triple labeling

[ronlab]

Hi!
We routinely do EGFP +PI double staining on various Becton -Dickinson 
and Coulter machines. Compensation is required, but is never a problem. 
We also did EGFP-PE- PI on FACSotr (B.-D.), but it took a rather 
complicated and problematic compensation. However, we achieved very good 
results using EBFP (UV-excitable, blue)+EGFP (488 excitation, green) + 
PI (488 exited, red). 
If you have an access to a UV-equipped machine, I would suggest you to 
use EBFP instead of EGFP. It should not interfere with your other 
detectors. 
If you don’t - keep working on compensation. 
It may be helpful to have a construct that expresses EGFP at a lower 
level, or expresses other, less bright, red-shifted GFPs. I have quite a 
few of such constructs in my collection (mostly - retroviral vectors).
Finally, there are fluorescent dyes that emit far red light, that should 
distinguish them such common red-light emitters (e.g. PI). I believe, 
secondary Abs labeled with these compounds are already commercially 
available. I would anticipate no cross talk between these dyes and EGFP, 
although you may have to purchase special filter sets for your machine.

Good luck!

Eugene Kandel.
U09577 at uic.edu




Jordi Barquinero wrote:
> 
> Dear GFP netters,
> We are trying to analyze the simultaneous expression of two phenotypic
> markers (CD34 and CD38) in human hematopoietic cells transduced with the
> EGFP gene, but EGFP signal (FL1) is very bright and produces a level of
> background in the other channels (FL2, FL3 and FL4) that makes color
> compensation imposible. We are using a COULTER XL cytometer with an
> argon-ion laser (488 nm). Any hints to help us? Thanks.
> 
> Jordi Barquinero, M.D.
> Institut de Recerca Oncologica
> 08907 Hospitalet
> Barcelona, SPAIN
> Tel (93) 2633817
> Fax (93) 2632251
> jbarquinero at iro.es