Cd244 fitc

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).

The following reagents were used: PD-1^FITC, CTLA-4^APC, CD4^{FITC/APC-H7/V500}, CD14^PerCP, CD19^PerCP, CD3^PerCP, Via Probe, Monensin, functional grade anti-CTLA-4 (BD Biosciences); CD244^FITC, PD-1^APC, CD127^APC-Cy7, IL-2^FITC, IFN-γ^PE, TNF-α^PE-Cy7, anti-PD-L1/2 and anti-IgG1 isotype control (eBioscience); TIM-3^APC (R&D Systems); CCR7^FITC, CD57^PacificBlue, CD45RA^PacificBlue, functional grade anti-TIM-3 (Biolegend). Micro-Beads^anti-PE (Miltenyi). KLRG1^{AlexaFluor488} (clone: 13F12F2) was generated as described [14] (link).