Culture, Transfection, and Immunocytochemical Analysis of Primary Macrophages

Macrophages are the primary phagocytes of the body and found in every tissue; often with tissue specific subtypes, e.g., microglia or Kupffer Cells. These cells are essential players in host defense, immune regulation, tissue repair, and homeostasis. Consistent with their diverse functions, macrophages display a remarkable level of plasticity and undergo rapid changes in morphology and activation state in response to environmental cues. Polarization of macrophages towards pro-inflammatory (classically activated or M1) or anti-inflammatory (alternatively activated or M2) activation states is highly dependent on their environment. These activation states result in either tissue remodeling and repair (M2) or enhanced inflammation (M1). As macrophages are dependent upon environmental cues for changes in their activation state, primary cell culture offers the ability to study macrophages under highly controlled conditions in which activation states are easily manipulated with specific growth factors, cytokines, or other signaling molecules and are readily examined through powerful tools such as immunostaining, ELISA, and Ca²⁺ imaging. Additionally, this approach allows the researcher to manipulate gene expression in these cells to better understanding the underlying principles and mechanisms of macrophage biology. Unfortunately, macrophages are resistant to most forms of transfection and researchers have to use either macrophages isolated from transgenic mice or viral delivery of transgenes which slows the study of these diverse cells. In this chapter we describe methods for isolating, culturing, transfecting, and immunostaining primary macrophages. Particular emphasis is placed on culture conditions and transfection protocol as we found these significantly impacted the success of this protocol. Pairing these methods with functional Ca²⁺ imaging enables investigation of the effects of silencing or overexpressing specific proteins on the functional properties of primary macrophages.