Quantitative analysis of phagocytosis and phagosome maturation.

Phagocytosis and phagosome maturation lead to killing and digestion of bacteria by protozoans and innate immune phagocytes. Phagocytosis of particles expressing or coupled to various fluorescent reporters and sensors can be used to monitor quantitatively various parameters of this central biological process. In this chapter we detail different labeling techniques of bacteria and latex beads used to measure adhesion and uptake by FACS analysis. We also describe methods to use fluorescent reporter dyes (FITC or DQgreen) coupled to silica beads to measure the kinetics of acidification and proteolysis. Measurements can be performed either at the single-cell level, using live microscopy, or for a whole cell population, with a fluorescence microplate reader.