Fasted (16 hrs) plasma insulin levels were determined after free access to either chow or junk-food in the home cage for 4 weeks (OP-Junk-food N=6, OR-Junk-food N=6; OP-Chow N=4, OR-Chow N=4). Blood samples were collected via tail nick into tubes containing EDTA (1.6 mg/mL, Sarstedt), and plasma was then isolated by centrifugation (1000 x G, 4°C, 10 min) and stored (−20°C) for subsequent analysis. Plasma insulin levels were determined by double-antibody radioimmunoassay using an 125I-Human insulin tracer (Linco Research, St. Charles, MO), a rat insulin standard (Novo Nordisk, Plainsboro, NJ), a guinea pig anti-rat insulin first antibody (Linco Research), and a sheep anti-guinea pig gamma globulin-PEG second antibody (Michigan Diabetes Research Core). The limit of sensitivity for this assay was 1 μU/ml. Inter-assay and intra-assay variability were 11.2% and 3.2%, respectively, at 30.5 μU/ml.
Rat insulin standard
Manufactured by Novo Nordisk
Sourced in Denmark
The Rat Insulin Standard is a laboratory product used as a reference material for the quantification and analysis of insulin levels in rat samples. It provides a consistent and reliable standard for researchers to measure and compare insulin concentrations in their experiments.
Automatically generated - may contain errors
Lab products found in correlation
125i human insulin tracer, by Linco Research (3 mentions)
Guinea pig anti rat insulin first antibody, by Linco Research (3 mentions)
Ethylene diamine tetraacetic acid (edta), by Sarstedt (2 mentions)
Minispec lf90ii, by Bruker (1 mentions)
Cm1850 cryostat, by Leica Microsystems (1 mentions)
Wako nefa c, by Fujifilm (1 mentions)
4 protocols using rat insulin standard
1
Plasma Insulin Levels in Junk-Food vs. Chow Diet
2
High-fat Diet Impacts Rats' Metabolic Profiles
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Rats were given free access to 60% high-fat diet (Open Source Diets D12492) in the home cage for a total of 8 weeks. Controls had free access to standard lab chow throughout (Lab Diet 5001, 13% fat). Weight was measured twice each week. In addition, after 7 weeks of high-fat or control diet, body composition was determined by NMR (Minispec LF90II, Bruker Optics), and fasted blood samples (16 h) were collected and used to determine plasma insulin levels. Blood samples were collected via tail nick into tubes containing EDTA (1.6 mg/ml, Sarstedt), and plasma was then isolated by centrifugation (1000 × g, 4°C, 10 min) and stored (−20°C) for subsequent analysis as previously described (Vollbrecht et al., 2015 (link)). Plasma insulin levels were determined by double-antibody radioimmunoassay using a 125I-human insulin tracer (Linco Research), a rat insulin standard (Novo Nordisk), a guinea pig anti-rat insulin first antibody (Linco Research), and a sheep anti-guinea pig γ globulin-PEG second antibody (Michigan Diabetes Research Core). Blood collection and NMR were conducted at week 7 to avoid additional stress during the week of slice preparation or NAc tissue collection (week 8). Food was removed from the cage 1-2 h before slice preparation or NAc tissue collection.
3
Plasma Insulin Determination in Fasted Rats
4
Biochemical Profiling of Plasma Markers
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Commercially available analytical kits were employed to determine plasma glucose, triglyceride (TAG) and uric acid concentration (Wiener Lab., Rosario, Santa Fe, Argentina). Plasma free fatty acids (FFA) were determined using an acyl-CoA oxidase based colorimetric kit (Wako NEFA-C, Wako Chemicals, Neuss, Germany). Immunoreactive insulin was measured as previously described [23] . The immunoreactive insulin assays were calibrated against the rat insulin standard (Novo Nordisk, Copenhagen, Denmark). Plasma tumor necrosis factor-a (TNF-a) was measured using commercial ELISA kits (Thermo Scientific, Rockford, USA; Legend max TM Biolegend 1 Inc., San Diego, USA). The minimum detectable limit was 4.2 pg/mL, with an intra-and inter-assay CV of less than 7.4% and 10.5% respectively. All determinations were performed in triplicate.
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