Lung Immune Cell Isolation Protocol

The upper left lung lobe was cut into small pieces, transferred to a C-tube (Miltenyi Biotec, Auburn, CA) containing digestion buffer (1mg/ml of Collagenase D and 0.1 mg/ml DNase I [Roche, Indianapolis, IN] in HBSS) and homogenized using a GentleMACS dissociator (Miltenyi Biotec), according to manufacturer guidelines. Single cell suspensions were obtained by passing homogenates through 70 um nylon cell strainers. Red blood cells were lysed in ACK lysis buffer (Life Technologies, Grand Island, NY) and remaining cells were counted using trypan blue exclusion of dead cells. Non-specific binding to the Fcy II/III receptor was prevented by incubating 1×10⁶ lung cells with anti-CD16/32 (clone 93,eBioscience, San Diego, CA). Cells were immunostained with rat anti-mouse antibodies: CD45 e780 (clone30-F11, eBioscience), CD11b eFluor 450 (clone M1/70, eBioscience), Ly6G (Gr-1) PE Cy7-conjugated (clone RB6-8C5, eBioscience), CD206 PE, CD24 APC, CD64 PerCP, and MHC II v500. Antibody incubation was carried out for 30 minutes at 4°C. Cells were washed and fixed as described (16 (link), 17 (link)). Flow experiments were performed using a BD Fortessa cytometer (BD Biosciences, San Jose, CA) and data analysed using Flow Jo FCS software (Tree Star Inc., Ashland, OR). Experiments were performed at minimum of two times, n=4–6 per group. Data from one representative experiment are shown.