Cd141 apc

Leucocytes subsets were identified using an antibody panel containing CD45‐PO (Life Technologies) for lymphocytes; CD123‐PerCPCy5.5 (BD Pharmingen), CD203c APC (Sony), HLA‐DR‐PB (Sony), and CD41‐PE‐Cy7 for basophils; and CD45‐PO (Life Technologies) and CD14‐APC‐H7 (BD Pharmingen) for monocytes. To distinguish between the three different subsets of dendritic cells (DCs), an antibody panel containing HLA‐DR‐PE‐CY7 (BioLegend), CD11c‐PB (BioLegend), and CD123 PerCP Cy5.5 (BD Pharmingen) was used for plasmacytoid dendritic cells (pDCs). For two subsets of myeloid dendritic cells (mDCs), CD14‐V500 (BD), HLA‐DR‐PE‐CY7 (BioLegend), CD1c‐APC‐Cy7 (BioLegend), and CD141‐APC (Miltenyi) were used. Leucocyte subset quantities were depicted as the percentage of cells within the total leucocyte measures/numbers.

The following antibodies were used in different combinations: CD3-FITC (eBioscience); CD19-FITC, CD20-FITC, CD56-FITC, CD25-PE, CCR4-PECy7, CCR6-PerCp/Cy5.5,HLA-ABC-PE, CD3 - BUV395 (BD); CLEC9A-PE, CD1c-PE, CD1c-APC, CD141-PE,CD141-APC, CD278 (ICOS)-VioGreen, CD127 – APC, CD25 -PE and CD45 -VioBright515 (Miltenyi); CD3-FITC, PD-L1-PE and CD45-PECy7 (eBioscience); CD11c-PerCp/Cy5.5, CD4 -BV785, CD8a - BV650, CTLA-4 - BV605, FoxP3 - BV421 and Ki-67 - BV711 (BioLegend); CCR10-PE (R&D Systems); CD14-FITC, CD83-FITC, CD86-FITC, CD40-FITC and HLA-DR-PE (Invitrogen). Samples were stained in indicated antibodies combination for 20min on ice, washed with PBS supplemented with 15% (v/v) FBS, and acquired with a BD FACS Canto (BD) or BD LSRFortessa Cell Analyzer (BD) and data were analyzed by FlowJo (TreeStar) software.

Antibody staining was performed as described previously [14] . For the comparison of young adults, community-dwelling seniors and advanced-age, frail elderly, fluorochrome conjugated antibodies included: CD2-PE, CD3-PE, CD16-PE, CD19-PE, CD56-PE, NKp46-PE, CCR2-Alexa647 (BD Biosciences, NJ, USA); CD15-PE, CD1c-FITC, CD141-APC (Miltenyi Biotec, CA, USA); CD14-APC-Alexa750 (Invitrogen, ON, CAN); CX₃CR1-FITC (Biolegend, CA, USA); CD16-PE-Cy7, HLADR-PerCp-Cy5.5, CD45-eFluor605NC, CD123-PE-Cy7, TLR-4-Alexa700, TLR-2-eFluor450 (eBioscience, CA, USA). For monocyte staining, lineage cells were defined as CD2, CD3, CD15, CD19, CD56 and NKp46 positive, and CD16 thresholds were defined using a fluorescent-minus-one (FMO) with isotype control (Figure 1). For DC staining, lineage cells were defined as CD3, CD15, CD16, CD19 and CD56 positive (Figure 1). Thresholds to determine percentage of cells expressing CCR2, CX₃CR1, TLR-2 and TLR-4 were calculated using an FMO with isotype control or negative staining population where appropriate. The frequency of monocyte and DC subsets is presented as per µl of whole blood (calculated using CountBright absolute counting beads) as well as the percentage of CD45 expressing PBMCs. Proportions of monocyte and DC subsets were defined as the percentage of CD45 expressing PBMCs. All analyses were performed in FlowJo 7.6.4 (Treestar, OR, USA).