Cd137 apc

To analyze the phenotype and purity of the fresh isolated products and expanded cells, they were stained with the ST-PE complex. Briefly, 0.75 μg of PE-labelled Strep-Tactin and 0.5 μg of antigen-specific MHC I-Strep (HLA-A*02:01/NLVPMVATV Streptamer) were incubated during 45 min at 4 °C in the dark to form the ST-PE complex. 0.2 μg of this reversible multimer were added to 1 × 10⁶ cells. The incubation was carried out during 45 min at 4 °C in the dark and afterwards cells were stained with, CD8-FITC (BioLegend, San Diego, USA), CD3-PerCP, CD137-APC (Miltenyi Biotec), and CD4-APC-Cy7 (BD Biosciences, San Jose, USA).
During the culture period, specificity and phenotype of expanded cells were analyzed every 7 days by staining with the ST-PE complex and monoclonal antibodies as previously described, with the addition of CD69 PE-Cy7 and CD57 VioBlue (Miltenyi Biotec).
Furthermore, at the beginning and the end of the expansion the memory phenotype of the cells was analyzed by staining with CD45RA-V450 and CCR7-PE-Cy7 (both BD Biosciences) during 15 min at room temperature.