HAT selection problem

Tue Apr 30 16:58:33 EST 1996


     We've recently run into a problem with growing 
hybridomas (NS-1/Balb/c). The problem arose during 
HAT selection of hybridomas after a fusion. No
hybridomas were left alive after a week. We tested our
media components by growing an established
hybridoma line in (also NS-l/Balb/c). Cells were
thawed from liquid nitrogen and grown on 10% FCS
/DMEM and transfered to: 

1)  DMEM / 10% Fetal Calf Serum
2)  DMEM / 10% FCS / HT
3)  DMEM / 10% FCS / HAT
4)  DMEM / 10% FCS / Aminopterin

Result after 48 hours:

1)   Grew well, healthy cells.
2)   Dead
3)   Dead
4)   Dead

     "Dead" cells appeared rough, with non-uniform size
and were of low cell density. DMEM and HT stocks were
freshly made and the experiment was replicated with
two different lots of fetal calf serum. Any suggestions
as to why the cells did not grow in HT or HAT would be
appreciated. Thanks in advance. Les.

Les Willis
Agriculture Canada
Summerland, B.C.
V0H 1Z0
WillisL at em.agr.ca

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