Transendothelial electron resistance (TEER) measurements and the FITC-dextran permeability of 2D cultured HTM monolayer

TEER of the monolayered 2D cultured HTM cells were performed as described previously²² using a 12 well plate for TEER (0.4 μm pore size and 12 mm diameter; Corning Transwell, Sigma-Aldrich). Briefly, at approximately 80% confluence, 5 ng/mL TGFβ2 and ROCK-i, 10 μM Rip or KD025 were added the apical side of the wells (Day 1), and cultured until Day 6. At Day 6, the wells were washed twice with PBS, and TEER (Ωcm²) were measured using an electrode (KANTO CHEMICAL CO. INC., Tokyo, Japan)²⁰.

In terms of fluorescein isothiocyanate (FITC)-dextran permeability, a 50 μmol/L solution of FITC-dextran (Sigma-Aldrich) was added to the well basal compartments of the culture and the culture medium from the apical compartment was collected at 60 min for the above different experimental conditions. The concentrations of the FITC-dextran were measured using a multimode plate reader (Enspire; Perkin Elmer, MA USA) at an excitation wavelength of 490 and an emission wavelength of 530 nm. The fluorescence intensity of the control medium was used as the background concentration.