4.2. Intestinal Epithelial Cell Culture and IL-8 Measurement

The human intestinal epithelial cell line T84 was obtained from the American Type Culture Collection (ATCC^® CCL-248^™, Manassas, VA, USA) and maintained in an atmosphere containing 5% CO₂ at 37 °C in the culture medium recommended by ATCC. T84 intestinal epithelial cells were seeded in 24-well tissue culture plates at a density of 2 × 10⁵ cells/well and incubated at 37 °C for 24 h. To induce IL-8 production, T84 cells were treated with an AIEC LF82 bacterial lysate (overnight culture boiled for 5 min) or H₂O₂ (1 mM). At the same time, 25 µL or 50 µL of NMW1 and NMW2 or controlled water CW (distilled water) was added, and the IL-8 concentration in the cell supernatant was quantified after a 66-h period. Quantification of the amount of the pro-inflammatory cytokine IL-8 released by human intestinal epithelial cells was performed by ELISA according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA).