Evom2 ohm meter

Transepithelial electrical resistance (TEER) was measured using an EVOM² ohmmeter with STX-2 probe (World Precision Instruments (WPI), Sarasota, FL, USA). Permeability was assessed using Lucifer Yellow CH dilithium salt, 10 mM in dH₂O (Sigma Aldrich, Burlington, MA, USA). The apparent permeability (P_app) was measured using Equation (5) [17 (link)] where V_R is the volume of the receiver compartment, dC_R/dt is the change in the drug concentration of the receiver compartment over time, A is the surface area of the membrane (0.3 cm²), and C_D0 is the concentration of the drug in the donor compartment at time zero.
$$P_{app }(\raisebox{1ex}{$\mathrm{cm}$}\!\left/ \!\raisebox{-1ex}{$\min )$}\right.=\frac{V_R\ast d{C}_R}{dt\ast A\ast C_{D0}}$$

Electrical impedance (to restrict ionic conductance) imparted by differentiated epithelial cultures was measured to determine the integrity of tight junctions (TJ) as previously reported [21 (link)]. Culture plates containing transwell ALI cultures were allowed to acclimatise for 30 min on a 37 °C heating platform (Lecia Biosystems, Mt. Waverley, VIC, Australia) within a biosafety cabinet before reading electrical impedance using the EVOM2 Ohm meter (World Precision Instruments, Sarasota, FL, USA). Raw resistance values were converted to TEER values by subtracting the resistance of a blank transwell insert and factoring for the surface area of the membrane support (0.33 cm²). The data was analyzed using a linear regression model with standard errors adjusted for the clustering of results within participants. Marginal mean and contrast values were determined to identify the effect of the treatments relative to the control and 10% CSE exposures. The statistical analysis was performed using R statistical software (release 3.2.3), and results expressed as Ω.cm².

To assess the integrity of the epithelial barrier, the trans-epithelial electrical resistance (TEER) was measured with an EVOM2 ohmmeter (World Precision Instruments, Sarasota, USA) with STX2 electrodes. First, the culture medium was replaced by 1.5 mL and 0.5 mL Hanks’ Balanced Salt Solutions supplemented with calcium and magnesium (HBSS^Ca2+/Mg2+, ThermoFisher Scientific, France) in the basal and apical compartment, respectively. HBSS was warmed at 37 °C before addition. For each experiment, the TEER was also measured in a cell-free Transwell (blank) and subtracted to values measured in cell seeded Transwell. Final TEER values are multiplied by the surface area of the inserts and expressed in Ω cm². We considered that a tight epithelium was formed if TEER > 200 Ω cm² for MucilAir following the provider’s guidelines, and > 300 Ω cm² for Calu-3 cells. This latter value was determined by combining TEER and Lucifer Yellow permeability assay results in our cell culture conditions (Fig. S2).