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

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Peripheral Blood Mononuclear Cell Isolation

1. Obtain human whole blood from a healthy donor or a patient via venipuncture using vacutainers containing acid-citrate-dextrose (ACD) anticoagulant (yellow-top tubes).
   NOTE: Whole blood can be stored in ACD at room temperature (18-22 °C) for up to 36 hours before proceeding to the next step. Normally, 1-2 10-mL vacutainer tubes of whole blood are sufficient for the assay.
2. Dilute the whole blood 1:1 v/v in warm complete RPMI medium (RPMI-1640 supplemented with 10% fetal bovine serum, 20 mM HEPES, and 0.01 mg/mL gentamicin).
3. Isolate peripheral blood mononuclear cells (PBMCs) from the diluted whole blood using density gradient centrifugation, as recommended by the manufacturer (See List of Materials). Layer the diluted blood very slowly over the density gradient (warmed to room temperature, 18-22 °C).
   NOTE: Minimize the amount of mixing at the interface for the optimal separation of blood by carefully layering the blood mixture in a dropwise fashion or by using a pipette.
   1. Allow the blood mixture to slowly layer over the top of the density gradient by placing the pipet tip close to the density gradient and by enabling the blood mixture to run down the side of the tube very slowly.
   2. Depending on the scale of the experiment, layer 10 mL of blood mixture on top of 3 mL of density gradient (in a 15-mL tube) or layer 35 mL of blood mixture on top of 15 mL of density gradient (in a 50-mL tube). Typically, 10 mL of whole blood yields 10 million PBMCs, with some donor-to-donor variation.
      NOTE: It is very important that there is no mixing of the blood mixture with the density gradient. The blood mixture should layer over the density gradient and slowly rise until all the blood is on top of the density gradient.
   3. Centrifuge the layered mixture at 700 x g for 30 min without brakes. The centrifuged mixture should be separated into 5 layers (from top to bottom): plasma, buffy coat (containing PBMCs), density gradient material, granulocytes, and RBCs.
   4. Remove and discard the majority of the plasma and carefully retrieve the buffy coat (PBMC) content into a new 15-mL tube using a Pasteur pipette and a suction bulb.
      NOTE: The removal of the buffy coat layer is effectively done by applying suction on the Pasteur pipette while performing a circular motion around the outside of the layer, with the tip of the pipette against the tube.
4. Wash the isolated PBMCs three times in pH 7.3 phosphate-buffered saline (PBS) by centrifuging at 350 x g for 10 min (with full brakes) in between washes. Reconstitute the PBMC pellet in complete RPMI medium. Depending on the size of the pellet, 3-7 mL of medium is sufficient.
5. Count the PBMC using trypan blue and a hemocytometer. Only count those cells that are not stained by the trypan blue. Reconstitute the PBMCs to 1,750,000 cells/mL in a complete RPMI medium.
6. Seed 400 µL (700,000 cells) into each well of an 8-chamber slide. Incubate the slide in a 37 °C, fully humidified tissue culture incubator (supplemented with 5% CO₂) for 1 h to allow the monocyte/macrophages to adhere.

2. Pre-treatment of Adhered Monocytes

NOTE: This step is only necessary if looking for ways to inhibit or enhance phagocytosis.

1. Pre-treat adhered monocytes with any drug(s) or compound(s) of interest. Reconstitute the drug(s) or other test material to the desired concentration using a complete RPMI medium.
   NOTE: For example, 200 µg/mL of IVIG is typically used to yield a 95-100% inhibition of phagocytosis when using human monocytes.
2. Aspirate and discard the supernatant containing any non-adherent cells from the 8-chamber slide after the 1-h incubation (after step 1.6). Replace it with 400 µL of a drug or another treatment and incubate for 1 h at 37 °C.
   1. When aspirating and replacing solutions in the 8-chamber slide, make sure to control the flow of the fluids so that weakly adhered cells do not lift off. Also, work with only 2-3 empty wells at a time and avoid drying the wells.
      NOTE: Typically, each treatment is performed in technical triplicates.

3. Opsonization of R₂R₂ Red Blood Cells

NOTE: Opsonized R₂R₂ RBCs are used as a positive control for FcγR-mediated phagocytosis. Naïve R₂R₂ should be stored in Alsever's solution (to prolong shelf life) at 4 °C for up to 1 month. Alsever's solution is made in-house and composed of 0.8% w/v trisodium citrate (dihydrate), 1.9% w/v dextrose, 0.42% w/v sodium chloride, and 0.05% w/v citric acid (monohydrate). If R₂R₂ cells are not available, other Rh-phenotyped cells, such as R₁R₂, R₁R₁, R₁r, or R₂r, can be used.

1. Wash R₂R₂ (cDE/cDE) red blood cells in PBS a total of three times using centrifugation at 350 x g for 5 min each.
   NOTE: The amount of RBCs needed depends on the experimental size and can be back-calculated. Always wash an excess amount of RBCs, since RBCs are lost during each wash due to lysis or during the removal of supernatant. For example, in an experiment with 5 treatments and 2 controls, all conducted in technical triplicates, there is a total of 21 wells. 21 wells with 400 µL/well of 1.25% RBC mixture indicate that 105 µL of packed, opsonized RBC is needed. 200 µL of RBC should be initially washed and 150 µL should be opsonized to ensure that there is an adequate amount of RBCs for the phagocytosis step.
2. Opsonize the washed R₂R₂ pellet with 1:1 v/v polyclonal anti-D antibodies from human serum and incubate for 1 h at 37 °C, with intermittent mixing.
   NOTE: If polyclonal anti-D antibodies are not available, it is possible to use monoclonal anti-D antibodies, which are commercially available, or anti-D that are normally used for Rh immune prophylaxis, which can be obtained from blood banks or transfusion services. Optimization testing should be conducted at the beginning when setting up the assay, and whenever switching lots of un-titered polyclonal antibodies or switching to a monoclonal antibody. This is to identify the optimal concentration or volume of anti-D required for an antiglobulin test (IAT) result of between 3+ and 4+ and a phagocytosis result of between 70-90 phagocytosed RBCs per 100 monocytes counted.
3. Wash the opsonized R₂R₂ a total of three times in PBS using centrifugation at 350 x g for 5 min each.
   NOTE: Successful opsonization of R₂R₂ can be confirmed by performing an indirect IAT. Briefly, secondary polyclonal anti-human antibodies are added to bind primary opsonizing antibodies on RBC surfaces, and the amplified signal can be observed in the form of hemagglutination. A detailed manufacturer protocol can be found in the supplementary materials file.
4. Reconstitute the washed R₂R₂ pellet to 1.25% v/v using a complete RPMI medium.
   NOTE: Excess opsonized R₂R₂ can be stored in Alsever's solution at 4 °C for up to a week.

4. Fc Receptor-mediated Phagocytosis

1. Aspirate the drug or medium supernatant from the 8-chamber slide and add 400 µL of the 1.25% v/v R₂R₂ mixture. Incubate at 37 °C for 2 h.
2. After 2 h of incubation, remove the chambers using the manufacturer's adaptors. Dab off excess R₂R₂ on a paper towel. Make sure that the slide does not dry out.
3. Fill a 100-mL beaker with PBS. Submerge and wash the slide by slowly moving the slide back and forth (around 30-40 strokes) to remove the majority of the unphagocytosed R₂R₂.
4. Remove the slide from the PBS. Dab off excess PBS using a paper towel or tissue and air-dry the slide.
5. Fix the air-dried slide in 100% methanol for 45 s. Then, air-dry the fixed slide.
   NOTE: Slides can be fixed using another method that is more compatible with downstream staining, such as the Grünwald-Giemsa stain21 or the Wright-Giemsa stain.
6. Mount the slide using an in-house-made Elvanol mounting medium (or another commercially available mounting medium) and add coverslips.
   NOTE: Elvanol mounting medium is composed of 15% w/v polyvinyl alcohol resin and 30% v/v glycerin in PBS. The mixture is heated until all the resin has dissolved and the glycerin is homogenously mixed. This can be replaced with other commercially available mounting medium.
7. Allow the mount to dry overnight before quantification.

5. Quantification of Phagocytosis

1. Using a phase-contrast microscope and a 40X objective lens, manually quantify the amount of phagocytic events by counting at least 200 monocytes and the number of phagocytosed R₂R₂ within these monocytes. Have one counter in each hand to simultaneously quantify the number of monocytes and the number of phagocytosed R₂R₂.
2. Obtain the average phagocytic index by dividing the number of phagocytosed R₂R₂ by the number of monocytes and multiplying by 100. Express the data as the mean (average phagocytic index) ± the standard error of the mean (SEM).