Phagocytosis of yeast: a method for concurrent quantification of binding and internalization using differential interference contrast microscopy

In studies of phagocytosis there is a need to distinguish targets that are internalized by the cell from those that are bound to the cell surface. The present work describes a simple method by which internalized and surface-bound yeast particles can be identified by differential interference contrast microscopy, using trypan blue to stain surface-bound yeast particles. The method has the advantage that both internalized and surface-bound particles can be visualized without the need to switch the illumination source and/or filter sets, thus facilitating concurrent quantitation of binding and internalization. The method was evaluated with the phagocytosis-modulating agents horseradish peroxidase (HRP) and cytochalasin D, using adherent resident macrophages as phagocytic cells. When macrophages are challenged with a particular type of target, they usually bind many more targets than they ingest. It was shown that yeast particles were arrested in the initial binding phase of phagocytosis depending on the region of macrophage plasma membrane where binding sites were formed. Failure of surface-bound yeast particles to trigger internalization was not due to modifications of the yeast particle surface. Nor was it due to binding to non-phagocytic receptors, or low-affinity receptor-ligand interactions. The glycoprotein HRP inhibited only the binding stage of phagocytosis, whereas cytochalasin D, a drug that affects actin polymerization, inhibited both binding and internalization. However, when the yeast particles were pre-incubated in fresh mouse serum, cytochalasin D inhibited only the internalization step. The assay described here may be useful in studies concerned with the function and expression of phagocytosis-mediating surface lectins.