Figure 5. Ex vivo egress in toxoplasmic exudates.

(A) Washed exudate cells (‘host’) prepared from mice 5d post-infection with GFP-RH were labeled in situ with Hoechst 33342 and mixed 1:1 with ‘donor’ exudate cells prepared from mice infected with YFP-RH. Each preparation represents a pool of 2 - 3 mice. Co-cultures were carried out for 1 h in non-adherent dishes in the presence of 1 mM EDTA + 10 U/ml heparin to reduce aggregation. The fate of donor YFP-RH was assessed by flow cytometry. The identity of donor and host cells bearing YFP-RH was verified by monitoring the intensity of GFP and YFP signals as discussed for Fig. 2 (not shown). The value for donor cell-associated parasites at 0 h was set to 100. (B) An experiment similar to (A) was carried out, except that co-cultures (1.5 h) of washed exudate cells were compared to similar co-cultures of whole exudate (fluid + cells). Instead of Hoechst dye, one exudate was labeled with DDAO-SE. The donor:host ratio was 1:9. Each of the 3 mixes represents a separate pair of donor and host mice. Whole exudate samples were filtered before analysis to remove any coagulated material. The data in the figure display mean ± S.E. of 3 replicates and are representative of either 7 (A) or 2 (B) similar experiments.