Antimalarial Compound Screening Protocol

Following synthesis, compound powder was diluted to 10 mM in dehydrated, sterile DMSO (Tocris) and a duplicate-well, 12-point, 3-fold semi log dilution series was prepared at 1000x final concentration in 384 well plates (Greiner Bio-one) in DMSO using a Biomek 4000 (Beckman Coulter). Plates were sealed using foil sealing tape (VWR) and kept in a desiccator until used to inoculate P. falciparum assay plates. Assay plates were prepared by plating 20 μL of culture media to pre-wet all wells, followed by addition of compound using a 40-nL pin tool (V&P Scientific) as previously described.^{32 (link)} Plates were then inoculated with 20 μL P. falciparum culture at 2% parasitemia and 0.75% hematocrit, leading to dilution of all test compounds to 1x and DMSO to 0.1% in complete media. Assay plates were maintained in bioassay dishes with water cups to prevent edge effect due to evaporation for 72–96 hours as previously described.^{22 (link),25}