Quantifying Parasite DNA Content

To measure the parasite DNA content, a previously described protocol (64 (link)) was followed. In brief, HFF monolayers were infected with both WT and TgOTUD3A-KO parasites. At 24 h and 36 h postinfection, parasites were harvested, syringe passaged, and filtered through 10-μm filters (CellTrics; Partec, GmbH). For a negative control, host cells only were harvested and treated the same way. Parasites fixed with filtered ethyl alcohol (EtOH; overnight fixation at −20°C) were treated with RNase, stained with the DNA dye SYTOX green (Invitrogen), and analyzed with a flow cytometer (Sony SY3200) by collecting 50,000 events for each parasite line. Data were analyzed using WinList 8.0 (Verity Software House).