Total cholesterol assay kit

Serum total cholesterol, triglyceride, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol were determined with a Beckman Coulter AU480 automatic biochemistry analysis system (Brea, CA, USA) with reagent kits provided by the manufacturer. For lipid extraction, tissue were rinsed with ice cold PBS to remove excess blood thoroughly and small pieces and homogenized them in 100-200μL PBS with a glass homogenizer on ice. The resulting suspension was stored overnight at −20°C. To further break to cell membranes, two freeze-thaw cycles were performed. After that, the homogenates were centrifuged for 5 minutes at 5000 x g. The supernatant was used for assay. Triglyceride ELISA kit (Aviva Systems Biology, San Diego, CA, USA) and total cholesterol assay kit (BioVision Inc., Milpitas, CA) were used for assay. Insulin was measured using a mouse insulin ELISA kit (ALPCO Diagnostics, Windham, NH, USA) following the manufacturer's protocol.

To measure plasma levels of adiponectin and insulin, blood samples were collected directly from the heart and transferred to EDTA-coated tubes. The plasma fraction of whole blood was obtained using a refrigerated centrifuge (2,500 × g for 30 min). Assay kits (Mouse Adiponectin ELISA Kit, KMP0041, Thermo Fisher Scientific; Ultra Sensitive Mouse Insulin ELISA Kit, 90080, Crystal Chem; Total Cholesterol Assay Kit, K603, BioVision; Free Fatty Acid (FFA) Assay Kit, K612, BioVision, Triglyceride (TG) Assay Kit, ab65336, Abcam) were used according to the manufacturer’s instructions.