Cell Cycle Synchronization of Intracellular Parasites

Flow cytometric analyses was performed as described previously (Liu et al., 2020 (link)). Monolayers of HFF were infected with tachyzoites at MOI = 1. After 12 h, synchronization of cell cycle was performed with 80 μM pyrrolidine dithiocarbamate (PDTC, Cat #S1809; Beyotime) at 37°C and 5% CO₂ for 8 h. Then the medium was replaced with culture medium in the presence of 5 μM of each compound. After 8 h, the extracellular parasites were removed by cold PBS washing, and the intracellular tachyzoites were isolated from host cells through a 27-gauge needle and purified with a 3-μm filter. After centrifugation at 4°C for 10 min at 1,000 × g, parasites were fixed with 70% ethanol at −20°C for 24 h followed by cold 2% FBS washing. Parasites were stained with propidium iodide (PI; Cat # PH0530, Phygene) at 37°C for 30 min in the dark and then were filtered with a 5-μm pore filter. Harvested parasites were detected on FACSCanto™II flow cytometer (Beckman Coulter, CA, United States), and the results were analyzed using FlowJo 7.6.1 software (FlowJo LLC, Ashland, OR, United States). At least 50,000 events were collected per sample.