Fgm 2 growth medium

NHLF were resuspended in FGM-2 growth medium (Lonza), mixed with an equal volume of NHBE in BEGM growth medium (Lonza), and seeded into a 96-well, flat-bottom culture plate (Corning) at a density of 20,000 and 1,600 cells per well, respectively. Compound dilution series were added, and the coculture was incubated for 18 hours at 37°C/5 % CO₂. The medium was then replaced with Fibroblast Growth Basal Medium (Lonza) containing 0.1 % fatty acid–free BSA and the compounds at the indicated concentrations. The medium was supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, for the entire duration of the culture. If not indicated otherwise, the coculture was incubated at 37°C/5 % CO₂ for 96 hours.

To measure dynamic cell shape and adhesion changes during fibrotic transdifferentiation, NHLF were seeded at a density of 20,000 cells per well in FGM–2 growth medium (Lonza) containing 100 units / ml of penicillin and 100 μg / ml of streptomycin into E–plates with continued impedance signal measurement (xCELLigence system, Roche Applied Science). After overnight growth, the medium was exchanged for FBM (Lonza) supplemented with 100 units / ml of penicillin and 100 μg / ml of streptomycin and 0.1% faf BSA and cells were starved for 32 h. Compound dilution series were added and myofibroblast transition was induced by adding TGF–β1 at a concentration of 5 ng / ml, followed by continued impedance sampling for up to 48 h. For data analysis, impedance raw traces were normalized to the time point of compound addition and the baseline response (DMSO, non–TGF–β1 treated cells) was subtracted. In some cases, impedance values at defined time points were used to generate concentration–response curves to determine compound potency and efficacy using GraphPad Prism software version 7 (GraphPad Software Inc).