I'm trying to figure out a good starting concentration of 15mer peptides to stimulate mouse CD4+ and CD8+ splenocytes for cytokine staining. We are pooling 72 peptides for one of our conditions to look for TNFa, IL-2, and IFNg secretion as a vaccine response by flow cytometry.
At what concentration would you recommend for stimulating 10^6 cells/ml of splenocytes? Should I use 1 ug/mL of each peptide (total 72 ug/mL) or a total of 1ug/mL for all peptides? I've seen both used in the literature, but the 1ug/mL total I see is usually used with a single peptide.
Please advise. Any help is appreciated.
I used to do 1 µg/mL of the peptide pool, even when I was working with a mix of 100-250 peptides and I used to get really nice T cell responses. The optimal antigen concentration for most T cell responses are in the
If you want to pool this high number of peptides then you will be limited by solubility. I suggest creating different pools of 5-10 peptides and then titrating the dose.