Macrophages are phagocytic immune cells that play essential roles in homeostasis and orchestrate multiple responses to pathogens [1]. These cells acquire specialized phenotypes and functions depending on their microenvironment and, indeed, several macrophage subtypes have been identified and characterized over the last decade [2]. In the context of atherosclerosis, macrophages are crucial components of atherosclerotic plaques and contribute to the pathogenesis of the disease from its initial stages throughout the progression of the atheroma, and up to the final stages when the plaque becomes vulnerable and may rupture into the bloodstream [3]. During this process, macrophages contribute to a variety of functions depending on their phenotype, which in turns depends on their microenvironment. In an oversimplified classification, Th1 pro-inflammatory cytokines such as IL-2, IL-12, IFNγ, and TNFα and β lead to the activation of macrophages towards the so-called classical inflammatory phenotype (CAMs or M1 macrophages). In addition, Th2 cytokines such as IL-4 and IL-13 as well as anti-inflammatory molecules like IL-10 and TGFβ activate macrophages towards an alternative phenotype (AAMs or M2 macrophages). While M1s have been described to be involved in the secretion of inflammatory cytokines and vasoactive molecules contributing to atheroma growth, M2 cells participate in a wide range of processes including apoptotic cell clearance or the release of anti-inflammatory molecules and are generally considered anti-atherosclerotic [4, 5]. Although IL-4 is considered “the original” cytokine promoting M2 polarization, its role in atherosclerosis still remains ambiguous, as it has been shown to be involved in both, pro- and anti-atherosclerotic mechanisms [6].

The physiological and pathophysiological processes macrophages participate in make them a very interesting therapeutic target for inflammatory diseases, including atherosclerosis and cancer. Although in recent years there have been major advances in the use of noninvasive techniques to investigate the mechanisms underlying cardiovascular diseases, the systematic study of macrophages in a living organism, specifically within the aorta or a blood vessel by these techniques, is not yet technically feasible [7]. Thus, the isolation and culture of macrophages, which may be subjected to different stimuli ex vivo, has been a very useful and widespread tool to investigate the phenotype and functions of a particular subtype of macrophages. The number, purity and ease of culture of these cells make them a very attractive in vitro model. To investigate in culture the (dys)regulated pathways within a specific macrophage subtype that are playing a role in a particular pathological condition such as atherosclerosis, macrophages are usually activated with IFNγ plus LPS (CAMs or M1) or with IL-4, IL-13, and IL-10 (AAMs or M2) [8]. Due to the complex stimuli milieu present within the atheroma, it is not surprising that other macrophage subtypes have also been described in response to other molecules that affect the pathogenesis of atherosclerosis, including oxidized-LDLs (Mox macrophages) [9], CXCL4 (M4 macrophages) [10], or heme/haemoglobin (Mhem macrophages) [11].

In this chapter we describe the most common protocols used to isolate and differentiate cultured murine bone marrow-derived macrophages and peritoneal macrophages, and outline how these macrophages are traditionally “polarized” to obtain the M1 and M2 subsets that may be relevant to atherosclerosis and other inflammatory diseases.