Multi well plate

Caco-2 and THP-1 cells were grown in RPMI medium 1640 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), 2 mM L-glutamine, 100 U/mL penicillin, and 10 μg/mL streptomycin. Caco-2 cells were seeded at a density of 1 × 10⁵ cells/mL in 24 multiwell plates (Corning) and allowed to undertake spontaneous differentiation for 21 days with regular medium changes every 5 days. Finally, before the incubations with the fecal material, cells were washed twice with phosphate saline buffer (PBS) and the medium was replaced with RPMI supplemented as above descripted without antibiotics.
For the co-culture system, Caco-2 cells were seeded at a density of 5 × 10⁵ cells/mL in 0.4-µm pore polycarbonate (PC) membrane inserts (Corning) and allowed to differentiate for 21 days with medium changes on days 4, 8, 12, 16, and 18. THP-1 cells were seeded at a density of 3.8 × 10⁵ in 12 multiwell plates (Corning) and subjected to treatment with phorbol 12-myristate 13-acetate (PMA) (2 nM) for 5 days to allow differentiation of THP-1 monocytes into macrophages. Caco-2 and THP-1 cells were washed twice with PBS, the medium was replaced with RPMI supplemented with FBS and glutamine, but without antibiotics. Finally, the membrane inserts were placed in the wells with differentiated THP-1 cells and left overnight before the incubation with the fecal material.

The concentration of collagen in the supernatants was analyzed by ELISA. Treated and control cells were cultured with medium for 48 hours. Then the culture supernatants were collected, filtered and added to multi-well plates (Costar). After incubation overnight at 4°C, wells were blocked with 1% BSA in PBS and incubated with a rabbit anti-Type I Collagen antibody (1:2500) (Acris Antibodies), followed by a peroxidase-conjugated goat anti-rabbit IgG (Sigma) at 1:500 dilution. All incubations were performed for 1 h at 37°C and between each step washed three times with PBS wash buffer (PBS containing 0.05% Tween₂₀). Ortho-phenylene-diamine was used as a chromogen. Optical densities (OD) were measured at 492 nm in an Easy Reader (Bio-Rad).

The concentration of collagen in the supernatants was analyzed by ELISA. Treated and control cells were cultured with medium for 48 hours. Then the culture supernatants were collected, filtered and added to multi-well plates (Costar). After incubation overnight at 4°C, wells were blocked with 1% BSA in PBS and incubated with a rabbit anti-Type I Collagen antibody (1:2500) (Acris Antibodies), and then with a peroxidase-conjugated goat anti-rabbit IgG (Sigma) at 1:500 dilution. All incubations were performed for 1 h at 37°C and between each step washed three times with PBST. Ortho-phenylene-diamine was used as a chromogen. ODs were measured at 492 nm in an Easy Reader (Bio-Rad).