25 cm2 plastic culture flasks

Four aortic arteries from four adult healthy male Beagle dogs were donated from Elanco Animal Health, Monheim, Germany. Arteries were kept at 4 °C in sterile 0.9% HBSS-HEPES buffer (pH 7.4; Gibco) supplemented with 1% penicillin (500 U/mL; Sigma-Aldrich) and streptomycin (500 µg/mL; Sigma-Aldrich, Darmstadt, Germany). For isolation of aortic endothelial cells, 0.025% collagenase type II (Worthington Biochemical Corporation, Lakewood, NJ, USA) was infused into the vessel lumen, the aorta was ligated with clamps and incubated for 20 min at 37 °C in 5% CO₂ atmosphere. After gently massaging aortas, infused collagenase II-cell suspension was collected and immediately supplemented with 1 mL sterile fetal calf serum (FCS; Gibco, Langenselbold, Germany) to inactivate collagenase II. After two washing steps (400× g, 10 min, 4 °C), the cells were suspended in endothelium cell growth medium (ECGM; PromoCell, Heidelberg, Germany), plated in 25 cm² plastic culture flasks (Nunc, Roskilde, Denmark) and kept at 37 °C in 5% CO₂ atmosphere until reaching confluent cell layers. Culture medium was changed every 2–3 days.

Primary bovine umbilical vein endothelial cells (BUVEC) were isolated as previously described by Taubert et al. [27] . Briefly, umbilical cords obtained from calves born by sectio caesarea were kept at 4°C in 0.9% HBSS–HEPES buffer (pH 7.4; Gibco, Grand Island, NY, USA) supplemented with 1% penicillin (500 U/ml; Sigma-Aldrich, St. Louis, MO, USA) and streptomycin (500 μg/ml; Sigma). For preparation of endothelial cells, 0.025% collagenase type II (Worthington Biochemical Corporation, Lakewood, NJ, USA) was infused into the lumen of the isolated and ligated umbilical vein and incubated for 20 min at 37°C in 5% CO₂. After gently massaging the umbilical vein, the collagenase-cell suspension was collected and supplemented with 1 ml FCS (Gibco) to inactivate the collagenase. After two washings (400×g, 10 min, 4°C), the cells were resuspended in ECGM (endothelial cell growth medium; PromoCell, Heidelberg, Germany), plated in 25 cm² plastic culture flasks (Nunc, Roskilde, Denmark) and kept at 37°C in 5% CO₂.
B. besnoiti (strain Bb1Evora04) tachyzoites were maintained by serial passages in BUVEC. Tachyzoites were collected from BUVEC supernatants, centrifuged, washed thrice with PBS, counted and suspended in RPMI 1640 medium (Gibco) until further use.