Normal human bronchial epithelial cells nhbe

Normal Human Bronchial Epithelial Cells (NHBE) and Bronchial Epithelial Cell Growth Medium were obtained from Lonza (Switzerland). Primary Human Nasal Epithelial Cells (HNEpC) and Airway Epithelial Cell Growth Medium were obtained from Promocell (Germany). Grindelic acid (purity ≥95% (LC/MS-ELSD)), clarithromycin (purity ≥95% (HPLC)), budesonide (purity ≥98% (HPLC)), LPS (from Escherichia coli 0,111:B4), Ficoll Hypaque gradient, GM-CSF, HEPES buffer, l-glutamine, RPMI 1640 medium, Fetal bovine serum (FBS), Cell Dissociation Solution (non-enzymatic), Accutase™, propidium iodide and Calcein-AM were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). TLR-4-(PE)-conjugate (cat no. HTA125) was obtained from eBioscience. Human Quantikine ELISA Kits were purchased from R&D System (Minneapolis, USA). Anti-Human CD54-(-APC) (cat no. 559771), Anti-Human CD106-(-PE) (cat no. 555647), Anti-Human CD62E-(-PE) (cat no. 551145) were purchased from BD Pharmingen.

Seven‐ to eight‐week‐old BALB/c mice were purchased from Jackson Laboratory (Bar Harbor, ME), maintained in a specific pathogen free facility at Cincinnati Children's Hospital Medical Center (CCHMC) and handled under Institutional Animal Care and Use Committee‐approved procedures. Following isoflourane anesthesia, animals underwent allergen immunization as previously described 25 with the following modifications: mice were serially immunized intratracheally every 48 h for 2 weeks with low‐endotoxin, low‐dose HDM extract (D. pteronyssinus, Greer Labs; Lenoir, NC) (3 μg total protein/dose in 50 μL of sterile phosphate‐buffered saline plus vehicle [PBS]). Myriocin (Cayman Chemical Company; Ann Arbor, MI) stock solutions (5 mM) were prepared in methanol, and aqueous solutions were prepared fresh before each experiment (1.2 μg/intratracheal dose). Ceramide C6 (N‐hexanoyl‐D‐erythro‐sphingosine) was obtained from Avanti Polar Lipids (Alabaster, AL), mouse GM‐CSF was acquired from PeproTech(Rocky Hill, NJ), and RPMI and serine‐free MEM were purchased from Gibco (Carsbad, CA). Normal human bronchial epithelial cells (NHBE) were obtained from Lonza (Walkerville, MD) and cultured per the manufacturer's instructions. Murine bone marrow‐derived dendritic cells (BMDCs) were generated as previously described 26.

Human lung adenocarcinoma cell lines [NCI-H1650, NCI-H1792, NCI-H1838, NCI-H1975, NCI-H2009, NCI-H2291, NCI-H2444, HCC-95, HCC-193, EKVX and SK-Lu-1] were provided by Max Planck Institute of Biochemistry and authenticated using STR profiling by John Hopkins Fragment Analysis Facility (April 2013). All cells were maintained in Roswell Park Memorial Institute medium supplemented with L-glutamine and 10% fetal bovine serum, except for SK-Lu-1 cells which were maintained in Minimum Essential Medium with L-glutamine and 10% fetal bovine serum. Normal Human Bronchial Epithelial Cells (NHBE) were purchased from Lonza (Basel, Switzerland), and cultured in serum-free medium as described by Clonetics. All cells were incubated in a 37°C incubator with 5% CO₂.