Generating Quiescent Malaria Parasites

Quiescent parasites were generated using the modified Teuscher, et al. protocol as described by Breglio, et al.^{22 (link),30} . Briefly, synchronized P. falciparum cultures were treated with 700 nM dihydroartemisinin (Sigma Aldrich, St Louis, MO) in DMSO for 6 h. Cells were washed three times with RPMI to remove drug before being resuspended in media and returned to culture conditions. Cultures were then treated with D-sorbitol every 24 hours for the following 72 hours to remove any actively growing parasites that did not enter quiescence. Immediately following the final sorbitol treatment, flasks were split into two aliquots. One aliquot was left unenriched to serve as the control, while the second aliquot was SLOPE enriched with 55 SLO units as described above. A portion of both untreated and SLOPE samples was then stained with SYBR-Green and MitoProbe DiIC1(5) as described above for flow cytometry analysis. The remainder of each untreated and SLOPE sample was stained for fluorescence microscopy as described below.