Re: BRDU background

Hello Stuart,

The negative control I would use is not unstained cells, but rather a 
parallel culture (or animal) unexposed to BrdU.  Then permeabilize and 
stain as you do the stained sample.  If you still see such a difference, 
there is indeed a problem.

Good luck,
Neal

--
Neal Benson
Interdisciplinary Center for Biotechnology Research
University of Florida

Stuart Berzins wrote:

> Hi everyone,
> 
> We want to analyse BRDU incorporation by FACS using anti-BRDU antibodies 
> to staining mouse lymphocytes. Although we are getting a clear positive 
> peak, the background staining of cells we know to be negative is very 
> high (2 logs higher) when compared to unstained cells. Advice from 
> colleagues is that this sort of thing is reasonably common and that we 
> shouldn't worry about high, or variable, backgrounds as long as the 
> positive peak is clear. This seems a little 'unscientific' and we'd 
> prefer to know what is going on and remedy the situation if possible. If 
> anyone has experience with anti-BRDU analysis by FACS and has solved or 
> knowingly avoided the problem of high background staining by anti-BRDU 
> antibodies, we'd love to hear from you. FYI, we are currently using a BD 
> anti-BRDU staining kit, but have encountered similar problems with 
> home-made buffers.
> 
> Regards,
> 
> Stuart