Quantitative Phagocytosis Assays in Primary and Cultured Macrophages.

This chapter describes methods to induce and quantify phagocytosis in primary macrophages and in myeloid cell lines. To this end, we initially detail the isolation of primary human monocytes and their differentiation into macrophages. Because primary cells are comparatively refractory to molecular manipulation, we also describe the culture of RAW 264.7 cells-an immortalized monocyte/macrophage cell line, which is more tractable. The chapter also includes methods for preparation of phagocytic targets, specifically sheep erythrocytes opsonized with immunoglobulin G (IgG), as well as means of distinguishing bound from internalized targets, using fluorescently labeled secondary antibodies.