We are currently have great problems maintaining an untransformed rat
T cell culture.
Briefly,
Rat inguinal/popliteal lymph node cells (CFA+ovalbumin) grown in RPMI
+ 1% NRS + 10µg/ml ova at 1e07 for 3 days fine...then into RPMI+ IL-2
(rat= 10%CAS) and 10% FCS for 5/6 days seem to be fine with T-cell
blasting visible. A restimulation of the cells is then set up after
Ficoll-Paque seperation of live lymphocyte cells with
RPMI+1%NRS+10µg/ml ova + a ratio of 25 thymocytes(for presentation) :
1 Tcell, aiming for 4e06 T cells. On various occasions either we have
had difficulty maintaining cell numbers between stages, the cells have
simply not been restimulated or all apoptose at this stage. Obviously
we wish to be able to keep an ovalbumin specific rat T cell line
grwoing with this cycling of IL-2 and ova.
Any thoughts?
R Maile
University of Bristol, UK
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