The article provides a comprehensive guide on the isolation and characterization of murine macrophages, which are mononuclear phagocytes crucial for both innate and adaptive immune responses. Macrophages can be derived from monocytes or local proliferation of tissue-resident macrophage colony-forming unit cells. The peritoneal cavity is a readily accessible site for harvesting resident macrophages, and the yield can be increased by injecting eliciting agents like thioglycollate broth or proteose peptone. Bone marrow-derived macrophages can be isolated by culturing bone marrow progenitor cells in the presence of macrophage colony-stimulating factor (M-CSF), yielding a homogeneous population of macrophages. Alveolar macrophages, found in the lung, are important for host defense and can be isolated by instilling saline into the lungs and collecting the alveolar fluid. The article also describes methods for characterizing macrophages using flow cytometry with fluorescently labeled monoclonal antibodies, which can distinguish macrophages from other cell types based on specific surface markers. Key reagents and solutions, critical parameters, and expected results are provided, along with detailed protocols for each isolation and characterization method.The article provides a comprehensive guide on the isolation and characterization of murine macrophages, which are mononuclear phagocytes crucial for both innate and adaptive immune responses. Macrophages can be derived from monocytes or local proliferation of tissue-resident macrophage colony-forming unit cells. The peritoneal cavity is a readily accessible site for harvesting resident macrophages, and the yield can be increased by injecting eliciting agents like thioglycollate broth or proteose peptone. Bone marrow-derived macrophages can be isolated by culturing bone marrow progenitor cells in the presence of macrophage colony-stimulating factor (M-CSF), yielding a homogeneous population of macrophages. Alveolar macrophages, found in the lung, are important for host defense and can be isolated by instilling saline into the lungs and collecting the alveolar fluid. The article also describes methods for characterizing macrophages using flow cytometry with fluorescently labeled monoclonal antibodies, which can distinguish macrophages from other cell types based on specific surface markers. Key reagents and solutions, critical parameters, and expected results are provided, along with detailed protocols for each isolation and characterization method.