Propagation and Isolation of P. falciparum Gametocytes

P. falciparum strain NF54 was maintained in RPMI [7.4] supplemented with 10% human serum and 5% haematocrit using standard culturing technique^{87 (link)}. NF54/iGP2 strain was additionally supplemented with 2.5 mM D-(+)-glucosamine hydrochloride (Sigma #1514) for maintenance. For induction, parasites were synchronized with 5% sorbitol^{88 (link)} and glucosamine was omitted from the medium for 48 h. From day 4–8 gametocyte cultures were treated with 50 mM N-acetylglucosamine (Sigma #A6525) to eliminate ABS parasites^{89 (link)}. For WT-NF54 gametocyte induction the same procedure was followed except that instead of glucosamine induction, parasites were overgrown for 5 days with only medium exchanges every 48 h prior to N-acetylglucosamine treatment. At day 14 after induction, gametocytes were magnet-purified according to the procedure described previously^{90 (link)}. ABS parasites were not further enriched prior to processing. Infected red blood cells were freed from host material through 10 min incubation in 10× pellet volume of 0.05% (w/v) saponin in phosphate-buffered saline (PBS; pH 7.4) and subsequent centrifugation at 3000 × g for 5 min. The parasite pellet was washed twice with PBS and the dry pellet was flash frozen and stored at −80 °C.