Anti cd83 pe cy5

Dendritic cells were harvested, and 1 × 10⁶ cells were washed with cold 1× DPBS and then incubated with anti-CD40-APC (BD Biosciences; San Jose, CA, USA), anti-CD80-PE (eBioscience; San Diego, CA, USA), anti-CD83-PE-Cy5 (BD Biosciences; San Jose, CA, USA), anti-CD86-PE (eBioscience; San Diego, CA, USA), anti-HLA-DR-PE (eBioscience; San Diego, CA, USA), and anti-CCR7-PE-Cy7 (BD Biosciences; San Jose, CA, USA) labeled antibodies (BD Biosciences; San Jose, CA, USA) for 30 min on ice. After washes, cells were fixed with 1% paraformaldehyde (Fisher Scientific; Waltham, MA, USA) and data were collected on an LSRII flow cytometer (BD Biosciences; San Jose, CA, USA). Expression of cell surface markers was analyzed on gated GFP positive cells using FlowJo 6.3.4 software (FlowJo; Ashland, OR, USA).

The phenotypic analysis of the endotoxin-treated moDCs was performed by labelling the cells with the following monoclonal antibodies conjugated with the respective fluorescent dyes: anti-CD40-FITC, anti-CD83-PE-Cy5 (both from BD Pharmingen, San Diego, CA, USA), anti-CD80-FITC, anti-HLA-DQ-PE (both from BioLegend, Uithoorn, The Netherlands), anti-CD86-PE (R&D System, Minneapolis, MN, USA) and isotype-matched control antibodies, all from BD Pharmingen. Fluorescence intensities were detected via multicolor flow cytometry using a FACS Calibur Flow Cytometer (Becton Dickinson), and the data were analyzed by FlowJo v. 5.7.2 Software (Treestar).