Cells were enriched by negative MACS selection. Differentiated in growth media supplemented with 5 ng/ml GM-CSF. GM-CSF included throughout feedings. Cells seemed to crash around day 14, or rather began to grow on top of one another. Seeding density was 5.5x10^5 per well in a 12 well dish. Media replenished every 3 days. Cells will be utilized for HIV related experiments.
Your assistance is very much appreciated. I have no macrophage experts to consult. Much obliged.
Dear Jason,
most protocols use monocyte-derived macrophages on day 5-7 (Martinez, J Immunol. 2006 Nov 15;177(10):7303-11.; Gleissner et al., J Immunol. 2010 May 1;184(9):4810-8. doi: 10.4049/jimmunol.0901368. Epub 2010 Mar 24.; Waldo et al., Am J Pathol. 2008 Apr;172(4):1112-26. doi: 10.2353/ajpath.2008.070513. Epub 2008 Mar 5.).
I am not aware of many papers using longer time spans. The question is what exactly you want to look for after a longer time span?
Christian
Primary cells are by definition not cell lines and naturally enclined to apotosis. In my opinion, you have to plan your HIV related experiment and start differentiating your monocytes a week before you need them. Also to my knowledge, monocytes are not supossed to replicate in the presence of GM-CSF but only differentiate so you should not have confluence issues if loaded at the right density on day 0. By waiting such a long time you might well be observing some pluripotent cell expanding... Hope this helps.
Thanks everyone. The Macrophage population looks textbook in terms of morphology. Flat with a defined cell body....absolutely beautiful. The culture was turning the media yellow in 1.5 days after day 10. I am now addressing optimal plating density with the current monocyte PBMC enrichment. These are my very first attempts at working with primary macrophages. I have worked with primary T-cells for decades, but they are easier to work with. I tried the adherence method once and that was enough.
I do not want the Macs phagocytosing beads and so used negative selection. Most labs seem to utilize negative selection procedures and so I went that route.
One direct question though....does the culture need to be continuously supplemented with GM-CSF after day 7????? Do they need the GM-CSF at 5 ng/ml throughout???? The prior lab procedure was to use 1 U/ml of GM-CSF, but that took too long, say 10+ days before differentiation. I need faster turn around and a consistent culture. So I can pump out data and move this project along. It is one i am very excited about.
Thanks again for the responses and willingness to share your expertise with me.
with care, HIV-infected monocyte derived macrophages can be kept viable in culture for 6-7 weeks. we use Iscoves medium with 10% human serum. They will change their morphology and some will detach, however they continue to produce HIV. The production of virions declines due to limitation of Tat expression and can be partially rescued by addition of exogenous Tat: see Sonza et al J Virol. 2002 December; 76(24): 12611–12621.
Any cytokines in your media???? Can you provide more details of your culture methods???? I am thinking that perhaps I can tone down the GM-CSF. The previous post-doc had his cells in culture for much longer and they remained viable. My culture crashed...went from perfect to dead in 2-3 days. No contamination was observed. The Macrophages just died and debris of some kind accumulated in the center of the dish. The ECM remained behind and the cell bodies detached, I assume apoptotic bodies. Still seems odd that they died off so rapidly. Same observed in subsequent cultures, so something is aberrant with my methods.
Hi Jason,
I have cultured GM-CSF and M-CSF derived macrophages for 14 days after differentiation (i.e. 20 days from initial isolation) without any problems, including infecting them with HIV for 14 days. The longer you leave them infected with HIV, the more synciitium will form. I also found that over such a long period of time the polarisation of the cells was lost. Ie the effects of GM-CSF is lost a few days after removal. The effects of M-CSF last longer. However, if it is the effect of HIV you are interested in, then leave that as your stimulus, rather than keeping GM-CSF in the medium. As long as you infect then shortly after removal of GM-CSF the effect will still be present. This paper might be useful.
Cassol, E., Cassetta, L., Rizzi, C., Alfano, M., & Poli, G. (2009). M1 and M2a Polarization of Human Monocyte-Derived Macrophages Inhibits HIV-1 Replication by Distinct Mechanisms. The Journal of Immunology, 182(10), 6237–6246. doi:10.4049/jimmunol.080344
Jason
we do not put growth factor in the medium when we culture macrophages for HIV infection. In fact, we and others have shown that GM-CSF inhibits HIV replication in human MDM (AIDS. 2000 Aug 18;14(12):1739-48.). We use 10 % pooled human serum in Iscoves medium plus pen/strep and glutamine; nothing more. take care to exclude possible sources of LPS contamination. Culture adherent at 3-5x10^6 per 10 cm dish, change medium every 3 days. I would also recommend reading guido poli's paper as mentioned by Anna, if you want to study the effect of macrophage polarisation on replication.
Hi Jason,
I work with macrophages and lentiviruses, they can live even more than 15 days. I differentiate the monocytes in regular RPMI with 10% of bovine serum and 100ng of rhM-CSF (I not use GM-CSF). I let the differentiate among 5-7 days and them infect them when they are already differentiated. I hope this help you ;)
Pilar
Hi Jason I work in primary macrophages and HIV. Depending of the donor, the cells last at least 25-30 days, after that they start apoptosis. HIV infection enhances survival ......(we can talk if you want) allowing longer experiments.
Hi Jason,
I agree with Damien and Eliseo, your macrophages should be fine at least for 15 days (that is the furthest I did experiments). About the GM-CSF, I did not remove the whole media, I used to take half of the media and add fresh one every 2-3 days. This was my protocol: PBMCs isolation, wait until cell attachment for 2h, was the plate with PBS and get rid of the floating cells. Add GM-CSF and wait for differentiation. Once cells are differentiated in macrophages, start the experiment.
I hope this help! Good luck!
Pilar
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