Human recombinant murine il 33

Four to five-week-old female nude BALB/c mice (Beijing HFK Bioscience Co., Ltd) and five to six-week-old female NSG mice (Shanghai Model Organisms) were used for subcutaneous xenograft models. All experiments were approved by the Ethics Committee of Harbin Medical University (HMU, Harbin, Heilongjiang, China) according to the Guidelines for the Care and Use of Laboratory Animals. Ln229 glioma cells were subcutaneously transplanted into back flanks of BALB/c nude mice. After transplanted for 2 weeks, human recombinant murine IL-33 (carrier-free, R&D Systems) or PBS was administered intraperitoneally (i.p.) into mice at 1 μg per injection for 10 days. All nude mice were killed in the fourth week. Ln229 glioma cells were subcutaneously transplanted into back flanks of NSG mice. After transplanted for 3 weeks, PBS, Anti-ST2, TMZ and Anti-ST2+TMZ were administered intraperitoneally (i.p.) into mice for consecutive 7 days. All NSG mice were killed in the sixth week.

Cell proliferation was evaluated using the Cell counting kit-8 (CCK-8) assay according to the protocol. Briefly, cells were seeded in 96-well plates and incubated with various concentrations of TMZ (Selleck), IL-33 (Human recombinant murine IL-33, R&D Systems) or Anti-ST2 (Human ST2/IL-33R Antibody, R&D Systems). Subsequently, CCK-8 solution was added, and the viable cells were quantified using IMARK microplate reader at 450 nm of absorbance. Each experiment was performed in triplicate and repeated for three times.