In vivo parasite invasion assay. Blood samples were taken from P. chabaudi adami DS infected SJL/J mice 30 mins and 3 hrs after injection with labelled RBCs and stained with JC-1, Hoechst 33342, anti-CD45 APC eFluor780, anti-CD71 PerCP eFluor710 and Streptavidin PE-Cy7. Cells were gated to G3 as described in Figure1 and then gated based on their respective label (A). Cells from Q1 (Biotin labelled) were gated based on JC-1 and Hoechst fluorescence and the parasitaemia was determined as Q1b divided by Q1 (B). Similarly, parasitaemia of cells from Q3 (Atto 633 labelled) was determined as Q3b divided by Q3 (C), and endogenous parasitaemia was determined based on the cells from Q4 (unlabelled). As a control experiment the two labelled RBC populations were untreated (D). To quantify the effect of protease treatment on invasion one labelled RBC population was treated with trypsin, chymotrypsin and neuraminidase, while the other was left untreated (E). Parasitaemia ratio calculated as the parasitaemia of the labelled RBCs of interest divided by the parasitaemia of the labelled control RBCs for each individual sample (F). Results are from five or six infected mice with RBC labels switched between treated and untreated to account for any invasion difference due to the RBC label. Error bars represent SEM, **p-value < 0.01, ***p-value < 0.001.