This article describes the isolation and characterization of murine macrophages, focusing on three main methods: peritoneal, bone marrow-derived, and alveolar macrophages. Macrophages are mononuclear phagocytes that play critical roles in immune responses, tissue remodeling, and wound healing. They can be activated by environmental stimuli and exhibit varied physiological responses depending on their location and the surrounding conditions. The article outlines protocols for isolating macrophages from different tissues, including peritoneal cavity, bone marrow, and lungs, and provides methods for characterizing them using flow cytometry and other biochemical markers. For peritoneal macrophages, mice are injected with eliciting agents like Brewer's thioglycollate broth or proteose peptone to increase macrophage yield. The peritoneal cavity is then accessed to collect macrophages, which are then centrifuged and resuspended in appropriate media. Bone marrow-derived macrophages are isolated by culturing bone marrow cells in the presence of macrophage colony-stimulating factor (M-CSF). Alveolar macrophages are isolated from the lungs using a method involving the instillation of saline into the lungs and subsequent centrifugation of the collected fluid. The article also describes the use of fluorescently labeled monoclonal antibodies for phenotypic characterization of macrophages using flow cytometry. Surface markers such as F4/80, CD11b, and CD68 are used to identify and characterize macrophages. The protocols emphasize the importance of using sterile techniques and endotoxin-free reagents to ensure the accuracy and reliability of the results. The article concludes with a discussion of the physiological differences between various macrophage populations and the importance of proper handling and storage of macrophages for experimental purposes.This article describes the isolation and characterization of murine macrophages, focusing on three main methods: peritoneal, bone marrow-derived, and alveolar macrophages. Macrophages are mononuclear phagocytes that play critical roles in immune responses, tissue remodeling, and wound healing. They can be activated by environmental stimuli and exhibit varied physiological responses depending on their location and the surrounding conditions. The article outlines protocols for isolating macrophages from different tissues, including peritoneal cavity, bone marrow, and lungs, and provides methods for characterizing them using flow cytometry and other biochemical markers. For peritoneal macrophages, mice are injected with eliciting agents like Brewer's thioglycollate broth or proteose peptone to increase macrophage yield. The peritoneal cavity is then accessed to collect macrophages, which are then centrifuged and resuspended in appropriate media. Bone marrow-derived macrophages are isolated by culturing bone marrow cells in the presence of macrophage colony-stimulating factor (M-CSF). Alveolar macrophages are isolated from the lungs using a method involving the instillation of saline into the lungs and subsequent centrifugation of the collected fluid. The article also describes the use of fluorescently labeled monoclonal antibodies for phenotypic characterization of macrophages using flow cytometry. Surface markers such as F4/80, CD11b, and CD68 are used to identify and characterize macrophages. The protocols emphasize the importance of using sterile techniques and endotoxin-free reagents to ensure the accuracy and reliability of the results. The article concludes with a discussion of the physiological differences between various macrophage populations and the importance of proper handling and storage of macrophages for experimental purposes.