Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022:2455:85-91.
doi: 10.1007/978-1-0716-2128-8_8.

Mouse Bone Marrow Cell Isolation and Macrophage Differentiation

Rachel Mendoza  1 , Indranil Banerjee  1 , Debashri Manna  1 , Saranya Chidambaranathan Reghupaty  2 , Rajesh Yetirajam  1 , Devanand Sarkar  3   4   5
Affiliations

Mouse Bone Marrow Cell Isolation and Macrophage Differentiation

Rachel Mendoza et al. Methods Mol Biol. 2022.

Abstract

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.

Keywords: Bone marrow cell; F4/80; L929 cells; Macrophage; Macrophage colony-stimulating factor (M-CSF); Nonalcoholic steatohepatitis.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Harvesting bone marrow from mouse femur and tibia. Clockwise from top left: Cleaned bones containing marrow, removal of marrow using 25G needle, bones after marrow removal
Fig. 2
Fig. 2
Cell pellets (arrows) before (left) and after (right) RBC lysis
Fig. 3
Fig. 3
Representative images of bone marrow-derived macrophages (BMDM). From left: bright-field image after three days of differentiation, bright-field image of fully differentiated BMDM, and fully differentiated macrophages stained with F4/80 antibody (green) and DAPI (blue)
See this image and copyright information in PMC

References

    1. Friedman SL, Neuschwander-Tetri BA et al. (2018) Mechanisms of NAFLD development and therapeutic strategies. Nat Med 24: 908–922 - PMC - PubMed
    1. Lefere S, Tacke F (2019) Macrophages in obesity and non-alcoholic fatty liver disease: Cross-talk with metabolism. JHEP Rep 1:30–43 - PMC - PubMed
    1. Pellicoro A, Ramachandran P, Iredale JP, Fallow-field JA (2014) Liver fibrosis and repair: immune regulation of wound healing in a solid organ. Nat Rev Immunol 14:181–194 - PubMed
    1. Fruhbeck G, Mendez-Gimenez L, Fernandez-Formoso JA et al. (2014) Regulation of adipocyte lipolysis. Nutr Res Rev 27:63–93 - PubMed
    1. Grove JE, Bruscia E, Krause DS (2004) Plasticity of bone marrow-derived stem cells. Stem Cells 22:487–500 - PubMed

Publication types

Substances