2.6. Cell Culture
ALDRICH
The Caco-2 cell line, a well-characterized intestinal in vitro model, was obtained from Sigma Aldrich (St. Louis, MO, USA). The cells were thawed in T75 tissue culture flasks and expanded in Caco-2/TC-7 Expansion Medium comprised of high-glucose DMEM supplemented with 1% L-glutamine and sodium pyruvate, 10% fetal bovine serum, 1% L-alanyl-L-glutamine and 1% non-essential amino acids (all reagents from Sigma Aldrich, St. Louis, MO, USA) and incubated at 37 °C in a humidified incubator with 5% COWhen the cultures reached 80% confluency, the cells were detached with 5 mL of accutase, resuspended in fresh Caco-2/TC-7 Expansion Medium, and reseeded in 60 mm plates at a density of 450 k cells/cmAfterward, cells were pre-treated either with vehicle (phosphate-buffered saline, PBS) or cocoa-derived polyphenols (25–50 µg/mL) for 1 h before stimulation with lipopolysaccharides from To verify the mechanism, the cells were also treated with TAK-242 (1 μM in PBS; InvivoGen, Toulouse, France), a specific inhibitor of TLR4 signaling [Conditioned media were collected to quantify soluble zonulin as described; pellets were analyzed by Western blot to verify barrier damage through occludin expression. The experiments were conducted on three different batches of Caco-2.