Hi everyone,
I am just starting some mitochondrial staining assays for flow cytometry which I'm not very familiar with, and I am struggling to interprate my results. I co stained my cells (hematopoietic stem cells) with TMRE (mitochondrial membrane potential) and MitoTracker Green (mitochondrial mass) according to the protocols used in my lab. Suprisingly to me, I obtained a majority (85%) of cells which were positive for TMRE but negative for MTG (see dotplot included). I do not really understand how cells which don't have mitochondria can be positive for membrane potential staining...
Can you please help me figure this out ?
Thank you very much
Mito tracker Green and similar dyes are supposed to produce fluoresce when they accumulate in lipid deposits. I can speculate that in your samples there are some liposomes or lipid micelle-like structures/ formations which are collecting up all the MTG stain and not allowing any of it to reach the mitochondria.
It is well known about mito trackers that the probe appears to be preferentially accumulated in mitochondria regardless of mitochondrial membrane potential. That property makes MTG a possible tool for determining mitochondrial mass. Although nothing is stated about possibility to accumulate in other organelles ( cell specific accumulation in say liposomes?) .
Another possibility: you do not gate the correct fluorescence. MTG emission maximum is blue-shifted. Check again your emission settings for the MTG according to the protocol from your manufacturer.
Your problem can be solved with other dyes (maybe mito tracker red) or mitochondria- specific antibodies.
I agree with Julie M Rumble: it is a possibility that TMRE influenced the MitoTracker emission. However, it can be easily checked. Do the parallel experiment and load the same amount of cells/the same conditions/ with MitoTracker alone, TMRE+MitoTracker, TMRE alone and compare the graphs.
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