see
http://www.cyto.purdue.edu/flowcyt/research/micrflow/gerhard/gerhard.htm
from the purdue CD rom for an example. Pure cultures look of
course much nicer and are a good system to ensure antibody
saturation, in particular when comparing unpurified culture
supernatants for signal intensity. Signal intensities can
vary significantly for both scatter and fluorescence
depending on microorganism, antibody clone, epitope
frequency and growth conditions e.g. cell volume.
I have also used combinations with a PE second step
fluorescence and CFDA for metabolically active cells and PI
for permeabilised cells, but the FITC/EB/PI combination was
more generally applicable. Fab2 fragments are required and I
actually used preadsorbed polyclonals from DAKO which showed
the lowest background at the time.
Counterstaining is very important as otherwise you detect
antibody negative events that are not necessarily bacteria.
Also make sure everything including the sheath and the
growth medium of your test culture is filtered before use to
avoid measuring unwanted bacteria.
The staining in 96 well filter plates described in the text
is a big improvement in sample handling as it makes the
washing steps quite easy and quick. We have now three
filterplate system in our department alone as the technique
has been proven very useful for other applications as well.
All the best
Gerhard
______________________________ Reply Separator _________________________________
Subject: FACS on gram+ bacteria
Author: Roederer@Stanford.edu at INTERNET
Date: 07/06/1999 20:31
We have a group that is trying to detect antibody staining of antigens on
the surface of gram-positive bacteria. Does anyone have experience with
this? (Or, more generally, analysis of gram+, or of surface antibody
staining on gram-negatives). We'd be interested in seeing some typical
methods (what colors do you use, do you use second step reagents) and to
see some typical profiles of both scatter and fluorescence.
Thanks for any information!
mr
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