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Real-Time Imaging of Macrophage Phagocytosis of IgG-Opsonized Red Blood Cells
Overview
This video demonstrates an assay to monitor independent macrophage-mediated red blood cell (RBC) phagocytosis events. Mouse immunoglobulin G (IgG)-opsonized human RBCs are incubated with mouse macrophages and observed under a confocal microscope. The RBCs are engulfed by macrophages via a phagocytic cup formation and targeted for lysosomal degradation.
Protocol
All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.
1. Seeding of Peritoneal Cells in Channel Slides
- After pipetting the peritoneal mouse macrophage cell suspension up and down to reduce clumping, pipette 100 µL suspension into the prefilled channel (volume, 100 µL) of a fibronectin-coated channel slide (a 100 µL channel has the dimensions (length x width x height): 50 mm x 5 mm 0.4 mm).
- Prefill the chamber by adding 1 mL serum-supplemented RPMI 1640 (Hepes) medium to one of the two reservoirs of the cha....
Representative Results
Figure 1: Handling of fibronectin-coated channel slides. (A) A channel slide consists of two reservoirs connected by a channel with the dimensions 50 mm x 5 mm 0.4 mm. Channel slides are initially prefilled by applying 1-2 mL medium to one of the two reservoirs and tilting the slide. (B) Caps can be placed onto the reservoirs prior to incubation. The caps can be conveniently used to pump out unwa.......
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Source: Horsthemke, M., et al. Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages. J. Vis. Exp. (2018)
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