In Vitro PhagosomeEndosome Fusion

Phagolysosome biogenesis plays a pivotal role in elimination of foreign particles and pathogens by leukocytes. This process is achieved by multiple cycles of membrane fusion between the phagosome and the endosomal system. In vitro reconstitution of phagosome fusion with endosomes is instrumental in studying this intricate process. Such an assay is also invaluable for the identification of effectors produced by intracellular pathogens that may hamper the pathway. In this chapter we describe a highly sensitive and relatively rapid method to measure fusion between phagosomes and early, as well as late, endosomal compartments. The assay is based on the formation of a stable biotin–streptavidin complex upon fusion between biotinylated–peroxidase loaded endosomes and magnetic streptavidin conjugated bead-containing phagosomes. Fusion is quantified using a fluorescence-based detection method that measures the peroxidase activity associated with the beads.