Isolation and Culture of Lung Cells

Lung cell suspensions were prepared after enzymatic digestion of the lungs using digestion buffer, containing DNase I and Collagenase A (Roche Diagnostics), for 30 min. The digestion was stopped by adding fetal calf serum (FCS, Hyclone Laboratories, Logan, USA). The lung pieces were passed through a 70 μm filter and rinsed with 10 mL RPMI. Cells were washed and resuspended in RPMI 1640 culture medium (Lonza, Allendale, USA) supplemented with 10% heat-inactivated FCS and 0.1% penicillin-streptomycin solution (Sigma-Aldrich). Lung cells (4 × 10⁵ cells/well) were cultured in medium with or without 50 μg/mL HDM (Greer Laboratories). The supernatant was harvested after 4 days of culture at 37°C in 5 % CO₂ and stored at −20°C until further analysis (31 (link)).