Fibroblast growth basal medium

Viability test was performed as previously described with slight modification [41 (link),42 (link)]. Briefly, 1 × 10⁴ cells were plated in a 96-well plate. Eight hours after perphenazine treatment, cells were incubated with a mixture (1:10) of EZ-Cytox cell viability assay kit (Dogen, EZ3000) and Fibroblast Growth Basal Medium (CC-3131, Lonza). Then, the plate was incubated for 30 min in the incubator and determined absorbance at 450 nm with reference to 655 nm wavelength (iMark, Biorad).

Human lung fibroblasts were isolated from BAL fluid of a 65-year-old male patient with IPF. The sample was filtered with 300 µm sterile gauze (Biosigma S.p.A. a Dominique Dutscher Company, Italy, Europe). The pellet was treated with 0.91 U/mg Dyspase (Gibco, ThermoFisher Scientific, Waltham, MA, USA) for 90 min to separate cells grown in vitro; the enzyme is very mild and does not damage cell membranes. It was also used to prevent clumping in culture. Then the cells were seeded in a 12-well plate at a concentration of 500,000/mL for 24 h with RPMI supplemented with 10% fetal bovine serum (FBS) (Gibco, ThermoFisher Scientific, Waltham, MA, USA) and 0.5% penicillin-streptomycin (Merck KGaA, Darmstadt, Germany). After 24 h, the supernatant was removed with non-adhering cells, which were washed with phosphate-buffered saline (PBS), adding 0.5 mL of Fibroblast Growth Basal Medium (Lonza, NC, USA) supplemented with 0.050 mL 1X insulin, 1X human fibroblast growth factor-2 (FGF-2) and 0.50 mL FBS, and 0.50 mL 1X Gentamycin1000 (all purchased from Lonza, NC, USA). The complete medium was changed once a week or when appropriate. Cells were maintained in 5% CO₂ at 37 °C.

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.