Cd3 apc r700

Whole blood was used to calculate absolute leukocyte counts by Trucount analysis (BD Biosciences) according to manufacturer’s protocol. The following antibodies were used: CD45-PerCP (BioLegend), and CD3-APC-R700, CD4-BV711, CD8-BV786, all purchased from BD Biosciences. Using the bead count of the Trucount tube and the CD3⁺ cell count, absolute cell numbers of the proliferation/apoptosis and senescence panel were also calculated. In addition, for all individuals, HLA typing on whole blood was performed by flow cytometry at the first visit. For all these HLA-types, we characterized the known immunodominant CMV epitopes for which commercial dextramers are available. Antibodies used were HLA-A1/36-biotin/strep-PE-CF594, HLA-A2-V450, HLA-A3-APC, HLA-A24-PE, HLA-B7-FITC, and HLA-B8-PE-Cy7. Cells were measured on a Fortessa flow cytometer (BD Biosciences) and analyzed with FlowJo V10 software.

PBMCs were resuspended in PBS supplemented with 0.5% wt/vol BSA, 2 mM EDTA, and 0.01% sodium azide and incubated with saturating concentrations of fluorescently labeled conjugated mAbs for 30 min at 4°C under constant agitation as described before (29 (link)). Cells were analyzed using a FACSCanto II flow cytometer and FACSDiva software (BD Biosciences). Using FlowJo software, proliferation of B and T cells was determined by measuring CFSE dilution, whereas activation and differentiation were assessed by the expression of CD25, CD27, CD38, CD138, and SLAMF7. The following mAbs (indicated as panel 1) were used for immunophenotyping: CD3 APC-R700 (557943; BD Biosciences), CD4 PE-Cy7 (348809; BD Biosciences), CD8 PerCP-Cy5.5 (341050; BD Biosciences), CD19 APC-R700 (564977; BD Biosciences), CD20 PerCP-Cy5.5 (332781; BD Biosciences), CD25 APC (340907; BD Biosciences), CD27 APC (337169; BD Biosciences), CD27 APC-eFluor 780 (47-0279-42; eBioscience), CD38 PE (345806; BD Biosciences), CD38 PE-Cy7 (335825; BD Biosciences), CD138 APC (347216; BD Biosciences), BAFFR APC (316916; BioLegend), BCMA PE (357504; BioLegend), CXCR4 PE-Cy7 (306514; BioLegend), IgD PE (555779; BD Biosciences), and SLAMF7 PE-Cy7 (331815; BioLegend).