From: Gerhard Nebe-von-Caron (Gerhard.Nebe-von-Caron@unilever.com)
Date: Thu Apr 23 1998 - 04:08:12 EST
I think you can also get other bacteria labeled by their
method on request.
Gerhard
______________________________ Reply Separator _________________________________
Subject: Re: Trypan Blue & Phagocytosis
Author: ngg1@stir.ac.uk at INTERNET
Date: 23/04/1998 00:54
Peter,
We had a similar problem some time ago and spend quite a lot of time
and energy trying to solve it. We were working with rainbow trout
macrophages and different bacteria. Our control test was performed with
Orpegen's 'Phagotest' kit (FITC-E. coli and a quenching solution -blue
dye-). This kit worked very well for FITC-E. coli, but not so well with the
bacteria we were labelling with FITC. The reason, we suppose, is that 'our'
bacteria had not been permeabilised so the quenching solution (Orpegen's and
others we tried -cristal violet and trypan blue) did not gain access to the
FITC-binding sites and therefore no significant fluorescence was being
quenched. We tried to permeabilise the bacterial membrane after FITC
labelling with tween 20, triton x-100 and other organic solvents, without
much success.
From our experience I would suggest to work with Orpegen's phagotest
kit (FITC-E.coli, quenching solution and propidium iodine) -if you don't
mind doing your phagocytosis with E. coli. I think Molecular probes also
sell their kit with the quenching solution. It might be that other active
components apart from trypan blue are present in this solution, making it work.
We tried different trypan blue, cristal violet and ethidium bromide
concentrations and protocols -including low pH- and, in our system and our
bacteria, they did not work.
Please if you find out if and what we have been missing, let us
know. I am still very interested on this subject.
Orpegen Pharma Phagotest: http://www.orpegen.com/html3/phago/
They also have a very useful leaflet explaining the test.
Good luck
Natalio Garcia-Garbi, PhD student
Institute of Aquaculture
University of Stirling
Scotland, UK
At 03:11 PM 4/20/98 -0500, you wrote:
>
>
> Dear Group,
>
> We have been trying to measure the phagocytosis by isolated monocytes
>by using FITC labeled bacteria (Molecular Probes) and quenching the
>reaction with trypan blue 70% dye (0.5 to 3 mg/ml, Sigma) to discriminate
>between attachment and internalization of bacteria by quenching the FITC
>fluorescence of surface bound bacteria leaving the fluorescence of
>internalized particles unaltered. All the literature makes this sound very
>straight forward but we are having a hard time convincing ourselves that we
>are quenching the FITC.
>
>- We can quench the fluorescence of the FITC beads used in the set-up of
>the FACsCaliber. (BDIS)
>
>- The FITC labeled bacteria were added to a tube of fixed monocytes (1%
>PFA) that had been suspended in a solution of 1mg/ml trypan blue. We then
>measured the same mean fluorescence of the monocyte gated region as when we
>did not add trypan blue but suspended in 1% PFA. No quenching!
>
>- The fluorescence of just the bacteria in suspension is not changed by
>the addition of our trypan blue. No quenching
>
>Is there a trick we are missing..a certain pH, a brand or purity of trypan
>blue? The literature also lists ethidium bromide and crystal violet but if
>I can make it work I'd rather work with trypan blue.
>
>Thanks For Any Comments.
>Best Regards,
>
>Peter Schroeder
>Center for Biomaterials Research
>Medtronic, Inc.
>6800 Shingle Creek Parkway
>Brooklyn Center, Minnesota 55430
>peter.schroeder@medtronic.com
>
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