Mouse il 6 elisa kit

Plasma samples were analyzed for total cholesterol (TG), triglyceride (TC), low density lipoprotein-cholesterol (LDL-C), free fatty acid (FFA), tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), monocyte chemoattractant protein 1 (MCP-1), adiponectin and leptin by using Cholesterol Quantitation Kit (Sigma), Triglyceride Quantification Kit (Abcam), HDL and LDL/VLDL Cholesterol Assay Kit (Abcam), Free Fatty Acid Quantification Kit (Abcam), Mouse Tumor Necrosis Factor α ELISA Kit (Sigma), Mouse IL-6 ELISA Kit (Sigma), Mouse MCP-1 / CCL2 ELISA Kit (Sigma), Mouse Adiponectin ELISA Kit (Abcam) and Mouse Leptin ELISA Kit (Abcam) according to the manufacturers’ instructions.

Enzyme-linked immunosorbent assay kits (mouse TNF-α ELISA kit, Mouse IFN-γ ELISA kit, mouse IL-1β ELISA kit, mouse IL-6 ELISA kit; Sigma) were used to determine the serum levels of the key pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-1β and IL-6, in accordance with the manufacturer’s instructions.

Mice were perfused with phosphate-buffered saline under anesthesia. Harvested organs (300 mg) were homogenized in RIPA buffer using the Next Advance Bullet Blender (Next Advance, Inc. Troy, NY). Homogenized samples were centrifuged at 18,800 x g for 10 min to obtain clear lysates. All tissue processing was carried out at 4°C. Heart IL-6 concentration was measured using the mouse IL-6 ELISA kit (Sigma-Aldrich) following the manufacturer's instructions. Total protein was quantified in the lysates using the BCA assay kit (ThermoFisher Scientific, #23225).

To determine levels of 8-OHdG, BDNF and IL-6 in the slice culture medium and the extracts of OBSCs, samples were analyzed using commercially available ELISA kits (New 8-OHdG Check ELISA kit, JaICA Co. Ltd. (Shizuoka, Japan), Human BDNF ELISA kit, RAB0026, Sigma-Aldrich, Mouse IL-6 ELISA Kit, RAB0308-1KT, Sigma-Aldrich, Tokyo, Japan). Culture medium was obtained during and after the cultivation of OBSCs. At the end of cultivation, OBSCs (2 slices) from a single culture membrane were carefully removed from the culture insert without containing matrigel. The OBSCs were then homogenized in 100 μL RIPA lysis buffer (WSE-7420 EzRIPA Lysis kit, ATTO Co., Tokyo, Japan) to obtain the extracts. Protein concentrations of the extracts were measured with a protein quantification kit (Protein Quantification Kit-Rapid, Dojindo Molecular Technologies, Inc., Kumamoto, Japan). For each ELISA, culture medium or OBSCs extracts (with same concentration of protein) were diluted to bring the expected concentration within the range of the standard curve and reacted with first and secondary antibodies, streptavidin-HRP and detection solution according to the manufacturer’s instruction. The reaction was stopped using the stop solution (from the ELISA kits) and absorbance read at 450 nm using a microplate reader (SH-9000Lab, Hitachi, Tokyo, Japan).

Serum IL-6 levels were quantified using a commercial mouse IL-6 ELISA kit (RAB0308; Sigma) according to the manufacturer’s instructions and analyzed using spectra-iMAX with SoftMaz Pro V6 software (Molecular Devices, USA).

IL-6 was measured in the cerebellum, heart, and serum by ELISA (Mouse IL-6 ELISA Kit; Sigma) following the manufacturer’s instructions. Briefly, tissue samples (∼20 mg) were homogenized with 1× phosphate-buffered saline (PBS) containing 1% protease inhibitor cocktail (Sigma) and 1% Triton X-100 (Sigma) to obtain total protein extracts. Protein concentrations in the target tissues and serum were analyzed using the Pierce BCA protein assay kit (Thermo Scientific). All protein samples were run in duplicate. Serum and tissue IL-6 concentrations were analyzed by SoftMax Pro 5.2 (Molecular Devices).