Isolation of Lung Cells from Bronchoalveolar Lavage

Bronchoalveolar lavage (BAL) was performed using three aliquots of saline via a tracheal cannula (28 (link)). BAL fluid was centrifuged, supernatants removed, and cell pellets resuspended in 0.5 ml complete media (RPMI/10% FCS/2 mM l-glutamine/100 U/ml penicillin/streptomycin). One lobe of lung tissue was mechanically chopped and incubated at 37°C for 1 h in complete media containing 0.15 mg/ml collagenase (Type D; Roche Diagnostics, West Sussex, UK) and 25 μg/ml DNAse (Type 1; Roche Diagnostics). Recovered cells were filtered through a 70-um nylon sieve, washed twice and resuspended in 1 ml complete media.