Encyclopedia of Experiments: Immunology
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Transcript
Specific growth factors and cytokines regulate the differentiation of monocytes — innate immune system cells — into pro-inflammatory M1 or anti-inflammatory M2 macrophages.
To study monocyte differentiation, seed peripheral blood mononuclear cells in a plastic multi-well plate containing a suitable medium. Incubate to allow the monocytes to attach to the plate surface while the non-adherent mononuclear cells remain suspended. Remove the non-adherent cells.
Add medium containing growth factor granulocyte-macrophage colony-stimulating factor, GM-CSF, to one set of wells and medium containing macrophage colony-stimulating factor, M-CSF, to the other.
GM-CSF binds to its heterodimeric receptor on the monocyte cell surface, leading to monocyte expansion and differentiation into M1-like macrophages, coupled with pro-inflammatory cytokine release.
M-CSF binds to its homodimeric receptor on the monocyte cell surface, triggering monocyte proliferation, differentiation into M2-like macrophages, and anti-inflammatory cytokine release.
Incubate the M1-like macrophages with lipopolysaccharide, LPS, and the cytokine Interferon‐gamma, IFN‐γ.
LPS and IFN‐γ treatment yields fully polarized and mature pro-inflammatory M1 macrophages with enhanced pro-inflammatory cytokine and reactive oxygen species release.
Conversely, incubation with interleukin-4 stimulates the maturation and polarization of M2-like macrophages into the anti-inflammatory M2 phenotype, along with enhanced anti-inflammatory cytokine production.
Visualize the macrophages under a light microscope. M1 macrophages appear elongated and stretched, while M2 macrophages exhibit a rounded morphology.
