Lhc 9 medium

Antibody against p-Stat-3(pS727)(sc-8001) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Antibodies against p-Stat-3(pY705)(#9145), p-NFκB-p65(pS536)(#8242), and NFκB -p65(#3033) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies against Stat-3(GTX15523), IL1-β(GTX22105), TNF-α(GTX110520), and IL-6 (GTX110527) were purchased from GeneTex, Inc. (Irvine, CA, USA). Antibody against visfatin(ab58640) was purchased from Abcam plc. (Cambridge, UK). Antibody against β-actin(A5411) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). LHC-9 medium was purchased from Invitrogen (Carlsbad, CA, USA). FK866, lipopolysaccharide, hematoxylin, and eosin were purchased from Sigma-Aldrich (St Louis, MO, USA).

Non-small cell lung cancer cell lines, A549, NCI-H838, NCI-H1299 and NCI-H460 and two small cell lung cancer cell lines, H69 and H146 were purchased from Amercian tissue collection center (ATCC). All of the cell lines, except A549, were cultured in RPMI-1640 medium (Invitrogen, Carlsbad, CA) containing 10 % fetal bovine serum (FBS) (Invitrogen) at 37 °C with 5 % CO₂. A549 is cultured with the same conditions as for other cell lines, except that it is cultured in F12K medium (Invitrogen). The immortalized cell line BEAS-2B was purchased from ATCC and was cultured in LHC-9 medium (Invitrogen). HBEC3 cell was generally provided by Dr. Jerry W. Shay (University of Texas Southwestern Medical Center). This cell line was cultured in Keratinocyte-SFM supplemented with 50 μg/mL bovine pituitary extract and 5 ng/mL epidermal growth factor (Gibco) at 37 °C on porcine gelatin-coated tissue dishes as previously described [22 ].

The bronchial epithelial cell line VA10 was previously established at the laboratory [25] (link). The VA10 cell line is available upon request for all academic investigators for non-commercial purposes. Cells were cultured in LHC9 medium (Invitrogen, NY, USA) supplemented with 50 IU/ml penicillin and 50 µg/ml streptomycin (Invitrogen). For air-liquid interface cultures, cells were seeded on the upper layer of Transwell cell culture filters (Corning®Costar®) pore size 0.4 µm, 12 mm diameter, polyester membrane) (Sigma-Aldrich, St. Louis, USA) at density of 2×10⁵ cells per well. The cultures were maintained on LHC9 for 5 days, 0,5 ml in the upper chamber and 1.5 ml in the lower chamber. After 5 days medium was changed to DMEM/F-12 (Invitrogen), supplemented with 2% Ultroser G (Cergy-Saint-Christophe, France) for another 5 days. For air-liquid interface culture, the medium was aspired from the apical side and rinsed with PBS. Primary cells were isolated from patient material with enzyme digestion and cultured in chemically defined bronchial cell growth medium as previously described [29] (link) Isolated cells were maintained on collagen coated flasks (VitroCol, Advanced BioMatrix) and cultured in chemically defined bronchial epithelial cell medium (BEGM, Life Technologies/Sigma) as previously described [30] .

Escherichia coli ATCC8739, Staphylococcus aureus ATCC6538P, Pseudomonas aeruginosa ATCC15692 (PAO1), Candida albicans ATTC10231 and Candida albicans SC5314 strains originated from Jan Kochanowski University, Poland collection. The eukaryotic cell lines: adenocarcinoma human alveolar basal epithelial A549 cells (ATCC® CCL-185™) and normal human bronchial epithelium BEAS-2B cells (ATCC® CRL-9609™) were provided by American Tissue Cell Collection. A549 cells were cultured in F-12K medium (Sigma–Aldrich Chemicals, USA) supplemented with 10% fetal calf serum (Invitrogen, CA, USA), 2 mM L-glutamine (Sigma–Aldrich Chemicals, USA) and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin (Invitrogen, CA, USA) at 37 °C. BEAS-2B cells were cultured in LHC9 medium (Invitrogen, CA, USA) at 37 °C. Both cell lines were cultured in a humidified 5% CO₂ atmosphere.

50.000 starved cells were seeded in DMEM/F12 basic medium on Falcon® (BD) cell culture inserts with 8 µm pore size in triplicates. LHC9 medium (Invitrogen) was used as a chemoattractant in the lower chamber. After 12 h incubation, cells in upper chamber were removed with a cotton swab and migrated cells in lower chamber fixed with 3,7% formaldehyde, stained with 0,1% crystal violet, rinsed with PBS and counted.