Mitosox and DCF -ROS. I measured ROS by exposing cells to a oxidative stressor. ROS measured by DCF increased dosis dependent. However, with the same conditions, mitosox increase under low stimuli vs. control and then super oxide return to control values (in the condition were I measure high total ROS). Please people could someone help me to interpret these findings?
Hello Agustina,
DCF can only penetrate the outer mitochondrial membrane whereas MitoSOX is able to enter the mitochondrial matrix. As such results from the two different approaches can not be directly compared. Moreover, it is not recommended to use DCF for measuring oxidative stress by some authors (please refer to the attached publication).
Regards, Christian
Article What does the commonly used DCF test for oxidative stress really show?
i hope that my comment will be help to you. high contents of ROS occasionally can induce SOD activity decrease. the possible explanation may be due to the effect of ROS on amino acid residue such as cystein and so on, while exact reason is unclear. concerning difference between DCF and MitoSOX, it is assumed that the ROS induction may be related with cytosolic events not by mitochnodrial....
Yes, MitoSox is more to the mitochondrial superoxide formation and depends upon mitochondrial oxidative stress. I do not know whether your stressor actually disturbed mitochondrial activity or not. But this may be the reason for your observation.
I would need more information about the experiment. Do you introduce first the dyes and then treate with the stressors? Or do you incubate with the stressors for several hours and then test ROS levels during 10-20 min incubation with the dyes? If the stressor increases SOD activity, then you will have low superoxide but high H2O2. This can be the reason. However, as it has been indicated previously, both dyes check ROS in different compartments.
There are some major differences between DCF and MitoSOX and it would not be surprising that the results are not directly comparable.
DCF responds nonselectively to several ROS, notably hydroxyl radical, peroxynitrite and ROO* radical. It is relatively unstable and auto-oxidizes. See this paper by the original development group: http://www.jbc.org/content/278/5/3170.full.pdf+html
DCF does not detect superoxide directly or even H2O2, according to groups who characterized it. DCF fluorescent products will basically distribute anywhere in the cells.
On the other hand, MitoSOX is derived from HE (hydroethidine); both have some selectivity for superoxide but suffer artifacts from nonspecific DNA binding which exaggerates the fluorescence. MitoSOX contains a triphenylphosphonium moiety for mitochondrial targeting, which is mitochondrial membrane potential (MMP) dependent. When the MMP is low (including cases of profuse ROS production), you will not be able to properly load your probe into mitochondria, confounding your results.
If the effects of a drug are unclear, one may need to perform extra tests to validate if the MMP is intact before using MitoSOX. Lastly, a mito-targeting scavenger group should be included. You can try Mito-TEMPO or MnSOD chemical mimetics.
Guillermo,
I treated the cells with my stressor. After 24hs I measured total ROS with DCF because I understand that is not only specific for H202. Idem with Mitosox.
What I found is that when I increase the oxidative stress condition, ROS incremented as I it was expected (measured by DCF). On the other hand, when I used Mitosox the curve of " O2*- " increase at first vs control and then decrease towards more oxidative stress condition.
If mitocondria loss it delta pi maybe Mitosox could not enter in this organelle and this could explain why I detected a decrease in mitosox oxidation in more oxidative stress condition. However, I know that there are other ROS in the cytoplasm and thiese culd oxidized Mitosox in this compartment...I can imagine this situation. Hence, Is mitosox a good probe???
Hi, I have the same question and my theory is that you should measure mt membrane potential togeather with mitosox. If your treatment does not change mt potential with respect to controls, mitosox should detect ROS in mitochondria in both cases. If the potential changes, you should consider whether changes in mitosox do not simply mimic changes in membrane potential or whether mitosox is not simply oxidized in cytosol...
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