Mouse Bone Marrow Cell Isolation and Macrophage Differentiation.

The rapid increase in the incidence of obesity contributes to a parallel increase in nonalcoholic steatohepatitis (NASH). Monocyte-derived macrophages, recruited from the bone marrow to the liver, promote NASH-related inflammation and fibrosis. In addition, adipose tissue macrophages (ATMs) release pro-inflammatory cytokines (PICs) which stimulate adipose tissue lipolysis liberating free fatty acids (FFAs) that can accumulate in the liver as triglycerides (TGs), thereby inducing steatosis. As such, bone marrow-derived macrophages (BMDMs) function as an essential tool to study the pathogenesis of NASH. BMDMs are primary bone marrow-derived cells which are differentiated into macrophages in vitro in the presence of growth factors. Macrophage colony-stimulating factor (M-CSF) is required for the proliferation and differentiation of committed myeloid progenitors into cells of the macrophage/monocyte lineage. Here, we describe a protocol for the isolation of mouse bone marrow cells and subsequent macrophage differentiation in which bone marrow cells are cultured in the presence of M-CSF, supplemented either by conditioned medium from L929 cells or in purified form. The efficiency of the differentiation is confirmed by immunofluorescent staining of macrophage surface antigen F4/80. The BMDMs serve as an excellent ex vivo model for a variety of studies, including hepatocyte-macrophage and adipocyte-macrophage cross-talk regulating NASH.