Parasite Culture in Human Red Blood Cells

Parasites were cultured in human red blood cells (Australian Red Cross Blood Bank) at 4%
hematocrit in RPMI media (Sigma-Aldrich, UK), supplemented with 0.2% NaHCO₃(Thermo Scientific, Australia), 0.25% Albumax II (Gibco, New Zealand), 0.37 mM
hypoxanthine (Sigma, USA), 25 mM HEPES (Gibco, USA), and 31.25 mg/L gentamicin (Gibco,
USA) (cRPMI) and incubated at 37 °C with 1% O₂, 5% CO₂, and 94%
N₂. The parasite lines used in this study includes the 3D7 African wild-type
strain parasite transfected with a Nanoluciferase reporter proteins targeted to the RBC
compartment (Hyp1-Nluc)^{24 (link)} and Dd2 strain parasite with a mutated
exonuclease domain within the DNA polymerase δ (Dd2-Polδ).^{35 (link)} Hyp1-Nluc was cultivated under 2.5 nM WR99210 (Jacobus Pharmaceutical Company)
selection to maintain episomal expression of the Hyp1-Nluc gene.^{57 (link)}To produce synchronous parasites for protein export and invasion assays, they were first
synchronized with 5% sorbitol to enrich for ring-stage parasites.^{58 (link)} The
parasite were grown until late schizonts and layered over a 67% Percoll density gradient
buffer in cRPMI (Percoll, 10 mM NaH₂PO₄, 143 mM NaCl (GE Healthcare
Bio- Sciences, Sweden)) and centrifuged (1500g/15 min).^{59 (link)} The dark-colored synchronous schizont layer was removed and used for the
downstream experiments.