irisgzz

Hi all!

I have a T-cell hybridoma cell line, that is supposed to recognize the OVA 323(327)-339 peptide bound to I-A^b . I would like to use it in order to assess changes in antigen presentation (so, to have them in co-culture with dendritic cells+ OVA or OVApeptide and measure IL-2 production at the supernatant by ELISA as a read-out).

The thing is, although they proliferate very well, the amount of IL-2 that I am getting is way too low (in the order of 2-10 pg/mL). I have been playing with different amount of cells per well (using U-bottom 96-well plates), different ratio of DC:T-hyb, and immature vs. matured DCs (by immature I mean 8-days old BMDCs right out of the plate, and by mature I first pre-stimmulated them for 4hrs with LPS, then washed and added to my co-cultures). Still, I am not able to get a decent IL-2 production. I am using the culture medium that my DCs like (RPMI +10%FCS+HEPES+mercaptoethanol+antibiotics).

I have just thawed a new frozen batch of these cell lines, and I am thinking on including some positive controls to see whether this cell line is really able to produce IL-2. I've thought about using some anti-CD3, but I don't know if it's better to use it soluble, or coating a plate.

Do you have any experience with T-cell hybridomas? What would you recommend me?

Thanks a lot in advance!