Microvette cb 300

Cytokine profiling of WT and Tg mice fed a low‐fat or a high‐fat diet was carried out as described previously (Petersen et al. 2014). In brief, mouse blood samples (~200 μL) were collected by tail vein bleed and separated by a Microvette^® CB 300 (SARSTEDT, Numbrecht, Germany). Serum cytokine levels (N = 8–9 per group) were measured in a Luminex Instrument (Luminex, Austin, TX) using a multiplex bead‐based assay (EMD Millipore, Billerica, MA) and analyzed by XPonent 3.1 Software (Millipore, Billerica, MA). Five separate multiplex assays, based on the known dynamic range of each cytokine, were carried out to cover a total of 71 cytokines. Some of the cytokines’ receptors are synthesized in membrane‐bound form, and proteolytic cleavage generates a soluble version that circulates in plasma. Thus, sCD30, sIL‐1RI, sIL‐1RII, sIL‐2Ra, sIL‐4R, sIL‐6R, sTNFRI, sTNFRII, sVEGFR1, sVEGFR2, sVEGFR3, sgp130, and sRAGE were also measured as part of the 71 cytokines profiled. Standards were provided for each mouse cytokine, from which standard curves were generated. Concentrations were determined for each of the 71 mouse cytokines relative to an appropriate 6‐point regression standard curve in which the mean fluorescence for each cytokine standard was transformed into known concentrations (pg/mL or ng/mL). Any sample below the detection limit of the assay (3.2 pg/ml) was excluded in analysis.

Mouse serum was harvested by tail bleed and at the time of euthanasia (~14 weeks old). Serum samples were separated by a Microvette^® CB 300 (Sarstedt, Nümbrecht, Germany) and centrifuged at 10,000 g for 5 min. TNF‐α (Millipore), IL‐1β (R&D Systems), and IL‐6 (Abcam) levels were determined using commercially available ELISA kits. Due to high circulating levels of these cytokines, samples had to be diluted 1:50 for the IL‐1β and IL‐6 assays, and 1:25 for the TNF‐α assay kit to meet the limits of the standard curve. For IL‐6 and IL‐1β, we included samples from mice treated with saline alone; however, the serum levels of TNF‐α in the saline‐injected control mice were below the detection limit of the standard curve and therefore its values were set at the detection limit of 2.3 ng/mL.

All metabolic tests were performed in age‐matched, sex‐matched cohorts. All mice were bred on a pure C57Bl6/J genetic background. Glucose tolerance test (GTT) was performed by intraperitoneally injecting 2 g of glucose/kg body mass of an aqueous 20% glucose solution to overnight fasted mice. Prior to injection, fasting glycemia was measured using the Wellion® LUNA glucometer with the corresponding Wellion® LUNA test stripes GLU by collecting a blood drop from the tail. Glycemia was measured the same way at 15, 30, 60, 90, and 120 min after glucose injection. When applying, blood sampling from the tail for glucose‐stimulated insulin secretion (GSIS) was performed using Sarstedt Microvette CB300. Insulin tolerance test was performed in a similar way as the GTT, injecting 0.75 IU/kg body mass in 6 h fasted mice.