A Phased Strategy to Differentiate Human CD14 ⁺ Monocytes into Classically and Alternatively Activated Macrophages and Dendritic Cells

Abstract

There are currently several in vitro strategies to differentiate human CD14+ monocytes isolated from peripheral blood mononuclear cells into M1 or M2 macrophage cell types. Thereafter, each cell type is then verified using flow cytometric analysis of cell surface markers. Human CD14+ monocytes have the potential to differentiate into M1 and M2 macrophages, both of which demonstrate varying degrees of cell surface antigen overlap. Using multiple surface markers with current macrophage polarization protocols, our data reveal several limitations with current methods, such as highly ambiguous cell types that possess cell surface marker overlap and functional similarities. By utilizing interleukin-6 and two phases of cytokine exposure, we have developed a protocol to differentiate human monocytes into M1, M2, or dendritic cells with a greater efficiently and fidelity relative to macrophages and dendritic cells that are produced from commonly used methods. This is achieved via alterations in cytokine composition, dosing, incubation times, and improvements in verification methodology. This report provides a reliable method to reproduce human in vitro monocyte derived dendritic cells and macrophage models that will aid in defining and understanding innate and adaptive immunity better as well as several pathologic states.

Recovery and assessment of leukocytes from LR Express filters

“…In comparison, purified CD14 + cells cultured with GM-CSF and IL-4 were relatively smaller in size, nonadherent, and exhibited limited protrusions. These observations are consistent with the established properties of moMϕ and moDC, respectively [16, 17]. Upon analysis by flow cytometry (Fig 6B), moDC exposed to LPS exhibited substantial increases in the cell surface expression of HLA-DR, CD83, and CD86, indicating that they had developed into mature dendritic cells [18].…”

Dual-peptide functionalized acetalated dextran-based nanoparticles for sequential targeting of macrophages during myocardial infarction

“…For both MØ and LPS/IL-4 stimulated macrophages of human origin, CD86 and CD206 were used as M1 and M2 markers, respectively. 60,61 Murine MØ macrophages were recognized by the expression of F4/80 and CD11b, 62 whereas M1-and M2-like macrophages, were distinguished by the expression of CD206 and CD11c. 63 Unstained cells were used as controls.…”

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