After TER measurement, the basal medium was replaced with phenol red-free DMEM (Life Technologies) supplemented with 10% FBS. The apical medium was replaced by the medium with 1 mg/ml FITC-dextran of molecular mass of 3–5 kDa (FD4; Sigma-Aldrich), 10 kDa (FD10S; Sigma-Aldrich), or 250 kDa (FD250S; Sigma-Aldrich). The cells were incubated in a 5% CO2 incubator at 37°C for 2 h, and the basal medium was collected. Fluorescence intensity of the medium was measured by microplate reader equipment (VARIOSKAN LUX; Thermo Fisher Scientific) in a black-bottomed 96-well plate (Corning). Standard curves were determined by measuring the fluorescence intensities of a serial dilution series of the FITC-dextran. Blank measurement of the medium without FITC-dextran was subtracted from the sample values. The apparent permeability coefficient (Papp) was calculated using the following equation:
where dQ is the amount of tracer transported to the acceptor (basal) compartment during the time period dt, A is the area of the filter, and C is the initial concentration of the donor (apical) compartment.
where dQ is the amount of tracer transported to the acceptor (basal) compartment during the time period dt, A is the area of the filter, and C is the initial concentration of the donor (apical) compartment.