P falciparum infects erythroblasts at all stages of terminal differentiation. (A) Schematic diagram summarizing our primary cell culture system for ex vivo erythropoiesis. (B) Schematic diagram depicting positive selection of late-stage P falciparum using a magnetic column before infection of erythroblast cultures. (C) May-Grünwald Giemsa staining showing ring-stage parasites in the cytoplasm of erythroid cells at multiple stages of terminal differentiation. Samples were collected from 18 to 20 hours after mixing with schizonts. Scale bars represent 10 μm. Arrows indicate ring-stage parasites. (D) Percentage of GFP⁺ erythroblasts measured via flow cytometry from 18 to 20 hours after mixing with schizonts from a GFP-expressing parasite strain, D10-PfPHG. Erythroblasts were infected on day 10 (left) or day 14 (right) of differentiation. (E) Gating for FACS of GFP⁺ erythroblasts 18 or 20 hours after infection with D10-PfPHG at multiplicity of infection = 1. Erythroblasts were infected on day 7 of differentiation. Fluorescence microscopy showing localization of GFP in an infected erythroblast sorted from the GFP⁺ population. Arrow indicates Hoechst staining overlapping the GFP. (F) Flow cytometry analysis showing transition of infected, day 7 erythroblasts into ProE and BasoE. (G) Flow cytometry analysis showing the transition of infected, day 14 erythroblasts into PolyE and OrthoE. EPO, erythropoietin; IL-3, interleukin-3; MOI, multiplicity of infection; RBC, red blood cell; SCF, stem cell factor; SSC, side scatter.