Flow cytometry of transiently-transfected COS cells
Kevin A. Mulcahy
K.Mulcahy at sheffield.ac.uk
Fri Mar 14 10:46:53 EST 1997
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,
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