Cd45 e780 clone30 f11

The upper left lung lobe was removed and cut into small pieces with a razor. The lung tissue was then transferred to a C-tube (Miltenyi Biotec, Auburn, CA) and processed using digestion buffer containing 1mg/ml of Collagenase D and 0.1 mg/ml DNase I (Roche, Indianapolis, IN) in HBSS and a GentleMACS dissociator (Miltenyi Biotec), according to manufacturer's instructions. The homogenates were then filtered through 70 um nylon cell strainers to obtain a single cell suspension. Red blood cells were lysed using ACK lysis buffer (Life Technologies, Grand Island, NY). Cells were counted using trypan blue to exclude dead cells. To assess neutrophil numbers, 1×10⁶ lung cells were first incubated with anti-CD16/32 (clone 93,eBioscience, San Diego, CA) to block unspecific binding to the Fcy II/III receptor. Cells were then immunostained with rat anti-mouse antibodies: CD45 e780 (clone30-F11, eBioscience), CD11b e450 (clone M1/70, eBioscience), and Ly6G (Gr-1) PE Cy7-conjugated (clone RB6-8C5, eBioscience). Antibody incubation was carried out for 30 minutes at 4°C. Cells were washed and fixed as described (Boehmer, Goral, Faunce, & Kovacs, 2004 (link); Murdoch et al., 2011 (link)). Samples were run on a BD Fortessa cytometer (BD Biosciences, San Jose, CA). Data analysis was performed using Flow Jo FCS analysis software (Tree Star Inc., Ashland, OR).

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.