Cd4 alexafluor488

Cells were stained using fluorochrome-coupled monoclonal antibodies (mAbs). The following mAbs were used in this study. CD3-PECy7, CD3-FITC, CD4-AlexaFluor488, CD4-PerCP/Cy5.5, PD-1-BrilliantViolet421, CD8-APC CD8-PerCP/Cy5.5, CD45RO-PE, CCR7-PE, and CD45RA -AlexaFluor700 were all obtained from BD Bioscience. CD8-FITC, PD-1-PE, PD-1-APC, and CD3-APC were obtained from eBioscience. CD244-PE, CD4-PE/Cy5, CD8-PE/Cy5, CD3-FITC, CD19-FITC, CD19-AlexaFluor700 and CD19-BrilliantViolet421 were obtained from BioLegend. Flow cytometry analyses were performed on a LSRII flow cytometer (BD Biosciences) and subsequently analyzed using Flowjo, version 9.0.2 software (ThreeStar).

To determine the cell phenotypes within the purified samples, we purchased anti-human CD1c-APC, CD127-APC, CD25-PE, CD45RO-FITC, CD123-FITC and CD303a-APC monoclonal antibodies from eBioscience (San Diego, CA, USA); anti-human CD45-PE, CD141-PE, CD11c-FITC, CD11c-PE-Cy7, CD8-PE, CD4 Alexa Fluor 488, CD4-APC and HLA-DR-PerCP-Cy5.5 antibodies from BD Pharmingen™ (San Diego, CA, USA); and anti-human CD16-BV421 and CD45RA-BV421 from BD Horizon™ (San Jose, CA, USA). To detect the various immune cells, PBMCs were stained with 3 µL of the above mentioned monoclonal antibodies for 30 min at 4 °C, washed twice in PBS containing 0.5% BSA and fixed in 1% paraformaldehyde (PFA, Sigma-Aldrich, St Louis, MO, USA). For quantification purpose, in each test 20000 PBMCs were counted by FACS.