Hladr pe clone g46 6

PBMCs were washed with PBS and re-suspended in 100 µl flow cytometry staining buffer (PBS with 1% FCS and 0.1% sodium azide). Fc receptors (FcR) were first blocked using FcR Blocking Reagent (Miltenyi Biotech, Bergisch Gladbach, Germany). 7-AAD viability dye or Fixable Viability Dye eFluor™ 780 (eBioscience, San Diego, USA) was used to gate live cells. Cells were then stained with cell surface antibodies against CD33-FITC (clone HIM3-4; BD Biosciences, Oxford, UK), HLA-DR-PE (clone G46-6; BD Biosciences), TIM-3-BV711 (clone 7D3; BD Horizon), PD-1-PE-CF594 (clone EH12.1; BD Pharmingen), galectin-9-PerCP (clone 9M1-3; Biolegend) and PD-L1-APC (clone MIH1; BD Pharmingen), and incubated at 4°C for 30 min. Cells were then washed twice with flow cytometry staining buffer and data were acquired using BD LSRFortessa X-20 SORP flow cytometer (BD Biosciences).
For sorting, cells stained with 7-AAD, CD33 and HLA-DR were re-suspended in Pre-Sort buffer (BD Biosciences). Cell sorting was performed on BD FACSAria III SORP cell sorter with BD FACSDiva software (BD Biosciences). Applicable measures were taken to ensure minimal sorter-induced cell stress (SICS). Data analyses were performed on FlowJo V10 software (FlowJo, Ashland, USA).