Hi,
I am trying to detect phospho-syk in murine macrophage cell lines upon stimulation with a bacterial antigen and the level of phospho-syk in unstimated cells is hindering any stimulation effect. People suggested to use serum depletion but I couldn't find a detailed protocol. I tried 1%FBS for 2 hours but that did not seem to help. Also, do I need to change back to 10%FBS before stimulation? Do the cells respond normally in serum-free conditions? Thank you.
If you indicate the name of murine cell line that you propose to use, it may be possible to address the question more specifically. In general, serum "depletion"- induced effects take longer than couple of hours. Further, "depletion" is rarely complete and the extent varies among the various cell lines.
Thanks for your reply Divaker. These are immortalized bone-marrow derived macrophage cell lines. As you suggested I will try overnight depletion. Do I need to switch back to normal FBS levels before stimulating the cells?
Is your macrophage cell line CSF-1 dependent? If so, overnight removal of CSF-1 should be sufficient to make them quiescent, even if the media contains 10% FCS.
Thanks for your reply Fiona, I do not add CSF-1. I am trying 1%FBS overnight today. I'll see if it works.
I culture BMDM and find that overnight 1% FCS works well to decrease background phosphos, if you leave them in FCS free media they will just keel over the die and that's never fun to deal with in the morning. Also don't change the media when you add you stimulant. I always add it directly to the plate, introducing new FCS will likely stimulate something and give you a big background.
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