What is your negative control? Negative cells, non-specific antibody,
blocked Ab? How you reduce unspecific binding? With Blotto, BSA, detergents,
...? This all can also be critical. As for flow cytometry itself, did you
try detection at a longer wavelength or using a different laser? You
probably know that Alexa 488 fluoresces from ~500nm to ~600nm and excites by
450-550...
Emir
"John M Neary" <neary at acsu.buffalo.edu> wrote in message
news:ai27lg$mjc$1 at prometheus.acsu.buffalo.edu...
> Emir,
>> In reply to your questions, my target is an outer membrane protein located
> on the surface of the organism and I am using live cells. I've had some
luck
> with that new conjugate I mentioned. It is called Alexa-fluor 488 and
it's
> made by Molecular Probes. The fluorophore is a FITC analog that excites
at
> 488nm (which matches the frequency of the argon laser most cytometers
use).
> I've only done one experiment with it so far, but it dramatically reduced
> the background in the negative control.
>> John
>> "Emir Khatipov" <khatipovNO at NOuchicago.edu> wrote in message
> news:OEK%8.44$X4.10409 at news.uchicago.edu...> > 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.
> > - Emir
> >
> > "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
> > an
> > > Alexa-488 labeled conjugate today. I heard good things, but haven't
> tried
> > > it yet.
> > >
> > >
> >
> >
>>
