In Vitro Macrophage Phagocytosis Assay

The key roles of macrophages in atherosclerosis include the phagocytosis of apoptotic and necrotic cells and cell debris, whose accumulation in atherosclerotic lesions exacerbates inflammation and promotes plaque vulnerability. Evidence is accumulating that macrophage phagocytic functions peak at the early stages of atherosclerosis and that the reduced phagocytosis at the late stages of disease leads to the generation of necrotic cores and a defective resolution of inflammation, which in turn promotes plaque rupture, thrombus formation, and life-threatening acute ischemic events (myocardial infarction and stroke). The impaired resolution of inflammation in advanced lesions featuring loss of macrophage phagocytic activity may be in part due to an imbalance between M1 and M2 subsets of polarized macrophages. A better understanding of the mechanisms that regulate macrophage phagocytic activity in the context of atherosclerosis may therefore help identify novel therapeutic targets. This chapter presents a protocol for establishing primary mouse macrophage cultures, a method for polarizing macrophages to the M1 and M2 states, and a method for the in vitro study of macrophage phagocytosis of IgG-opsonized or IgM/complement component 3-opsonized erythrocytes.M.R.H. is supported by an FPI predoctoral fellowship from the Spanish Government (BES-2011-043938) and R.V-B. by a Juan de la Cierva postdoctoral contract from the Spanish Government (JCI-2011- 09663). Work in V.A.’s laboratory is supported by grants from the Spanish Ministry of Economy and Competitivity (MINECO) (SAF201346663-R), Fondo Europeo de Desarrollo Regional (FEDER), Instituto de Salud Carlos III (RD12/0042/0028), the Progeria Research Foundation (Innovator Award 2012, Established Investigator Award 2014), and the European Union (Liphos, Grant Agreement 317916). The Centro Nacional de Investigaciones Cardiovasculares (CNIC) is supported by the MINECO and the Pro-CNIC Foundation.S