0.4 μm pore size filters

A253 cells were cultured to confluence in the inner chambers of 12 mm transwell inserts with 0.4 μM pore-size filters (Corning Life Sciences). The TEER was measured using an EVOM voltameter with an ENDOHM-12 (World Precision Instruments, Sarasota, FL, USA) on a heating plate (Fine, Tokyo, Japan) adjusted to 37 °C. The values were expressed in standard units of ohms per square centimeter and presented as the mean ± S.D. For calculation, the resistance of blank filters was subtracted from that of filters covered with cells.

Paracellular permeability was done as previously described^{69 (link)}. 4-kDa fluorescein isothiocyanate-labeled (FITC)-dextran (70 kDa; 200 µg/ml) (Sigma, Cat. FD4) and tetramethylrhodamine isothiocyanate (TRITC)-dextran (4 kDa, 200 µg/ml) (Sigma Aldrich, Cat. T1037) was assessed in control, Cldn23 OE, and KD confluent monolayers grown on Transwell filters (0.4 μm pore-size filters, Corning, Cat. 3460). After TEER measurement, upper and lower Transwell compartments were washed twice with calcium-containing PBS and placed in pyruvate buffer (10 mM HEPES, pH 7.4, 1 mM sodium pyruvate, 10 mM glucose, 3 mM CaCl₂, and 145 mM NaCl₂) for 1 h at 37 °C. A freshly prepared solution of 4 kDa TRITC-dextran and 70 kDa FITC-dextran dissolved in pyruvate buffer was added to the top chamber of the Transwells and incubated for 3 h at 37 °C. Samples from the bottom chamber of the Transwells were collected every 30 min, and the fluorescence intensity was measured with a fluorescent plate reader. For TRITC-dextran excitation was achieved at 555 ± 10 nm and emission detected at 580 ± 20 nm. For FITC-dextran excitation was achieved at 490 ± 10 nm and emission was recorded at 525 ± 20 nm. The apparent permeability (P_app) was determined by calculating the rate of change of the paracellular flux of TD4 and FD70 every 30 min for 3 h per sample.