Ncl h441

NCl-H441 (American Type Culture Collection, HTB-174) cells were obtained from LGC Standards (Teddington, UK) and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 1mM sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin (all from Fisher Scientific-UK Ltd, Loughborough, UK) at 37 °C in a humidified air atmosphere containing 5% CO₂. For establishment of an epithelial barrier, the H441 cells (1.5*10⁵ cells/insert) were cultured on the apical side of cell culture inserts in the presence of dexamethasone (Sigma-Aldrich, Poole, UK) with 1% insulin-transferrin-sodium selenite (ITS) supplement (Roche Diagnostics Limited, West Sussex, UK). The membrane supports of the cell culture inserts (0.33 cm² cell culture area) were either the PLLA polymer or PET (Transwell Clear inserts (pore size 0.4 μm), Corning, VWR, Dublin, Ireland). Responses of H441 cells to the pro-inflammatory cytokine TNFα (10ng/ml; Sigma-Aldrich, Poole, UK) were assessed on day 4 post-seeding by challenging the apical epithelial surface with the cytokine or vehicle control; after 24h the cell-free culture supernatants from the apical and basal compartments were collected and stored for ELISA, while the cells were fixed for immunofluorescence staining.

NCl-H441 (American Type Culture Collection, HTB-174) cells were obtained from LGC Standards (Teddington, UK) and cultured at 37 °C in air supplemented with 5% CO₂ in Gibco RPMI-1640 medium (Life Technologies, Paisley, UK) containing 10% fetal bovine serum (FBS), 1% sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin (all from Sigma-Aldrich, Dublin, Ireland).

The human distal lung epithelial cell line NCl-H441 was obtained from the American Type Culture Collection (ATCC) and cultured as previously described [54 (link), 55 (link)], using RPMI-1640 medium (ATCC, Manassas, VA) supplemented with 10% fetal bovine serum (Gibco, Waltham, MA), 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/l glucose, 1.5 g/l sodium bicarbonate and antibiotics (100 IU/ml penicillin and 100 μg/ml streptomycin). Cells were incubated in a humidified atmosphere of 5% CO₂ and 95% O₂ at 37°C. For Ussing chamber assays, cells were grown in Costar Snapwell culture cups, until reaching confluency at 24 h, and then the culture medium was changed every other day for air-liquid interface cultures. We measured transepithelial resistance with an epithelial volto-hmmeter (WPI, Sarasota, FL) and selected the confluent filters for measuring Isc levels with resistance > 500 Ωcm².