2.4. FITC-dextran permeability assay

To assess barrier formation in differentiated Caco-2 cells, the permeability of FITC Dextran was determined [23]. For this, Caco-2 cells were differentiated on Transwells for 10 days. FITC-dextran (FD4, Sigma Aldrich, St. Louis, Missouri, USA, Cat No: 60642-46-8) was dissolved in complete EMEM medium containing 10% FBS at 500 μg/mL concentration and then applied to the apical side of the cell monolayers on the Transwells. FD4 permeability was determined by sampling the basolateral solution for every 30 min for 3 h, while replacing the sampled amount with fresh media without FD4 at each time point. The amount of FD4 that passed the Caco-2 barrier across the Transwell membrane was quantified by fluorescence spectroscopy at 535 nm using a calibration curve using a range of concentrations of FD4 between 10 μg/mL⁻¹ mg/mL. As a control, proliferating Caco-2 cells that did not form the barrier were used.