Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

This protocol allows to measure the ability of antibodies to opsonize and induce the phagocytosis of plasmodium falciparum infected erythrocytes. Antibodies present in the serum of individuals naturally exposed to parasite infection, as well as those induced by immunization with parasite antigens, can be tested. Demonstrating the procedure with me will be Maiken Visti, a technician from my laboratory.

To set up a THP-1 cell culture for a phagocytosis assay, the day before the experiment seed the cells in a 25-centimeter squared culture flask and a concentration of 2.5 times 10 to the fifth cells per milliliter in THP one cell culture medium. On the day of the experiment, at least one hour before the experiment block to round-bottom 96-well plates with 150 microliters of sterile 2%FBS in PBS per well. For mid-to late-stage trophozoite-infected erythrocytes harvest and purification, prepare around 1.2 milliliters of packed P falciparum parasite culture in 10 milliliters of parasite culture medium, with at least 5%parasitemia and add to a magnetic column.

Wash the column with 50 milliliters of parasite culture medium. To elute the infected the erythrocytes, remove the column from the magnet and flush it with 50 milliliters of parasite culture medium. Then, collect the infected erythrocytes by centrifugation and resuspend the pellet in one milliliter of fresh parasite culture medium for counting.

Adjust the purified infected erythrocyte suspension by 3.3 times 10 to the seventh cells per milliliter in parasite culture medium containing ethidium bromide, to a final concentration of 2.5 micrograms per milliliter. Next, remove blocking solution from one 96-well plate by flicking the plate into a waste container. Then remove any excess over a paper towel and add 30 microliters of the ethidium bromide label infected erythrocyte suspension to all but one well in the upper half of the plate.

Add 30 microliters of parasite culture medium to the empty well, and incubate the plate for 10 minutes at room temperature, protected from light. At the end of the incubation, add 170 microliters of parasite culture medium to each well and sediment the infected erythrocytes at the bottom of the plate by centrifugation. Remove the supernatant by flicking the plate into a waste container, and wash the ethidium bromide-labeled infected erythrocytes two more times, with 200 microliters of parasite culture medium per well, as just demonstrated.

After the second wash, use a multi-channel pipette to carefully remove the entire volume of supernatant from each well, without disturbing the pellets. And resuspend the ethidium bromide-labeled infected erythrocytes in 30 microliters of antibody solution, including the appropriate controls and test samples previously prepared in parasite culture medium. Then, place the plate at 37 degrees Celsius for 45 minutes, protected from light.

While the infected erythrocytes are being opsonized, collect the THP-1 cells by centrifugation, and resuspend the pellet in 12 milliliters of fresh, pre-warmed THP-1 cell culture medium. After a second centrifugation, resuspend the pellet in one milliliter of THP-1 cell culture medium for counting, and adjust the cell concentration to five times 10 to the fifth cells per milliliter in THP-1 cell culture medium. Remove the blocking solution from the second 96-well plate and add 100 microliters of THP-1 cells to each well.

Then, place the plate in the cell culture incubator. At the end of the opsonization incubation, add 170 microliters of parasite culture medium to each well of the opsonization plate, and sediment the infected erythrocytes to the bottom of the plate by centrifugation. Wash the opsonized infected erythrocytes two times with 200 microliters of parasite culture medium per well, using a multi-channel pipette to carefully remove the supernatant after the second wash.

Resuspend the opsonized infected erythrocytes in 100 microliters of pre-warmed THP-1 cell culture medium per well, and transfer 50 microliters of each opsonized infected erythrocyte suspension to a corresponding well in the phagocytosis plate. When all of the cells have been added, place the plate in the cell culture incubator for no more than 40 minutes, protected from light. At the end of the incubation, stop the phagocytosis by centrifugation at four degrees Celsius.

Remove the supernatant and resuspend the pellets with 150 microliters of room-temperature ammonium chloride lysing solution per well. After exactly three minutes, add 100 microliters of ice cold at 2%FBS in PBS to stop the lysis, and sediment the intact cells by centrifugation. Next, wash the cells three times with 200 microliters of fresh ice cold 2%FBS in PBS per well, per wash.

After the final wash, resuspend the cell pellet in 200 microliters of fresh ice cold 2%FBS in PBS per well. For flow cytometry acquisition and analysis, immediately load the cells onto a flow cytometer and use the linear forward versus linear side scatter plot for the wells without infected erythrocytes to gate the THP-1 cells. Acquire 10, 000 events in this gate, and use the THP-1 cells with the infected erythrocytes opsonized with a positive control to set up a histogram plot to measure the ethidium bromide fluorescence intensity.

For analysis, open the linear forward versus linear side scatter plot for the wells without infected erythrocytes, and gate the THP-1 cells. Then, set up a positive gate in an FL-3 histogram and copy these gates onto all of the other sample wells to determine the percentage of ethidium bromide-positive THP-1 cells. For each sample tested, phagocytosis can be reported as the absolute value or as the relative phagocytosis calculated as a percentage using the positive control as the maximum.

The THP-1 cells should be periodically checked for FC gamma receptor surface expression by flow cytometry. The cells should be negative for CD16, and positive for CD32 and CD64. In this opsonization experiment, the THP-1 cells were gated first according to their forward and side scatter profiles, to allow quantification of the percentage of ethidium bromide-labeled cells, indicative of cells that have phagocytized at least one antibody opsonized infected erythrocyte.

The negative control samples should all generate a single negative peak in the FL3 channel, with few events observed in the ethidium bromide marker. Accordingly, the mean phagocytosis values, both as absolute ethidium bromide-positive THP-1 cells and as relative phagocytosis percentages, should be very low. In contrast, the positive control should generate traces with two peaks, a negative peak and a clearly positive and well-separated peak located within the ethidium bromide marker.

The mean phagocytosis values are normally highest for the positive control, followed by the malaria-exposed female pool and the single malaria-exposed woman controls. Although this methodology generates consistent results, deviations in the slope coefficient of the adjusted lines have been observed. Therefore, the use of relative values is recommended, especially if it's not possible to run all of the samples in a single experiment.

Be sure to carefully plan the experiment in advance, preparing both the THP-1 cells and the parasites accordingly, using only trophocytes with a purity greater than 80%In addition, be sure to always include the appropriate controls, to allow the quality of the experiment to be assessed and facilitate data analysis.