Anti cd73 apc

Transduced and parental wild type (wt) cells were stained with anti-CD45/VioBlue, anti-CD33/PE, anti-CD90/VioBlue, anti-CD105/PE and anti-CD73/APC (all Miltenyi Biotec, Bergisch-Gladbach, Germany) monoclonal antibodies (mAbs) to analyze the hMSC marker profile. To monitor for transgenic 4-1BBL expression, SCP-1 cells were stained with an anti-CD137L/PE (BD Bioscience, Heidelberg, Germany) mAb. Samples were analyzed using a MACSQuant Analyzer and the MACSQuantify software (both Miltenyi Biotec).

The phenotype of hESC-MSCs was characterized using flow cytometry and the following monoclonal antibodies: anti-CD31-APC (Miltenyi Biotec), anti-CD44-FITC (BD Pharmingen), anti-CD45-FITC (BD Pharmingen), anti-CD73-APC (Miltenyi Biotec), anti-CD90-FITC (BD Pharmingen), anti-CD105-PE (eBioscience), HLA-ABC-APC (BD Pharmingen), and HLA-DR-FITC (BD Pharmingen). Briefly, the cells were dissociated and suspended in FACS buffer (1x PBS/0.5% BSA) and nonspecific binding blocked with FcR blocking agent (Miltenyi Biotec) for 10 minutes at 4°C. For labeling cell surface antigens, the cells were incubated with the abovementioned fluorescent conjugated antibodies for 10 minutes at 4°C. After antibody labeling, data was acquired using Dako Cytomation CyAn ADP and analyzed using FlowJo v7.6.5 (Tree Star).

For surface marker immunophenotyping, cells were stained with the following conjugated antibodies: anti-CD45-FITC, anti-CD34-PE, anti-CD14-PE, anti-CD73-APC, anti-CD90-FITC, anti-CD105-Vioblue, and relevant isotypes (Miltenyi-Biotec). 7AAD and Annexin V/PI were used to assess cell viability and apoptosis. At least 20,000 events for test samples were acquired. The MACSplex Cytokin12 kit was used for the cytokines analysis. Supernatants were mixed to capture specific beads for each cytokine: granulocyte/macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-α, IFN-γ, interleukin (IL)-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-17A, and tumor necrosis factor (TNF)-α. PE-conjugated antibodies were added and incubated for 2 h at room temperature and away from light. After centrifugation, the pellets containing beads were resuspended; flow cytometric acquisition and data analysis were performed by the MACSQuant® Express Mode. Background signals were determined by analyzing beads incubated with the cell culture medium alone. The background signals were subtracted from the signals obtained for beads incubated with supernatants.