An overview of the assay methodology. Thin smears stained with new methylene blue (A-B) or Giemsa (D-G) illustrating the key methodologic steps of the ex vivo P vivax invasion assay. (A) Cord blood at collection contains a mixture of normocytes, reticulocytes, and leukocytes. (B) After 2 rounds of leukocyte depletion on a CF-11 cellulose column, reticulocytes form the majority of the cells present in the band (seen at the 5.5-mL mark on the tube pictured in panel C) obtained by enrichment on a 70% Percoll cushion. (D) Thin smear showing mature trophozoites (black arrows) and leukocytes typically observed from P vivax isolates collected from patients. (E) Typical yield of concentrated mature P vivax parasites obtained on a 45% Percoll cushion (from a band similar to the one depicted in panel C after a single round of leukocyte depletion on CF-11 and 20 hours ex vivo maturation). (F) The concentrated reticulocyte target cells (B) and mature P vivax schizonts (E) are mixed at a ratio of 6:1 for the invasion assay. (G) After culturing for ∼ 24 hours, the invasion assay mixture shows remnants of ruptured schizonts (*), and RBCs newly invaded by one (black arrow) or multiple merozoites (orange arrowhead). Black scale bar on the micrographs corresponds to 10 μm.