Quantitative live-cell fluorescence microscopy during phagocytosis

Abstract

Phagocytosis is a receptor-mediated process whereby professional phagocytes internalize invading pathogens and apoptotic bodies into an intracellular vacuole or phagosome, leading to their degradation. During the formation and maturation of the phagosome, several lipids undergo changes and effector proteins are recruited on the nascent phagosome in a concerted manner. These highly localized, dynamic, and transient processes can only be studied by methods capable of high spatial and temporal resolution. The use of genetically encoded chimeric constructs coupled with fluorescence confocal microscopy enables the continuous, noninvasive analysis of the distribution and metabolism of lipids and effector proteins during phagocytosis. Here, we describe a method where the mouse macrophage cell line, RAW 264.7, and primary macrophages are transiently transfected with fluorescent chimeric probes to analyze and quantify phagocytosis of immunoglobulin-opsonized particles, using confocal microscopy.