Fig. 3

Flow diagram of in vitro oocyst culture. Gametocyte culture was seeded from an asexual culture at 2% parasitaemia and 5% hematocrit in 30 mL of complete media (RPMI with 10% A + human serum). Two flasks of 30 mL cultures were used for each experiment. Media changes were performed daily and percent gametocytaemia was determined. When one or more exflagellation events in 4000 RBC was reached, the culture was considered mature. Once the culture was matured, two flasks were combined, pelleted, and resuspended in 5 mL of ookinete media. Zygotes formed after 5 h with incubation at 27 °C and shaking at 50 RPM. Ookinetes transformed after a total of 24 h of incubation at 27 °C and shaking at 15 RPM. Ookinetes were purified using two sequential magnetic columns. Purified ookinetes were resuspended in oocyst media and plated into 8-well-chamber slides that were pre-coated with basal lamina components. Oocysts were incubated at 27 °C for up to 6 days in oocyst media