Nafamostat mesylate

Mouse brain endothelial bEND.3 cells (5 × 10⁵ cells in 500 µL DMEM media, passage number 4 or 5) were grown for 1 day on 3 μM transwell inserts (BD Biosciences; Corning, #353492) until confluency and tight junctions were well-established. Media were removed from the transwells and the supernatants harvested from various experimental conditions of BMMCs were added. Barrier function of the endothelial monolayers was measured as TEER using a Millicell ERS Ohmmeter with the probe included by the manufacturer (Millipore, Burlington, MA; MERS00002). TEER readings were taken at baseline, 3h, 6h, and 24 h after introduction of stimulus. For transwell assays using BMMCs, an MOI of 1 was used. For some groups, chymase-specific inhibitor (TY-51469; 100 μM) and tryptase-specific inhibitor (nafamostat mesylate; 10 μM, Sigma-Aldrich, St. Louis, MO; #82956-11-4) were incubated with BMMCs during JEV activation. At 24 h after bEND.3 cell stimulation with the various experimental supernatants, FITC-dextran was added to the upper compartments at 0.5 mg/mL for 1 h, then the medium was removed from the lower compartments for fluorescence measurement. Fluorescence was measured using a plate reader (Tecan Infinite M200; excitation wavelength of 492 nm and emission wavelength of 520 nm).

Bafilomycin A1 was obtained from InvivoGen (San Diego, CA, USA). Stock solutions of 100 μM (160 μl DMSO added to a 10 μg Bafilomycin A1) Bafilomycin A1 were prepared. In control experiments, equal amounts of DMSO (vehicle control) as for the Bafilomycin A1-treated cells were added. The pH probe Lysosensor Blue DND-167 was purchased from ThermoFisher Scientific (Waltham, MA, USA). Nafamostat mesylate was obtained from Sigma-Aldrich (Steinheim, Germany).