Murine Organ and Fluid Collection

After lung function measurement, bronchoalveolar lavage fluid (BALF) was collected by flushing the lungs with 1 mL phosphate buffered saline (Gibco PBS, pH = 7.4, Thermo Fisher Scientific, Waltham, MA) through the tracheal cannula. BALF was transferred to a 10 mL glass centrifuge tube and centrifuged at 1500 rpm at 4°C for 10 min to pellet BAL immune cells. BALF supernatant was removed and stored in a new 10 mL glass centrifuge tube at −80°C until further downstream processing. Pelleted BAL immune cells were reconstituted in 500 μL phosphate buffered saline (Gibco PBS, pH = 7.4, Thermo Fisher Scientific) and counted as previously described (Manni et al., 2014 (link); Manni et al., 2016 (link)). Blood was collected via cardiac puncture and transferred to serum separator tubes on ice. Serum separator tubes were thawed at room temperature for 30 min and then centrifuged at 10,000 × g for 90 seconds. Murine lung, cecum, and mid-colon tissue were collected immediately after sacrifice and flash-frozen with liquid nitrogen. All BALF, serum, lung, cecum, and mid-colon tissue samples were stored at −80°C until further downstream processing.