Human collagen 1

To assess the blocking capacity of NC410, a titration assay with LAIR-1 reporter cells was performed as previously described (Lebbink et al., 2006 (link)). Black Falcon clear flat bottom 96-well plates were coated with 5 μg/mL human collagen I (Sigma-Aldrich) in 2 mM acetic acid (Merck), anti-mouse-CD3 (BD), anti-human-LAIR-1 antibody (clone 8A8) in PBS (Sigma-Aldrich) or isotype control (eBioscience) in PBS by spinning down for 3 min at 1700 rpm and incubating overnight at 4°C. The next day, plates were washed with PBS and pre-incubated with the indicated concentrations of NC410 or isotype control (NextCure) in culture medium by spinning down for 5 min at 1500 rpm at room temperature (RT) and incubating for 2 hr at 37°C.
WT and hLAIR-1 reporter cells were harvested and seeded at 1 × 10⁶ cells/mL in 50 μL/well on top of the collagen and fusion protein-treated wells and spun down for 3 min at 1700 rpm at RT. Plates were incubated overnight, approximately 16 hr, at 37°C, and GFP expression was measured on a LSRFortessa (BD Biosciences).

For biofilm assays, bacterial overnight cultures in THY were suspended in fresh BHI supplemented with 0.5% glucose, adjusted to 10⁴ CFU/ml and inoculated into 96-well microtiter plates. Wells were coated overnight at 4 °C with 2 μg/well human collagen I, collagen IV, or fibronectin (Sigma) in PBS. After incubation for 24, 48 or 72 h as standing cultures at 37 °C in a 5% CO₂ / 20% O₂ atmosphere, the biofilms were quantified in a Spectramax M2 plate reader after staining with crystal violet as described previously [17 (link)].

Processed yrEnCL1, yrEnCL3 and yrEnCB (2.5 μg each) were incubated at 37 °C with 50 μg of macromolecular substrates (bovine haemoglobin and albumin, human collagen I, IgG and fibrinogen; Sigma-Aldrich) dissolved in 100 μl of 50 mM/100 mM CPB with 2 mM DTT in pH 4.5–6. Aliquots (10 μl) for SDS-PAGE analysis in 12% gels were taken at various intervals (0, 30, 60 and 120 min and 16 h).