A novel flow cytometric phagocytosis assay of malaria-infected erythrocytes.
Monocytes play a crucial role in controlling malaria infection. To facilitate our research into the development of antibody-mediated immunity against pregnancy-associated malaria we have established several novel malaria-specific flow cytometric phagocytosis assays based on ethidium bromide staining of DNA present in blood stage trophozoites. The first assay quantifies the ability of sera to opsonise trophozoites and promotes phagocytosis by the monocytic cell line THP1. This measures the levels of functional antibodies to the chosen strain of parasite. The second assay is a whole blood phagocytosis assay which measures the phagocytic ability of patient monocytes ex vivo. The third assay employs simultaneous labelling of trophozoites with ethidium bromide and erythrocytes with fluorescein isothiocyanate to compare phagocytosis of both non-infected and parasitised erythrocytes to assess possible bystander effects on uninfected erythrocytes. These assays have the advantage over other malaria phagocytosis assays in that they are rapid, simple and specific to malaria-infected cells and avoid potential bias associated with manual counting.
Tippett
Emma
E
AIDS Pathogenesis and Clinical Research Program, Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Australia. emma@burnet.edu.au
Fernandes
Liselle A
LA
Rogerson
Stephen J
SJ
Jaworowski
Anthony
A
D000818:Animals; D000954:Antigens, Surface / Q000276:immunology; D002460:Cell Line; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D004912:Erythrocytes / Q000737:chemistry / Q000276:immunology* / Q000469:parasitology; D004996:Ethidium / Q000737:chemistry; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007700:Kinetics; D008288:Malaria / Q000097:blood / Q000276:immunology*; D009000:Monocytes / Q000166:cytology / Q000276:immunology; D009895:Opsonin Proteins / Q000276:immunology; D010587:Phagocytosis / Q000276:immunology*; D010963:Plasmodium falciparum / Q000235:genetics / Q000276:immunology; D011247:Pregnancy; D012156:Reticulocytes / Q000737:chemistry / Q000276:immunology / Q000469:parasitology; D053058:Trophozoites / Q000276:immunology
D000954:Antigens, Surface; D016054:DNA, Protozoan; D007106:Immune Sera; D009895:Opsonin Proteins; D004996:Ethidium; D016650:Fluorescein-5-isothiocyanate