Mononuclear cells from lungs were isolated as previously described, with minor modifications [7 (link), 48 (link)]. Briefly, each set of lung lobes was dissected and placed into GentleMACS C tubes (Miltenyi Biotec, Auburn, CA) containing RPMI 1640 medium (HyClone Laboratories), 300 U/ml Clostridium histolyticum type I collagenase (Worthington Biochemical, Freehold, NJ), 50 U/ml DNase (Sigma-Aldrich, St. Louis, MO), 5% FBS (Hyclone Laboratories), 10 mM HEPES (Fisher Scientific, Pittsburgh, PA), and antibiotic/antimycotic solution (Cellgro, Manassas, VA). Lung samples were homogenized using a GentleMACS (Miltenyi Biotec) on the provided setting for mouse lungs, protocol 2. Homogenates were then incubated at 37°C while mixing on a Nutator (Fisher Scientific) for 20 minutes. Subsequently, the homogenates were filtered through a 250-μm nylon mesh. Cells were then purified through density-gradient centrifugation using Lympholyte M (Cedarlane Laboratories, Burlington, NC).
Spleen and lower respiratory lymph nodes were pushed through a 250-μm nylon mesh, and cells isolated through centrifugation. This was followed by red cell lysis using ammonium chloride-potassium carbonate lysis buffer, or ammonium chloride-Tris lysis buffer. Total cells were counted using a Cellometer Auto T4 cell counter (Nexcelom Bioscience, Lawrence, MA).
Spleen and lower respiratory lymph nodes were pushed through a 250-μm nylon mesh, and cells isolated through centrifugation. This was followed by red cell lysis using ammonium chloride-potassium carbonate lysis buffer, or ammonium chloride-Tris lysis buffer. Total cells were counted using a Cellometer Auto T4 cell counter (Nexcelom Bioscience, Lawrence, MA).