In article <1994Jun16.141707.18047 at galileo.cc.rochester.edu>,
PHIP at BPHVAX.BIOPHYSICS.ROCHESTER.EDU (RICHARD P. PHIPPS) wrote:
> 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
