Portal and systemic blood glucose levels were measured at each time point using a glucometer (LifeScan OneTouch). For hormonal analysis, blood was centrifuged at 4°C, 4,000g, for 15 min, the plasma aspirated, and stored at −80°C. Plasma GLP-1 (active), glucose-dependent insulinotropic peptide (GIP) (total), insulin, and peptide YY (PYY) levels were measured in duplicate using the Milliplex rat metabolic hormone panel (cat. no. RMHMAG-84K; Millipore, Darmstadt, Germany). Coefficient of variation was <20% for all samples.
Rmhmag 84k
Manufactured by Merck Group
Sourced in United States
The RMHMAG-84K is a laboratory equipment product manufactured by Merck Group. It is designed for general laboratory use. The core function of the RMHMAG-84K is to perform specific laboratory tasks. No further details can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Milliplex map rat metabolic hormone magnetic bead, by Merck Group (1 mentions)
Xponent software, by Merck Group (1 mentions)
Luminex magpix system, by Merck Group (1 mentions)
Amylin, by Merck Group (1 mentions)
Ghrelin, by Merck Group (1 mentions)
Luminex 200, by DiaSorin (1 mentions)
Meso quickplex sq 120 instrument, by Mesoscale (1 mentions)
P2714 1btl, by Merck Group (1 mentions)
Dpp4 010, by Merck Group (1 mentions)
High capacity cdna archive kit, by Thermo Fisher Scientific (1 mentions)
Mouse and rat fgf 21 elisa, by BioVendor (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Bio plex multiplex system, by Bio-Rad (1 mentions)
6 protocols using rmhmag 84k
1
Plasma Metabolic Hormone Analysis
2
Rat Plasma Biomarkers Analysis
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On PND 36–40, the rats that were previously evaluated for their behavior were decapitated, and their trunk blood was collected in conical tubes that contained 10% ethylenediaminetetraacetic acid (EDTA). The samples were centrifuged (3,500 RPM, 15 min, 15°C), and the plasma was obtained. Using the Luminex/Magpix system (RSH69K03, Millipore, Billerica, USA), biomarkers related to autism and/or pioglitazone were studied, including the following: IL-6, TNF-alpha, MCP-1, insulin, and leptin (part of the Milliplex map rat metabolic hormone magnetic bead panel-metabolism multiplex assay, catalogue no. RMHMAG-84K; Millipore, Billerica, USA) according to the manufacturer's instructions. The range of detection of the peptides was 1.6–400.000 pg/ml. The concentrations were estimated using a five-parameter polynomial curve (Xponent software, Millipore). All results are expressed in pg/ml.
3
Multiplex Profiling of Rat Metabolic Hormones
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In vivo rat study: Plasma concentrations of peptide hormones were determined using a customized xMAP based multiplex-assay (Milliplex map rat metabolic hormone magnetic bead panel – metabolism multiplex assay, cat. no. RMHMAG-84 K, Millipore), for the following selected analytes: Amylin (Active), C-Peptide, Ghrelin (Active), GIP (Total), GLP-1 (total), Glucagon, IL-6, Insulin, Leptin, MCP-1, PP, PYY (Total), TNF-α. The assay is reported to have no significant cross-reactivity with other hormone tested. After the analytes were bound to the antibody-coupled beads, a biotinylated detection antibody was added, followed by incubation with streptavidin-phycoerythrin conjugate. Subsequently, the beads were subjected to a flow cytometry-based detection method using a Luminex laser-based analyzer (catalog no. Luminex 200, Luminex, Austin, TX 78727, USA). The manufacturer's instructions were closely followed. Reported intra- and inter-assay variation was 1–8% and 7–29%, respectively. The median fluorescent intensities from the eight calibrators were used to interpolate concentrations in plasma samples using 5-parameter logistic regression.
4
Multiplex Assay for Rat Metabolic Hormones
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Blood samples were collected at termination in EDTA-coated tubes containing DDP-IV inhibitor (DPP4-010, Millipore, Denmark) and protease inhibitor cocktail (P2714-1BTL, Sigma-Aldrich, Denmark). Plasma was separated and stored at −80 °C. Leptin, C-peptide, peptide YY (PYY) and ghrelin were determined in a rat multiplex assay (#RMHMAG-84K, Merck Millipore, Darmstadt, Germany) on a Luminex Flexmap 3D machine (Luminex, Austin, TX). Total and active GLP-1 was determined using single-plex assays (K150JVC/K150JWC, Meso Scale Discovery, Rockville, MD) on a MESO QuickPlex SQ 120 instrument (Meso Scale Discovery, Rockville, MD).
5
Quantifying FGF21 and Insulin Plasma Levels
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Plasma FGF21 was determined using an enzyme-linked immunoassay (Mouse and Rat FGF-21 ELISA, BioVendor). Plasma insulin was measured with a Luminex assay (RMHMAG-84 K, MILLIPLEX Rat Metabolic Magnetic Bead Panel, Merck-Millipore).
Total RNA was extracted from duodenal, liver, muscle, brown adipose tissue, epididymal adipose tissue and the hypothalamus using TRIzol reagent (Invitrogen). RNA concentration was measured using a nanodrop spectrophotometer at 260 nm, and RNA integrity was verified by electrophoresis on agarose gel. Retrotranscription was performed on 0.4 µg of RNA using the High Capacity cDNA Archive Kit (Applied Biosystems). To measure gene expression, real time PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems) on the Step One (Applied Biosystems) with 5 ng of cDNA. Gene expression was calculated as 2−ΔCT, where ΔCT = CTGene − CT18S. In order to detect potential contamination, negative controls were used (control without RT or RNA). Primer Express was used to design the primer sequences of genes, and the sequences of primers used are described in Supplementary Table2 .
Total RNA was extracted from duodenal, liver, muscle, brown adipose tissue, epididymal adipose tissue and the hypothalamus using TRIzol reagent (Invitrogen). RNA concentration was measured using a nanodrop spectrophotometer at 260 nm, and RNA integrity was verified by electrophoresis on agarose gel. Retrotranscription was performed on 0.4 µg of RNA using the High Capacity cDNA Archive Kit (Applied Biosystems). To measure gene expression, real time PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems) on the Step One (Applied Biosystems) with 5 ng of cDNA. Gene expression was calculated as 2−ΔCT, where ΔCT = CTGene − CT18S. In order to detect potential contamination, negative controls were used (control without RT or RNA). Primer Express was used to design the primer sequences of genes, and the sequences of primers used are described in Supplementary Table
6
Intestinal Hormones and Liver Markers in Rats
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At the end of the experiments, the rats were anesthetized by an intraperitoneal injection of 30 mg/kg pentobarbital following a 12-h fasting period. Blood samples were collected from the orbital plexus and the hepatic portal vein and centrifuged (2,000 × g for 10 min at 4°C) to obtain plasma for further biochemical analyses. LPS concentration in portal plasma was determined using a kit utilizing Tachypleus amebocyte lysate (Endosafe; Charles River Laboratories International, Inc., Wilmington, MA, USA) and estimated using the kinetic turbidimetric method. Intestinal hormone levels in portal plasma [total GIP, total pancreatic polypeptide (PP) and PYY] were determined in triplicate using a rat metabolic hormone kit (cat. no. RMHMAG-84K; Merck KGaA) and Luminex technology (Bio-Plex Multiplex system; Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer's protocol. Total portal GLP-1 and GLP-2 levels were determined using ELISA kits. Plasma alanine triglycerides, cholesterol aminotransferase (ALT) and aspartate aminotransferase (AST) were determined using an automatic biochemistry analyser (HITACHI 2000; Hitachi, Ltd.).
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