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[Preprint]. 2024 Aug 29:2024.01.03.574077. Originally published 2024 Jan 4. [Version 2] doi: 10.1101/2024.01.03.574077

Figure 1.

Figure 1.

FACS gating strategy and Giemsa-stained cytospin images of control- and DHA/sorbitol-treated NF54-SFG parasites. Synchronized 6–9 h rings were exposed to the drug vehicle for 6 h on day 0 and mock-selected with iRPMI on day 1 (Control), or they were treated with 700 nM DHA for 6 h on day 0 and selected with 5% sorbitol on day 1 (DHA/Sorbitol). The samples were harvested on day 2 (t= 50 h), stained with Hoechst 33342 (HO) plus MitoTracker Deep Red FM (MT). Parasitized erythrocytes were collected and imaged after two sequential steps of purification by the yield and 4-way purity modes of a BD Aria™ Fusion Flow Cytometer. (A) Panels labeled ‘Yield’ show the parasitemias of culture samples and gates employed for the first step of high-throughput yield sorting. Panels labeled ‘Purity’ show the distributions of cells obtained after yield sorting and delineate the P1 – P4 gates used to collect the purity-sorted populations. Uninfected red cells are represented in blue and HO+/MT+ cells in purple. The vehicle-, iRPMI mock-treated population containing 3.7% HO+ cells was yield-purified, providing an enriched distribution of 0.4%, 2.8%, 27.6%, and 5.5% HO+ cells in the P1 – P4 windows for purity-collected controls. Similarly, the population of 1% HO+ cells from the DHA/sorbitol-treated culture was yield-enriched to provide 1.7%, 8.3%, 4.1%, and very rare cells (0.0% reported) in the P1 – P4 windows for purified DHA/sorbitol-treated cells. (B) Images of Giemsa-stained parasites from cytospin preparations of P1–P4 subpopulations. Late rings and trophozoites predominate in the P3 subpopulation of control-treated samples; smaller percentages of late-stage schizonts and early rings are explained by loss of synchrony from experimental manipulations or naturally arising cell-to-cell variability (96). In DHA/sorbitol-treated samples, persisters were most frequent in the P2 window. Although FACS detected almost no events in the DHA/Sorbitol P4 window, our searches identified rare mature forms in highly concentrated cytospin preparations (two parasitized cells shown).