I am trying to establish a long-term culture of OT1 T cells using OVA. I've been trying to use T-cell depleted splenocytes and BM derived DCs as accessory cells. This works fine for the first stimulation, but most of the cells die during re-stimulation on day 5. I would like to know if anyone has experience with this and could give me some advice?
Stimulate your T cells with minimal peptide (actually peptide-pulsed APC at 10e-9M). Use A low APC ratio (1 APC for 10 T cells). Use low IL-2 as well and wait for enough time (10-15 days) before restimulation. Follow our method (attached)
Cheers
Weisan
I have tried to do it using BMDCs loaded with the peptide in the first stimulation and then splenocytes plus peptide and IL-2 (25 U/ml) in the second restimulation (6-8 days later) and then weekly. In my experience, restimulation with BMDCs does not work. On the other hand, compared to polyclonal memory CTL lines derived from vaccinated/infected mice, naïve OT-i cells can only be restimulated with splenocytes several times and not indefinetely. Good luck!
I cloned DO11.10 T cells using OVA protein and was successful for several months but eventually the clones died. I used unsorted splenocytes that were irradiated as APC. 2 x 10^5 T cells per ml with 1 x 10^7 APC per ml and a range of OVA protein (from 3 ng/ml to 3 ug/ml) and EL-4 supernatant presumably containing IL-2 but I'm convinced there are other "goodies" in it as recombinant IL-2 did not work. I restimulated every 14 days.
(EL-4 supernatant is tissue culture medium from the EL-4 thymoma cell line that has been stimulated with PMA overnight.)
Good luck!
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