This chapter describes techniques to identify macrophages and measure their endocytic and phagocytic capabilities. These methods can assist in the analysis of the physiological and pathological situations in which macrophages play a central role including homeostasis, wound repair, and immunity.
The expression of cell surface proteins and other molecules varies greatly among macrophage populations as a result of heterogeneity in their differentiation, activation state, or response to external influences in their microenvironment or cell culture conditions (1-3). Immunochemical and fluorescent techniques can be used to investigate the basal expression of antigens, the changes induced in response to a particular stimulus, and to detect antigens in tissues.
The function of cellular proteins can also be investigated using immunochemical and fluorescent techniques. In the case of endocytic receptors, appropriately labelled ligands can be added to the cell, their intracellular trafficking traced, and compared with that of other macrophage and non-macrophage receptors. Changes in activity of known receptors and potential functions of novel molecules can be tested in a range of assays with soluble and particulate ligands.
In vivo and in vitro investigation of macrophages or specific molecules is essential for an overall understanding of their biological function, as is highlighted in the analysis of knockout and transgenic mice that lack, under-, or overexpress selected molecules.
This chapter details basic techniques used to detect macrophage-specific anti gen markers in tissue and in vitro, and to assay endocytic and phagocytic abilities of isolated cells, with emphasis on selected plasma membrane receptors involved in innate immune recognition. The techniques described can be applied to a variety of macrophage-like cell lines such as RAW 264.7 murine macrophages, THP-1 human monocytic-like cells, and isolated murine or human primary macrophage populations. These include: murine resident peritoneal macrophages or cells elicited by Bio-Gel polyacrylamide beads, thioglycollate broth, or Mycobacterium bovis BCG; murine bone marrow-derived macrophages and dendritic cells; human blood monocytes and monocyte-derived macrophages and dendritic cells (see Chapters 1 and 2). Methods used to examine other characteristic macrophage functions, including release of secretory products, are discussed elsewhere in this volume (see Chapters 5-7) and in useful general compendia (4).
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