Bovine collagen 1

For 3D cultures, both cell lines, CCArly CAF and MUG CCArly, were harvested from 2D cultures. Single suspensions of these cell lines alone and in combination were then seeded in ultra-low attachment 96-well-plates (Corning, NY, USA) at a cell density of 5000 cells/well in 100 μL media/well to build 3D spheroid cultures. MUG CCArly single-culture spheroids were supplemented with 28 μg/mL bovine collagen I (Gibco, Life Technologies, Darmstadt, Germany) for stable spheroid formation. Half media changes were performed every second to third day. For immunocytochemistry staining, spheroids were harvested on day six, fixed in 4% formalin, and embedded in paraffin (FFPE). Supernatants were collected on cultivation days three and seven for Luminex xMAP technology. In total, 50 μL per well and condition was pooled for 20 biological replicates, and stored at −80 °C until further use.

hCECs were seeded onto commercial micropatterned glass substrates (CYTOO, Grenoble, France) which have been customized to contain an array of circular micropatterns (65 and 80 µm diameter) on which cells can adhere. The area surrounding the circular micropatterns was selectively passivated to attenuate cell and protein adhesion. Before cell seeding, the micropatterned substrates were coated with a mixture consisting of Fibronectin (Sigma-Aldrich, Burlington, MO, USA F1141 0.03 mg/mL), Bovine Collagen I (5 mg/mL Gibco, Agawam, MA, USA, A1064401, 0.01 mg/mL) and Bovine Serum Albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA, A331, USA, 0.01 mg/mL) in 1× HBSS, and incubated at 37 °C for 24 h. Coated micropattern substrates were transferred to a 35 mm dish (Thermo Scientific, China, 150460), which was passivated with 10% Pluronic acid (Sigma-Aldrich, St. Louis, MO, USA, P2443) in 1× HBSS overnight. Cells were seeded onto the micropatterned substrates at a density of 300,000 cells/mL. Unattached cells were removed after 2 h by washing with 1× HBSS 5 times, followed by passivation with 0.2% Pluronic acid for 10 min, and finally replaced with 2 mL of complete medium. The cells were cultured on the micropattern substrates for 24–48 h based on the assay being performed.

The assay was performed as previously described (Kanatani et al., 2015 (link)). Briefly, a collagen layer was prepared using bovine collagen I (0.75 mg/ml, GIBCO) in a 96-well plate (80 μl/well). Microglia were challenged with freshly egressed T. gondii tachyzoites (MOI 3) in CM for 4 h. The cell suspension (5 × 10⁴ cells) was applied to the collagen layer and incubated for 18 h at 37°C and 5% CO₂. Gels were fixed and DAPI-stained. Image stacks were generated (200 optical sections) by confocal microscopy (LSM 780, Zeiss) and migrated distances by cells were analyzed using Imaris x64 v.8.1.1 software (Bitplane AG, Zurich, Switzerland).

A collagen layer was prepared by mixing bovine collagen I (0.75 mg/ml; Gibco) in PBS and pH was adjusted to 7.0–7.5. Cold collagen solution was poured into a 96-well plate (80 μl/well) avoiding air bubble formation. Gel formation at 37°C for 1 h was allowed before start of the assays. DCs were challenged with freshly egressed T. gondii tachyzoites in CM for 4 h at multiplicity of infection (MOI) 3. The infection frequency of DCs was determined by epifluorescence microscopy (S2 Fig). The cell suspension (5×10⁴ cells) was applied to the collagen layer and incubated for 18 h at 37°C and 5% CO₂. The medium was gently aspirated and the gels were fixed by addition of 100 μl of 4% paraformaldehyde in PBS. Gels were then washed once with PBS, stained with 57.2 nM DAPI in PBS and stored at 4°C.
To establish a chemotaxis gradient, 40 μl of collagen solution containing 3 mg/ml CCL19 (Peprotech) was casted into a 96-well plate, allowed to polymerize, and 40 μl of collagen solution was overlaid. The migration assay was performed for 24 h at 37°C and 5% CO₂.

Wells of a 96-well plate were coated with 2 µg bovine fibronectin (Sigma-Aldrich), 5 µg bovine collagen I (Gibco), or were left uncoated. Free binding sites were blocked with BSA. Hoechst 33342-stained (Life Technologies) cells were seeded at 1×10⁵ cells per well and incubated for 1 h at culture conditions. Non-adherent cells were washed off and fluorescence intensity of attached cells was measured with the microplate reader Infinite M200 (Tecan).