Quantification of Merozoite Phagocytosis

The process for quantification of merozoite-phagocytosis has been described in detail^{10 (link)}. Leukocytes were distributed in 96-well U-bottom plates containing 5 × 10⁴ cells in 100 μl of cell medium per well. Ethidium bromide-stained merozoites were resuspended in cell medium, opsonized with immune and non-immune plasma at a 1:100 dilution, and 100 μl were added to each well. After a 30 min incubation at 37 °C, cells were washed thrice with cold FACS buffer. Cells were resuspended in cold FACS buffer containing the following antibodies diluted 1:800: BV421 anti-human CD66b (clone G10F5, BD Biosciences 562940); APC anti-human CD45 (clone HI30, BD Biosciences 555485); and APC-AF750 anti-human CD14 (clone TuK4, Thermo Fisher Scientific MHCD1427) (Supplementary Fig. 1). After a 30 min incubation at 4 °C, cells were washed thrice with cold FACS buffer. Samples were quantified with a CytoFLEX S flow cytometer and analyzed with Kaluza Analysis Software version 2.1. Phagocytosis was measured using the 610/20 nm detector to quantify the ethidium bromide signal.