A Simplified, Sensitive Phagocytic Assay for Malaria Cultures Facilitated by Flow Cytometry of Differentially-Stained Cell Populations

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Abstract
Phagocytosis of infected and uninfected erythrocytes is an important feature of malaria infections. Flow cytometry is a useful tool for studying phagocytic uptake of malaria-infected erythrocytes in vitro. However, current approaches are limited by the inability to discriminate between infected and uninfected erythrocytes and a failure to stain the early developmental ring stages of infected erythrocytes. The majority of infected erythrocytes in circulation are of the ring stage and these are therefore important targets to study. In vitro P. falciparum cultures comprising infected and uninfected erythrocytes were labeled and exposed to cells derived from the human monocytic THP-1 cell line. Phagocytosis was assayed by flow cytometry. Dual labeling of Plasmodium DNA and erythrocyte cytoplasm with dihydroethidium and CellTrace™ Violet respectively allowed, for the first time, the detection and enumeration of phagocytes with ingested erythrocytes from both early ring- and late schizont-stage P, falciparum cultures. The sensitivity of the method was tested using varying conditions including phagocyte type (monocytes versus macrophages), parasite stage (rings versus schizonts), and negative (incubation with cytochalasin D) and positive (incubation with immune sera) effectors of phagocytosis. The current assay clearly demonstrated uptake of infected and uninfected erythrocytes exposed to phagocytes; the extent of which was dependent on the conditions mentioned. We describe a simple, sensitive and rapid method for quantifying phagocytosis of P. falciparum-infected erythrocytes, by flow cytometry. This approach can be applied for studying parasite-phagocyte interactions under a variety of conditions. The investigation of phagocytosis of P. falciparum-infected erythrocytes can extend from looking solely at late-staged infected erythrocytes to include early-staged ones as well. It does away with the need to purify infected cells, allowing the study of effects on neighboring uninfected cells. This method may also be translated for use with different types of phagocytes.

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