Milliplex mouse metabolic hormone magnetic bead panel mmhmag 44k

Serum adiponectin was assayed by ELISA (KMP0041, Life Technologies, US), according to manufacturer’s protocol. Leptin, resistin, C-Peptide 2, glucose-dependent insulinotropic peptide (GIP) and anorexigenic peptide PYY were simultaneously assayed on serum samples added with antiproteases (Complete EDTA Free, Roche, Meylan, France) using the Milliplex mouse metabolic hormone magnetic bead panel (MMHMAG-44K, Merck Millipore, Fontenay sous Bois, France). Circulating cytokine levels were analysed using the 10-spot V-plex kit Proinflammatory Panel 1 Mouse (Meso Scale Discovery, Rockville, USA). Data acquisition of multiplex analyses was performed at the Cochin Cytometry and Immunobiology Facility (France) using lectors Bioplex 200 (Biorad) and Sector Imager 2400 (Meso Scale Discovery). Lipid profile, hydroxybutyrate and total antioxidant status (TAS) were assayed on trunk serum using an Olympus AU-400 multiparametric analyzer at 'Plateforme de Biochimie' (INSERM U1149, France). Values for triglycerides, total cholesterol, high-density lipoprotein-cholesterol (HDL) and non-esterified fatty acids (NEFA) allowed the calculation of very low density lipoprotein-cholesterol (VLDL) and low density lipoprotein-cholesterol (LDL) following the Friedewald's equation^{39 (link)}.

PANKO-C57 and WT mice were subjected to fasting overnight before plasma collection. Whole blood from mice that had been subjected to fasting was obtained through submandibular vein puncture and free flow collection into BD MicroContainer^® (Becton, Dickinson and Company, Franklin Lakes, NJ). Whole-blood samples were immediately placed on ice before being centrifuged for 5 minutes at 13,200 r.p.m. in an Eppendorf Microfuge 5430 for plasma separation into a new Eppendorf (Eppendorf, Hauppague, NY) collection tube. The endocrine hormone panel was performed using the commercially available Milliplex Mouse Metabolic Hormone Magnetic Bead Panel MMHMAG-44K (EMD Millipore Corporation, Billerica, MA) and Luminex Multiplex Platform (MAGPIX^®). All procedures regarding the metabolic hormone panel adhered to the manufacturer’s protocol included in the assay kit. Corticosterone EIA kit (Enzo Life Sciences, Farmingdale, NY) and Triglyceride Quantification Kit (Abcam^®, Cambridge, MA) were utilized for the determination of corticosterone and plasma triglyceride concentrations, respectively, and both were performed in accordance with the manufacturers’ suggested protocols.