Parasitemia Measurement by Flow Cytometry

Ring-stage parasites at 5% to 10% parasitemia were washed twice in the culture medium to remove ATc and resuspended in the culture medium, and the hematocrit was adjusted to 2%. Parasites were divided into 2 cultures grown in the culture medium supplemented with 0.5 μM ATc or grown in the medium without ATc. The growth assays were continued for 4 reinvasion cycles. At the schizont stage of each cycle, parasitemia was measured by flow cytometry starting at 24 h (0 reinvasions) after ATc removal. Culture aliquots were incubated with 17 μM dihydroethidium (Thermo Fisher) for 15 min at room temperature (RT) to stain nuclei, and 50,000 events were analyzed on a BD Accuri C6 flow cytometer. Cultures were diluted with fresh culture medium with red blood cells at 2% hematocrit so that the parasitemia under the +ATc condition was 1% and the −ATc condition was diluted by the same factor; ATc was added as required. Aliquots of culture for Western blotting were taken at 24 h and 72 h (0 and 1 reinvasion) after ATc removal.