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Hi Peter
In response to your email on phagocytosis assays in macrophage cells we have
done some similar work here in J774 cells. However, we haven't quantitated the
data on the Flow Cytometer so I'm not sure what the results would appear like
on Flow. We've studied the uptake of FITC labeled E.coli cells in murine
macrophage and found that the fluorescent bacteria are taken up very
efficiently at 37C. If you cool the temp down to 4C most of the bacteria are
found adhering to the cell surface. They're not phagocytosed very efficiently
at this temp or if cells were pretreated with a protein synthesis inhibitor
such as cycloheximide. We've also looked at fluorescent beads such as Nile red
beads and FITC-beads using the fluorescence microscope. Another way to
distinguish between surface bound bacteria/yeast and internalized material is
by using antibodies to FITC if the material is conjugate to fluorescein. You
could quench the surface bound fluorescence with antibody and look at the
fluorescence of the internalized ligand alone. The problem here is that the
anti-fluorescein antibodies could be taken up by the macrophage cell and
actually quench the internal fluorescence also.
The only time we tried to study uptake of FITC-E.coli on the Flow Cytometer it
turned out the cells were too sticky and clogged the machine. So once again
we're not very sure of how you would study this problem by Flow but we have
looked at some similar stuff on a fluorescence microscope. We haven't yet
published any of this but a manuscript is in preparation.
Hope this onformation helps you a little at least.
Anu Cherukuri
Graduate student in Edward Voss's lab
Dept. of Microbiology
University of Illinois, Urbana-Champaign
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