Murine bone marrow-derived macrophages (BMDMs) have emerged as a valuable model system for studying macrophages due to their physiological relevance, ease of manipulation, and availability. Here Creative Biolabs presents a comprehensive guide for culturing and maintaining murine BMDMs.

The protocol encompasses bone marrow isolation, differentiation of bone marrow cells into macrophages, and subsequent characterization of the cultured BMDMs. This protocol has been optimized to yield high-quality, functional BMDMs suitable for various downstream applications, including immunological studies, drug screening, and infection models.

Materials

• Murine bone marrow
• Dulbecco's Modified Eagle Medium (DMEM)
• Conditioned medium/M-CSF
• Phosphate-buffered saline (PBS)
• Hematopoietic growth factor
• Cell surface markers and antibodies

Methods

1. Isolation of Murine Bone Marrow Cells
   • First, harvest the mouse bone marrow samples and rinse the marrow into sterile tubes with sterile PBS.
   • Then centrifuge to collect the precipitates.
   • Resuspend the precipitate in erythrocyte lysis buffer, incubate and then wash with PBS.
   • Count the cells and adjust the concentration to the desired cell density using complete DMEM medium.
2. Differentiation of Bone Marrow Cells into BMDMs
   • Load bone marrow cells into culture dishes or cell culture flasks at the desired cell density and incubate.
   • 24 h later, remove non-adherent cells by gently aspirating or washing the cultures with PBS. Add conditioned medium to the adherent cells to induce their differentiation into macrophages.
   • Replace the medium with fresh medium every 2-3 days until the desired maturation stage. To differentiate into specific macrophage subpopulations, the medium can be supplemented with specific hematopoietic growth factors.
3. Characterization of BMDMs
   • Perform cell counts to determine cell yield.
   • Assess the phenotype and purity of macrophages by flow cytometry, using specific cell surface markers and antibodies.
   • Assess the phagocytic capacity of BMDMs by incubating them with appropriate phagocytic substrates and analyzing uptake using flow cytometry or microscopy.
   • Other methods can be used for various characterizations.

Notes

• The age, strain, and gender of the donor mice can influence the characteristics of BMDMs. Therefore, standardize the experimental conditions using mice of the same background.
• Culturing BMDMs for an extended period may lead to the loss of certain macrophage functions or induce polarization towards a specific phenotype. Optimize the differentiation time based on the intended experimental requirements.
• Maintain aseptic techniques throughout the protocol to prevent contamination.
• You can consider the specific requirements of your experimental design and adjust the protocol accordingly.

The culture of murine bone marrow-derived macrophages provides an invaluable tool for studying macrophage biology and immune responses in vitro. This comprehensive protocol will enable researchers to establish a reliable and reproducible system for investigating the diverse functions of macrophages in immunological studies, infection models, and drug screening assays.

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Reference

1. Murray P J, et al. Macrophage activation and polarization: nomenclature and experimental guidelines. Immunity, 2014, 41(1): 14-20.