Facscantoii

Lung tissue was minced on ice and incubated in lysis buffer (2.5 mg/ml Collagenase D, 0.01 mg/ml DNase I in RPMI) at 37 °C for 30 min with constant agitation. Bone marrow was isolated by centrifugation from mouse tibia and femur at 1000 × g for 5 min at 4 °C. Single cell solutions were prepared using a 70-µm cell strainer (Corning) in FACS buffer (2% FCS in PBS). Red blood cells were lysed using RBC lysis buffer (BioLegend) for 5 min on ice. Life/dead staining was performed using Zombie Nir Fixable Viability Kit (BioLegend; catalog number: 423105) for alveolar macrophages or DAPI for other populations following the manufacturer’s instructions. Antibody staining was performed for 30 min at RT in the dark with an antibody concentration of 6 µl/ml. For antibody details see key resource table. Stained cells were resuspended in FACS buffer containing 2% BSA for subsequent analysis or sorts. Flow cytometric analysis as well as FACS was performed using a FACSAria II Fusion SORP (BD), analysis was performed on a FACSCantoII and data analysis was done using FACSDiva software v 8.0 as well as FCS Express 6 (DeNovo Software, USA). A representative example gating strategy that was used for sorting AMs and the confirmed purity of the sort (re-analysis) is shown in Supplementary Fig. 9. Representative gating strategies used for the data shown in Fig. 4 are shown in Supplementary Fig. 10.

For cell cycle analysis experiments using propidium iodide (PI), cells were pelleted and fixed in 80% ice-cold ethanol and stored at 4°C until further processing. Cells were stained with PI at 50 μg/ml in PBS containing RNase A and analyzed by flow cytometry on the BD FACSCanto II analyzer (BD Biosciences). The percentage of cells in G0/G1, S and G2/M phases were determined using Modfit 3.0 software.
For cell cycle analysis using 5-bromo-2′-deoxyuridine (BrdU) incorporation, cells were labeled with 10 μM BrdU for 30 min, washed twice with PBS, collected and harvested as above. Cells were pelleted and incubated in 1 mL of 2N HCl containing 0.5% (v/v) Triton X-100 for 30 min then pelleted and washed in 1 mL of 0.1M Na₂B₄O₇.10H₂O (pH 8.5). Cell pellets were sequentially incubated for 30 min with anti-BrdU and Alexa Fluor 488 anti-mouse IgG antibodies (Supplemental methods, Table 2) in PBS containing 2% FBS and 0.5% Tween-20. Cells were washed with PBS- 2% FBS then incubated in RNaseA containing 10 mg/mL PI solution at 37°C for 15 min. Cells were analyzed on the FACSCanto II and cell-cycle analysis was performed using FCS Express software (De Novo, Los Angeles, CA, USA).
For Annexin-V analysis cells were stained with Annexin-V-APC (BD Pharmigen 550474) and 10 μg/mL PI and analyzed with the BD FACSCanto II. Flow cytometry data was analysed with FCSExpress software.