Can you recommend a good LPS brand etc that you know works well or has been well established in publications? Can you tell me what medias I need to use to culture the splenocytes and IP cells. I have RPMI 1640 and DMEM currently available in the lab. What do I supplement them with? I've seen so many variations of the media in literature that I am confused. Also, if measuring cytokines after stimulation, best to measure secreted cytokines in media, or block secretion and measure cytokines in cell lysate?
Will be harvesting after stimulation and run through flow to characterize. Will also harvest some to use for mouse cytokine array.
I agree with Isabella. You might also try Sigma for the LPS. As for measuring cytokine production using the supernatants for quantitating cytokine production by ELISA, multiplex, or other is good. Using intracellular staining/flow cytometry to look at single cell production is not quantitative for cytokine produced, but provides the frequency of cytokine producing cells. They are different data points and both may be useful depending on the context of your experiments or model.
Hey Cori,
things mentioned above are all fine. For your splenocytes take care about the following: If it's short-term (couple of hours) then regular medium as describe by Isabella is fine, if it's long-term (days) I usually add 2-Mercaptoethanol, NEMs and Na-Pyruvate. Stimulation time is determined by the cytokine of interest.
For stimulation with CD3/CD28: Both of them together are a strong stimulus and will trigger your T cells to proliferate massively after 2 days. For short-term stimulation of splenocytes I prefer to use only plate-bound CD3. If you go for Flow analysis, definitely block secretion. For splenocytes I can highly recommend plates from Nunc - before I had those my assays were often crap.
Good luck!
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