Cd103 pe

Mucosal immune cell subsets were characterized by FACS analysis as previously described (figure 1).^{25 (link),26 (link)} The cell suspension was washed once with staining buffer (phosphate-buffered saline containing 3% FCS and 2 mM EDTA), and the cells were stained with 100 μL staining buffer for 20 minutes at room temperature in the dark. For enumeration of LPDCs, directly labeled monoclonal antibodies for the following markers were used: lin (lineage) 1-FITC (CD3, CD14, CD16, CD19, CD20, CD56, and CD34), HLA-DR-PerCP-Cy5.5, CD11c-APC, and CD103-PE. LPDCs were identified as lin1-/HLADR+ cells. For determination of Tregs, anti-CD3-APC-Cy7, anti-CD4-V450, anti-CD8-FITC, anti-CD25-PE, and anti-CD127-Alexa Fluor 647 antibodies were used. With the exception of CD103-PE (eBioscience, San Diego, CA), all antibodies were purchased from BD Bioscience (San Jose, CA). FMO (fluorescence minus one) controls were used to set the boundaries for gating of positively stained cells. After the staining reaction, the cells were washed once with staining buffer and resuspended in 100 μL staining buffer. For the exclusion of dead cells, propidium iodide was added to the samples immediately before acquisition on an LSR II (BD Bioscience) flow cytometer. The data files were analyzed using FlowJo (FlowJo, LLC, Ashland, OR) software.