This article describes a method for studying the phagocytosis of opsonized red blood cells by mouse macrophages using confocal microscopy. The process involves labeling macrophages and red blood cells with fluorescent antibodies to visualize the phagocytic events in real-time.
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.
To label the peritoneal macrophages, replace the bicarbonate medium in each channel slide with complete medium supplemented with HEPES, and tilt the slide to allow 100 microliters of the fluorescently-tagged mouse macrophage-specific antibody of interest to be added drop-wise to the opening of the 100-microliter channel of the slide. Aspirate the medium that flows into the downstream reservoir, and incubate the cells for 20 minutes in a moist chamber at 37 degrees Celsius in the absence of carbon dioxide.
While the peritoneal cells are being labeled, gently mix the 4 to 2,000 red blood cell sample, and transfer 400 microliters of the cells into a new 2-milliliter microcentrifuge tube in a heated aluminum block inside a laminar flow hood for about 5 to 8 minutes.
When the cells have warmed to 37 degrees Celsius, mix 0.4 microliters of an appropriate red fluorescent plasma membrane stain with the cells, and place the cells back in the heat block. After 5 minutes, wash the cells by adding 1.6 milliliters of fresh HEPES-supplemented complete medium and centrifuge. Rotate the tube to identify the red blood cell pellet.
Using a 1- to 1.5-milliliter pipette tip, carefully remove the supernatant in two volumes without disturbing the pellet. Then, mix 2,000 microliters of fresh HEPES-supplemented complete medium with the cells. Centrifuge and resuspend the pellet in 400 microliters of medium. Then, label the tube PMS for Plasma Membrane Stain. At the end of the 20-minute peritoneal cell labeling incubation, wash the slide with 1 milliliter of fresh complete HEPES-supplemented medium.
To opsonize the red blood cells, add 1 microliter of mouse IgG2b monoclonal anti-human CD235a antibody to the PMS sample tube. After 8 minutes at 37 degrees Celsius with mixing once per minute, transfer 100 microliters of the plasma membrane-stained, IgG-opsonized, human red blood cells to the peritoneal macrophage-containing channel slide. As soon as the human red blood cells have been added, mount the slide on a spinning-disk disk confocal microscope with the stage incubator set to 37 degrees Celsius, and immediately, begin imaging the cells.
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