Culturing Fluorescent Malaria Parasites

Green fluorescent protein (GFP) expressing P. falciparum (D10-PfPHG) (Wilson et al., 2010 (link)) and P. knowlesi (PkYHI) parasites (Lim et al., 2013 (link)) were cultured in human O⁺ red blood cells (RBCs) (Australian Red Cross Blood Service) in RPMI-HEPES culture medium (Thermo Fisher Scientific) supplemented with 0.5% v/v Albumax (Gibco), 52 μM gentamycin (Gibco), 367 μM hypoxanthine (Sigma-Aldrich), and 2 mM sodium bicarbonate (Thermo Fisher Scientific), adjusted to a pH of 7.2-7.4. Cultures were grown in sealed containers with 1% O₂, 5% CO₂ and 94% N₂ (BOC Gases) at 37°C (Trager and Jensen, 1976 (link)). Synchronization of D10-PfPHG parasites for growth inhibition assays was achieved using heparin sodium (Pfizer) as previously described (Boyle et al., 2010 (link); Wilson et al., 2013 (link)). PkYH1 parasites were passaged over a gradient of 70% Percoll (Sigma-Aldrich) to purify late stage schizonts which were then allowed to rupture for ~4 hrs in the presence of fresh RBCs prior to ring-stage treatment with 5% w/v sorbitol (Sigma-Aldrich), enabling effective synchronisation of 0-4 hrs old rings.