Evom2 system

iBMECs plated on a Transwell were washed with Transport Buffer. For paracellular permeability assays, 1 μM sodium fluorescein was added to the upper chamber. Aliquots were then extracted from the bottom chamber every 15 min and replaced with fresh buffer and fluorescence (485 nm excitation and 530 nm emission) was quantified at the end of the experiment on a plate reader. For efflux transporters experiments, cells were pre-incubated with 10 mM cyclosporin A (Sigma), 1 mM Ko143 (Sigma), or 10 mM MK571 (Sigma), depending on the experiment, for 1 hr at 37°C on a rotating platform, then co-incubated with the inhibitor and compound of interest (rhodamine 123 (R123), carboxymethyl-2′,7’-dichlorofluorescein diacetate (DCFDA) or FL-prazosin) and was quantified using a plate reader. For radiolabelled TH transport assays, 1 nM (2 × 10⁵ cpm) [¹²⁵I] T₃ or [¹²⁵I] T₄ (Perkin Elmer) was added to the upper chamber and measured in the bottom chamber using a gamma counter. The rate of accumulation was used to calculate Pe values. Monolayer fidelity was confirmed at the beginning and at the end of each experiment by measuring TEER by an EVOM2 system and a Chopstick Electrode (World Precision Instruments).

TEER was measured on 24-well Transwell inserts with a 0.4 µm pore size (Corning 3470), as previously described (24 (link)). An EVOM2 system (World Precision Instruments) with a STX2 probe was used to measure the resistance (Ω). TEER values were reported as Ω cm² following subtraction of the resistance of an acellular Transwell insert and correction for the membrane area (0.33 cm²). All TEER experiments were performed with at least 2 independent differentiations. We used TEER rather than solute permeability as a proxy for barrier function since the time dependence is much easier to measure and permeability coefficients have limited value in cases where defects in the monolayer lead to focal leaks.