Mouse tnf α and il 6 elisa kits

The mouse MPO ELISA kit was purchased from Abcam (Cat. No. ab275109), PYY ELISA kit from Aviva Systems Biology (Cat. No. OKCD05062, USA), mouse TNF-α and IL-6 ELISA kits from Biolegend (Cat. No. 431,301 and 430,901, USA). The protocols were performed as the manufacturers described. In brief, 96-well plates were coated with the corresponding capture antibody. After incubation with blocking buffer (PBS + 1% bovine serum albumin), a 100-µl aliquot of either standards, serum, or colonic protein extracts (5 mg tissue per mL PBS supplemented with protease inhibitors) was added into each well individually and the plates were incubated for 2 h at room temperature. The detector antibody was added to the wells, the plate was incubated, and unbound detector antibody was removed by washing. The avidin-peroxidase conjugate was added, the plate was incubated, and unbound conjugate was removed by washing. The enzymatic reaction catalyzed by horseradish peroxidase (HRP) was initiated by adding the substrate, 3,3’,5,5’ tetramethylbenzidine dihydrochloride (TMB). The absorbance was measured by using a microplate reader at a wavelength 450 nm after the reaction was stopped with 2 M H₂SO₄.

Taraxasterol was supplied from Chengdu Fenruisi Biotechnology Co. Ltd. (Chengdu, Sichuan, China), and its purity was 99.5% based on HPLC analysis. Tiopronin (TPN, no. 036140404) was purchased from Xinyi Pharmaceutical Co. Ltd. (Xinyi, Henan, China). Absolute ethanol was purchased from Sigma-Aldrich (St. Louis, MO, USA). Liber-DeCarli liquid diet was obtained from Tropic Animal Feed High-Tech Co. Ltd. (Nantong, Jiangsu, China). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) commercial reagent kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). Mouse TNF-α and IL-6 ELISA kits were purchased from BioLegend Inc. (San Diego, CA, USA). Anti-CYP2E1, I kappa Bα (IκBα), nuclear factor-κB (NF-κB) p65, nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). β-Actin, horseradish peroxidase- (HRP-) conjugated goat anti-mouse IgG, and goat anti-rabbit IgG were purchased from Santa Cruz (Santa Cruz, CA, USA). Diaminobenzidine (DAB), bicinchoninic acid (BCA), and chemiluminescent (ECL) reagent kits were purchased from Beyotime Biotech. (Shanghai, China).

Cultured BV-2 cells (2 × 10⁵ cells/mL) were treated with skimmianine (10, 20 and 30 μM) for 30 min, followed by stimulation with LPS (100 ng/mL) for 24 h. Levels of TNFα, IL-6 and IL-10 in culture supernatants were measured with mouse TNFα and IL-6 ELISA kits (Biolegend), respectively, according to the manufacturer’s instructions.