Cd274 pe

For phenotypic analysis BM-MSCs, UCT-MSCs and AT-MSCs were harvested and stained for 15 minutes at room temperature with the following monoclonal antibodies (mAbs): anti-human CD90 APC, CD73 PE, CD34 PE, CD200 PE, CD273 APC, CD274 PE, CD71 FITC, CD44 PE (BD Pharmingen, Heidelberg, Germany), anti-human HLA-DR PE, HLA-A,B,C FITC, CD144 (VE-cadherin) APC, CD31 FITC, CD105 PE (Biolegend, San Diego, CA, USA), CD45 FITC (Tonbo, Biosciences, San Diego, CA, USA) or with the appropriate fluorochrome-conjugated isotype-matched mAbs to establish background fluorescence. After incubation cells were washed with PBS, centrifuged at 1400 RPM for 5 minutes and suspended in PBS for flow-cytometry analysis. Samples were acquired using a FACSCalibur (Becton Dickinson, San Josè, CA, USA) and the data were analysed with CellQuest software (Becton Dickinson).

Ten to fifteen milliliters of heparinized peripheral blood were collected for CIK in vitro expansion from health volunteers with informed consent. Peripheral blood mononuclear cells (PBMC) were separated by Ficoll density gradient centrifugation (GE Healthcare) and seeded at a concentration of 1.5×10⁶ cells/ml in culture medium (Gibco, Life Technologies) with the addition of 1000 U/ml interferon-γ (Pepro Tech) and 50ng/ml anti-human OKT-3 (eBioscience) on day 0 and 300U/ml interleukin (IL)-2 (Huaxin High Biotechnology, Shanghai) on day 1. Phenotype and immune profile of CIK cells were characterized by multiparameter flow cytometric analysis using following monoclonal antibodies: CD3-FITC, CD56-PC7, CD3-PC7, CD4-FITC, CD8-FITC, CD314-PC7 (anti-NKG2D), CD152-PE (anti-CTLA-4), CD223-APC (anti-LAG-3), CD226-PE (anti-DNAM-1), CD244-FITC (anti-2B4), CD274-PE (anti-PD-L1), CD279-APC (anti-PD-1), Foxp3-PE (mAbs were from BD Biosciences). Flow cytometric analysis was performed using the Coulter FC500 flow cytometer (Beckman Coulter). Data were further analyzed by Flow Jo 7.6.5 software (Tree Star).