Goal: I am trying to activate CD4+ or CD8+ using EG7-OVA cell line in vitro. I have only been successful with activating them using the EG7 vs CD8s. I tried OVA protein (whole protein not peptide) as a control but no matter the concentration (up to 20 uM), I don't have CD4+ or CD8+ proliferate. Now I have read CD8+ are only activated via cross presentation at concentration 20mg/ml. I haven't gone up to that much.
1) Is that how much you need to activate CD8s?
2) Does anyone know how much whole OVA protein one needs to add to activate CD4+ in vitro?
3) I would also be interested if anyone knows any cell line that secretes OVA peptide for OTI or OTII? Note: when I say CD4+ or CD8+, they are part of the splenocytes and APCs are not removed.
4) Would BMDC be better help than splenic DCs with activate CD4+ (need more protein) or CD8+ (need more efficient cross presentation)?
Thank you.
If you use soluble OVA for CD8+ T cell (OT-I) stimulation, you need either more OVA as soluble antigen to load DCs in your system or preferentially use an adjuvent such as ISCOMATRIX, QS21, etc to help the soluble protein to gain cross-presentation pathway in DCs. Check out these papers Schnurr Blood p2465 2005 CP ISCOM and Zhao pone.0044707 2012 B18 ESO T cells. Not sure about the OT-II. Maybe you could in vivo (or even in vitro) prime them and then use them as a memory population. In that case all the B cells could potentially become APC once you provide soluble OVA
i am asking about a cell line that secretes soluble PEPTIDE, do those exist? el-4 ova is whole protein
Absolutely not and most peptides if not bound to MHC (protected) will be degraded in no time either within the cells or in the medium containing serum
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining