Parasite growth assay using [3H]-hypoxanthine

Semi-synchronous 3D7, Dd2, FVO/FCR1, and HB3 in trophozoite/schizont stages at 2% hematocrit were plated in 96-well plates with a starting parasitemia of 2.5–3.0% and the cultures were kept shaken throughout the experiment to mimic in vivo conditions and limit the number of multiple invasion events into a single RBC. Purified RII and/or antibodies were immediately added to cultures after plating. These cultures were allowed to progress through 1.5 cycle (72 hr) in assay medium (90% hypoxanthine-free rich medium mixed with 10% rich medium). [³H]-hypoxanthine (0.5 μCi/well) was then added and parasites were incubated for a further 18–24 hr in assay medium. The assay duration (96 hr) corresponds to approximately two intraerythrocytic developmental cycles. The assay is stopped by harvesting the well contents onto a filter paper (Skatron FilterMAT 11731) using a semiautomatic cell harvester (Skatron Instruments, Sterling, VA). After air dried, the mats were analyzed using Beckman LS6000 IC liquid scintillation counter. Each [³H]-hypoxanthine incorporation measurement was performed in duplicate (in accordance with ALARA for the use and disposal of radioactive waste) for three independent times.