Plasmodium falciparum in vitro culture

3D7 (BEI Resources Repository, NIAID, NIH: Plasmodium falciparum, Strain 3D7, MRA-102, contributed by Daniel J. Carucci) and FCB2 parasite strains were cultured with O+ human erythrocytes in RPMI 1640 culture media, supplemented with human inactivated serum, in a 90% N₂, 5% O₂, 5% CO₂ atmosphere [30]. The maintenance protocol of asexual and sexual forms of Delves et al. was followed [22]. Briefly, asexual parasite form culture was checked every 48 h, maintaining 0.5% culture parasitaemia in 4% haematocrit until gametocytes became mature (gametocyte stage V) after 12 to 15 days culture involving daily medium replacement, without adding fresh erythrocytes. The gametocytes were then tested regarding their sexual differentiation capability in vitro, using 100 µL gametocyte culture (stage V) at 27 °C for 20 min [31]. Samples were then spun at 2500 rpm for 3 min, pellets were analysed by Giemsa staining and gametes were visualized at 40× using a Primo Star Carl Zeiss microscope [32].

An exflagellation test was made on gametocyte culture; 50 µL mature gametocyte culture mixed with human serum was kept at room temperature for 10 min. The cells were visualized in a Neubauer chamber at 40x using a Primo Star Carl Zeiss microscope. Exflagellation centres were counted and exflagellation percentage calculated using the following equations [22]: