Anti ccr7 pe

After thawing PBMCs in PBS containing the endonuclease benzonase (Novagen/Merck Milipore Schwalbach, Germany), cells were directly stained in PBEA buffer (PBS, 0.5% BSA, 2 mM EDTA and 0.01% NaN₃ ) for 30 min on 4°C. An overview of the B cell-, T cell- and NK cell/regulatory T cell (Treg)-phenotyping panels including fluorochromes is provided in Table S2. Cells were washed with PBEA buffer and fixed in PBS containing 1% formaldehyde. The following monoclonal antibodies were used after initial testing and titration: anti-CD3 Alexa-eFluor 780, anti-CD4 PE-Cy7, anti-CD19 Pe-Cy7, anti-CD27 APC, anti-CD28 FITC, and anti-CCR7 PE (eBioscience, SanDiego, USA), anti-CD4 PerCP-Cy5.5, anti-CD8⁺ PerCP-Cy5.5, anti-CD16 PerCP-Cy5.5, anti-CD20 FITC, anti-CD25 V450, anti-CD38 PerCP-Cy5.5, anti-CD45RA HV450, anti-CD69 FITC, anti-CD86 V450, anti-IFN-g APC, anti-IgD PE, anti-TNF-a FITC (Becton Dickenson GmbH, Heidelberg, Germany), anti-IL-2 PacificBlue (BioLegend,); anti-CD56 PE (Miltenyi, Bergisch Gladbach, Germany).

Fluorochrome-coupled antibodies specific for human antigens: anti-CD4-FITC, anti-CD127-PE-Cy7, anti-CD62L-PE, anti-CCR4-PE, anti-CCR7-PE, anti-CCR6-PE, anti-CCR10-PE, anti-CXCR3-PE-Cy5, anti-HLA-DR-FITC, anti-CD25-PE-Cy7, anti-CD39-PE, anti-CD44-PE-Cy7, anti-CD45RA-PE-Cy5, anti-GITR-PE, anti-CD45RO-PE, and anti-CD278-PE (all from eBioscience, USA) were used for cell surface staining. Monoclonal antibodies (mAbs)anti-Foxp3-PE, anti-CTLA-4-PE-Cy7, anti-Helios-FITC, and anti-IDO (indolamine-2,3-dioxygenase)-PE-Cy7 (all from BD Pharmingen, USA) were used for intracellular staining. Quantitative analysis was performed using FlowJo software.