Hla dr bv711

PBMC were thawed at 37°C, transferred into pre-warmed complete RPMI10 culture medium, pelleted by centrifugation, resuspended in RPMI10, and left to rest at 37˚C for 1.5 h. Cells were stained with a cocktail of cell surface antibodies in FACS buffer (PBS + 2% FCS) for 30min at room temperature (RT) in the dark. Cells were washed and resuspended in FACS buffer, 7AAD viability dye (Biolegend) was added, and cells were acquired on a BD FACSymphony A5 flow cytometer (BD Biosciences). Data were analysed by FlowJo V10 (BD Biosciences). The following anti-human flow cytometry antibodies were used: TCR-gd-FITC (B1, Biolegend), CD19-PerCP-Cy5.5 (HIB19, Biolegend), CD14-PE (M5E2, Biolegend), CD56-PE-CF594 (NCAM16.2, BD Biosciences), CCR7-PE-Cy7 (G043H7, Biolegend), PD-1-AF700 (EH12.2H7, Biolegend), CD45RO-APC-Cy7 (UCHL1, BD Biosciences), CD127-BV421 (A019D5, Biolegend), CD28-BV480 (CD28.2, BD Biosciences), CD20-BV605 (L27, BD Biosciences), CD16-BV650 (3G8, BD Biosciences), HLA-DR-BV711 (L243, Biolegend), CD4-BV786 (SK3, Biolegend), CD3-BUV395 (UCHT1, BD Biosciences), CD8-BUV496 (RPA-T8, BD Biosciences), CD25-BUV737 (2A3, BD Biosciences).

Cell surface staining was processed using whole blood lysis method. Freshly drawn heparinized peripheral blood samples were labeled with monoclonal antibody (mAb) panel for T lymphocyte subsets [anti-CD3-AlexaFlour700, -CD4-APC, -CD8-APC/CY7, -CD45-BV510, -CD45RA-FITC, -CD45RO-PE, -CCR7-PE and -HLA-DR-BV711 (all from Biolegend, San Jose, CA, USA)], mAb panel for B lymphocyte subsets [anti-CD19-APC, -CD24-PE, -CD27-PE/CY7, -CD38-AlexaFlour700, -CD45-BV510, -CD138-BV421 and -IgD-FITC (all from BD-Biosciences, San Jose, CA, USA)], and with mAb panel for Treg [anti-CD3-AlexaFlour700, -CD4-APC, -CD25-PE, -CD45-BV510, -CD127-BV421 (all from BD-Biosciences, San Jose, CA, USA)]. Following incubation, erythrocytes were lysed using FACS Lysing Solution (BD Biosciences, San Jose, CA, USA), and at least 10.000 cells were collected in CD45⁺ lymphocyte gate. The data were acquired on a NovoCyte flow cytometer (Agilent Technologies, USA) and analyzed using the NovoExpress operating system software (Agilent Technologies, USA).