Permeability Assay for HUVEC Monolayers

The permeability through HUVEC monolayers was assessed by the passage of albumin-FITC (Sigma-Aldrich) as previously described.^{20 (link)} Briefly, HUVECs (2 × 10⁵) were cultured for 2 days in transwell chambers with fibronectin-coated 3 µm pore-size polycarbonate membrane inserts (Costar, Cambridge, MA). For the assay, SFM containing albumin-FITC (100 µL, 40 mg/mL) was applied to the upper chamber. Endothelial monolayers were treated for 4 hours at 37°C with 2 µg/mL rCD177 or rPR3 or rCD177/PR3 followed by mAbs (2 µg/mL) against CD177 (MEM166) or PR3 (PR3 D1) as described earlier. In some experiments, before stimulation with protein, the endothelial monolayer was pretreated with PECAM1.2 F(ab′)2 fragments. The albumin-FITC concentration in the lower chamber was screened every 15 minutes over 1 hour in triplicate in a fluorescent microtiter plate reader (BioTek, Bad Friedrichshall, Germany). Thrombin (0.2 U/mL) was included as a positive control.