After using mitotracker green from life technologies to stain the mitochondria of A20 cell line(Lymphoma murine), when i observe the cells at confocal microscopy the fluorescence appears as diffused and not localized. I can't see the mitochondria as they are but the fluorescence seems to stain all the cytoplasm. the concentration used is 150 nM.
Possible solutions?
Thanks in advance.
The cytosolic staining is usually associated with overdosing the stain. If you are absolutely certain that your final working concentration is 150 nM then the next thing I would check is whether your cells have had the stain loaded into them for too long. Although it is a "vital" stain, MitoTracker will cause some mitochondrial damage after 3-4 hours and the cells/mitochondria will begin to lose both their morphology and viability. How long after loading the cells do you look at them?
Also, are your cells kept in either an incubation chamber or imaging solution during their time on the confocal?
photobleaching is a big problem with MT green. Perhaps switching to MT red would help with that, and it also has an electrophilic functionality that crosslinks it to mitochondria and diminishes leaking.
Did you fix the cells? I have recently read that MT green is not compatible with fixation, it should rather be used for live imaging. MT red works fine also after fixation.
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