I don't know if you had a look at the Phagotest assay sold
by BD (Orpegen now has the manual on their internet site).
It gives very reproducible results which is very important
for longer studies and you do not have to do all the reagent
QC. I think they also sell the bacteria separately, but
there should be no lumps as they cause artefacts.
If you analyse the cells life you might also want to try pH
quench of your FITC on the bacteria outside the cell,
assuming that the intracellular pH remains constant for some
time. Unfortunately the same effect will take place inside
your cell in the phagosome and I would expect some loss of
fluorescence because of that and due to degradation after 1
hour ingestion.
Regards
Gerhard
______________________________ Reply Separator _________________________________
Subject: RE: Phagocytosis by Flow
Author: koratich@sri.org at INTERNET
Date: 02/06/1999 20:52
This is a Thank You to all the kind Flowers that responded to my original
question found below. What I have now adopted is using Opsonized
FITC-E.coli which I mix with the cells in a tube and rock for 1 hour at 37C.
The cells are washed and analyzed immediately. By using a cell control and
a bacteria control it is possible to exclude the clumped bacteria from
analysis. I have tried using an anti-FITC to quench the bacteria that may
be stuck to the outside of the cell but have had very little luck. When
examining the cells under the scope I find very few free bacteria and those
that are present are clumped and easily removed from analysis. I have also
seen very few bacteria stuck to the outside of cells. Further, the cells
that have bacteria stuck to them also have bacteria inside. While I still
have some work to do on this assay, I have a very good jump start now.
Thanks Again,
> Mike Koratich
> Cell Biology and Immunology Group
> Southern Research Institute
> Birmingham AL 35205
>
> ----------
> From: Koratich, Mike
> Sent: Thursday, May 20, 1999 1:24 PM
> To: Cytometry Mailing List
> Subject: Phagocytosis by Flow
>
>
> Hi all,
>
> Does anyone have a good protocol for doing phagocytic activity of
> peritoneal
> macrophages using FITC beads by flow. We are currently harvesting the
> macrophages by lavage and adherence to 6-well TC plates for 2 hours in
> DMEM
> w/o serum. We then wash and add DMEM w/ 10% FBS at let them incubate ON.
> We then wash and add 1E8 beads, which is roughly a 100:1 bead to cell
> ratio. This is incubated for 90 mins. and then washed to remove most of
> the
> free beads. This all works fairly well and the cells look good under the
> scope. Our problem is how to get the cells to detach so we can run them
> on
> the flow without causing disruption of the membrane and loss of beads. We
> are currently using 0.4% EDTA with poor results. We have found a protocol
> that uses dispase and will try that next. Any ideas on other things to
> try
> would be appreciated. Further, any suggestions on the method in general
> are
> also welcome.
>
> TIA,
>
> Mike Koratich
> Cell Biology and Immunology Group
> Serquest
> Southern Research Institute
> Birmingham AL 35205
>
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:53:34 EST