High-resolution quantitation of discrete phagocytic events by live cell time-lapse high-content microscopy imaging

Phagocytosis is a dynamic process central to immunity and tissue homeostasis. Current phagocytosis quantitation methods largely rely on indirect or static measurements, such as target clearance or dye uptake, and thus provide limited information about engulfment rates or target processing. Improved kinetic measurements of phagocytosis could provide useful, basic insights in many areas. We present a live-cell, time-lapse, high-content microscopy imaging method based on the detection and quantitation of fluorescent dye “voids” within phagocytes that result from target internalization to quantitate phagocytic events with high temporal resolution. Using this method, we measure target cell densities and antibody concentrations needed for optimal antibody-dependent cellular phagocytosis. We compare void formation and dye uptake methods for phagocytosis detection and examine the connection between target cell engulfment and phagolysosomal processing. We demonstrate how this approach can be used to measure distinct forms of phagocytosis, and changes in macrophage morphology during phagocytosis related to both engulfment and target degradation. Our results provide a high-resolution method for quantitating phagocytosis that provides opportunities to better understand the cellular and molecular regulation of this fundamental biological process.