RE: Phagocytosis Assays
Dan Smith (DJS@ALLP.COM)
Tue, 25 Jun 96 16:50:00 PDT
Another way to differentiate between 'cell association' and phagocytosis is
to add cytochalasin B to the cells and they will not be able to phagocitize
the particulate.
Dan Smith
djs@allp.com
----------
{From: owner-cyto-sendout
{To: Cytometry Mailing List
{Subject: Phagocytosis Assays
{Date: Monday, June 24, 1996 3:36PM
{
{
{REGARDING Phagocytosis Assays
{
{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
{
{
CD ROM Vol 2 was produced by staff at the Purdue University Cytometry Laboratories
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