Bronchial Alveolar Lavage Sampling Protocol

Bronchial alveolar lavage (BAL) samples were obtained and processed as previously described^{26 (link)}. Lungs were flushed with 2 mL total (0.5 mL per wash, 4×) of HBSS supplemented with 0.3% FBS and 300 μM EDTA. Collected BAL was separated into three separate aliquots: 1.0, 0.5, and 0.5 mL. Cell count and viability measurements using trypan blue stain were performed using Countess Automated Cell Counter (Invitrogen). Cells were blocked with anti-CD16/CD32 (BD Biosciences, San Jose, CA) for 20 min on ice and processed for flow cytometry. For protein lysate samples, one of the 0.5 mL BAL aliquots was treated with 10 μL protease inhibitor cocktail set III, EDTA-free (Calbiochem, MA) and 20 μl of 10% Triton X-100 (Sigma, MA) and mixed by inversion for 20 minutes at 4 °C. Protein lysate samples were used for mouse neutrophil elastase/ELA2 and MPO ELISA (R&D Systems, MN) per manufacturers protocol. The other 0.5 mL BAL aliquots were centrifuged, and cell-free supernatant was collected and used for lactate dehydrogenase (LDH) release assay according to manufacturer’s instructions (Sigma).