Bio plex pro mouse diabetes 8 plex assay

Serum levels of triglycerides, total cholesterol, and LDL cholesterol were measured using automated equipment. Leptin, insulin, GIP, resistin, and PAI-1 were determined in serum using Bio-Plex Pro Mouse Diabetes 8-Plex Assay (BIO-RAD, USA), and adiponectin was determined using Bio-Plex Pro Mouse Diabetes Adiponectin Assay (BIO-RAD, USA). A pathologist blinded to the study performed a histological analysis in hematoxylin and eosin (H&E)-stained epididymal fat tissue.
The adipocyte size (μm²), cell hyperplasia, cell hypertrophy, and inflammatory infiltrates were analyzed. To assess the adipocyte size, at least 10 microscopic fields/mice at 20X magnification were analyzed using Image-Pro software (16 (link)).

Blood was terminally collected from 30–34-week-old mice and serum was taken. Serum samples were analyzed in duplicates using magnetic bead-based multiplex immunoassays (Bio-Plex Pro Mouse Diabetes 8-Plex Assay, #171F7001M and Pro Mouse Diabetes Adiponectin Assay, #171F7002M, Biorad) according to the manufacturer’s instructions. Analyses were performed using the Bio-Plex200 suspension array system (Biorad). Analyte concentrations (pg/ml) were calculated from the appropriate optimized standard curves with the help of the Bio-Plex-Manager-Software version 6.0 (Biorad).
Non-esterified fatty acids (NEFA) were measured using the Wako HR NEFA Kit (Fujifilm) according to the manufacturers’ protocol. A standard series comprising 0, 0.125, 0.25, 0.5, and 1 mM NEFA Standard was prepared and 4 µl serum or standard and 200 µl Fujifilm NEFA (HR) 1 were incubated for 5 min at room temperature. Samples were measured using the Synergy microplate reader (BioTek) at 550 nm. Subsequently, 100 µl NEFA (HR) 2 was added and samples measured after another 5-minute incubation at room temperature.

Blood parameters were measured at 17 weeks of age (n = 8). Blood glucose was measured with Freestyle™ and leptin, insulin as well as glucagon levels were determines using quantitative Bio-Plex Pro Mouse Diabetes 8-Plex Assay (Bio-Rad, Munich, Germany) according to the manufacturer’s instructions. Data were collected and analyzed using a BioPlex 200 instrument equipped with BioManager analysis software (Bio-Rad). To determine insulin resistance and pancreatic β cell function the surrogate parameters HOMA-IR (homeostatic model assessment of insulin resistance) and HOMA-β (homeostatic model assessment of β cell function) were used. Body composition was measured using nuclear magnetic resonance (n = 9–23/per genotype, Whole Body Composition Analyzer; Echo MRI, Houston, TX, USA).

Mice had been fasted ~ 5 h prior to euthanasia and blood collection. During euthanasia, blood was collected via cardiac puncture, mixed with 5 μL of EDTA on ice, and spun at 1500×g for 10 min at 4 °C to collect plasma. Plasma samples were stored at − 80 °C until assayed. Diabetes-associated markers and cytokine levels in the plasma were assessed using the Bio-Plex Pro Mouse Diabetes 8-Plex Assay (Cat# 171F7001M; Bio-Rad, Carlsbad, California) and the Bio-Plex Pro Mouse Cytokine 23-Plex Assay (Cat# M60009RDPD; Bio-Rad, Carlsbad, California), respectively, according to the manufacturer’s instructions.

Blood was collected from fasted (4–16 h) or re-fed animals (15 min to 1 h). Re-feeding was performed by injecting a bolus of glucose (1 g/kg of BW) IP or by oral gavage as indicated. For active GLP-1 measurement, blood was collected on Dpp4 inhibitor-coated tubes (BD P700). Serum glucose, triglycerides, and cholesterol levels were assessed using Infinity reagents (Thermo Scientific) as per the manufacturer's instructions. Serum endocrine panel (ghrelin, GIP, GLP-1, glucagon, insulin, leptin) was performed using the Bio-Plex Pro Mouse Diabetes 8-Plex Assay (Bio-Rad) as per the manufacturer’s protocol.

The plasmas' level of metabolic hormones (ghrelin, leptin, insulin, glucagon, glucagon‐like‐peptide and resistin) were measured using a commercially available suspension of magnetic bead array immunoassay kit following manufacturer's specifications (BIO‐RAD Bio‐Plex Pro Mouse Diabetes 8‐Plex Assay #171f7001m).