I am currently trying to determine whether IL2 conjugates designed in our lab maintain their functionality. Currently I'm working with mouse splenocytes (seeded at 1x10^6 cells/mL) in a 96 well plate and I treat them with IL2 ranging from 50-200U concentration as well as similarly concentrated conjugates. I then culture them at 37C for 2-4 days and run flow for them, testing with CD3, CD4, and CD8 antibodies. So far I have not been able to observe a significant different between Il2 treated cells and cells just in RPMI. Does anyone have advice for how to adapt my protocol or test methods to obtain a significant different and be able to determine whether IL2 is functioning properly in the conjugates we have designed?
I would do a short-term assay. IL-2 signalling in CD4 cells as measured by Stat5 phosphorylation peaks around 15 minutes and remains increased until around 12 hours. So, either an intracellular staining and FACS analysis or pStat5 WB coupled with half-log dilutions should be able give an indication whether your IL2 conjugates are functioning as well as non-conjugated IL2.
There is a murine cell line (CTLL-2; TIB-214 ATCC) that is dependent on IL2 for its growth and can be used to determine the potency of your different IL2 formats. Alternatively, if using primary mouse T cells, you will have to stimulate them with an anti-CD3 antibody (2C11 clone works well). The trick would be to titrate the 2C11 antibody so that you find a concentration where it alone induces very little proliferation but where the addition of IL2 will give rise to a robust proliferation. Once having set up this system with native IL-2, you can test your variants.
- Badges
- Science topic
- Similar topics
- Chemistry, Organic
- Organic