Cultivation of Genetically-Modified Cambodian Malaria Parasites

Cam3.II K13^WT, Cam3.II K13^R539T (RF967), and Cam3.II K13^C580Y are laboratory-adapted Cambodian isolates that were genetically engineered at the K13 locus as described in the literature ^{14 (link)}. Cam3.II K13^C580Y parasites expressing β2 C31Y, β2 C31F, or β5 A20S were obtained from in vitro selection studies with WLL and WLW as described in the literature ^{36 (link)}. Parasite cultures were propagated in O+ red blood cells (RBCs) (anonymous donors, purchased from Interstate Blood Bank, Memphis, Tennessee). RBCs were stored at 50% hematocrit in ADSOL (2 mM adenine (Alfa Aesar, Haverhill, Massachusetts), 111 mM dextrose (Fisher BioReagents, Pittsburgh, Pennsylvania), 41.2 mM mannitol (Acros Organics, Fair Lawn, New Jersey), and 154 mM sodium chloride (Fisher BioReagents) for prolonged RBC vitality as described in ref ^{47 (link)} at 4^oC. Parasites were cultured in complete media (RPMI 1640 media supplemented with 0.01 mg/mL gentamicin (Gibco, Dun Laoghaire, Co Dublin, Ireland), 50 mg/mL hypoxanthine (Acros Organics), and 0.5% Albumax II (Invitrogen, Carlsbad, California)) at 5% hematocrit, and were maintained at 37^oC in a Heracell™ VIOS 160i CO₂ Incubator (Thermo Fisher Scientific, Waltham, Massachusetts) under hypoxic conditions (5% O₂, 5% CO₂, 90% N₂). Gas was purchased from Matheson Gas (Irving, Texas).