To perform the 0–3 h RSA protocol developed by Witkowski, Amaratunga, and colleagues [
8] (henceforth designated as the Percoll gradient RSA), 300 µL of packed parasitized RBCs containing 2–3 % mature schizonts (>10 well-segmented nuclei) were collected from a
P. falciparum culture previously synchronized twice with 5 % sorbitol (Sigma-Aldrich, MO, USA) dissolved in distilled tissue culture-grade water (Gibco, MD, USA), 10 min, 37 °C, over two parasite intra-erythrocytic cycles. The parasites were then incubated in RPMI-1640 containing 15 U/mL heparin (Sigma-Aldrich), but no other supplements, for 15 min at 37 °C. After incubation, parasites were placed onto a 35/65 % Percoll (GE Healthcare Life Sciences, Pittsburgh, PA, USA) gradient, centrifuged at 3000 rpm for 10 min (966×
g, Eppendorf Centrifuge 5810, rotor A-4-81, no brake). The inter-phase band of late-stage schizonts was collected and incubated in media with fresh RBCs for 3 h, at 37 °C under 90 % N
2, 5 % CO
2, and 5 % O
2 gas mixture (see Additional file
1). This gradient is slightly modified from the original Percoll gradient RSA, as per recommendation of Chanaki Amaratunga to improve separation (personal communication). After incubation, some schizonts had yet to invade; thus, parasites were treated with 5 % sorbitol again to remove remaining schizonts and isolate 0–3 h ring-stage parasites. The parasite mixture was adjusted to 1 % parasitaemia in 20 µl with fresh packed RBCs, and suspended in 1 mL of cRPMI in a 24-well plate containing either 0.1 % dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) or 700 nM DHA (Sigma-Aldrich, MO, USA) dissolved in 0.1 % DMSO for 6 h. After drug pulse, cells were washed twice with 10 mL cRPMI with 3 min centrifugation at 2500 rpm (671×
g, 9 acceleration, 5 de-acceleration, Eppendorf Centrifuge 5810, rotor A-4-81). Parasites were further incubated with drug-free cRPMI for 66 h, when thin blood smears were prepared, methanol fixed, and stained with 20 % Giemsa (Sigma-Aldrich, MO, USA) for 15 min for independent survival assessment by at least two experienced microscopists. Evaluation consisted of counting the number of parasitized cells in an estimated 10,000 RBCs and comparing survival to DMSO drug-free incubation.