An IVET assay indicates normal invasion but increased clearance of Sptb-mutant erythrocytes. (A) Host mice were infected with 1 × 104P chabaudi parasites. At day 9 to 10 of infection, at the peak of schizogony, labeled blood from the 2 mutant lines and WT (+/+) mice was transfused into host mice (n = 5). Labels were switched to account for any dye effects. (B) Representative flow cytometry plots from a host blood sample are shown. Gating of whole cells (Bi), single cells (Bii), mature RBCs (Biii), and the 3 labeled cell populations (Biv) was performed, with other populations removed from analysis. The percentage of infected cells in each population was determined by JC-1/HO dual positive staining (Bv). The percentage of infected cells (ratio to +/+) was determined by dividing the percentage of infected cells (labeled mutant) by percentage of infected cells (labeled WT) in each host animal (in the example plot, for MRI26194/+, the ratio is 14.7/17.8 = 0.83) (Ci). Remaining cells (ratio to +/+) were determined by dividing the percentage remaining of all labeled mutant cells (compared with 2-minute time point) by the proportion remaining of all labeled donor WT cells in each host animal (Cii). A 1-sample t test was used between the sample values and the hypothetical value of 1 to test for ratio significance. *P < .05, **P < .01, ***P < .001. pRBC, parasitized RBC; WBC, white blood cell.
