Hello everybody,
I have to induce M1 activation with IFN gamma and LPS on murine macrophages. Do you advise to proceed in serum or serum-free conditions? I have read different protocols about this topic but I can't find a standard procedure.
Thank you very much in advance.
To generate M1 macrophages, we routinely use normal culture media (i.e. with serum) to incubate the cells with LPS & IFNgamma for 24hrs.
To generate M1 macrophages, we routinely use normal culture media (i.e. with serum) to incubate the cells with LPS & IFNgamma for 24hrs.
Thanks a lot. And what concentration of LPS and IFN gamma do you use?
We polarize our macrophages in medium supplemented with serum as well. But for polarization with LPS and IFNg we employ lower levels (as described by Al Faraj in PMID PMID:23281292 ): 1 ng/ml LPS and 10 ng/ml IFNgamma. But probably both protocols work fine. Good luck!
use serum. LPS requires serum proteins for proper recognition.
i would also go for the lower conc. of LPS and IFNg.
and unfortunately there is no standard procedure. good luck with your exp.
We routinely use culture medium supplemented with serum to induce M1 polarization of monocytes with LPS (100ng/mL) and IFNg (100U/mL).
I've tried both as I was going on to use the macrophages in western blotting and wanted to see if serum starving made a difference. If your incubation are longer than two hours you should be using serum as any longer and I find the primary BMDM I work with start to loose viability in culture. For polarisation I normally use LPS at 100ng/ml and INFg 10ng/ml.
Just be careful with your concentrations that you are using, if you are going on to look at pathways and signalling etc. I do a lot of LPS pathway westerns and I do not use INFg with the LPS as of course this will introduce another pathway and you can't go above 100ng/ml or you start to activate pathways outside of TLR4. There are a lot of variables to think about with concentration and it is dependent on what you want to do really downstream of your stimulation.
Use serum as this provides medium factors for the activation to take place.
We change the normal culture medium (10% FBS) by medium supplemented with 2% serum before the polarization. For polarization we use LPS at 200 ng/ml and IFNg at 200 ng/ml.
It was further demonstrated that pre-differentiation with GM-CSF enhances final M1 activation status. Good luck!
It will depend what will be your read out: TNF production, surface markers expression... the time of incubation, and the source of macrophages (peritoneal cavity, bone marrow derived MØ...). In our hands, TNF production was maximum without serum or very low amounts of FCS (0.2%). Then you can ask yourself, if it makes sense to offer bovine proteins to your mouse cells. The problem being that if you use mouse serum, it will inhibit the cells (Warren et al. J Infect Dis. 2010 Jan 15;201(2):223-32). In other words do you want to set up an articifial model or something with some vague relevance with true life ;-). But an M1 cell derived from a peritoneal MØ has nothing to deal with an M1 cell derived from bone marrow derived MØ (Shuyi Zhang et al. Plos Negl. Trop. Dis. 2010, 4, e648). Should we talk about M1 cells when they are so much difference in terms of gene expression ? Back to the question about serum, the true best approach will be to do a dose response curve to define the appropriate % in your own hands (it depends also on the batches of FCS you have... All batches don't behave similarly). Good luck.
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