Interleukin 13 il 13

Neutrophils were isolated from peripheral blood using an EasySep direct human neutrophil isolation kit (Stemcell, Vancouver, BC). Cells were then counted and cultured at 10⁷ cells/mL in RPMI supplemented with 5% fetal bovine serum and 1% penicillin/streptomycin. Neutrophil cultures were treated with GM-CSF (1–25 ng/mL, R&D Systems, Minneapolis, MN), FSTL1 (100–1000 ng/mL, R&D Systems), Interferon-γ (IFNγ) (10 ng/mL, R&D Systems), lipopolysaccharide (LPS) (1 μg/mL, Enzo Life Sciences, Farmingdale, NY), Interleukin 4 (IL-4) (20 ng/mL, R&D Systems), Interleukin 13 (IL-13) (20 ng/mL, R&D Systems), Interleukin 25 (IL-25) (10–100ng/mL, R&D Systems), Interleukin 33 (IL-33) (10–100 ng/mL, R&D Systems), thymic stromal lymphopoietin (TSLP) (10–100 ng/mL, R&D Systems) or Leukotriene C₄ (LTC₄) (10⁻⁶ –10⁻⁷ Cayman Chemical, Ann Arbor, MI) for 20 hours, cell culture supernatants were collected for protein analysis and cell lysates were collected to isolate RNA. Plates were coated with E-selectin, ICAM (50ng/well, Peprotech, Rocky Hill, NJ) or bovine serum albumin (BSA) (50ng/well, Sigma Aldrich) in pH 8 tris buffered saline (TBS) overnight at 4 degrees. Neutrophils were seeded into the wells and the culture supernatants were harvested at 20 hours.

Cell culture reagents and supplies including Dulbecco’s Modified Eagle’s Medium/F12 (DMEM/F12), trypsin and Antibiotic-Antimycotic (AbAm) were purchased from Invitrogen (Carlsbad, CA). Charcoal stripped fetal bovine serum (FBS) was from Sigma-Aldrich (St. Louis, MO). α7nAChR antibody was from Novus Biologicals (Littleton, CO). Other antibodies for α4nAChR, β2nAChR, caldesmon, α-SMA, calponin-1 and transgelin/SM-22 were obtained from Abcam (Cambridge, UK). β-actin antibody was from ABM Biological Materials (Richmond, Canada). Pro-inflammatory cytokines, tumor necrosis factor alpha (TNFα) and interleukin-13 (IL-13) were from R&D Systems (Minneapolis, MN). RIPA cell lysis/extraction buffer and A/G PLUS Agarose beads for co-immunoprecipitation techniques were obtained from Thermo Fisher Scientific (Waltham, MA) and SantaCruz Biotechnology (Dallas, TX) respectively. Pharmacological inhibitors for signaling pathways NFkB (SN-50) and STAT6 (AS1517499) were from SantaCruz Biotechnology (Dallas, TX) and Millipore Sigma (Burlington, MA) respectively. STAT3 (SD 1008), AP1 (SR 11302), CREB (666-15) and MAPK (PD 98059) were from Tocris Bioscience (Bristol, UK). Fluorescent secondary antibodies were from Li-Cor Biosciences (Lincoln, NE). Other chemicals/drugs/antibodies were from Sigma-Aldrich unless otherwise specified.