and were housed in a pathogen-free animal housing facility at Beijing University Cancer Hospital and Institute at 233C, a relative humidity of ~50%, 12 h light/dark cycle and a standard sterilized rodent diet from Vital River Laboratories Co., Ltd (Bejing, China) and sterilized water ad libitum. 96-well plates, at a density of 103 cells/well, with 50 l RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.). Subsequently, an equal volume of effector cells or control medium was added to each well to ensure an effector-target ratio (E/T ratio) of 25:1, 12.5:1, 6.2:1, 3.1:1, 1.6:1, or 0.8:1. Following an 8-h incubation, cell supernatants were obtained by centrifugation at 800 g for 10 min at room temperature and collected for cytokine measurements of IL-2 and interferon (IFN)- concentrations using an ELISA kit (BioLegend, Inc., San Diego, CA, USA), according Mouse monoclonal to BMPR2 to the manufacturer’s protocols. Xenograft mouse model of GC Ten NOD/SCID mice (male; 7 weeks old; 18C20 g) were purchased from Vital River Laboratories Co., Ltd. and were housed in a pathogen-free animal housing facility at Beijing University Cancer Hospital and Institute at 233C, a relative humidity of ~50%, 12 h light/dark cycle and a standard sterilized rodent diet from Vital River Laboratories Co., Ltd (Bejing, China) and sterilized water ad libitum. The animal experiments were approved by the Animal Ethics Committee of Peking University Cancer Hospital and Institute. A total of 100 l 1106 3H11-antigen-positive MGC-803 cells, were injected subcutaneously into NOD/SCID mice on day 0. Tumor-bearing mice were randomly assigned to the CAR-T cell and control T cell groups prior to treatment, with five mice in each group. The Refametinib tumor volume (TV) of each mouse was measured twice weekly using a vernier caliper and was calculated according to the following formula: TV = 1/2 length width2. On day 14, when the mean TV reached ~100 mm3, 200 l 2107 CAR-T cells or control T cells, were infused into the tumors of the mice twice weekly by multipoint injection. Immunohistochemical examinations Humane endpoints were used in accordance with Peiking University Cancer Hospital and Institute standard operating protocols. Tumor tissue samples from sacrificed mice on the 35th day, according to the humane endpoints of diameter of the tumor mass (i.e., greater than 1.5 cm diameter in mice) were fixed in 10% formaldehyde solution for 24 h, dehydrated in ethyl alcohol, and embedded in paraffin, prior to being cut into 6 m thick sections using a microtome. Immunohistochemical (IHC) staining was performed according to standard procedures. Briefly, slides were immersed in xylene to remove paraffin, washed in a graded series of ethanol, immersed in citrate buffer at pH 6.0 and then incubated in a high-pressure sterilization oven for antigen retrieval at 100C for 3 min. Endogenous peroxidase activity was blocked in a blocking solution with 3% H2O2 in PBS for 10 min at room temperature, and then the slides were incubated with PBS containing 1% bovine serum albumin (Amresco, Solon, OH, USA) Refametinib for 10 min at room temperature to block non-specific binding. The tissue sections were incubated at room temperature for 1 h with a primary rabbit anti-human CD3 antibody (1:200; cat. no. ab5690; Abcam), followed by incubation with horseradish peroxidase-conjugated goat anti-mouse IgG (1:500; cat no. A4416; Sigma-Aldrich; Merck KGaA) for 1 h at room temperature. Then, the slides were visualized with 0.1% 3,3-diaminobenzidine (Sigma-Aldrich; Merck KGaA) for 2 min, and counterstained with one drop of 1% hematoxylin Refametinib for 10 min at room temperature. Statistical analysis Statistical analyses were performed using Prism V5.00 for Windows (GraphPad Software, Inc., La Jolla, CA, USA). The differences between Refametinib two groups were assessed using independent samples t-test. Dunnett’s multiple comparison.