Flow cytometry of transiently-transfected COS cells

[Kevin A. Mulcahy]

Hi,

I've been trying to transiently transfect COS-7 cells with cDNA for 
HLA-A3 cloned into an SV40-ori containing expression vector. I have been 
harvesting the cells 72 hours post-transfection and analysing them by 
flow cytometry to determine the percentage of cells expressing HLA-A3 at 
the cell surface.

However, I have run into difficulties. Compared to untransfected COS-7, 
the "transfected" cells have a really high background of staining (i.e. 
when just staining with the second FITC-labeled antibody). In addition, 
I can't detect any HLA-A3 positive cells in the population, possibly 
because of the extremely high background (?).

Has anyone got any ideas as to why I'm having this problem? I have tried 
both electroporation and DEAE-dextran/chloroquine mediated methods but 
both give the same results. Is it that the cells are made more "sticky" 
or "leaky" so that much more of the 2nd antibody binds to them?

Many thanks for your help,

Kevin Mulcahy.