I'm infecting human macrophages with an intracellular bacteria and I have performed the infection always under serum-free conditions. I differentiate the macrophages from blood CD14+ monocytes culturing them during 5 days in RPMI 10% FBS and M-CSF. After differentiation, I always discard supernatant and replace it with the bacteria diluted on serum-free RPMI, spin to synchronize infection, and then measure cytokine production at early times (6h). As I want to do a more extended kinetic, I'm worried about the health of the macrophages under serum-free conditions during this prolonged time (up to 48h), as I don't want to have cytokines in the supernatants as a product of apoptotic events. I'm thinking to develop the infection in RPMI 1%FBS, and I want to know if somebody has experience about the effect of serum in cytokine production using in vitro models of infection.
I usually use 10%FBS for everything (mostly viruses, sometimes bacteria) as default, but whether this works depends on your pathogen. If your FBS is heat-inactivated (inactivates complement) and if your pathogen naturally does not infect cows (otherwise maternal IgG against it could be in your FBS), serum most likely won't hurt.
I would recommend to perform the infection as you described and then change the media to RPMI 10% FBS at a timepoint when most bacteria have entered the cells (1% is better than nothing and usually keeps them happy for about 12 hours, but for longer time macrophages usually like a little more serum).
Good luck with your experiment :-)
I usually use 10%FBS for everything (mostly viruses, sometimes bacteria) as default, but whether this works depends on your pathogen. If your FBS is heat-inactivated (inactivates complement) and if your pathogen naturally does not infect cows (otherwise maternal IgG against it could be in your FBS), serum most likely won't hurt.
I would recommend to perform the infection as you described and then change the media to RPMI 10% FBS at a timepoint when most bacteria have entered the cells (1% is better than nothing and usually keeps them happy for about 12 hours, but for longer time macrophages usually like a little more serum).
Good luck with your experiment :-)
I use RPMI with 10% FBS to infect human monocyte-derived macrophages with Mycobacterium avium paratuberculosis and works well. I am able to use the cells until 7 days after infection.
I have done work with macrophages and dendritic cells and we found that FCS sometimes, depending on the batch, could contain enough TNF-alpha to alter the activity of both cell lines. This was more evident with Dendritic cells but did affect macrophages as well. This would tend to suggest that serum free would be better but think about cell culture medium that does not need FCS in it at all instead.
I infect MDMs with Mtb with 5%FCS without any problems. I detect plenty of cytokines across a 30plex panel following infection, supernatants of uninfected cells give no signals and uninfected cells are perfectly fine up to 72h. Lowering down the density, you could even push up to 5 days. Hope this helps. (As Timothy recalled below, I also meant "heat-inactivated" FCS...)
You should consider if there are intering factors in the serum; and/or cytokines or chemokines in the serum that your assay measures regardless of the % serum used for the 48 hr challenge. Also, serum has growth factors and cytokines of itself that may effect/stimulate other monocyte production giving false positives. DMEM:F12 (1:1) alone may keep them happy for 24 hrs, but I don't know about 48hrs, but might be worth a shot to save the aggravation of evaluating serum. You can perform cell viability assays and/or apoptotic assays across your 48 hr time course. With serum you likely will have growth/metaboliism due to FBS. RPMI is a lousy medium and I don't know why anyone uses it anymore, except it is cheaper than other media. Most of your growth and pH balance is due to FBS and RPMI has little to do with it.
Please note also that all lots of serum are not the same and if you use a different lot then that needs to be evaluated for interfering cytokines and /or chemlkines of interest; and the ability to stimulate monocytes. The evaluation should be against serum free conditions for as long as they support your cells to know if the levels are important.
See poster describing measurement in FBS:
http://www.perkinelmer.com/content/Featured/SBS2009/pdfs/P7012%20-%20ANU-AlphaLISA%20vs%20CisBio_15apr09.pdf
Hi, you can also used free serum media such as x-vivo15. I've perform these culture in the past for long time culture of dc alone or during mlr to measure soluble protein affected by protease contained in sera or two perform immunoprecipitation of secreted protein. Let me know
FBS inhibits the M1 - Killer phenotype. This is because of TGF-b mainly that is present in very high amounts because of lysed platelets (how serum is made - clot). So, macs function better in serum - free. Not for as long in vitro because "Killer" phenotype also kills macs themselves (e.g., Nitric Oxide). So, you can differentiate them in FBS, then serum - free best for assaying. Understand that human monocyte - derived "macs" are not the same as real tissue macs. See Mills C, J Immunology, 164:6166, or Mills C, Crit Rev Immunol, 32:463 (can send full article to you). See recent article by Carl Nathan in JCI about culturing human monocytes. Good Luck
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