The overall goal of this essay, is to prepare macrophages and opsonized red blood cells for the visualization of Phagocytosis by three dimensional time lapse microscopy. This method can help answer key questions in the field of immunology, such as how do phagocytes capture and ingest particles. This technique allows the visualization of particle uptake in real time, unlike in more traditional end-point assays that only allow the assessment of whether but not how, a particle has been ingested.
Generally, individuals new to this method may struggle until they become accustom to the cell isolation procedures. We first had the idea for this method when we realized the advantages of spinning this confocal microscopy for 3D time-lapse imaging of Phagocytosis. To isolate peritoneal macrophages, place a 24 gauge plastic catheter into the peritoneum of a three to four month old mouse, and use a plastic five milliliter syringe to lavage the cavity two times with 4.5 milliliters of ice-cold HBSS without calcium and magnesium per lavage.
Transferring the aspirated suspension into a 14-milliliter polypropylene round bottom tube as it is collected. When both samples have been harvested, centrifuge the aspirate, and resuspend the peritoneal cells in one milliliter of complete bicarbonate free RPMI 1640 medium to achieve about a six times 10 to the sixth cells per milliliter concentration. Next, to pre-fill slides add one milliliter of complete HEPES supplemented medium to one reservoir of a fibronectin coated channel slide, and tilt the slide to allow the medium to be aspirated from the downstream reservoir.
To remove unwanted air bubbles, add one to two milliliters of fresh medium to one of the two reservoirs, and tightly apply a reservoir cap. Then apply rhythmic thumb pressure to the cap to pump any air out of the slide, tilting the slide as necessary. When all of the air has been expelled, tilt the slide toward the uncapped medium containing reservoir, and remove the cap to avoid air being sucked back into the channel.
Now pipette the cell solution a few times to reduce clumping, and add 100 microliters of the cells into one channel of the slide for a two hour incubation in a moist chamber at 37 degrees celsius in the absence of carbon dioxide. At the end of the incubation replace the HEPES containing medium in each slide with complete bicarbonate supplemented medium as just demonstrated. Then place the cells in a 37 degrees celsius humid incubator with 5%carbon dioxide for their overnight culture.
The next morning transfer one milliliter of freshly obtained healthy donor blood into a two milliliter round bottom polypropylene microcentrifuge tube. Then centrifuge the sample. Remove the plasma and buffy coat containing supernatant.
And carefully transfer 100 microliters of the sedimented red blood cells into a two milliliter mircocentrifuge tube containing 100 microliters of complete HEPES supplemented medium. Label the tube 1:1 and transfer four microliters of the diluted red blood cells into a new two milliliter microcentrifuge tube containing two milliliters of complete HEPES supplemented medium. Then label this tube 4:2000 and place both diluted red blood cell sample tubes on ice.
To label the peritoneal macrophages replace the bicarbonate medium in each channel slide with complete mediums supplemented with HEPES. Then 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 4:2000 red blood cells sample and transfer 400 microliters of the cells into a new two milliliter microcentrifuge tube, in a heated aluminum block inside a laminar flow hood for about five to eight 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. Then place the cells back in the heat block.
After five 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 one to 1.5 milliliter pipette tip carefully remove the supernatant in two volumes without disturbing the pellet and mix 2000 microliters of fresh HEPES supplemented complete medium with the cells.
Centrifuge and re-suspend 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 one milliliter of fresh complete HEPES supplemented medium.
To opsonize the red blood cells add one microliter of mouse IGG2B monoclonal anti human CD235A antibody to the PMS sample tube. After eight 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 disc confocal microscope.
With the stage incubator set to 37 degrees celsius and immediately begin imaging the cells. Time lapse 3D-imaging of green fluorescent macrophages presented with red fluorescent human red blood cells enables the visualization of single particle phagocytic events. For example, the capture of a mouse IGG opsonized human red blood cell by a macrophage filopodium can be observed.
As well as, the squeezing of a human red blood cell during phagocytic cup formation. After watching this video you should have a good understanding of how to isolate mouse resident peritoneal macrophages, fluorescently labeled cells, and opsonized particles.
