Mouse CTL cloning - Culture conditions and method (Jan/03/2006 )

Hi
Happy New Year
I am looking for a strait forward method to clone CTL lines from peptide vaccinated mice.
Does anyone have a clue for this.
My previous assays with Con A, IL-2 and limit dilution didn't work.

Thanks

pmsalves

Yes I have.
I have done limiting dilution in Teraski plates (20µL volume/well - 60 wells per plate).
I plate 1 T cell per well with 10000 irradiated spleenocytes, 50IU/mL IL-2 and ConA 2µg/mL.
Nothing grew in more than 60 plates done.
I am thinking to increase the number of irradiated feeders to 50000, as the feeders die very fast.

Thanks for replying.

Pedro