining results are confounded.
**
Lastly, some dyes have phototoxicity (they could either induce it directly or sensitize other molecules to become photoreactive and damaging). The deep red core is different from that of the red and green trackers. Therefore, there's no sure way to say that one dye's behavior will predict that of another.
I recommend you reduce the deep red tracker dose and do the staining in live cells (followed by fixing if needed, and check if the the localization is retained).
Good luck
Yes, Mitotrackers are cyto- and photo-toxic.
It is advised to use the lowest possible concentration and assay / image cells quickly.
BTW,: Mitotracker CMXROS is fixable.
If you want to know more about the toxicity of Mitotracker dyes have a look at this paper: http://www.ncbi.nlm.nih.gov/pubmed/11164242
Maybe the illumination intensity / laser power settings were changed in the microscopy system you use? This can also lead to quick degradation of cellular structures. Check if the values for these parameters are not set at too high levels.
With best regards,
Christian
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