An Assay for Quantifying Antibody-Mediated Phagocytosis of Parasite-Infected Erythrocytes
Transcription
Take Plasmodium falciparum-infected erythrocytes. Label the parasite DNA with a fluorescent dye for parasite visualization.
Incubate these parasite-infected erythrocytes with sera from malaria-exposed individuals. These cells display parasite antigens on their surface, which attach to naturally acquired human IgG antibodies in sera, opsonizing cells for phagocytosis.
Transfer the opsonized parasite-infected erythrocytes to a protein-coated multi-well plate containing a suspension of human-derived monocytes — or phagocytic cells.
During incubation, the Fc-gamma receptors on monocytes interact with the IgG-opsonized erythrocytes, triggering the engulfment of erythrocytes into phagosomes.
Post-incubation, centrifuge the plate to prevent further cellular interactions and stop phagocytosis.
Discard the supernatant. Treat with ammonium chloride lysing solution, selectively rupturing non-phagocytosed infected erythrocytes without affecting monocytes.
Add serum-containing buffer to stop the lysing process. Centrifuge and discard the supernatant containing lysed erythrocytes.
Resuspend the monocytes in a buffer.
Perform flow cytometry to quantify monocytes that display fluorescence from the parasite-infected erythrocytes within them, confirming their antibody-mediated phagocytosis.
