Hello everyone,
I am looking for an established protocol to macrophage polarization using PBMC from mice.
But I don't know the density and proportion of monocytes after PBMC isolation.
All the papers that I read use bone marrow from mice to perform the macrophage polarization assay.
Is anyone have the protocol with these number, for example, for PBMC from human we use 9.6 cm2 - 1.5x106 monocytes - 80-90% macrophage.
Any protocol and support will be appreciated.
All the best,
Dear Aline!
You also look at this article and protocol, please:
Article Manipulation of macrophage polarization by peptide-coated go...
C57BL/6 mice
For M1 polarization, mature BMDMs were co-stimulated with 10 ng/mL LPS (InvivoGen, USA) and 10 ng/mL IFN-γ (PeproTech, USA) for 24 h; for M2 polarization, they were alternatively co-stimulated with 10 ng/mL IL-4 (PeproTech, USA) and 10 ng/mL IL-13 (PeproTech, USA) for 24 h.
Hello. I am only a student, but I'll leave this just for some reference.
I have never taken PBMC from mouse blood. I have looked at murine bone marrow cells on FACS after CD11b staining, and primary bone marrow cells seem to have very few macrophage population, probably less than 5%.
This site might help in case you haven't seen it already :https://www.biolegend.com/ja-jp/blog/expected-cell-frequencies
The protocol to generate macrophages in my lab are to harvest the mouse and take the bone marrow and culture all cells in 20ng/ml M-CSF, in a plastic adherent dish. All the non-adherent cells will be washed the next day in order to remove cells of non-monocytic lineage. Monocytes should differentiate into macrophage in about 3-5 days. However, cells cultured in M-CSF will predispose toward a M2-like phenotype(https://www.frontiersin.org/articles/10.3389/fimmu.2014.00554/full).
I think it would be difficult to gain murine PBMC using the Ficoll gradient method as in human blood, since mouse blood is low in volume.
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