A549 cells (American Type Culture Collection, Rockville, MD, USA) and NHBE cells (CC-2541, Lonza, Walkersville, VA, USA) were maintained in, respectively, Dulbecco’s modified minimal essential medium (DMEM; Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Invitrogen) and bronchial epithelial growth medium (BEGM; Lonza) in a humidified incubator (5% CO2 at 37°C).
The toxin-producing H. pylori strain, ATCC 49503, was the source of VacA, which was purified as described previously [12 (link)].
To quantify cytokine production, A549 cells were seeded in 6-well plates at a density of 3.0 × 105 per well and NHBE cells in 12-well plates at a density of 1.0 × 105 per well. At subconfluence, serum-free medium was added to the wells. Cells were subsequently stimulated for 3, 6, 12, and 24 h with 1, 10, 30, 60, or 120 nM VacA or heat-inactivated VacA (100°C, 10 min; iVacA control). These VacA concentrations have been used for the purpose of investigating the effects of toxin on gastric epithelial cells [13 (link)]. Changes in cell morphology were assessed by phase-contrast light microscopy.
The toxin-producing H. pylori strain, ATCC 49503, was the source of VacA, which was purified as described previously [12 (link)].
To quantify cytokine production, A549 cells were seeded in 6-well plates at a density of 3.0 × 105 per well and NHBE cells in 12-well plates at a density of 1.0 × 105 per well. At subconfluence, serum-free medium was added to the wells. Cells were subsequently stimulated for 3, 6, 12, and 24 h with 1, 10, 30, 60, or 120 nM VacA or heat-inactivated VacA (100°C, 10 min; iVacA control). These VacA concentrations have been used for the purpose of investigating the effects of toxin on gastric epithelial cells [13 (link)]. Changes in cell morphology were assessed by phase-contrast light microscopy.