Nci h441 cells

The human lung adenocarcinoma NCI-H441 cells were purchased from ATCC and maintained in RPMI medium supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals, Flowery Branch, GA, USA), 2 mM L-glutamine, 25 U/mL penicillin, and 25 μg/mL streptomycin. The A549-hACE2 (HA-Flag) cells were obtained from BEI Resources (NIAID, NIH) and maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS (Atlanta Biologicals), 2 mM L-glutamine, 25 U/mL penicillin, and 25 μg/mL streptomycin and 1 μg/mL puromycin. The cells were grown at 37 °C and 5% CO₂ in a humidified chamber.

The use of embryos for cell culture was approved by the University of Queensland animal ethics committee (SCMB/002/18). Seventeen-day-old chicken and twenty-one-day-old Pekin duck embryonated eggs were purchased from Darling Downs Hatchery (Queensland, Australia). Primary aortic endothelial cells were cultured from the aortic arches of chicken and duck embryos as described previously using EGM-2MV medium (Lonza, Basel, Switzerland) with 10% FCS (Gibco, Waltham, MA, USA) [7 (link),8 (link)]. Primary chicken and duck embryo fibroblasts were cultured as described previously [9 (link),10 (link)]. Madin–Darby Canine Kidney (MDCK) cells were obtained from American Type Culture Collection (ATCC), and cultured in DMEM (Gibco, Waltham, MA, USA) supplemented with 10% FCS (Gibco, Waltham, MA, USA) and 1% penicillin/streptomycin (Gibco, Waltham, MA, USA). NCI-H441 cells (human lung epithelial cells) were obtained from ATCC and cultured in RPMI (Gibco, Waltham, MA, USA) medium supplemented with 10% FCS (Gibco) and 1% penicillin/streptomycin (Gibco). Primary human pulmonary microvascular endothelial cells were obtained from Sciencell (Carlsbad, CA, USA) and cultured in an endothelial cell growth medium (Sciencell, Carlsbad, CA, USA). All cell lines of mammalian origin were incubated at 37 °C 5% CO₂ whilst all avian cells were cultured at 40 °C 5% CO₂ unless otherwise stated.

We performed the in vitro transmigration assay of human PMNs through a monolayer of NCI-H441 cells (ATCC, USA) (n≥2) to separate the effects of the PDE4-inhibitors on PMNs and epithelium [23 (link)]. Epithelium or PMNs were incubated with rolipram or roflumilast for 60 min at indicated concentrations (n≥4). Until reaching confluence, human epithelial cells were cultivated on inserts of a transwell system (3.0μm pore size, 6.5mm diameter; Costar, Cambridge, MA, USA). Isolated human PMNs (Percoll gradient; GE Healthcare Bio-Sciences AB, Uppsala, Sweden) migrated through the monolayer of epithelial cells along a chemotactic gradient (CXCL2/3; 200ng/ml; Pepro Tech, Hamburg, Germany). By determination of myeloperoxidase were migrated PMNs quantified (absorption length: 405 nm).