Real-Time Imaging of Macrophage Phagocytosis of IgG-Opsonized Red Blood Cells
Transcription
Take a channel slide containing mouse macrophages. The channel connects to media-filled reservoirs, supplying nutrients to macrophages.
Replace media with media-containing fluorescently tagged antibodies, which bind to specific surface glycoproteins, labeling macrophages.
Take human red blood cells, RBCs, with fluorescently labeled cell membranes. Opsonize RBCs with mouse IgG antibodies, which bind specifically to the glycophorin A.
Add the IgG-opsonized RBCs to the macrophage-containing slide. Macrophage Fc receptors bind the Fc region of IgGs on RBCs, resulting in the clustering of Fc receptors around the contact area and marking the RBCs for phagocytosis.
Binding triggers intracellular signaling pathways and activates GTPases. Activated GTPases drive actin reorganization and polymerization, forming thin membrane protrusions termed filopodia.
Further, filopodia extend around the RBC, enclosing it within the developing phagocytic cup. The edges of the cup fuse, forming a phagosome that internalizes the opsonized RBC for degradation.
Using a confocal microscope, obtain time-lapse images of single phagocytic events.
