Cell Culture and CCL20:LUC Reporter Assay

Caco-2 cells stably transfected with a luciferase reporter construction under the control of the chemokine-ligand-20 (CCL20) promoter (Caco-2-CCL20:LUC) have been previously described (20). The cells were routinely grown in Dulbecco’s Modified Eagle’s Minimum Essential Medium (DMEM, GIBCO BRL Life Technologies, Rockville, MD, USA); supplemented with 15% (v/v) heat-inactivated (30 min, 60°C) fetal-bovine serum (FBS, PAA, GE Healthcare Bio-Sciences Corp., USA), 1% (v/v) non-essential amino acids (GIBCO BRL Life Technologies, Rockville, MD, USA) and the following antibiotics (Parafarm, Saporiti SACIFIA, Buenos Aires, Argentina): penicillin (12 IU/mL), streptomycin (12 µg/mL), and gentamicin (50 µg/mL). Caco-2-CCL20:LUC cells were used at 24 h post-confluence after 8 days of culture at subculture passages between 12 and 22 from the original stocks. All experiments were performed in serum-free medium.

Confluent Caco-2-CCL20:LUC cells cultured in 48-well plates were treated for 30 min with different concentrations of lactate pH 7.4 or different solutions of glycolysis inhibitors. The cells were then exposed to stimulation by flagellin (1 µg/mL), Il-1β (10 ng/mL), or TNF-α (100 ng/mL), during 6 h at 37°C in an atmosphere of 5% CO₂—95% air. A basal condition without any treatment was included as a control lacking stimulation; while flagellin, TNF-α, or IL1-β was added to cell that did not receive any treatment as control of 100% of induction of the proinflammatory response. The cells were next lysed with lysis Buffer (Promega, Madison, WI, USA), and luciferase activity was evaluated using the Luciferase Assay Kit (Promega, Madison, WI, USA) following manufacturer’s instructions and measured in a luminometer (Luminoskan TL Plus). Luminescence was normalized to the stimulated control cells and expressed as a percentage of the normalized average luminescence (% normalized luciferase activity) ± SD from at least three independent experiments.