Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Abstract

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

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“…Human tongue squamous cell carcinoma cell line Cal-27 and human gingival fibroblast (HGF) cell line were purchased from the American Type Culture Collection (ATCC). BMDCs were isolated from the bone marrow of C57BL/6 mice, as previously reported . All the cells were cultured in RPMI 1640 (SCC-7 cells and BMDCs) or high-glucose DMEM (Cal-27 and HGF cells) supplemented with 10% FBS and 1% penicillin–streptomycin and incubated in a 5% CO 2 , humidified incubator at 37 °C.…”

“…BMDCs were isolated from the bone marrow of C57BL/6 mice, as previously reported. 25 All the cells were cultured in RPMI 1640 (SCC-7 cells and BMDCs) or high-glucose DMEM (Cal-27 and HGF cells) supplemented with 10% FBS and 1% penicillin−streptomycin and incubated in a 5% CO 2 , humidified incubator at 37 °C.…”