Hi everyone, I hope everyone can help me with this.
First, I am measuring mitochondrial membrane potential by flow cytometry using TMRE and MTG dyes. I think we are not experiencing problems with my TMRE readings. However, I have problems with my MTG readings. Supposed to be that MTG is independent with the membrane potential. But, if I add uncouplers (CCCP), the values for MTG stained cells are significant lower compared to CCCP untreated samples (30-50% lower).
My procedure is, I stain the cells first with MTG and add the uncoupler (30uM) after 20 mins, and then incubate it for another 10mins (total of 30mins for the dye).
There are 2 possible scenarios that I am thinking.
First, the dye and CCCP interact with each other leading to a lower fluorescence intensity readouts.
or,
Second, the uncoupler cause severe mitochondrial damage that it initiates mitophagy.
but is 10 mins exposure to the uncoupler (plus few more minutes while waiting it to be read in flow cytometer) enough time already to be degraded by mitophagy? or is the first scenario a more logical explanation for the lower values in CCCP treated cells?
Choco Michael Pagayatan Gorospe
This could be elucidated with few experimental controls:
1. Cells with MTG w/o uncoupler for 20 mins
2. Cells with MTG w/o uncoupler for 30 mins
3. Cells with MTG for 20 mins + uncoupler for 10 mins (your test sample)
Now, if there is difference between 2 and 3 (i.e 2 is giving you higher fluorescence than 3), then it is possible that your second hypothesis is correct.
If you find no difference between 2 and 3, then it might be due to longer exposure, in this case you should get normal results with 1.
These experimental controls would help you in analysing your results.
mitophagy hardly kills all mitochondria at a time. Given that each yeast cell has apx 30+ mitochondria, it is unlikely that missing a couple of organelles would significantky affect your measurements. btw, in higher, fast-dividing eukaryotic cells mitophagy can last from 15 min to an hour to see full "digestion" of a single fragmented mitochondrion (and mitochondria divide apx once in 2 hours).
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