I read two references regarding this question. Reference 1 says that FM4-64 majorly stains inner membrane. But reference 2 says FM4-64 stains outer membrane. Anyone knows how does FM4-64 exactly work? Thanks
http://onlinelibrary.wiley.com/doi/10.1046/j.1365-2958.1999.01425.x/full
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035205
As FM4-64 is highly hydrophobic, it stains the very first membrane it encounters, in your case the outer membrane of the bacterial envelope.
But with time it can diffuse throughout the whole cell by membrane traficking. I have no idea how fast these exchanges occur between the inner and outer membrane in E coli.
@Olivier Catrice Thank you very much for your reply. Following the question above, do you know whether washing cells after FM4-64 staining would wash out the FM4-64 staining in the outer membrane?
I have seen FM4-64 fluorescence decreases pretty significantly when I wash stained cells with medium
.
Do you plan to wash cells with alcohols or oil ?
If no FM4-64 won't move.
Hi, guys!
May I ask, how FM4-64 impair cell growth? Can E.coli grow normally in the presence of FM4-64? Is that correct, that the dye gives low background if not bound to membrane, so washing is no necessary?
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