I would also be interested to hear opinions on that subject. I myself try to
do similar things with mammalian cells, and ran out of ideas on how to
reduce background. I guess in case of bacteria the problem should be even
worse because of the cell wall or maybe other reasons. I also know that FITC
itself binds pretty strongly to membranes. However, it is obviously not the
case with fixed and permeabilized cells. Are you fixing the cells
((para)formaldehyde) before running FACS or work on live cells? What is the
target, surface or intracytoplasmic? If your Ab works in immunomicroscopy of
fixed cells, then it should work in FACS after minor protocol adjustments.
"John M Neary" <neary at acsu.buffalo.edu> wrote in message
news:ahnnpu$r2p$1 at prometheus.acsu.buffalo.edu...
> Is there anyone on this board familiar with flow cytometry on indirectly
> immunostained (primary Ab - labeled conjugate) bacteria? I'm getting high
> background and low signal with a FITC-labeled conjugate. I just recieved
> Alexa-488 labeled conjugate today. I heard good things, but haven't tried
> it yet.