Stability of the monolayers was assessed using phase contrast microscopy (DMi1, Leica), TEER measurements, and Papp assays. The details of each technique have been described elsewhere [39 (link),40 ]. Briefly, the TEER (Ω*cm2) was measured daily using epithelial Volt-Ohm Meter (Millicell ERS-2, Millipore AG, Burlington, MA, USA), subtracting the blank value measured in Ω and multiplying by the cell culture surface area (0.33 cm2) [37 (link)]. The Papp (cm/s) of 4 kDa FITC-dextran was determined on day 1, 3, and 5 of culture by measuring the fluorescence intensity of the basolateral culture medium over 100 min following apical application of 0.5 mg/mL FITC-dextran to each well. The measurements were obtained with excitation and emission wavelengths of 495 and 535 nm, respectively, using a SpectraMax i3x microplate reader (Molecular Devices) [37 (link)]. The monolayers were considered stable once cells grew to confluence and TEER and Papp values reached a plateau. At least two technical replicates per experiment and more than three biological replicates were assessed to ensure the repeatability. Subsequent experiments were conducted using monolayers which had reached a stable state.
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