*4.5. Caco-2 Cells*

Human colon epithelial cells, Caco-2 (ATCC®HTB-37™), were purchased from the American Type Culture Collection (Rockville, MD, USA). In our experiments, the Caco-2 cells were cultured in Dulbecco's Minimal Essential Medium (DMEM) supplemented with 10 mM nonessential amino acids, 10% (*v*/*v*) fetal bovine serum (FBS), 100 μg/mL streptomycin, 100 U/mL penicillin, and 2 mM glutamine (growth medium). Cells were incubated at 37 ◦C and 5% CO2, and sub-cultured at 80–90% confluence every 3–4 days.

For differentiation, Caco-2 cells were seeded at a density of 1 × 105 cells/well in 12-well trans-well plate (12 mm, with 0.4 μm pore polycarbonate membrane Insert, Corning) and differentiated for 21 days in DMEM growth medium. The medium was replaced every 2–3 days for both the apical (AP) and basal (BL) sides of the trans-well filters. The integrity of cell monolayer was checked by measuring the trans-epithelial electrical resistance (TEER) before and after the experiments with an Epithelial Volt/Ohm Meter (EVOM).

## *4.6. Cell Viability Assay*

Cell viability in Caco-2 cells treated in different experimental conditions was analyzed following the enzymatic reduction of 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to MTT-formazan, catalyzed by mitochondrial succinate. Briefly, 100 μL of MTT solution (5 mg/mL) was added to each well. After 2 h, the incubation buffer was removed and the blue MTT–formazan product was extracted with DMSO (dimethyl sulfoxide). Supernatants were collected in a 96-well plate and the absorbance was measured at 540 nm (Microplate Rader) [37].
