Recombinant human il 17b protein

Recombinant human IL-17B protein was purchased from R&D Systems (1248-IB-025), and was resuspended in PBS solution under sterile conditions. MGC-803 or HGC-27 cells were adherently cultured in the 6-wells plate, meanwhile add rIL-17B solution into the plate to the corresponding concentration, then incubator at 37 °C in an atmosphere of 5% CO₂ for 48 h. To evaluate the effect of rIL-17B on the sphere-formation ability of GC cells, MGC-803 and HGC-27 cells treated with PBS or rIL-17B for 48 h, then the spheroid formation assay were operate.

DCs were generated from the bone marrow cells of 8 weeks old female BALB/c mice. The bone marrow cells were centrifuged, washed and cultured in roswell park memorial institute 1640 medium (Hyclone, Utah, USA) containing 10% fetal bovine serum (Sciencell, CA, USA), 1% penicillin/streptomycin (Beyotime), 10 ng/ml recombinant human IL-4 protein (R&D systems, Minnesota, USA) and 20 ng/ml granulocyte macrophage-colony stimulating factor (PeproTech, NJ, USA) and incubated at 37 °C and 5% CO2 for seven days. Cells were cultured with recombinant human IL-17B protein (500 ng/mL) (R&D systems) for 24 h. Cells cultured with medium provided a negative control while cells cultured with LPS (10 ng/mL) (Sigma, Missouri, USA) provided the DC activation positive control. DC phenotypes were analyzed via surface expression of specific markers. Live DCs were immunolabeled with APC-CY7-livedead (Thermo Fisher Scientific, Massachusetts, USA), eFluor450-CD11C (Thermo Fisher Scientific), PE-CD80 (Thermo Fisher Scientific), APC-CD86 (Thermo Fisher Scientific), FITC-MHC II (Thermo Fisher Scientific) at 4 °C for 30 min. Resuspend cells were detected on Beckman Cytoflex LX and the result was analyzed by FlowJo 10.