This article describes a method for opsonizing red blood cells (RBCs) with polyclonal antibodies and their subsequent phagocytosis by monocytes. The process involves incubating RBCs with antibodies, followed by interactions with monocyte monolayers, leading to actin cytoskeleton reorganization and phagocytosis.
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.
To opsonize R2R2 red blood cells, first, wash the red blood cells three times in PBS. Dilute the R2R2 pellet after the third centrifugation at a one-to-one ratio with polyclonal anti-D antibodies from human serum for a one-hour incubation at 3 degrees Celsius with intermittent mixing. At the end of the opsonization, remove the cells from the incubator, and then wash them three more times in PBS as just demonstrated.
Resuspend the pellet to a 1.25% volume-to-volume in complete RPMI medium. Next, replace the supernatant from each well of the eight-chamber slide with 400 microliters of the opsonized R2R2 cells for two hours at 37 degrees Celsius. At the end of the incubation, use the slide adapters to remove the chambers, dabbing the excess R2R2 with a paper towel.
Now, fill a 100-milliliter beaker with PBS, and slowly dip each slide 30 to 40 times in the salt solution to remove the majority of the unphagocytosed R2R2. After drying, fix the slides in 100% methanol for 45 seconds, and allow the cells to dry before mounting the samples with coverslips.
The next day, load each slide onto a phase contrast microscope with a 40x objective lens, and manually count at least 200 monocytes and the number of phagocytosed R2R2 within each monocyte using one counter in each hand to simultaneously quantify the number of monocytes and the number of phagocytosed R2R2 cells per sample.
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