NF54 P. falciparum Culture and Gametocyte Isolation

P. falciparum isolate NF54 [32] (link),[33] (link) was cultured using the automated tipper-table system of Ponnudurai et al [34] (link) as implemented in the CEPIA mosquito infection facility of Institut Pasteur. Briefly, a subculture of thawed NF54 stabilate was grown in 10 ml RPMI 1640 medium supplemented with 25 mM HEPES and L-glutamine, 10% heat-inactivated human serum, and sodium bicarbonate at 0.2% final concentration under a constant gas regime (5% CO2, 1% O2, 94% N2). Fresh anonymous erythrocytes obtained from blood banks were added to 7% final concentration. Fourteen days after initiating the subculture, gametocyte maturity was tested by exflagellation of microgametes, and parasitemia and numbers of mature male and female gametocytes were counted on Giemsa stained slides.
Ten ml of culture was centrifuged at 2000 rpm, and the cell pellet was resuspended in an equal volume of normal type AB human serum. The infected erythrocytes were added to fresh erythrocytes in AB human serum and were transferred to a membrane feeder warmed to 37°C. Mosquitoes were allowed to feed for 15 minutes, and only fully engorged females were used for further analysis. Bloodfed mosquitoes were maintained on 10% sucrose solution supplemented with 0.05% para-amino benzoic acid.