For the differentiation of human primary monocytes to macrophages, I use human serum added to RPMI. Due to some problems with the human serum, the cells isolated from a lot of blood samples couldn't differentiate fully and died half way. So I would like to ask if someone knows the existence of a serum free medium to differentiate the monocytes to macrophages?
Serum results from the lysis of platelets. And platelets contain a lot of TGF-b. TGF-b is the strongest inhibitor of macrophage activation. Try plasma or the serum- free media described in my paper (M1/M2 Macrophages... Mills et al. The Journal of Immunology, 2000, 164: 6166–6173.) You might also search Carl Nathan (I believe J. Clin Invest.). He has worked on differentiating monos to macs, and is an expert in this area. Good luck.
Does it have to be human serum? I usually derive macrophages in a media with a combination of FBS and horse serum, but perhaps we have different end goals. Regardless, I wouldn't anticipate that monos would do very well differentiating in a serum-free media.
You can try with PANSERIN 401, a serum-free medium specifically designed for using in adherent cells like macrophages avoiding the use of serum.
http://www.pan-biotech.com/content/index.php?option=com_content&view=article&id=20&Itemid=190&lang=en
We have been using with CD14+ monocytes isolated with magnetic CD14beads from human blood, and they attach and spread as macrophages in Panserin 401, but I cannot say you about surface markers.
Serum results from the lysis of platelets. And platelets contain a lot of TGF-b. TGF-b is the strongest inhibitor of macrophage activation. Try plasma or the serum- free media described in my paper (M1/M2 Macrophages... Mills et al. The Journal of Immunology, 2000, 164: 6166–6173.) You might also search Carl Nathan (I believe J. Clin Invest.). He has worked on differentiating monos to macs, and is an expert in this area. Good luck.
Dear Aravind,
The first steps of monocytes-macrophage differentiation are identical to the differentiation protocols used for monocytes-derived DC. Therefore, you can use the same medium, i.e. X-VIVO-15 from Lonza Belgium, GMP quality, serum-free or the comparable DC medium from Cellgenix, Freiburg). These media are complete serum free and clinical proved.
Detailed protocols are published by several groups.
>Due to some problems with the human serum, the cells isolated from a lot of blood samples couldn't differentiate fully and died half way.
As suggested you have to heat serum at 56°C for 30 minutes. It is true for bovine serum but also for human serum!
The problem of human primary cell death in human serum is found also in rodent cells. Any success of primary cell culture with alogenic serum (or species specific serumO is desirable.
Note, in the standard protocol of human monocyte-derived DC there is no serum or AB serum. It is heat-inactivated plasma (supernatant after ficoll gradient centrifugation). Importantly, first heat inactivation of complement and afterwards centrifugation with 1000g, use the platelet free supernatant only (1-2%). However, with X-VIVO-15 from Belgium there is no need for any serum/plasma addition.
anyone tried the X-VIVO-15 in BMDMs in a co-culture (transwell) setting? thanks!
https://www.promocell.com/fileadmin/promocell/PDF/Macrophage_Generation_Detail.pdf
https://cdn.stemcell.com/media/files/poster/SP00175-A_Serum-Free_Medium_for_Differentiation_of_Monocytes_to_Macrophages.pdf?_ga=2.125799112.338247338.1586269455-126264093.1586269455
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