Fibroblast growth factor 21 mouse rat elisa

Mouse plasma samples were analyzed using ELISA assays to measure insulin (Ultra-Sensitive Mouse Insulin ELISA Kit, #90080, Crystal Chem, USA), leptin (Mouse/Rat Leptin Immunoassay, #MOB00B, R&D Systems, USA), total ghrelin (Rat/Mouse Total Ghrelin, #EZRGRT-91K, EMD Millipore, USA), GDF15 (Rat/mouse GDF15 Quantikine ELISA kit, #MGD150, R&D Systems, USA) and FGF21 (Fibroblast Growth Factor 21 Mouse/Rat ELISA, #RD291108200R, BioVendor R&D, Czech Republic) using manufacturer instructions. Plasma was diluted 1:5 for total ghrelin measurement, 1:20 (1:60 for ExpOF d14 samples) for leptin measurements, 1:10 for FGF21 (1:3 for controls) and 1:5 for GDF15 measurements. Blood glucose was measured from tail blood in awake mice using a glucometer. Plasma total cholesterol (Infinity Cholesterol Liquid Stable Reagent, #TR13421, Thermo Fisher Scientific, USA), total glycerol (triglycerides) (Infinity Triglyceride Liquid Stable Reagent, #TR22421, Thermo Fisher Scientific, USA), and free fatty acids (HR Series NEFA-HR(2), #434-91795, #436-91995, #270-77000, Fujifilm Wako Chemicals Europe) concentrations were measured by using commercially available kits.

Circulating NNPA and NPPB were assessed in blood serum obtained when animals were sacrificed (4–6 h within the light cycle; random feeding condition). Starting serum volumes and dilutions were optimized for each peptide, and measures were performed in duplicates according to the manufacturer’s instructions. Essentially, NPPA was assessed in a 2-fold dilution of 25 μL of serum using the Atrial Natriuretic Peptide kit (Phoenix Pharmaceuticals, Burlingame, CA). NPPB was assessed in a 5-fold dilution of 42 μL of serum using the RayBio^® Mouse BNP EIA Kit (RayBiotech, Norcross, GA). Preliminary studies demonstrated equivalent sensitivity and reproducibility using 17.5 to 35 μL of serum when assessing FGF21 using the Fibroblast Growth Factor 21 Mouse/Rat ELISA (BioVendor, Asheville, NC). Therefore, FGF21 was assessed both at random feeding (6-fold dilution of 35 μL of serum) and fasting conditions (after an 18 h overnight fast; 9-fold dilution of 23 μL of serum) according to the manufacturer’s instructions.