I have bone marrow derived GM-CSF differentiated DCs pulsed with ova & stimulated with TLR ligand. Used these DCs to co-culture OT1 T-cells. After 3 days when I check T cell proliferation by CFSE I do not see too many activated CD8 T cells. CD44 vs CFSE, CD69, and CD25 vs. CFSE have been used by people to gate and analyze proliferating T cells. Has anyone tried these gating strategies? What is the ideal percentage one should get after a 3 day coculture?
Ideally, you would expect a significant percentage (e.g., 30-60%) of the CD8 T cells to be CFSE^low and positive for activation markers like CD44, CD69, or CD25 after 3 days of co-culture.
Wow 30-60% seems quite ideal. Somehow I am unable to achieve that high a percent. I usually keep a ratio of DC: T cell at 1:2. You think this could be an issue?
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