Assessment of phagocytic activity of cultured macrophages using fluorescence microscopy and flow cytometry

Phagocytosis is the process by which phagocytes, including macrophages, neutrophils and monocytes, engulf and kill invading pathogens, remove foreign particles, and clear cell debris. Phagocytes and their ability to phagocytose are an important part of the innate immune system and are critical for homeostasis of the host. Impairment in phagocytosis has been associated with numerous diseases and disorders. Different cytokines have been shown to affect the phagocytic process. Cytokines including TNFα, IL-1β, GM-CSF, and TGF-β1 were found to promote phagocytosis, whereas high mobility group box-1 (HMGB1) inhibited the phagocytic function of macrophages. Here, we describe two commonly used methods to assess the phagocytic function of cultured macrophages, which can easily be applied to other phagocytes. Each method is based on the extent of engulfment of FITC-labeled latex minibeads by macrophages under different conditions. Phagocytic activity can be assessed either by counting individual cells using a fluorescence microscope or measuring fluorescence intensity using a flow cytometer.