Bebm media

Human Het-1A squamous esophageal cells were obtained from American Type Culture Collection (ATCC, CRL-2692). Cells were cultured at 37°C and 5% CO₂ in BEBM media (Lonza/Clonetics Corporation) according to ATCC recommendations. Culture flasks and plates were precoated with a mixture of 0.01 mg/mL fibronectin, 0.03 mg/mL bovine collagen type I and 0.01 mg/mL bovine serum albumin dissolved in culture medium. Cells were seeded in 96-well plates (1×10⁴ cells per well), and 24 h later the analyzed compounds were added to cells. Next, cells were incubated for 48 h, examined under microscope, and characterized using WST-1 assay and fluorescent Hoechst/propidium iodide assay as described elsewhere [19 (link),25 (link)]. Hoechst/propidium iodide assay allows evaluation of cell viability by staining all cell nuclei with Hoechst 33342 and dead cell nuclei with propidium iodide. For inhibition of nAChRs and muscarinic acetylcholine receptors (mAChRs), 1 h preincubation of keratinocytes with 1 μM α-bungarotoxin (α-Bgtx, Tocris), 10 μM mecamylamine hydrochloride (Mec, Sigma), 1 μM atropine (Sigma), or 1 μg per 50 μl anti-α7-nAChR antibody (#ab10096, Abcam, Cambridge, UK) was performed before rSLURP-1 application. Antibodies at the same concentration were additionally added to the cells after 24 and 40 hours of incubation with rSLURP-1.

Ethical approval for the study was granted by the Research Ethics Committee (10/HO720/12 and 15/SC/0101). Consent was obtained from all surgical patients for tissue donation for biomedical research purposes. Control participants had no history of respiratory diseases. Characteristics of these cells are detailed in Additional file 1: Table S1. NHBEs and DHBEs were isolated from bronchial brushings from healthy controls and IPF patients undergoing bronchoscopy at the Royal Brompton Hospital (London, UK). For cell isolation, the bronchial brushes were agitated in media to detach the cells and centrifuged at 315 ×g for 5 min to pellet the cells. Cells were cultured in Lonza BEBM media + BEGM bullet kit. Media was changed upon reaching 80% confluency and conditioned media removed for sEV collection after 72 h. Cells were used between passages 1 and 3.