Pacific blue anti ly6g

Spleen, blood, bone marrow (BM) and BAL cells were harvested and single cell suspensions were created by passing the cells through 70 μm pore sized cell strainers (BD Falcon, Durham, NC). Erythrocytes were then lysed using ammonium chloride lysis buffer and washed two times using phosphate-buffered saline (PBS) without calcium, phenol red, or magnesium. Cells were stained with the following antibodies for flow cytometric studies: PE Cy7 anti-CD11b, APC anti-Gr-1, and Pacific Blue anti-Ly6G (BD Pharmingen, Billerica, MA). Additional antibodies used were anti-lineage mixture (Lin; BD Biosciences, San Jose, CA), anti-ckit, anti-Sca-1, anti-CD135, anti-CD150 (eBioscience, San Diego, CA). Sytox Blue (Invitrogen, Carlsbad, CA) was used for cell viability analysis and samples were acquired and analyzed using an LSRII flow cytometer (BD Biosciences) and FACSDiva^™ (BD Biosciences)(37 (link), 38 (link)).

The trachea was cannulated and lavaged four times with 800 μl cold PBS containing 2 mM EDTA. The BAL cells were counted using a hematocytometer (Hausser Scientific, Horsham, PA). BAL single cell suspensions were created by passing the cells through 70 μm pore sized cell strainers (BD Falcon, Durham, NC). Cells were stained with the following antibodies for flow cytometric studies: PE Cy7 anti-CD11b, APC anti-siglec, and Pacific Blue anti-Ly6G, FITC anti CD11c, and PE anti-F4/80 (BD Pharmingen, Billerica, MA). Sytox Blue (Invitrogen, Carlsbad, CA) was used for cell viability analysis and samples were acquired and analyzed using an LSRII flow cytometer (BD Biosciences) and FACSDiva™.