Phagocytosis of yeast: a method for concurrent quantification of binding and internalization using differential...

Understanding phagocytosis requires distinguishing between surface-bound and internalized particles. Prior research has shown that macrophages bind more targets than they ingest. However, no prior work had resolved how to quantify both processes simultaneously. This gap motivated the development of a method using differential interference contrast microscopy. Existing techniques often require switching illumination sources or filters, which complicates concurrent quantification. This paper introduces a new approach using trypan blue to stain surface-bound yeast. No prior work had resolved how to distinguish internalized from surface-bound particles without additional steps. The method aims to simplify and streamline phagocytosis studies.

The study aimed to develop a method for distinguishing surface-bound and internalized yeast particles during phagocytosis. This is important because macrophages often bind more targets than they ingest. The goal was to enable concurrent quantification without switching illumination sources. The method uses trypan blue staining with differential interference contrast microscopy. The researchers sought to evaluate the method using known modulators of phagocytosis. They tested horseradish peroxidase and cytochalasin D. The study also aimed to assess whether binding site location affects internalization. The purpose was to provide a reliable and efficient assay for phagocytosis research.

The method uses differential interference contrast microscopy to visualize yeast particles. Trypan blue was used to stain surface-bound yeast particles. Internalized particles remain unstained and visible. The method avoids switching illumination sources or filter sets. The researchers evaluated the method using macrophages and yeast particles. They tested the effects of horseradish peroxidase and cytochalasin D. The study also examined the role of macrophage plasma membrane regions in binding. Pre-incubation with mouse serum was used to assess cytochalasin D effects. The method was validated by comparing binding and internalization rates.

The method successfully distinguished surface-bound and internalized yeast particles. Trypan blue staining allowed clear visualization of surface-bound particles. Internalized particles were visible without additional steps. Horseradish peroxidase inhibited only the binding stage of phagocytosis. Cytochalasin D inhibited both binding and internalization. Pre-incubation with mouse serum altered cytochalasin D effects. Surface-bound yeast particles failed to trigger internalization. This failure was not due to yeast surface modifications or non-phagocytic receptors. The method provided concurrent quantification of binding and internalization. The results suggest that binding site location affects internalization.

The method described here allows concurrent quantification of binding and internalization. Trypan blue staining with differential interference contrast microscopy is effective. The method avoids switching illumination sources or filters. Horseradish peroxidase inhibits only the binding stage of phagocytosis. Cytochalasin D affects both binding and internalization. Pre-incubation with mouse serum modifies cytochalasin D effects. Surface-bound yeast particles do not trigger internalization. This may be due to binding site location on macrophage plasma membranes. The method may be useful for studying phagocytosis-mediating surface lectins. The findings suggest that binding site location influences internalization.