An Assay for Studying Fcγ Receptor-Driven Phagocytosis in Monocyte Monolayers

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.