Hi,
I was wondering, what are the controls one would use for pulsing peptides into naive in vitro derived dendritic cells? How can I verify that the pulsed peptide is actually being presented on the MHC class I in DCs? Also, if I pulse post-translationally modified peptides, can they undergo further modification/lose modifications by DCs endogenous PTM machinery?
Best,
Martyna
It depends on what cells you are expanding. For CD8 T cells, we use CEF pool as a positive control and test CEF response on expanded and non-expanded cells. We do not use a negative control for expanding cells but test that expanded T cells with peptide-pulsed DCs have no response against media alone. If you use post-translationally modified peptides a fraction of them will likely loose the modifications but hopefully not all. You can verify that peptides are presented by DCs using tandem mass spectroscopy. See attached paper.
https://www.ncbi.nlm.nih.gov/pubmed/12960169
You can stain the OMVs as well as stain the DCs and then go for fluorescent microscopy. Where you will be able to see the surface markers that shows the maturation of DCs. You can follow the below link which will be useful for you:
https://www.nature.com/articles/srep27206
Dear Martyna ,
Search works of A de Groot with tregitopes and verification of tregitopes in vitro, hope its help in your works,
Best regards,
Tomasz
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