Fibroblast blues

RICHARD P. PHIPPS PHIP at BPHVAX.BIOPHYSICS.ROCHESTER.EDU
Sat Jul 24 14:14:22 EST 1993


> >I have a hybridoma cell line contaminated with fibroblasts.
> >Is there any way of selectively getting rid of these fibroblasts?
> >
> >Thanks for any help and comments,
> >Chris R.
> >
> 
> Chris,
> 
> 1) culture medium with d-valine substituted for l-; fibroblasts lack the
> enzyme to utilize the d- isomer.  Available from Gibco.
> 
> 2) anti-Thy1.1 followed by complement-mediated cell lysis.  Don't know if
> this would work in a hybridoma culture.  Thy1.1 is a fibroblast-specific
> cell surface marker.  I can look up the antibody source if you want.
> 

The d-valine idea sounds pretty good; I wasn't aware this was the case.

There's a problem with the anti-Thy1.1 approach, though.  I assume this 
fibroblast line is from a mouse (I didn't see the original posting-
someone please correct me if I'm wrong).  If this is the case, Thy is 
not fibroblast specific,as it is expressed on T-cells (and maybe neurons?)
-shouldn't be a problem here.
  
BUT - I think that most commonly used mouse strains express Thy1.2, not 
Thy1.1.  The antibody for this is called 30H12-the hybridoma is available
from ATCC (or supernatant from me, if you decide to go with this 
and if I get my boss's permission).  

BUT - not all fibroblasts express this marker (in human, rat or mouse),
in fact we sorted mouse fibroblasts on the basis of Thy1.2 and found
functional subsets.  So it still might not work.

I think I would try growing the culture in a tissue culture treated flask
or dish-the fibes will adhere better in these-and remove the hybridoma 
containg media as gently as possible so as not to purturb the fibes too 
much.  Then run the cell suspension on a Percoll or Lympholyte-M gradient,
which separates cells on the basis of size quite sensitively-we use it to
separate activated and resting B cells, and a tech I know actually 
"decontaminated" a T-cell hybridoma contaminated with yeast (along with
plenty of fungizone).  This method assumes that the fibroblasts are of 
a sufficiently different size-I'm sure they are.

Tell me what you think,

Rick Willis
University of Rochester Cancer Center



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