Facsariar

FACSAriar (BD Bioscience) was used to perform fluorescent expression analysis. Cells were harvested the following days after transfection and stained with mouse anti-human antibody labeled by fluorescence for 30 m in 4 °C in darks as follows: CD3-PerCP-CY5.5(OKT-3, eBioscience) or CD3-FITC (HIT3a, BD Bioscience), CD4-APC (RPA-T4, BD Bioscience), CD8-PE (HIT8a, BD Bioscience), PD-1-PerCP-CY5.5 (EH12.1, BD Bioscience), CD25-PE (BC96, eBioscience), CD62L-FITC (DREG-56,e Bioscience), CD27-PE (0323, eBioscience), CD28-PE (CD28.2, eBioscience), CD45RO-PE (UCHL1, BD Bioscience), CD69-PE (FN50, BD Biolegend), HLA-DR-PE (G46-6, BD Bioscience).

Cell Counting Kit-8 kit was purchased from DOJINDO (CK04, Kumamoto, Japan). Cancer cells (5000) or PBMCs (50,000) were placed on 96-well plates (100 μl/well). The experiments were performed as previously described [21 (link)]. After co-culture of cancer cells with PBMCs for 48 h, cells were harvested and stained with the antibodies labeled by fluorescence for 30 min in 4 °C in darks. Propidium Iodide (PI) (556463, BD, New Jersey, USA), FITC Annexin V (556420, BD, New Jersey, USA) were used. Fluorescence intensity was assayed by FACS Ariar (BD, New Jersey, USA), and analyzed using FlowJo 7.6.1 software (FlowJo, RRID:SCR_008520).