Macrophage phagocytosis assay with reconstituted target particles.

Macrophage phagocytosis can be triggered by diverse receptor-ligand interactions to clear pathogens and dead cells from a host. Many ways of assaying phagocytosis exist that utilize a variety of phagocytic targets with different combinations of receptor-ligand interactions, making comparisons difficult. To study how phagocytosis is affected by specific changes to the target surface, we developed an in vitro assay based on reconstituted membrane-coated target particles to which known molecules can be added. The targets are made by coating glass beads with supported lipid bilayers followed by coupling proteins and other ligands of interest. Composition of the lipid bilayer can be varied to bind and orient specific proteins, incorporate signaling and reporter lipids, and control bilayer fluidity. To quantify phagocytosis, the reconstituted target particles are incubated with macrophages in vitro for a defined period of time, imaged with fluorescence microscopy and analyzed with software that measures the amount of target particle fluorescence within each macrophage. A multi-well plate format can be used for multi-parameter studies (e.g., to investigate how phagocytosis is affected by specific receptor-ligand interactions, ligand density, lipid charge, membrane fluidity and other molecular details). As an example, we demonstrate that antibody-dependent phagocytosis is more efficient for targets with fluid membranes than non-fluid membranes. The assay protocol takes approximately 6 h and requires basic molecular biology, mammalian cell culture and fluorescence microscopy skills. This assay can also be used with other phagocytic and non-phagocytic cells to study the individual or collective roles of receptors and ligands in immune effector function.