Malaria Parasite Inhibitor Assay

Inhibitor IC₅₀s were calculated in parasite assays following the protocol described by Desjardins et al. (52 (link)). Briefly, cultures were synchronized to ring stage by D-sorbitol lysis, using a 5% D-sorbitol solution. The parasites were diluted to a final parasitemia of 1%. Hypoxanthine-free medium was added to give a hematocrit level of 4%. A 2-fold dilution series of the inhibitors in hypoxanthine-free medium was prepared, and 100 μL of each inhibitor concentration was seeded in a 96-well plate, with 5 technical replicates of each included. Two no-inhibitor controls, i.e., hypoxanthine-free medium only, were included. Plates were incubated at 37°C in 5% CO₂ for 24 h. After 24 h, [³H]hypoxanthine was added to each well to a final concentration of 0.5 μCi/well, and the plates were incubated for a further 24 h. Plates were then freeze-thawed to lyse the cells, and the [³H]hypoxanthine uptake was measured in a Beckman scintillation counter.
Parasite calcium homeostasis was measured as described by Pandey et al. (25 (link)), using 25 μM artemisinin derivative artemisone, and compared to results with the inactive derivative deoxyartemisone. Three biological replicates were carried out, after an initial blinded experiment.