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

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.