Trypsinised

hF were obtained from lung explants. Lung slices (1 mm) were cut and placed in Petri dishes of 90 mm². The small pieces were separated by 2–3 cm and incubated with RPMI 1640 medium (Gibco, USA) supplemented with 10% inactivated foetal bovine serum (FBS) (Gibco, USA), 1 mM L-glutamine, penicillin-streptomycin (50 U/ml, 0.05 mg/ml, respectively), 0.025 mg/ml Vancomycin (Pfizer, Spain) and 0.1 mg/ml cefotaxime (Normon, Spain) and 1 mM HEPES. The explants were removed 21 days later, and fibroblasts grew until confluence. Fibroblasts were used between passages 2 and 7. The cells at 85% confluence were trypsinised (Sigma, Spain) and seeded at a density of 5 × 10⁴ on 24-well plastic dishes.
HF cytospin preparations were stained with Diff-Quick kit following manufacturer’s protocol. Moreover, hF cells were fixed in paraformaldehyde and the immunofluorescence protocol described above was performed. Antibodies used were mouse anti-rat ACTA2 (1:100) (Proteintech, USA) and goat anti-rabbit IgG-TR (1:500) (Santa Cruz Biotechnology, USA) (Fig. 1).

A cell death assay was performed using the Annexin V-FITC and propidium iodide (PI) apoptosis and necrosis detection Kit (Clinisciences, France). All cell types (hAM, hATII and hF) were injured with LPS and treated with heparin, as previously described. Cells were trypsinised (Sigma, Spain), washed with PBS 1X and 2 × 10⁷ cells were resuspended in 1 ml of binding buffer (Clinisciences, France). Ten microliters of Annexin V-FITC solution and 15 μl of PI were added to each treatment condition and incubated for 45 min at 4 °C in the dark. Three hundred microliters of PBS 1X were added, and the stained cells were analysed with flow cytometry (FACSCanto).