Parasite strains, culturing and resistance selection

Protocol for selection of compound resistant parasites

A lab-adapted clone of the Plasmodium falciparum parasite clone 3D7 was used for selections. This clone, called 3D7-A10, has a 38 hrs life cycle (http://www.pnas.org/cgi/doi/10.1073/pnas.1209823109). Asexual parasites were grown stationary with 3% haematocrit in human erythrocytes at 37°C under 5% O₂, 5% CO₂, and 90% N₂ in RPMI 1640, supplemented with 27 mM sodium bicarbonate, 11 mM glucose, 0.37 mM hypoxanthine, 0.5mM sodium pyruvate, 10 μg/ml gentamicin, and 2.5 g/L Albumax II. For selections, 5x10e8 parasites were cultured at 1% parasitemia with 3x EC₅₀ concentrations of compound. Compounds were maintained during the selection process. Parasites were diluted 1:1 weekly and fresh erythrocytes were added. Blood smears were performed daily to monitor the clearance of parasites for the first week of selection. After the initial clearance of parasites, blood smears were done weekly. Viably parasites appeared after approximately 3-4 weeks. Once parasites grew well in compound-containing growth media, they were cloned by limited dilution without compound. Resistance of several clones from each selection was assessed. gDNA was harvested from resistant parasites and was submitted to whole genome sequencing. 

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