In this chapter, we have described the main procedures for macrophage purification currently used in various laboratories and we have reviewed some critical points to be considered in approaching the separation of these cells from different tissues.

Monocytes/macrophages are extremely responsive to a variety of stimuli, which can markedly affect their phenotype and functional activities. Thus, slight variations in the protocols or reagents used for purification may result in major differences in the experimental results, especially when the cell cultures are to be tested shortly after their isolation. Each of the different methods used for macrophage purification has its advantages and disadvantages (Table 1) and the specific choice may depend on the macrophage functions to be studied as well as on practical considerations. In general, methods based on velocity sedimentation, elutriation, or isopycnic sedimentation are more suitable than those based on cell adherence, if the cells have to be maintained in a non-activated state at early times after separation. Especially when the same research objective is faced by different groups, it is highly recommendable that reference protocols for macrophage purification are followed by the different groups involved in the study. Notably, most of the currently used standard methodologies for macrophage purification are somehow deficient in one or more of the general criteria desirable for an ideal separation (high purity and yield, functional integrity, maintaining of the correct proportions of monocyte subsets). In order to obtain a 100% pure population of monocytes/macrophages, the use of combined methods of purification should be considered. In this regard, immunomagnetic procedures based on the use of antibody-coated magnetic beads would be useful for removing specific subsets of contaminating cells.

Research interest in macrophages is likely to increase because of the accumulating evidence on their crucial role in the regulation of the host response in physiological and pathological conditions. The need for purified macrophage cultures for experimental studies increases the need for a comparison of the results obtained in different laboratories. It is to be 'hoped' that these factors will stimulate experimental efforts aimed at improving macrophage purification procedures as well as at defining reference protocols for specific research purposes.

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