Jagged1

Collagenase digestion of mouse lung tissue was previously described in ^{30 (link)}. In short, mice were euthanized by CO₂ asphyxiation after which perfusion was carried out by injecting 3–5 mL of phosphate buffered saline (PBS) through the right ventricle of the heart. Lungs were resected, minced with scissors, and digested in complete DMEM containing 15 mg/mL collagenase A (Roche, Indianapolis, IN), and 2500 units of DNase I (Sigma-Aldrich, St. Louis, MO) for 30 m at 37°C. Digested tissue was disrupted through a 10 mL syringe, filtered through a 100 μM pore size nytex screen and centrifuged through a 20 % Percoll solution in serum free media. 10x10⁶ cells were then stained with appropriately diluted fluorophore-conjugated antibodies for 30 m. For intracellular cytokine staining (ICS) cells were diluted to 1x10⁶/mL and stimulated for 4 h with PMA (10 ng / mL), ionomycin (10 μM), and Golgi-stop reagent (BD Bioscience, San Jose, CA). Antibodies used in this study are as follows: anti-CD45, CD11b, CD4, IL-17A, MHCII (I-A^b), CD103, SiglecF (BD Bioscience, San Jose, CA), CD11c, IFNγ (eBioscience San Diego, CA), DLL1, DLL4, Jagged1, Jagged2, and CD64 (Biolegend, San Diego, CA).