4.5. Barrier Intergrity Assay (BI Assay) and Calculation of Aperent Permeability (P-App)

The barrier integrity assay was performed to assess the barrier integrity of a boundary tissue and has been described in detail previously [6,41,64]. In short, basal media was added to the middle and bottom inlets and outlets. The top inlets or the lumens of the intestinal tubules were perfused with media containing high 0.5 mg/mL FITC-dextran (150 kDa, Sigma No. 46946) and low molecular weight TRITC-dextran (4.4 kDa, Sigma No. T1037). Plates were imaged using ImageXpress XLS Micro HCI system with an interval of every 3 min for total time of 15 min. The permeability of the fluorescent particles was quantified by determining the fluorescence levels in the basal gel region and normalizing it to the fluorescence in the lumen of the tubule to compensate for bleaching effects. The ratio between the fluorescent signal in the basal and apical region of the tube was analyzed using Fiji. Cell-free chips were taken as negative controls. These data were then used to calculate the apparent permeability value (P-app) according to van Duinen et al. [65].