Flow cytometry of transiently-transfected COS cells

Ian A. York iayork at panix.com
Fri Mar 14 11:44:30 EST 1997


In article <332972ED.67EA at sheffield.ac.uk>,
Kevin A. Mulcahy <K.Mulcahy at sheffield.ac.uk> wrote:
>
>However, I have run into difficulties. Compared to untransfected COS-7, 
>the "transfected" cells have a really high background of staining (i.e. 

This is a chronic problem in transient transfection, especially with the
DEAE-dextran method.  I'm a little surprised you also see this with
electroporation; we see very little background increase with
electroporation.  With the DEAE-dextran, I know of no way to greatly
improve the background.  You might get some improvement by trypsininzing
the cells aggressively about 24 hours after the transfection and then
re-plating them and allowing them to recover for 48 hours.

Unless the background you get is dramatically higher than we see, I don't
know that the failure to observe the HLA-A3 is a function of the
background, though.  We have used several cell-surface molecules as
positive controls, and we see high-level expression (i.e. comfortably
higher than background) for all of them; this includes ICAM, H-2Db,
HLA-B27, and so forth.

You might want to confirm that both your A3 plasmid and your antibody are
working, and then try playing with electorporation conditions, because as
I say we see much less background with that than with the DEAE-dextran. 

Ian
-- 
      Ian York   (iayork at panix.com)  <http://www.panix.com/~iayork/>
      "-but as he was a York, I am rather inclined to suppose him a
       very respectable Man." -Jane Austen, The History of England



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