Elastase

PBS-perfused lungs were inflated with 2 ml of digestion cocktail containing 50 U/ml dispase (Corning), 250 U/ml collagenase type I (Worthington), 5 U/ml elastase (Worthington), and 60 U/ml DNAse I (Roche). The trachea was clipped distally and lungs were dissected in a petri dish on ice to remove extrapulmonary airways (trachea and main bronchi). Lung lobes were placed in a C tube (Miltenyi) containing 3 ml of digestion cocktail, and the m_lung_01 program was run on gentleMACS (Miltenyi). C tubes were placed in a rotating incubation oven at 37 °C for 30 min. The m_lung_02 program was run again, and the tubes were placed on ice for the next steps. The lung cells were passed through a 70 µm cell strainer (Corning) and centrifuged at 400 g for 5 min. The pellet was resuspended in a red blood lysis buffer solution (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA), incubated for 2 min on ice, and washed with EasySep buffer (STEMCELL Technologies) at 400 g for 5 min. To isolate mesenchymal cells (Fig. 2, Supplementary Fig. 2), a milder digestion cocktail was used containing only 375 U/ml collagenase and 60 U/ml DNAse I. In all experiments, lung single-cell suspensions from several mice were pooled before fluorescence-activated cell sorting was performed.

Single-cell suspensions were prepared as described previously (12 (link), 28 (link)). Briefly, mice were deeply anesthetized and intracardially perfused with 20 ml of ice-cold PBS to eliminate blood cells. The hearts were minced with fine scissors and placed into a cocktail of 1 mg/ml collagenase II (Worthington, Lakewood, NJ, USA), 100 U/ml elastase (Worthington), and 100 U/ml DNase I (Sigma-Aldrich) and shaken at 37°C for 1 h. Tissue samples were then triturated through a 70 μm cell strainer and centrifuged (5 min, 400 g, 4°C). The obtained cells were counted after erythrocyte lysis and washed using PBS for further analysis. For staining, 5 × 10⁶ cells were pre-incubated for 5 min on ice with anti-CD16/CD32 antibody (2.4G2, BD Bioscience, San Jose, CA, USA) to block the non-specific antibody and then stained with directly conjugated antibodies for 30 min at 4°C in the dark in PBS. For intracellular cytokine staining, single-cell suspensions were stimulated with 50 ng/ml (PMA, Sigma-Aldrich), 1 μg/ml ionomycin (Sigma-Aldrich), and Golgi-PlugTM (BD Biosciences) for 4 h. Surface staining was performed first. After fixation and permeabilization using the Cytofix/Cytoperm Soln kit (BD Biosciences), intercellular proteins were stained. All experiments were performed on an Attune NxT flow cytometer (Invitrogen) and analyzed using FlowJo version 10 software.

Mice were deeply anaesthetized and intracardially perfused with 40 ml of ice‐cold PBS to exclude blood cells. The heart was dissected, minced with fine scissors, and enzymatically digested with a cocktail of type II collagenase (Worthington Biochemical Corporation, Likewood, NJ, USA), elastase (Worthington Biochemical Corporation), and DNase I (Sigma‐Aldrich) for 1.5 hrs at 37°C with gentle agitation. After digestion, the tissue was triturated and passed through a 70‐μm cell strainer. Leucocyte‐enriched fractions were isolated by 37–70% Percoll (GE Healthcare, Pittsburgh, PA, USA) density gradient centrifugation as described elsewhere 11. Cells were removed from the interface and washed with RPMI‐1640 cell culture medium for further analysis.