Il 23 elisa kit

C57BL/6 mice were purchased from Shanghai SLAC Laboratory Animal. All mice were housed at the Zhejiang University Laboratory Animal Center under specific pathogen-free conditions. All the animal experiments were performed with relevant guidelines and regulations approved by the Institutional Animal Care and Use Committee at Zhejiang University. This study was approved by the Ethics Committee of Sir Run Run Shaw Hospital of Zhe Jiang University School of Medicine (approval no. 20200717-015).
IMQ (tlrl-imq) was purchased from Invivo Gen. Recombinant mouse GM-CSF (315-03) and IL-17A (200-17) were from PeproTech. TRIzol (15596018) was purchased from Thermo Fisher. ReverTra Ace qPCR RT Kit (FSQ-201) was from Toyobo. SYBR Green master Rox (04707516001) was from Roche. IL-23 ELISA kit (433704) was purchased from Biolegend and CXCL1 kit (ab216951) was from Abcam.

For detection of IL‐23 and IL‐23R expression, MLE‐12 cells (SV40‐transformed mouse‐derived alveolar epithelial cell line; American Type Culture Collection) were grown in Dulbecco's modified Eagle medium:Ham's F‐12 with 2% fetalbovine serum in a humidified atmosphere at 37°C with 5% CO2. The cells were then stimulated with low‐dose Dp (10 μg/ml) or PM (0.1 μg/ml) for overnight. The expression of IL‐23 was detected in cytosol using NE‐PER Extraction Reagent (Thermo Fisher Scientific) and IL‐23 ELISA kit (Biolegend). For expression of IL‐23R, cells were stained with PE‐conjugated anti‐IL‐23R (Thermo Fisher Scientific). For IL‐23 siRNA experiment, MLE12 cells were treated with IL‐23p19 siRNA (Santa Cruz, 30 nM/well) or normal control (NC, Santa Cruz) siRNA using transfectamine (Invitrogen) and after 5 hours, the cells were washed and stimulated low‐dose Dp (10 μg/ml) or PM (0.1 μg/ml) for overnight. The protein level of IL‐23, GMCSF in cytosol and IL‐33 in the nucleus was measured using NE‐PER Extraction Reagent (Thermo) and ELISA kit (R&D). For detection of intracellular IL‐33 expression from MLE‐12 cells, the cells were permeabilized and incubated with anti‐IL‐33 (1:100, R&D), 647‐conjugated anti‐goat IgG (1:500, Invitrogen). Each sample was read on LSRII (BD Biosciences).