Murine Bone Marrow-Derived Macrophages

The molecular phagocytic lineage comprises, in order of increasing maturity, the committed macrophage precursor cell, the monoblast, promonocyte, monocyte and the macrophage. Methods for the preparation and culture of bone marrow-derived macrophages, developed by Stanley and colleagues (1 –3 ), provide large numbers of mononuclear phagocytes that are capable of extensive cell proliferation. Since their proliferation can be stimulated by colony stimulating factor-1 (CSF-1), granulocyte macrophage colony stimulating factor (GM-CSF), or interleukin-3 (IL-3), they represent an important primary cell source for studies of the actions and interactions of these three growth factors. The principles underlying the method are: (1) to generate and expand primitive mononuclear phagocyte precursor cells by culturing bone marrow cells in a combination of partially purified CSF-1 and IL-3 for a period of 3 d, (2) to remove contaminating red cells, fibroblasts and mature macrophages and disrupt aggregates of proliferating cells, by proteolytic digestion of the nonadherent cells at d 1 and 3 of culture and, (3) to obtain a population of mononuclear phagocytes that is relatively homogeneous with respect to their state of differentiation by recovering only those cells (i.e., mono-blasts, promonocytes) that acquire the capacity to adhere to tissue culture plastic during d 4–5 of culture.