Quenching FITC

Mike Herron herro001 at maroon.tc.umn.edu
Thu Jun 16 20:00:38 EST 1994

In article <1994Jun16.141707.18047 at galileo.cc.rochester.edu>,

> In <2tnt72$hc8 at news.duke.edu> mjtino at acpub.duke.edu writes:
> > I am having trouble with an assay for phagocytosis in which I would like to 
> > examine fluorescence of ingested FITC-labelled bacteria by macrophages.
> > In order to do this, I must quench the FITC outside of the macrophages to
> > distinguish between extra- and intracellular fluorecence.  I've
> > tried crystal violet and trypan blue, but with no success.  Any suggestions?
> > 
> > Michael Tino
> > michael_tino at cellbio.duke.edu
> > 
> > 
> I seem to remember a protocol where the cells you wish to assay 
> (macrophages in this case) are labelled red (don't remember whether
> they used PE or EtBr or something like that).  Under a fl. microscope,
> extracellular bacteria is green and phagocytosed bacteria is sort of
> yellow-orange.  If I remember the name of the author I will repost.
> Rick Willis
> University of Rochester Cancer Center

The dye used is acridine orange.  It is more a a live/dead thing (as
opposed to phagocytosis) as I recall the dead bugs DNA uncoils and that
causes a shift in the color of the acridine flourescence.  I saw it work a
few years ago...pretty impressive actually.  I tried the quenching stuff
too...with no luck.

Mike Herron
herro001 at maroon.tc.umn.edu

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