RO was calculated using the following formula: Data were analyzed using FCAP Array ver software. According to the manufacturer’s instructions. Cytokine measurementĪfter performing the cytotoxicity assay described above, the remaining culture medium was collected and used to measure the levels of the cytokines IL-2, IL-4, IL-6, IL-10, TNFα and IFNγ using the human cytokine CBA Th1/Th2 group II (BD) with FACSVerse. 7.6.5 (Digital Biology Tomy) to calculate CD25 + or CD69 + The ratio in CD3 + Population. Data were analyzed using FlowJo ver software. Live/dead cell staining (Fixable Viability Dye eFluor 780 Thermo Fisher Scientific, Waltham, MA) was used to quantify the number of live cells. After washing the cells with PBS, samples were analyzed with FACSVerse (BD). Next, antibodies against CD3 (APC-conjugated) (BD), CD25 (PE-conjugated) (BD), and CD69 (FITC-conjugated) (BD) were added, and samples were incubated on ice for 30 min. Samples were incubated at room temperature for 10 minutes. Cells were washed with 0.5 w/v% bovine serum albumin (BSA)/CellWASH (FACS/PBS) (BD, Franklin Lakes, NJ), and then diluted Fc blocking reagent (Miltenyi, Gladbach, Germany) was added. T cell activation assayĪfter cytotoxicity examination, residual CD3 + Cells were collected and used to measure the expression of CD69 and CD25. The cytotoxicity of ERY974 was calculated according to the manufacturer’s instructions. LDH derived from lysed tumor cells was measured using an LDH Cytotoxicity Detection Kit (TAKARA Bio, Shiga, Japan). 5/96 well), and ERY974 at different concentrations were in a round-bottom 96-well plate and incubated for 24 h at 37 ☌ with 5% CO 2. ABC was calculated using a calibration curve. After further washing, cells were analyzed with FACSlytic (BD, Franklin Lakes, NJ). After washing, cells were incubated with the secondary antibody (included in QIFIKIT) for 30 min at 4 ☌. Cells were incubated with 20 μg/ml murine anti-GPC3 mAb (internal preparation) for 30 min at 4 ☌. The ABC of GPC3 in each cell line was determined using QIFIKIT (DAKO, Glostrup, Denmark) according to the manufacturer’s instructions. After centrifugation (2300 rpm, 30 min) at room temperature, the PBMCs were collected as described above. #NOD SCID MICE PLUS#Cynomolgus monkey PBMCs were also isolated by Ficoll–Paque PLUS by density gradient centrifugation without Leucosep. After centrifugation (2300 rpm, 10 min) at room temperature, PBMCs were washed twice with PBS (1100 rpm, 10 min) at room temperature, and cells were counted. Briefly, heparin blood was diluted twice with PBS and transferred to Leucosep (Greiner Bio-One, Frickenhausen, Germany). For species comparison studies, human PBMCs were isolated from heparinized blood obtained from healthy volunteer donors by Ficoll–Paque PLUS density gradient centrifugation (GE Healthcare, Chicago, IL), following the manufacturer’s instructions. For various in vitro assays with huH-1, frozen human cryopreserved PBMCs were purchased from 10 different donors from Cellular Technology Ltd. Informed consent was obtained from all donors through Chugai Pharmaceutical Co Ltd, or Cellular Technology Ltd. preparationĪll studies using human PBMCs were approved by the Chugai Ethical Committee, and the study was conducted in accordance with its policy. We did not validate these cell lines ourselves. All cell strains were cultured according to the manufacturer’s instructions. PC-10, a human lung cancer cell line, was purchased from Immunology Biological Laboratories (IBL) (Gunma, Japan). SK-pca31a SK-pca13a and SK-pca60 are derivatives of SK-HEP-1 expressing GPC3 slightly, moderately or strongly, respectively. HepG2, a human hepatoblastoma cell line, and SK-HEP-1, a human hepatoma cell line, were purchased from the American Type Culture Collection (ATCC) (Manassas, VA). HuH-1, a human hepatocellular carcinoma cell line, was purchased from the Japanese Research Biosource Group (JCRB) (Osaka, Japan).
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