Anti cd19 af647

Bone marrow cells were collected from the femurs and tibiae of mice by flushing the opened bones with Iscove’s Modified Dulbecco’s medium (IMDM) as previously described (Dyer et al., 2008 (link)). Spleen cell suspensions in HBSS supplemented with 1% FBS and 10 mM HEPES were prepared as described (Dyer et al., 2011 (link)). Red blood cells were lysed with ACK lysing buffer (Lonza). Live/dead stain (Invitrogen) was added to the cells and non-specific antibody binding to Fc receptors was blocked with anti-mouse CD16/CD32 (BD Biosciences). For analysis of T and B cells, cell suspensions were incubated with anti-CD45-eF450 (eBioscience), anti-CD3-AF700 (eBioscience), anti-CD4-FITC (eBioscience), anti-CD8a-PE (BD), and anti-CD19-AF647 (BD) in phosphate-buffered saline with 0.1% bovine serum albumin (PBS/BSA) at 4°C for 30 min and washed with this buffer prior to analysis. For evaluation of myeloid cells, cell suspensions were incubated with anti-CD45-AF700 (BD), anti-CD11c-AF488 (BD), anti-SiglecF-PE (BD), anti-GR1-V450 (BD) and anti-MHCII-APC (eBioscience). At least 100,000 events were collected on an LSR II flow cytometer (BD Biosciences) and findings were analyzed in FlowJo 9.2.

Lung tissue was harvested and single-cell suspensions were prepared as previously described (Garcia-Crespo et al., 2013 (link); Percopo et al., 2014a (link)). Live/Dead stain (Invitrogen) was added to the cells and Ab binding to Fc receptors was blocked with anti-mouse CD16/CD32. For analysis of T and B cells, lung suspensions were stained with anti-CD45-eF450 (eBioscience), anti-CD3-AF700 (eBioscience), anti-CD4-FITC (eBioscience), anti-CD8a-PE (BD), and anti-CD19-AF647 (BD) in PBS with 0.1% BSA at 4 °C for 30 min and washed with this buffer prior to analysis. For myeloid cell analysis, cells were stained with anti-CD45-AF700 (BD), anti-CD11c-AF488 (BD), anti-SiglecF-PE (BD), anti-GR1-V450 (BD) and anti-MHCII-APC (eBioscience). Natural killer cells were characterized by staining with anti-CD45-eF450 (eBioscience), anti-CD3-AF700 (BD), and anti-CD49/DX5-PE (BD). A minimum of 100,000 events were collected on an LSR II flow cytometer (BD Biosciences) and findings were analyzed in FlowJo 9.2.