Figure 2. Transfer of parasite from donor to host cells in vivo.

Donor peritoneal exudate was obtained by paracentesis from mice infected 5d earlier with either GFP-RH or YFP-RH and transferred immediately (0.1 ml/recipient) to host mice bearing a comparably progressed infection with the opposite strain. Donor cells were labeled in situ with Hoechst 33342 1 h prior to transfer. Host mice were sampled by paracentesis at the indicated times and the samples were analyzed by flow cytometry. (A, B) A sample taken 1 h after a GFP→YFP transfer is used to illustrate gating to distinguish host- and donor-derived parasite (gates R1 and R2, respectively, in (A)) and donor and host cells (gates R3 and R4, respectively, in (B), gated on R2). The dot plot in panel A is gated on total cells, exclusive of free parasites. In untransferred donor cells, the percentage of R2-gated cells present in R4 is < 1%. The uniformity and stability of Hoechst 33342 labeling is detailed in Fig. S5. (C) At each timepoint, the fraction of total donor parasite burden that remained donor cell-associated was calculated (solid lines). Each line represents sequential sampling of a single animal. The data are representative of 5 mice. The corresponding recoveries of total donor parasite (intracellular + extracellular, as a fraction of total parasites) are shown as dashed lines (the value at 1 h post-transfer is set to 1). (D) GFP-RH histograms of donor (unshaded, solid line) and host cells (shaded) 1 h after GFP→YFP transfer. A histogram of untransferred donor cells is shown for comparison (dashed line).