L glutamine and sodium pyruvate

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% CO₂.
When 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/cm². The passage number of the cells used in the experiments was between 2 and 6. The culture medium was replaced every 48 h.
Afterward, 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 Escherichia coli 0111:B4 (Sigma Aldrich, protein contamination <1%) in a range of concentrations (30–120 pg/mL) in PBS for 48 h.
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 [26 (link)], before stimulation with lipopolysaccharides from Escherichia coli 0111:B4.
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.

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% CO₂.
When 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/cm². The passage number of the cells used in the experiments was between 2 and 6. The culture medium was replaced every 48 h.
Afterward, 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 Escherichia coli 0111:B4 (Sigma Aldrich, protein contamination <1%) in a range of concentrations (30–120 pg/mL) in PBS for 48 h.
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 [26 (link)], before stimulation with lipopolysaccharides from Escherichia coli 0111:B4.
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.

All the experiments were performed with MDA-MB-231 (ECACC 92020424) and MCF-7 (Sigma-Aldrich, St. Louis, MO, USA, ECACC, #92020424) cells. Cells were cultured in a Dulbecco’s modified Eagle medium (DMEM-High Glucose), with L-glutamine and sodium pyruvate (Sigma-Aldrich, St. Louis, MO, USA), and supplemented with 10% (v/v) of fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA) and 1% (v/v) penicillin-streptomycin (EuroClone Spa, Pero (MI), Italy). Both cell lines and cells seeded on the substrates were cultured in the incubator at 37 °C, 5% CO₂.

DMEM (Dulbecco’s Modified Eagle’s Medium, 4500 mg/L glucose and sodium bicarbonate, without L-glutamine and sodium pyruvate, Sigma), 15% fetal calf serum (Lonza), penicillin (100 units)/streptomycin (100 µg) (Life Technologies), Glutamax (1X, Life Technologies), 0.1 mM beta-mercaptoethanol, non-essential amino acids (1X, Life Technologies), sodium pyruvate (1 mM, Life Tecnologies), leukemia inhibitory factor (LIF, ESGRO 1000 U/ml, Chemicon).