NHBE cells (Lonza Walkersville, Inc; Walkersville, Maryland) were plated at 3,500 cells/cm- in culture dishes in bronchial epithelial cell growth medium supplemented with the single-quot kit (Lonza Walkersville, Inc) without antibiotics and cultured at 37°C in a 5% CO- incubator. We used endotoxin-free media (< 0.005 endotoxin units/mL) and second-passage cells for all experiments. Cells were grown to confluence for 6 days. Cultures without antibiotics were then transferred to six-well or 35-mm dishes coated with type 1 rat-tail collagen and seeded at 3,500 cells/cm2. The medium was changed every 24 h. To avoid influence of growth factors on cell signaling and IL-8 secretion, cells were cultured in supplement-free bronchial epithelial cell basal medium for 24 h before stimulation. We evaluated cell response at the time of cell confluence rather than normalized to the relative number of cells because cell maturation could affect cell signaling and cytokine secretion, and at confluence, all cells are at similar growth stages.
For NHBE cell differentiation, cells were plated at 2.0 X 105 cells/cm2 onto polycarbonate inserts of 6.5-mm diameter, 0.4-pm pore size, and 10-pm thickness (Transwell Clear; Corning Costar; Cambridge, Massachusetts); coated with type 1 rat-tail collagen; and cultured with serum-free Dulbecco modified eagle medium:nutrient mixture F-12 medium containing 1.0% ITS-A (Invitrogen Corp; Carlsbad, California), 0.5 ng/mL recombinant human epidermal growth factor (Invitrogen Corp), 10 ng/mL triiodothyronine (MP Biomedicals LLC; Solon, Ohio), 0.5 mg/mL hydrocortisone (MP Biomedicals LLC), 1.0 X 102? M all-trans retinoic acid (Sigma-Aldrich Corp), 2.0 mg/mL bovine serum albumin (Sigma-Aldrich Corp), and 30 mg/mL bovine pituitary extract (Invitrogen Corp). After achieving confluence, the apical medium was removed, and cells were cultured with an air-liquid interface (ALI) method Canadian Generic viagra. The culture medium was changed every 48 h, and cells were maintained at 37°C in 5% CO- for 10 to 14 days.