Isolation and Analysis of Lung Leukocytes

Lungs were perfused with PBS containing EDTA (0.5 mM), minced, and digested with Liberase TM (100 µg/ml; Roche, Indianapolis, IN), together with collagenase XI (250 µg/ml), hyaluronidase 1a (1 mg/ml), and DNase I (200 µg/ml; Sigma, St. Louis, MO) for 1 h at room temperature (44 (link)). Dead cells were stained with Pac-Orange Live/Dead fixable, dead staining dye (Invitrogen). Cells were filtered through a 70 µm strainer, treated with RBC lysis buffer (BD Biosciences, Franklin Lakes NJ), and kept on ice in media containing 10% serum. Lung cells were then stained with fluorescently labeled antibodies against various leukocyte surface markers (CD45, CD11b, CD11c, F4/80, and CCR2). Appropriate isotype-matched controls and Fluorescence Minus One (FMO) controls were used in all experiments. Antibodies were purchased from EBiosciences (San Diego, CA) or Biolegend (San Diego, CA). Cells were fixed and analyzed on a Fortessa (Becton-Dickinson, San Jose, CA) or FACSAria II (BD Biosciences) flow cytometer. Results were analyzed using FlowJo software (Tree Star, Ashland, OR).