Isolation and culture of lung cells

Lung cell suspensions were prepared by cutting the lung into small pieces and by adding digestion buffer, containing DNase I and Collagenase A (Roche Diagnostics, Germany), for 30 min. The digestion was stopped using fetal calf serum (FCS; Hyclone Laboratories, USA). The lung pieces were transferred toward a 70-µm nylon cell strainer (BD Biosciences, The Netherlands) and rinsed with 10 mL RPMI. Cells were washed and resuspended in RPMI-1640 culture medium (Lonza, USA) supplemented with 10 % heat-inactivated FCS and 0.1 % penicillin–streptomycin solution (Sigma-Aldrich). Total number of cells was calculated using a Beckman Z1 coulter^® Particle Counter (Beckman, USA). Lung cells (4 × 10⁵ cells/well) were cultured in a Greiner bio-one CellSTAR 96-well U-bottom plate (Greiner Bio-One B. V., The Netherlands) in medium with or without 50 µg/mL HDM (Greer Laboratories, USA). The supernatant was harvested after 4 days of culture at 37 °C in 5 % CO₂ and stored at −20 °C until further analysis [13 (link)].