Fgm 2

A normal human lung fibroblast (NHLF) cell line (cat # CC2512) was purchased from Lonza (Lonza, Walkersville, MD, USA). We used 3 different batches of NHLF cells collected from 3 different donors as follows: Batch # 0000343490 was collected from a 24-year-old female; batch # 0000369145 was collected from a 43-year-old male and batch # 0000608197 was collected from a 67-year-old male. NHLFs were cultured in fibroblast growth medium (FGM-2) (Lonza), and cells at passage 3 to 7 were used for all experiments. Three human IPF lung fibroblast cell lines (CCL-134 and CCL-191) were purchased from American Type Culture Collection (ATCC) (ATCC, Manassas, VA, USA), and IPF lung fibroblasts (cat # CC-7231) purchased from Lonza at passage 2 to 5 were used for all experiments. IPF lung fibroblasts from ATCC were cultured in F12K medium, while those from Lonza were cultured in FGM-2 (Lonza). Early passages of IPF fibroblasts were used in this study as IPF-derived fibroblasts are known to display morphological features of replicative senescence even at early passage, unlike age-matched control fibroblasts at the same passage [30 (link), 31 (link)]. Human recombinant IFN-γ (Sigma-Aldrich, St. Louis, USA), PFD (TCI America, Montgomeryville, PA), TGF-β1 (Cell Signaling Technology, Danvers, USA) and PDGF-BB (R&D Systems, Minneapolis, USA) were used in our experiments.

GFP-HUVEC (Neuromics, USA) or Non-GFP-HUVEC (Lonza, USA) were cultured up to passage 6 in endothelial growth medium (EGM-2, Lonza). HLFs were cultured up to passage 10 in fibroblast growth medium (FGM-2, Lonza). Cell cultures were grown to 80% confluence before passage and use in experiments. DPBS (Gibco, USA) was preheated at 37 °C for 2 h. The fibrinogen solution (15 mg/ml) was prepared by dissolving 15 mg bovine fibrinogen (Sigma, USA) in the preheated DPBS. Thrombin (100U, Sigma, USA) solution was dissolved in 1 ml DPBS which contained 0.1% BSA.