Evom2 voltmeter

HBEs were seeded on 0.4 μm pore size Millicell inserts (Millipore Sigma, PIHP01250, Billerica, MA) coated with collagen-I at a density of 4.15 × 10⁵ cells/cm². The cells were cultured in non-proprietary “UNC ALI” media (Marsico Lung Institute at the University of North Carolina, Chapel Hill), 300 μL in the apical compartment and 3 mL in the basal compartment. At 100% confluency, the cultures were switched to ALI culture by aspirating the apical media and providing only basal media to the culture. Confluency of the cultures were confirmed with trans-epithelial electrical resistance (TEER) using STX2 chopstick electrodes connected to an EVOM2 voltmeter (World Precision Instruments, FL, USA). The apical surface of the culture was washed with PBS once a week to clear mucus.

For trans-endothelial electrical resistance (TEER) and permeability experiments, endothelial cells were seeded on pre-coated type I collagen Transwell^® filters (Corning^® Transwell^® Polyester membrane cell culture inserts, 24 well, 6.5 mm Transwell^® with 0.4 μm pore polyester membrane insert, Sigma, Australia) at a density of 50,000 cells/cm². Cell culture media were changed every 2–3 days, and experiments were performed when cells were approximately ~90% confluent, 5–6 days after seeding. AMS-6PB or PB was added together with 1 μg/mL LPS at the start of the experiment, and TEER measurements were taken by using an EVOM2 voltmeter with STX-2 electrodes (World Precision Instruments) at 2-, 4-, 6- and 24-h time points. To calculate the TEER (Ω·cm²), electrical resistance across a collagen coated insert without cells was subtracted from the TEER readings obtained on inserts with cells, and this value was multiplied by the surface area of the insert.