αcd57 fitc

Thawed PBMCs were pre-treated with 50 nM dasatinib before tetramer staining to enhance the detection of low avidity T cells as described previously (25 (link)). The following monoclonal antibodies (mAbs) were used for phenotypic analysis: (i) αCD3-H7APC, αCD4-V500, αCD45RO-PECy7, αCD57-FITC, αCD95-PE and αCCR7-PerCPCy5.5 (BD Biosciences); (ii) αCD8-QD705 (Life Technologies); and (iii) αCD27-PECy5 (Beckman Coulter). Dead cells were excluded from the analysis using the amine-reactive dye ViViD (Life Technologies); monocytes and B cells were eliminated in the same dump channel after staining with αCD14-PacBlue and αCD19-PacBlue, respectively (Life Technologies). Stained samples were acquired using an LSRFortessa^™ flow cytometer (BD Biosciences) and analyzed with FlowJo version 9.4 (TreeStar Inc.). The gating strategy is illustrated in Supplementary Figure 1. For quality control purposes, tetramers were batch-tested prior to experimentation using specific CD8 T cell clones where available (5 (link)). Assay variability was monitored throughout the study using aliquots of cryopreserved PBMCs drawn from a single healthy donor at a single time point (Supplementary Figure 2).