Hr series nefa hr kit

Tissue triglyceride levels and serum glycerol concentration were measured by Serum Triglyceride Determination Kit (TR0100, Sigma). NEFA was measured by HR series NEFA-HR kit (Wako Diagnostics). Commercial ELISA kits were used according to the manufacturer’s instructions to detect catecholamines (BAE5400, Rocky Mountain Diagnostics), insulin and corticosterone (98880 and 80556, Crystal Chem Inc.), and resistin (ELM-Resistin-1, Raybiotech). The limit of detection (LOD) for the insulin measurements is set at 0.05 ng/mL. Samples below the LOD are not included for statistical calculations. All other circulating cytokines and adipokines in serum were detected by Luminex Multiplex Assays at the University of Virginia flow core facility.

Blood was collected from tail veins in citrate-EDTA tubes, and plasma was isolated by centrifugation at 2000g for 10 minutes at 4°C. Plasma levels of TGs and cholesterol were measured using Infinity series kits (Thermo Fisher Scientific). NEFA levels were measured in duplicate or triplicate using the HR Series NEFA-HR Kit (Fujifilm Wako Diagnostics). Results were obtained using the Synergy Neo2 plate reader (BioTek Instruments). Plasma glucose levels were measured using Contour Blood Glucose Monitoring System (Bayer).
For fatty acid analysis of tissues, lipids were extracted from the blood (51 (link)) of the fasting and refed animals, derivatized to form methyl esters, and separated by gas/liquid chromatography using a Hewlett Packard 6890 Series GC system. The identity of the fatty acid methyl ester was determined by comparing the retention times with fatty acid standards (GLC-744; NU-Chek Prep). The abundance of each fatty acid was determined from the peak intensity and the internal standard. Fatty acid profiles were generated using a modified GC-MS method, as previously described (52 (link)).