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
If you dont - 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.
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