Parasite Invasion Susceptibility Assay

The assay was performed and analyzed as in^{25 (link)} using two different concentrations of CellTrace Far Red DDAO (Invitrogen Life Technologies/Molecular Probes): 1 µM (high) or 0.1 µM (low). Differentially labeled RBCs were combined in equal density in triplicate into a 96 well plate and seeded with MACS purified (Miltenyi Biotec) late stage parasites to achieve 1.5–2% pRBCs in 1% hematocrit. Plates were maintained for 12–18 hours to allow for schizont rupture and subsequent invasion of labeled RBCs. Invasion into high and low DDAO RBCs was detected by DNA dye Sybr Green I, and analyzed by flow cytometry as in^{1 (link)}, and the ratio of the prevalences of parasitized high to low DDAO RBCs was calculated to generate the “Susceptibility Index”. The parasite strains used were parent strain 3D7 and knockout strains ΔPfRh1, ΔPfRh2a, and ΔPfRh2b, all assayed in parallel for invasion into the same donor RBCs from non-anemic controls and anemic, pregnant subjects (n = 15). This allowed for comparison of invasion deficits of parent and transgenic parasite strains into anemic RBCs versus non-anemic RBCs.