An Assay for Studying Fcγ Receptor-Driven Phagocytosis in Monocyte Monolayers
Transcription
Begin with red blood cells, or RBCs.
Add polyclonal antibodies specific to RBC surface antigens.
Incubate. The antibodies opsonize RBCs by coating them through specific binding to surface antigens.
Centrifuge and remove the unbound antibodies.
Add the resuspended antibody-opsonized RBCs to a multi-chambered slide containing monocyte monolayers. Incubate.
The surface Fc gamma receptors on monocytes bind to the Fc region of polyclonal antibodies bound to RBCs.
This binding causes Fc receptor clustering around the contact area and triggers intracellular signaling, resulting in actin cytoskeleton reorganization in monocytes.
This cytoskeleton rearrangement induces pseudopod extensions and creates a phagocytic cup around opsonized RBCs.
The phagocytic cup progressively encloses opsonized RBCs, leading to their complete engulfment within a membrane-bound phagosome.
Post-incubation, wash the slide with buffer to remove non-phagocytosed RBCs. Fix the cells.
Mount the slide using mounting media.
Perform phase-contrast microscopy to visualize and quantify the phagocytosed RBCs within monocytes.
