3 replies to this topic

[cardosopedro]

Hi.

I used MitoTracker Red (CMXRos) to detect variations in mitochondrial membrane potential (MMP) in a cell line treated with a drug in comparison with a control.

I want to quantify the loss/gain of MMP. What I did was to establish two regions in the control sample histogram, one to the left and one to the right of the maximum peak of fluorescence, and then I copied these regions to the drug treated samples. Is this a correct procedure? If not, where should I establish the regions?

Also, I was expecting loss of MMP. However, in two of the samples I get gain of MMP. What's the meaning of MMP gain?

Thanks for any reply.

Edited by cardosopedro, 05 September 2009 - 03:38 AM.

[Binchen]

cardosopedro, on Sep 5 2009, 11:18 PM, said:

What's the meaning of MMP gain?

Just a thought:
MMP is the difference between amounts of protons in the mitochondrial matrix (low proton content) and intermembrane space (more protons there). It is established through electron transfer and coupled proton transfer at the membrane complexes of the respiratory chain. MMP is used to generate ATP and also to drive protein import into mitochondria across the inner membrane (through TIM complexes). These processes let the protons flow back out from the intermembrane space into the matrix. Ionophores can dissipate the membrane potential.
So I'd say if you see a gain in MMP you either are building a stronger proton gradient with more respiration going on or you're not using it up; maybe because you don't have enough ADP that can be made into ATP? So if your drug would inhibit the ATP synthetase your protons couldn't flow back and you would get an increase in MMP. But as I said there's other processes that interfere with MMP so it could be other reasons.

Edited by Binchen, 30 September 2009 - 06:24 PM.

[cardosopedro]

Thanks for the answer.

Another possibility came to my mind. With this drug I also get a G2/M arrest in the cell cycle. Maybe the cells are arrested after the organelle division (namely mitochondria division), but, because they are arrested, the don't divide into two daughter cells. But there is a quantity of mitochondria enough for 2 cells. In this way, I would get an increase in CMXRos staining, and erroneously, consider it as an increase in MMP...

What do you think?

[blotted]

its correct to gate the peak of the control. You can also put a negative and positive control.
I would suggest you to change the dye. I don't trust really in Mitotracker for quantify DeltaPsi. I would use JC-1, TMRE (tetramethylrodhamine ethyl ester) or Rhodamine 123 (people don't like this dye but I have got good results with it).
Equally than ROS production, DeltaPsi could be changing along the treatment. I suggest you again to perform a time course!