Hello Everyone,
I am trying to measure mitochondrial membrane potential (MMP) with specific mitochondrial dyes name as "MitoTracker Red CMXRos".
I am using CCCP to reduce MMP to induce mitophagy. After 4 hour of treatment, I am getting increased MMP instead of lower. I repeated nearly more than 5 times and I got the same results all the time. I did not understand the reasons.
On the other hand, in control sample I am getting two population having high and low MMP but in the CCCP induced one has same MMP. I am attached the images.
Can you please comment on it about the reason ?
Thank you very much.
Hello,
I haven't used a mito tracker dye, but I hear from the experience of my colleges that mito tracker dyes should be used with caution, because they may bind covalently to mitochondrial structures and therefore do not respond necessarily to changes in MMP. Could it be that this is your problem?
Gisela
Gisela Beutner , Thank you very much for your answer. When I did 30 minutes of CCCP treatment, I obtained a very good reduction of the intensity but after 4 hours, I am not getting the same. The cell may try to increase MMP somehow.
Besides, I am also feeling the same thing but I dont have any funding to buy another dye like TRME.
i also don't do this kind of thing. But you can check with concentration of Dye. may be concentration you are using may not sufficient for longer period of time.
Try with higher concentration.
MitoTracker Red CMXRos accumulates in polarized mitochondria and binds to mitochondrial proteins, therefore the signal remains after cell fixation. I may suggest to use MitoFluor 589 (Invitrogen) if you have to use similar Ex/Em wavelengths. Otherwise, TMRM (TMRE) or even JC-1 can be used instead.
In addition to changes in MPP, MitoTracker Red CMXRos is used to measure oxidative stress inside the cells (it reacts with superoxide). That is probably the reason, you see an increase in signal due to increase in oxidative stress after 4 h treatment with FCCP. Use TMRM or TMRE to monitor MMP. As other researchers mentioned before, many Mitotrackers are not sensitive to changes in MPP at all.
Article Detection of Mitochondrial Mass, Damage, and Reactive Oxygen...
- Badges
- Science method