PBECs were cultured at a density of 1 × 106 cells/mL in 96- or 6-well plates (Corning) pre-coated with collagen, fibronectin and BSA as described before [18] (link). After reaching near-confluence, these PBECs were pretreated with 0.5%, 1% or 2% GOS for 24 h or pretreated with MCC950 (10 μM; InvivoGen, San Diego, CA) for 6 h prior to stimulation with LPS (10 µg/mL; isolated from E. coli O111:B4, Sigma-Aldrich) for 6 or 24 h with or without ATP (5 mM; InvivoGen) for 0.5 h, or stimulation with leukotoxin A (10 ng/mL; Enzo Life Sciences, Bruxelles, Belgium) for 0.5, 1, 6, 12 or 24 h or stimulation with M. haemolytica (1 × 105 CFU/mL) for 24 h, or stimulation with rotenone (10 μM; Sigma-Aldrich) for 6 h. After stimulation, supernatants were collected and stored at −20 °C until analysis.
A549 cells were cultured at a density of 0.5 × 105 cells/mL in 96- or 6-well plates (Corning). After reaching near-confluence, A549 cells were pretreated with 2% GOS for 24 h prior to stimulation with LPS (10 µg/mL; E. coli O111:B4, Sigma-Aldrich) for 6 or 24 h with or without ATP (5 mM; InvivoGen) for 0.5 h. After stimulation, supernatants were collected and stored at −20 °C until analysis.
A549 cells were cultured at a density of 0.5 × 105 cells/mL in 96- or 6-well plates (Corning). After reaching near-confluence, A549 cells were pretreated with 2% GOS for 24 h prior to stimulation with LPS (10 µg/mL; E. coli O111:B4, Sigma-Aldrich) for 6 or 24 h with or without ATP (5 mM; InvivoGen) for 0.5 h. After stimulation, supernatants were collected and stored at −20 °C until analysis.
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