F1141 2mg

Human embryonic kidney (HEK293T) cells (632180; Takara) were cultured in DMEM (10-013-CV; Corning) supplemented with 10% FBS (1500-500; Seradigm) and 1% penicillin (25–512; GenClone). Human colorectal adenocarcinoma (SW480) cells were cultured in DMEM with high glucose (SH30081.02; HyClone) supplemented with 10% FBS (1500-500; Seradigm), 1x L-Glutamine (25–509; GenClone), and 1% penicillin (25–512; GenClone). SW480 cells were FACS-sorted based on surface marker ROBO-1, and ROBO + and ROBO- cells were used in Fig. 4 and Fig. 6, respectively. The cells were plated into 8-well chambers and then fixed. The eight-well plates (155409; Thermo Scientific) for HEK293-T and SW480 cells were coated with fibronectin bovine plasma (F1141-2MG; Sigma Aldrich) before seeding cells onto the 8-well plates. All cultures were grown at 37 °C with 5% CO₂.

Three BUVEC isolates were seeded in 12-well plates with coverslips precoated with fibronectin (1:400, Sigma-Aldrich, F1141-2MG) and infected either with T. gondii Me49 or NED tachyzoites at sub-confluency (MOI 1:2). At 24 h p. i., all samples were fixed in 4% paraformaldehyde (15 min, RT) and washed three times in sterile PBS. The samples were incubated in a blocking/permeabilization solution (PBS with 3% BSA and 0.3% Triton X-100) for 1 h at RT. Thereafter, they were incubated in primary antibody solutions (Table 1) at 4°C in a humidified chamber overnight. The samples were then washed three times with 1X PBS and incubated in secondary antibody solutions (Table 1) for 30 min at RT and darkness. Host cell nuclei were labeled with DAPI present in the mounting medium solution (Fluoromount G-DAPI, Thermo Fisher, cat. Number 495952). The samples were analyzed with ReScan Confocal instrumentation (RCM 1.1 Visible, Confocal.nl) combined with a Nikon Eclipse Ti2-A inverted microscope.