2.5. Paracellular Permeability Assay

Caco-2 cells grown at confluence were seeded at 1.5 × 10⁵ cells/mL in polycarbonate Transwell^® inserts (12 mm Ø, 0.4 µm pore size) (Costar; Corning Incorporated, Kennebunk, USA) and cultured as indicated above. After renewing the medium every 3 days, transepithelial electrical resistance (TEER) was measured with an Epithelial Volt/Ohm Meter (World Precision Instruments, Sarasota, FL, USA) to ensure that the cells had reached confluence and differentiation, which occurred at 21 days.

Differentiated cells were incubated with the digested-wine fluids (IDW or CDW) and pure phenolic compounds in two independent assays. In a first experiment, 500 µL of sample [the intestinal digested extract (IDW) or the simulated pancreatic juice (IDM) (both 1:40 v/v diluted in DMEM), or a quercetin solution (200 µM in DMEM), or DMEM (as control)] were added to the apical side of the Transwell^® inserts containing the Caco-2 cell monolayers. These plates were incubated at 37 °C for 4 h, simulating the time for which dietary compounds remain in the small intestine in in vivo conditions.

In a second experiment, in a similar way, 500 µL of sample colonic digested wine (CDW) or the colon nutrient medium (CDM) (both 1:40 v/v diluted in DMEM), or a 3,4-dyhydroxyphenylacetic acid (3,4-dhpa) solution (200 µM in DMEM), or DMEM (as control)] were added to cell monolayers and incubated for 16 h, which simulated the average time that dietary components remain in the colon. For both experiments, incubations were carried out in triplicate and experiments were repeated on three different days.

For both experiments, when the incubation time was over, the apical chamber solutions of the Transwell^® culture inserts were replaced by 500 µL of a DPBS (Dulbecco’s Phosphate-Buffered Saline) solution containing 1 mg/mL of fluorescein isothiocyanate (FITC-)-dextran 4 kDa and the basolateral sides of the plate inserts were filled with DPBS. Then, the plate was incubated at 37 °C for 30 min. Afterwards, 100 µL from the basolateral chamber of each well were taken in triplicate to measure the concentration of FITC-dextran by fluorescence intensity in a BioTek FL600 microplate reader (Biotek, Winooski, VT, USA) with an excitation wavelength of 480 nm and an emission wavelength of 520 nm.