Re: Phagocytosis Assay

Dave Coder (dcoder@u.washington.edu)
Fri, 17 Oct 1997 09:31:04 -0700

You may want to consider something other than FITC labeling for the
bacteria. Although it can work, the labeling does diminish the binding
capacity. This is not too surprising considering the high pH needed for FITC
labeling, and the potential for irreversibly affecting the confirmation of
the bacterial protein adhesins. (Bacterial binding was the first flow
project I ever did some 15 years ago, and a Scatchard analysis of the
binding indicated a diminution due to labeling.)

There are two alternatives: One is using a fluorescently tagged lipid
intercalator (the PKH reagents), and another is introducing the Green
Fluorescent Protein (GFP) into the bacteria. The later has the nice
advantage that you need to do subsequent labeling at all--the bacteria are
endogenously fluorescent.

This approach has been successful here in tracking the adhesion of E. coli
to exfoliated vaginal epithelial cells. Binding of GFP labeled bacteria is
better than FITC-labeled bacteria, but there is a problem with transfection
stability in that segmented colonies can appear (I don't have the
information on the vector and promoter used, but I can find it if needed.)

Dave Coder
dcoder@u.washington.edu

-----Original Message-----
From: carol walker <gwalk02@emory.edu>
To: cyto-inbox
<cytometry@flowcyt.cyto.purdue.edu>
Date: Thursday, October 16, 1997 5:08 PM
Subject: Phagocytosis Assay

>
>I'm interested in an assay for measuring the binding of bacteria to
>macrophages and bacterial killing by macrophages (phagocytosis).
>Peripheral blood monocytes from rhesus monkey will be used. I would
>like to FITC-label and analyze by flowcytometry. If you can assist in
>any manner please notify me. Thank you. Pamela
>
>Pamela L. Turner
>email - pturn01@emory.edu
>fax - (404) 894-8502
>phone - (404) 894-5986