The Conversion of Human Tissue-Like Inflammatory Monocytes Into Macrophages

Curr Protoc. 2021 Mar;1(3):e74. doi: 10.1002/cpz1.74.


Classical circulating LyC6high murine monocytes differentiate progressively from inflammatory tissue monocytes to mature macrophages (Mϕ) after entry into gut mucosa. This protocol provides a two-step in vitro culture method that replicates the human monocyte maturation cascade. First, purified circulating CD14+ CD16- monocytes exposed to granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFNγ), and interleukin 23 (IL-23) differentiate into tissue-like inflammatory monocytes. Next, addition of transforming growth factor beta (TGFβ) plus interleukin 10 (IL-10) promotes their maturation into tissue-like Mϕ. Methods to sort these cells after culture are also provided. The fine-tuning of this system might open therapeutic avenues for chronic inflammatory disorders. © 2021 Wiley Periodicals LLC Basic Protocol 1: Isolation of human monocytes from peripheral blood Basic Protocol 2: First step culture for generation of inflammatory monocyte-like cells Basic Protocol 3: Second step culture for differentiation of inflammatory monocyte-like cells into macrophages Alternate Protocol: Sorting and culturing of inflammatory monocyte-like cells.

Keywords: GM-CSF; IFNγ; IL-10; IL-23; TGFβ; differentiation; human tissue-like inflammatory monocytes; macrophages; monocytes.

MeSH terms

  • Animals
  • Cell Differentiation
  • Granulocyte-Macrophage Colony-Stimulating Factor / pharmacology
  • Humans
  • Interferon-gamma
  • Macrophages*
  • Mice
  • Monocytes*


  • Interferon-gamma
  • Granulocyte-Macrophage Colony-Stimulating Factor