eoe sub articles

EoE Sub Articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Seeding of Peritoneal Cells in Channel Slides
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	<li>After pipetting the peritoneal mouse macrophage cell suspension up and down to reduce clumping, pipette 100 &#181;L suspension into the prefilled channel (volume, 100 &#181;L) of a fibronectin-coated channel slide (a 100 &#181;L channel has the dimensions (length x width x height): 50...

real-time imaging of macrophage phagocytosis of igg-opsonized red blood cells

Source: Horsthemke, M., et al. <a href="https://app.jove.com/v/57566">Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages.</a> J. Vis. Exp. (2018)
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.

<img alt="Figure 1" src="/files/ftp_upload/21782/21782_Figure1.jpg" /> 
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...

Real-Time Imaging of Macrophage Phagocytosis of IgG-Opsonized Red Blood Cells

Source: Horsthemke, M., et al. Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages. J. Vis. Exp. (2018)
This video demonstrates an assay to monitor ...

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.