D sorbitol

To obtain synchronous 0–3 h ring-stage parasites prior to DHA exposure, cultures were treated twice with 10 ml of 5% D-sorbitol (Thermo Fisher Scientific, Ward Hill, MA) 46 h apart. Each treatment occurred at room temperature, lasted 10 minutes, and was followed by one wash with iRPMI (73 (link)). Directly after the second sorbitol treatment, cultures were diluted to 2% parasitemia and 5% hematocrit at a total volume of 10 ml. A stock solution of 700 μM DHA (Sigma-Aldrich, St. Louis, MO) dissolved in DMSO (Sigma-Aldrich, St. Louis, MO) was diluted in cRPMI to treat parasites at a final concentration of 700 nM DHA/0.1% DMSO for 6 h at 37°C. In parallel, a separate culture was treated with 0.1% DMSO diluted in culture medium as a control. Cells of both treatments were washed twice with cRPMI and returned to culture conditions. Blood smears were prepared prior to drug treatment and following the final wash. 24 h post-DHA, cells were subjected to 5% sorbitol for 30 minutes at 37°C to eliminate mature parasites that survived DHA treatment. Simultaneously, cells that were exposed only to DMSO underwent a mock-sorbitol treatment with iRPMI. Following two washes with iRPMI, cells were returned to cRPMI under normal culture conditions.