Plasmodium falciparum Synchronization and Culture

After donation, blood was immediately processed as previously described [21 (link)]. Briefly, RBCs were separated from plasma and leukocytes by three washings in wash medium [RPMI 1640 (Invitrogen) containing 2 mM glutamine, 25 mM HEPES, 20 mM glucose, 27 μg/mL hypoxanthine and 32 μg/mL of gentamicin (Sigma) (pH 7.2)]; P. falciparum strain Palo Alto was then cultured at 1–5% hematocrit [33 (link)] under a 1% O₂, 5% CO₂, and 94% N₂ atmosphere in complete media (CM) [wash medium supplemented with 0.5% Albumax II (Gibco)]. Parasites were synchronized using a Percoll gradient method, as previously described [24 (link)]. After allowing the synchronized parasites to mature and reinvade fresh RBCs, drug studies were performed at the desired times as hours post-invasion (hpi). To assess parasitemia and infected cell morphology, thin smears were prepared, labeled with Diff-Quick stain (Siemens), and examined by light microscopy.