Murine Lung Organoid Generation

Bronchoalveolar lung organoids (BALO) were generated from flow-sorted murine lung epithelial stem cells co-cultured with flow-sorted mesenchymal cells as previously described^{36 (link)}. In lung organoid cultures, 3 × 10⁵ WT BMDM (1 or 2) or 4 × 10³ BMDM2 from Plet1^flx/flx or CX₃CR1^iCre-Plet1^flx/flx) and organoids were cultured in Matrigel® (Corning) (α MEM, 10% FCS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM L-glutamine, 1× insulin/transferrin/selenium, 0.0002% heparin)^{37 (link)}. In selected experiments, anti-Plet1 Ab (20 ng/ml) or isotype control (20 ng/ml) was added to the organoid medium upon cell seeding. BALO numbers and size were analyzed manually, by light microscopy with an EVOS FL imaging system (Thermo Fisher Scientific) and data are provided as means per each well with single data points representing one well.