Hi colleagues,
I am trying to measure mitochondrial membrane potential and mitochondrial mass in S. cerevisiae with TMRE and mitotrackerGreen as dyes using flow cytometry. However, I always see two peaks when measuring them.
I have been trying to optimize my protocol to prevent or reduce the double peaks but still not successful.
Has anyone observed these same results in their experiments in yeasts? And how did you deal with it?
So far, what I have seen in other publications are single peaks, and I am not sure if they gated out the other peak and if I have to gate it out, which of those peaks have to be gated out?
I was also thinking it is probably because of doublets, or maybe unbudded cells, or aggregated cells. And I have tried to address them by sonication, adding Tween20 and EDTA, but it did not resolve the problem.
Article Loss of Mitochondrial Membrane Potential Triggers the Retrog...
I am not qualified in these experiments but I will just write my opinion.
In human this peaks are generaly a result of dividing cells.
Because in your analyze also there is a small population that has double intensity but low amount in terms of cell number.
Can you try to perform same experiment by sencronising yeasts? Such as by using tymidin analog or stressing with starvartion ( I am not sure if this works on yeast)
Best
It is easy to analysis the results of Flowcytomerty.
select the live cells only.
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