Hla dr percp

For cytofluorimetric analysis, cells were stained with surface antibodies in PBS 5% FCS for 20 min at 4 °C. The following antibodies were used: CD133-APC, EpCAM -VioBlue, CD105-PE, CD90-VioBlue, IFNγ-PE, CD107a-APC, CCR7-FITC, CD163-PerCP-Vio700, CD206-VioBlue, HLA-DR-PerCP, CD80-APC (MIltenyi Biotec); CD56-PC7, NKp30-PE (Beckman Coulter); CD29-PE (ImmunoTools); CD146-PC7, CD73-FITC, NKp46-V450 (BD Biosciences); E-cadherin-APC (Thermo Fisher Scientific); N-cadherin-PE (Abcam); DNAM1-PE (Biolegend).

For cytofluorimetric analysis, cells were stained with surface antibodies in PBS containing 5% FCS for 20 min at 4 °C. The following antibodies were used: IFNγ-PE, CD107a-APC, HLA-DR-PerCP, CD69-FITC, PD-1-APC (Miltenyi Biotec); CD56-PC7, NKp30-PE (Beckman Coulter); NKp46-V450, CD16 PerCP-Cy5,5, CD155-AF647, PD-L1 PE-CF59 (BD Biosciences); E-cadherin-APC (Thermo Fisher Scientific); N-cadherin-PE (Abcam); NKG2D-PE/Dazzle, DNAM1-PE, CD112-PerCP-Cy5,5; MICA/B PC7 (Biolegend); ULBP3 AF405, ULBP2/5/6 PE (R&D).

Peripheral blood was collected using EDTA and Heparin-coated tubes and samples were shipped to the Universitätsklinik für Kinder-und Jugendmedizin, Tübingen (Germany) at room temperature and analyzed within 24 h. MDSCs were characterized as previously described (21 (link), 22 (link)). In brief, peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by Ficoll density gradient centrifugation (Lymphocyte Separation Medium; Biochrom), washed with RPMI-1640 and cell viability was confirmed by trypan blue staining. The isolated PBMC, containing only low density granulocytes, were stained with specific antibodies for G-MDSC (CD66b-FITC, CD33-PE) and M-MDSC (CD14-FITC and HLADR-PerCP) (Miltenyi Biotec) and quantified by flow cytometry using a FACSCalibur (BD). G-MDSCs were phenotypically characterized as low-density fraction granulocytes CD33⁺CD66b⁺ cells (Figure 1). The percentage of G-MDSC was determined as ratio of CD33⁺CD66b⁺ cells (P2 in Figure 1) over total PBMCs containing the low density granulocyte fraction (P1 in Figure 1). Calculations were performed with BD CellQuest Pro analysis software and FlowJo V7.