Hla dr v450

Anti-human CD66bFITC, CD3, CD19, CD14, CD163,CD138, CD56, CD15 all PE conjugated, HLADRV450, CD45V500, CD16APC-H7, CD14PE-Cy7, CD38PE-Cy7, CD 206APC were obtained (BD Pharmingen, Stockholm, Sweden). CD34PE, CD235aPE, CD11bPerCp-eFlour710 (eBioscience, San Diego, CA, USA), CD192APC, and CX3CR1AlexaFlour647 were from BioLegend, San Diego, CA, USA. Human FcReceptor binding inhibitor, 7AAD, and Annexin V-Alexa647, were obtained from eBioscience, San Diego, CA, USA, Sigma–Aldrich, (St. Louis, MO, USA), and Molecular Probes, Eugene, OR, USA, respectively.

The orthotopic HCC tumor tissues or subcutaneous xenografts were dissected out and minced. Then tissues were digested with 0.8 mg/mL Collagenase IV (Sigma, USA) at 37 ˚C for 1 h. The cell suspensions were filtered through 70 μm strainer and resuspended in 36% Percoll (GE Healthcare, UK). PBMCs of anonymous human healthy donors were isolated by Ficoll reagent (Sigma, USA) according to manufacturers’ instructions. For cell surface staining, 1 × 10⁶ cells were incubated with anti-Fc receptor blocking antibody (2.4G2) at 4 ˚C for 15 min. Murine samples were stained with anti-mouse CD45 APC, CD3 FITC, Gr-1 V450, CD4 PE, CD8a V450, CD25 APC-CY7, Ly6C FITC, Ly6G PECY7, CD11 PE, and F4/80 APC-CY7 from BD Bioscience (USA). Human samples were stained with anti-human CD45 APC, CD11 PE, CD33 FITC, and HLA-DR V450 from BD Bioscience (USA). For intracellular staining of Arg1, Foxp3, and Ki67, cells were fixed and permeabilized by Fixation/Permeabilization solution (BD Biosciences, USA) at 4 ˚C for 15 min. Then cells were washed and stained with anti-mouse Arg1, anti-mouse Foxp3, and anti-Ki67 from BD Bioscience (USA). Flow cytometry was performed on a B.D. Influx cell sorter (BD Bioscience, USA). Flowjo software was used to analyze the data.