ciBECs were cultured on cell inserts with astrocytes on the bottom side of Transwell for 7 days. For the transport experiments, all media were removed from the upper chamber of the inserts and replaced with 50 μL of pre-warmed Hank’s balanced salt solution (HBSS) (Life Technologies) supplemented with 10 mM HEPES (Life Technologies) and 0.1% BSA (Sigma-Aldrich) by equilibration to 37°C for 15 min. At a time point 0 min, 50 μL of 2 μM fluorescein-Na or 2 mg/mL FITC-labeled dextran was added to the upper chamber of the inserts, which were then transferred to 24-well plates containing 1 mL of pre-warmed HBSS supplemented with 10 mM HEPES. Samples (100 μL) were collected at 15, 30, 45, and 60 min from the bottom chamber and transferred to a black-walled 96-well plate (Nunc). The fluorescence was measured with an Envision fluorescence plate reader (PerkinElmer). Concentrations were calculated using standard curves generated from the stock solutions of each compound. Permeability coefficients (Pe) that take into account the barrier to transport from both the endothelial monolayer and the cell-culture insert were calculated as described previously (Perrière et al., 2005, Nakagawa et al., 2009, Watson et al., 2013).
