Cd123 pe cy7

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).