[11] Assay of phagosome-lysosome fusion

This chapter discusses the assay of phagosome–lysosome (P–L) fusion. Particles ingested by cells are contained within a membrane-bounded vacuole or phagosome. In many cases, this vacuole undergoes fusion with lysosomes, resulting in exposure of the particle to both the acidic pH and hydrolytic enzymes of the lysosome. Electron microscopy has been used to look for the appearance of lysosomal markers, such as acid phosphatase or exogenously fed peroxidase, ferritin, colloidal gold, or thorium dioxide in the phagocytic vacuole. Lysosomes are labeled with a fluorescent marker, and fluorescence microscopy is used to follow transfer of the marker into phagocytic vacuoles. This assay is simple, rapid, enables detailed and reproducible rate studies, and makes possible the analysis of P–L fusion using intact and viable cells as a test system. Cultured cells are prelabeled with acridine orange (AO), a fluorescent vial dye that by its weakly basic nature becomes concentrated primarily in the low pH environment of lysosomes. Phagocytosis is essentially complete by the first time point of the fusion assay, and the rate of subsequent P–L fusion may be followed independent of the phagocytic rate.