Milliplex mouse adipokine magnetic bead panel

Plasma insulin and adipocytokines (leptin, monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), plasminogen activator inhibitor-1 (PAI-1)) were measured after treatment. Blood collection was performed by cardiac puncture using K₂EDTA as an anticoagulant. Then, samples were centrifuged at 3,000 g, 4°C, for 15 min to obtain the plasma and stored at −80 °C until the analysis. Plasma insulin and adipocytokines were quantitatively determined using an enzyme-linked immunosorbent assay (ELISA) kit (Milliplex^® Mouse Adipokine Magnetic Bead Panel; MilliporeSigma, Burlington, MA, United States) according to the manufacturer’s instructions. Briefly, plasma samples were mixed with magnetic beads coated with antibodies in a 96-black well plate and incubated with overnight shaking at 4 °C. Then, the biotinylated detection antibodies were added and incubated for 30 min. Next, Streptavidin-Phycoerythrin was added and incubated for a further 30 min. After the plate washing steps, Sheath Fluid was added to each well and incubated for 5 min on a plate shaker. Finally, the quantification of fluorescence signals was determined using the Luminex MAGPIX^® system (MilliporeSigma, Burlington, MA, United States).

Plasma levels for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and circulating levels of high-density lipoprotein (HDL), triglycerides, and cholesterol were obtained using an Abaxis Piccolo Xpress analyzer (Abbott; Abbott Park, IL, USA) with Lipid Panel Plus diskettes (catalog:400-0030; Abaxis Inc.; Union City, CA, USA). Circulating levels of plasma adipokines and cytokines were measured using a MILLIPLEX^® Mouse Adipokine Magnetic Bead Panel (Millipore Sigma, Burlington, MA, USA) for analytes interleukin 6 (IL-6), leptin, total plasminogen activator inhibitor 1 (PAI-1), tumor necrosis factor alpha (TNFα), and resistin. Data was obtained from a Luminex 100/200 System equipped with xPONENT Software (Luminex Corporate, Austin, TX, USA). Hepatic lipids were extracted using a modified Bligh and Dyer method with a 2:1 ratio of chloroform to methanol mixture (27 (link), 28 (link)). Extracted lipids were measured using Infinity Assay Reagents: Infinity Triglycerides reagent (TR22421, ThermoFisher Scientific, Middletown, VA, USA) and Infinity Cholesterol reagent (TR13421, ThermoFisher Scientific, Middletown, VA, USA). Sample absorbance at 500 nm was calculated from a standard curve constructed from clinical standards (catalog: T7531-STD, C7509-STD; Point Scientific; Canton, MI) for each analyte ran simultaneously with unknown samples.