4.12.1. Measurement of FITC-Dextran Flux across Monolayers of Cultured Epithelial Cells

Caco2 cells were seeded on top of transwell filter chambers on 12-well plates (0.4 μm pore size; Falcon, Heidelberg, Germany) and EGCs were seeded below them (Figure 2A) as previously described [18]. After reaching confluence, cells were rinsed with PBS three times. Afterwards, the cells were incubated with fresh DMEM without phenol red (Sigma) containing 10 mg/mL FITC-dextran (4 kDa). Paracellular flux was assessed by taking 100 μL aliquots from the outer chamber over 2 h of incubation. Fluorescence was measured using a Tecan Microplate Reader (MTX Lab systems, Bradenton, USA) with excitation and emission at 485 and 535 nm, respectively. For all experimental conditions, permeability coefficients (P_E) were calculated by the following formula (15): P_E = [(ΔCA/Δt) × VA]/S × ΔCL, where P_E = diffusive permeability (cm/s), ΔCA = change of FITC-dextran concentration, Δt = change of time, VA = volume of the abluminal medium, S = surface area, and ΔCL = constant luminal concentration.