Facscanto 2 flow cytometer

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.

Briefly, HUMSCs were obtained after normal deliveries following 38–40 week gestations. HUMSCs were cultured in Dulbecco’s modified Eagle medium (Gibco, Waltham, MA, USA) containing 10% fetal bovine serum (Gibco) and negative for mycoplasma contamination. All experiments were conducted using HUMSCs at passages 5–8. After reaching 80% confluency, various concentrations of MIONs@PDA (0, 10, 25, 50, 100, and 200 μg/mL) were added to the medium for 12 h. The Cell Counting Kit-8 (Sigma-Aldrich, St. Louis, MO, USA) assay was used to detect the cytotoxicity of MIONs@PDA. HUMSCs were stained with the Prussian blue iron staining kit (Solarbio, Beijing, China), according to the manufacturer’s instructions. The phenotype of HUMSCs was confirmed by the expression of surface markers (positive for CD44-fluorescein isothiocyanate (FITC) and CD105-phycoerythrin (PE), and negative for CD45-FITC) (all from BD Biosciences, San Jose, CA, USA) using a FACSC anto II flow cytometer (FC500; Beckman Coulter Brea, CA, USA), and the data were analyzed with the CXP software (Beckman Coulter). Differentiation capacity of the HUMSCs was assessed by inducing osteocyte and adipocyte differentiation using the StemPro Osteogenesis/Adipogenesis Differentiation Kit (Invitrogen, Waltham, MA, USA) and evaluated by Alizarin Red S, Oil red O, and Alcian blue staining, respectively.

Using PBS bufffer to collect and wash cell suspension (1 × 10⁶ cells/ml) twice. Cells were stained with Huh7 PE (BD, USA), Huh7 7-ADD (BD, USA), Huh7 PE and 7-ADD together, PE and 7-ADD together. A negative control was processed in parallel. Then, measured cell apoptosis by FACSCanto II flow cytometer (Beckman, USA).