An Assay for Quantifying Antibody-Mediated Phagocytosis of Parasite-Infected Erythrocytes

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

1. Parasite culture

NOTE: Follow the local regulations for human pathogens handling.

1. Maintain Plasmodium falciparum parasites according to the standard protocol as described before in the parasite culture medium (see Table of Materials).
2. Keep the parasites tightly synchronized using sorbitol treatment as previously described.
3. To ensure VAR2CSA expression on the surface of the Plasmodium falciparum-infected erythrocytes (IE), perform repeated rounds of immune-magnetic selection using an anti-VAR2CSA antibody (e.g., PAM1.4 antibody, a cross-reactive human monoclonal VAR2CSA-specific IgG) coupled to protein G-magnetic beads.
   1. In brief, incubate late-stage trophozoite-IEs with magnetic beads coupled to an anti-VAR2CSA antibody and positively select using a magnet.
   2. Expand the selected parasites in culture for a few cycles until parasitemia is at least 5%.
   3. Alternatively, select parasites that bind to plastic-immobilized CSA (the receptor for VAR2CSA) as previously described.
4. To verify VAR2CSA expression on the selected parasites perform flow cytometry as previously described.
   1. In brief, label the late-stage trophozoite-IEs with the same antibody used for the magnetic selection (step 1.3) followed by a fluorescently labeled secondary antibody.
   2. Measure the IE surface reactivity by flow cytometry. Successful antibody selection normally results in a parasite population that expresses VAR2CSA in most of the parasites (≈80%).
5. For the phagocytosis assay, use purified mid to late-stage trophozoite-IEs. Perform purification using magnetic separation as previously described.
   NOTE: To accurately determine the stage of the parasites, perform Giemsa staining on blood smears followed by light microscopy observation. Follow standard procedures and morphological guidelines as described before. Late-stage purification can also be performed using a density gradient medium. The IE yield, however, in our experience is lower, and, therefore, magnetic purification is preferable.
   1. For magnetic separation, spin the parasite culture (5-10% parasitemia) at 500 x g for 10 min at room temperature. Remove the supernatant and re-suspend the cell pellet in 10 mL of parasite culture medium.
   2. Add the parasite suspension into a Claricep Flash Silica, CS-column coupled to a magnet (see Table of Materials) and let it slowly pass through the column. Trophozoite-IEs are paramagnetic (due to the presence of hemozoin) and will stay in the column mesh.
   3. Wash the column with 50 mL of parasite culture medium and elute the IEs from the magnetic column using 50 mL of parasite culture medium.
6. Spin the eluate from 1.5.3 at 500 x g for 10 min at room temperature. Carefully discard the supernatant and re-suspend it in 1 mL of parasite culture medium.
7. Using a hemocytometer, count the number of IEs (easily identified by light microscopy as erythrocytes with dark hemozoin pigment) and total cell number to calculate the final parasitemia (percentage of IEs).
   NOTE: Only use parasites with at least 80% purity (80% IEs).
8. Based on the number of serum/antibody samples planned for testing, estimate the total amount of IEs needed and scale up the parasite cultures accordingly. A 75 cm² culture flask with 25 mL of culture at 5% hematocrit and 5-10% parasitemia should yield enough IEs to run a full 96-well plate. For a full plate (42 samples plus 6 controls in duplicate), a minimum of 1.5 mL parasite suspension at 3.3 x 10⁷ IEs/mL is needed.

2. THP-1 cells

NOTE: The THP-1 cell line is used in this assay. This monocyte cell line is derived from a patient with monocytic leukemia and can be purchased from the American Type Culture Collection (ATCC). Maintain the cell line according to the provider's instructions in the THP-1 culture medium (see Table of Materials).

1. For regular maintenance, start THP-1 cell culture at 2-4 x 10⁵ cells/mL and subculture when cell concentration reaches 8 x 10⁵ cells/mL.
   NOTE: Do not allow the cell concentration to exceed 1 x 10⁶ cells/mL and keep track of passage number. Avoid the use of cells that are beyond passage 25. The average doubling time of the THP-1 cell line varies between 19-50 h. Determine the doubling time of the cell batch before starting the phagocytosis experiments. This will help to roughly estimate the necessary amount of culture needed for a determined number of serum samples to be tested (e.g., for a full 96 well plate, 10 mL of culture at 5 x 10⁵ cells/mL are needed).
2. Periodically check the THP-1 cells for the surface expression of the Fcγ-receptors I (CD64), II (CD32), and III (CD16) as previously described.
   1. In brief, stain the THP-1 cells (separately) with anti-CD64, anti-CD32, and anti-CD16 fluorescently labeled antibodies (Table of Materials) for 30 min at room temperature (1:100 dilution prepared in 2% fetal bovine serum (FBS) in phosphate-buffered saline (PBS).
   2. Wash three times with 2% FBS in PBS and measure surface staining by flow cytometry.        
      NOTE: The THP-1 cells are negative for CD16 and positive for CD32 and CD64 as previously reported (Figure 1).
3. For the phagocytosis assay, set up a THP-1 cell culture flask with 2.5 x 10⁵ cells/mL the day before the experiment to yield around 5 x 10⁵ cells/mL on the day of assay.     
   NOTE: Ensure 4-6 x 10⁵ cells/mL are present on the day of the assay.

3. Phagocytosis assay (Figure 2)

1. Before starting the assay, block (>1 h) two round-bottom 96 well plates (one for the opsonization and another for the phagocytosis) using 150 μL per well of sterile 2% FBS in PBS.         
   NOTE: Label plate 1 as an opsonization plate and use it both for ethidium bromide (EtBr) staining and opsonization. Label plate 2 as a phagocytosis plate and use both for THP-1 cell plating and for the phagocytosis step.
2. Parasite staining and opsonization
   1. Take the IE suspension prepared in 1.6 and adjust cell count to 3.3 x 10⁷ IEs/mL by adding parasite culture medium and ethidium bromide (EtBr) to achieve a final concentration of 2.5 μg/mL (1:40 dilution, from a 0.1 mg/mL stock).
      NOTE: EtBr stains the parasite DNA, allowing detection by flow cytometry.
      CAUTION: EtBr is a mutagen and skin, eye, and respiratory irritant. Use appropriate protection and dispose of waste according to local regulations.
   2. Remove the blocking solution from one of the 96 well plates (opsonization plate), flicking the plate and removing liquid excess over a piece of towel paper.
   3. Add 30 μL per well of the IE suspension prepared above in the upper half of the plate. Leave one well empty and add 30 µL of parasite culture medium (without IEs for the THP-1 alone control). (Figure 3A). Incubate for 10 min at room temperature and protected from light.
   4. Add 170 µL per well of parasite culture medium. Spin the plate at 500 x g for 3 min at room temperature and remove the supernatant by flicking the plate over an appropriate waste container.
   5. Wash the EtBr-labeled IEs two more times using 200 µL per well of parasite culture medium spinning the plate at 500 x g for 3 min at room temperature. The supernatant from the first wash can be removed by flicking the plate. Carefully remove the supernatant from the second wash using a multichannel pipette to make sure the entire volume of the washing medium is removed. Do not disturb the pellet.
   6. Re-suspend the EtBr-labeled IEs in 30 μL of antibody/plasma/serum solution prepared at the desired concentrations in a parasite culture medium.
   7. Always include the following controls (Figure 3B): a control without IEs/THP-1 cells control (parasite culture medium); a control without any antibody or plasma/serum (un-opsonized control); a positive control using the commercially available rabbit anti-human erythrocyte antibody at 1:100 dilution (see Table of Materials); two negative plasma/serum controls at 1:5 dilution (a malaria-naïve pool and a pool from malaria-exposed males); and a positive serum control at 1:5 dilution (a pool from malaria-exposed women, who have previously been pregnant (preferably multigravidae)).
      NOTE: A 1:100 dilution for the positive control seems to work consistently across different batches of purchased antibodies (data not shown). However, it is recommended to rule out variations between batches by testing several dilutions every time a new batch of antibodies is used.
   8. Incubate for 45 min in the dark at 37 °C.
3. THP-1 cells preparation and phagocytosis
   1. While the IEs are being opsonized (3.2.8), begin preparing the THP-1 cells.
   2. Remove the THP-1 cells from the culture flask, spin them down (500 x g for 5 min at room temperature), decant the supernatant, and re-suspend the pellet in the THP-1 cell culture medium.
   3. Spin again (500 x g for 5 min at room temperature), decant the supernatant, and re-suspend the cell pellet in 1 mL of THP-1 cell culture medium.
   4. Determine cell count in the solution prepared above and add more medium to obtain a final concentration of 5 x 10⁵ cells/mL.
   5. Remove the blocking solution from the remaining 96-well plate (phagocytosis plate) by flicking the plate and removing liquid excess over a piece of towel paper.
   6. Add 100 μL per well of the THP-1 cell suspension prepared in 3.3.4 and put it back in the cell culture incubator (Figure 3C).
   7. Once the antibody/plasma/serum incubation time has finished, add 170 µL per well of parasite culture medium. Spin the plate at 500 x g for 3 min at room temperature and remove the supernatant by flicking the plate over an appropriate waste container.
   8. Wash the opsonized IEs two more times using 200 µL per well of parasite culture medium, spinning the plate at 500 x g for 3 min at room temperature. The supernatant from the first wash can be removed by flicking the plate. Carefully remove the supernatant from the second wash, using a multichannel pipette to make sure the entire volume of washing medium is removed. Do not disturb the pellet.
   9. Finally, re-suspend the opsonized IEs in 100 µL of pre-warmed THP-1 cell culture medium. Transfer 50 µL of opsonized IE suspension to each well in the phagocytosis plate. Since there is a total of 100 µL of IEs, each antibody/serum dilution can be run in duplicates in the phagocytosis plate (Figure 3D)   
      NOTE: The amount of IEs and THP-1 cells used in the assay correspond to a 10:1 ratio.
   10. Incubate for 40 min in the dark at 37 °C, 5% carbon dioxide (CO₂).
       NOTE: Do not allow the phagocytosis to proceed for more than 40 min.
   11. Stop the phagocytosis by centrifugation at 4 °C (500 x g, 5 min) and discard the supernatant by flicking the plate. Add 150 µL of room-temperature ammonium chloride lysing solution (Table of Materials) and mix by pipetting, incubate for exactly 3 min.
       NOTE: This step will lyse the erythrocytes that have not been phagocytosed by the THP-1 cells.
   12. Stop the lysis by adding 100 µL of ice-cold 2% FBS in PBS. Spin the plate at 4 °C (500 x g for 3 min) and remove the supernatant by flicking the plate over an appropriate waste container.
   13. Wash three times using 200 µL per well of ice-cold 2% FBS in PBS spinning the plate at 4°C (500 x g for 3 min). After the final wash, re-suspend in 200 µL of ice-cold 2% FBS in PBS and immediately, analyze by flow cytometry.
       NOTE: Previous publications have used cell fixation in 2% paraformaldehyde prior to flow cytometry³¹, but the results presented here were acquired immediately after assay completion. Postponing flow cytometry data acquisition by storing the plate at 4°C is not recommended, since the percentage of EtBr⁺ THP-1 cells decays rapidly (Figure 4).

4. Flow cytometry acquisition and analysis     

NOTE: Any flow cytometer supporting 96-well plate format and having the appropriate lasers/filters to measure EtBr fluorescence can be used.

1. For acquisition, gate on THP-1 cells using a linear forward-scatter (FSC) vs. linear side-scatter (SSC) plot using the wells where no IEs were added (Figure 2A) and acquire 10,000 events on this gate.
2. Measure fluorescence intensity for EtBr (FL3-Log) on the THP-1 gate using a histogram plot.
3. For gating, first gate on THP-1 cells in an FSC vs. SSC density plot, using the wells where no IEs were added (Figure 5A). Then set up a positive gate in an FL3 (EtBr) histogram, using the THP-1 cells (no IEs added) and the un-opsonized control (Figure 5B).
4. Copy these gates in all the other samples tested in the same plate and determine the percentage of EtBr-positive THP-1 cells (THP-1 cells that have phagocytized at least one IE). For each of the samples tested, phagocytosis can be reported as the absolute values (percentage of EtBr⁺ THP-1 cells) or as relative phagocytosis calculated as the percentage using the positive control as maximum.