When I try to polarize murine BMDMs to M1, Arg-1 is simultaneously upregulated with other M1 markers. On the other hand, in M2 polarization protocols, iNOS and IL-6 upregulation is seen. Has anyone seen similar reports or results?
The polarization of macrophages is not always distinct, but sometimes difficult to distinguish whether M1 or M2. Other than Arg-1 and IL-6, IL-1-beta, CD25 etc are well-recognized as M1 molecular markers. In contrast, Dectin-1, TGF-beta, and IGF-1 are known to be expressed exclusively in M2.
Hi,
Both treatments will upregulate very many genes. The reason why there are so few strictly M1 or M2 genes is that many of the genes that are upregulated are actually shared to some extent. Arg1 is definitely upregulated by crude LPS treatment, albeit not to the extent of the IL4 treatment. This just highlights the difficulty when analysing cells isolated from different inflammatory settings, i.e. the expression of one or a very few marker genes is not a very reliable determinant of the actual inflammatory pathways that are active within the cell.
Hi Arya. Do you use FBS in your culture medium? I have faced similar problems when I used high concentrations of FBS (up to 10%). FBS contains many cytokines and growth factors (like TGF-b) which can preactivate and polarize immune cells including macrophages. Now I work with 2% FBS and everything seems better.
Hi Arya. Those pappers "Macrophage plasticity and polarization: in vivo veritas
by Antonio Sica and Alberto Mantovani" and "Exploring the full spectrum of macrophage activation by David M. Mosser and Justin P. Edwards" explain better about your question. However is difficult to establish one panel of markers for macrophages polarization, because of the plasticity of these cells.
Hi Lina...thanks for that tip. I will try it. But do you need to use higher M-CSF conc. or frequent media changes while using lower FBS?
I use 20 ng/ml M-CSF or 20% L929 conditioned medium and medium change every 3-4 days.
Hi,
Yes, we have seen up-regulated of Arginase-1 in M1, but not the other way around.
CD206/MR and Arginase-1 have been classically considered M2 and iNOS, IL-1, TNF, have been classically classically considered M1. However, CD206/Mannose receptor and Arginase-1 protein expression was low for ideal flow. In addition, some M1 macrophages also up-regulated Arg-1. Since reliable markers of mac phenotype would be a huge benefit to the field, we looked for exclusive M1 and M2 markers. Our strategy: select genes up-regulated during M1 differentiation but down-regulated during M2 differentiation and vice versa. The results of this study and the new markers we have identified, including a new CD38/Egr2-based flow cytometry strategy to distinguish M1 and M2 macrophages, can be found in Jablonski et al. Plus One. 2015 (find link below).The markers described in this paper allowed to perfectly distinguish in vitro M1 and M2 macrophages and also identified inflammatory populations in vivo.
Hope these new markers are useful to you.
What are the best markers to differetiate M1 macrophages (bone marrow derived with m-csf) from M2 macropahges (bone marrow derived with gm-csf)? - ResearchGate. Available from: https://www.researchgate.net/post/What_are_the_best_markers_to_differetiate_M1_macrophages_bone_marrow_derived_with_m-csf_from_M2_macropahges_bone_marrow_derived_with_gm-csf2 [accessed Jan 15, 2016].
Article Novel Markers to Delineate Murine M1 and M2 Macrophages
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