Multiparametric Flow Cytometry Immunophenotyping

Blood samples were analysed by 8-colour flow-cytometry assays, which were performed similarly as previously described.^{2 (link)} Shortly, frozen PBMC aliquots were quickly thawed, and washed using an automatic cell washer. Following primary wash step, cell viability and cell numbers were analyzed using Vi cell counter. PBMC staining was performed with CD3-QDot605 and CD45RA-PacificBlue (both Invitrogen), CD4-AlexaFluor700, CD8-APC-H7, CCR7-PE/Cy7, CD27-APC, and CD28-PerCp/Cy5.5 (all BD Biosciences). Following three wash steps, LIVE/DEAD® Fixable Aqua Dead Cell Stain (Thermo Fisher Scientific, Waltham, MA, USA) was added to allow discrimination of viable and dead cells. Samples were measured on a BD LSR II cytometer using BD FACSDiva acquisition software. At least 100,000 viable cell events per sample were acquired. The gating scheme is depicted in Fig. 2a.