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Authors: K.C. Kent Lloyd
Group: Mouse Metabolic Phenotyping Centers
Summary: Basic Adipocyte Culture Protocol

Proper citation: K.C. Kent Lloyd 2019. UC Davis - Basic Adipocyte Culture Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.7bvhin6 Copy   

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Authors: Gary Cline, John Stack
Group: Mouse Metabolic Phenotyping Centers
Summary: Procedure used to determine the concentration of albumin in blood, plasma, and serum. Albumin is measured as its conjugate with bromocresol green monitored at 600 nm.

Proper citation: Gary Cline, John Stack 2019. Yale - Blood Albumin. protocols.io https://dx.doi.org/10.17504/protocols.io.y2cfyaw Copy   

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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: This experiment provides the quantification of multiple hormones using multiplexed-Luminex technology based on beads containing specific antibodies. Sample matrices include plasma, serum, tissue culture supernatants, and tissue or cell lysates. Resistin is an adipocyte-derived hormone (adipokine) that regulates insulin sensitivity and glucose metabolism. Serum resistin levels are altered in obesity, insulin resistance, and type 2 diabetes.

Proper citation: Jason Kim 2019. U Mass - Resistin. protocols.io https://dx.doi.org/10.17504/protocols.io.x4kfquw Copy   

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Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary: Glutathione reductase (GR, EC 1.6.4.2) is a flavoprotein that catalyzes the NADPH- dependent reduction of oxidized glutathione (GSSG) to glutathione (GSH). This enzyme is essential for the GSH redox cycle which maintains adequate levels of reduced cellular GSH. A high GSH/GSSG ratio is essential for protection against oxidative stress. The Cayman Chemical Glutathione Reductase Assay Kit measures GR activity by measuring the rate of NADPH oxidation. The oxidation of NADPH to NADP+ is accompanied by a decrease in absorbance at 340 nm. Since GR is present at rate limiting concentrations, the rate of decrease in the A340 is directly proportional to the GR activity in the sample. The Cayman GR Assay Kit is provided in a convenient 96 well plate format and can be used to measure GR activity inplasma, erythrocyte lysates, tissue homogenates, and cell lysates.

Proper citation: Peter Havel 2019. UC Davis - Glutathione Reductase. protocols.io https://dx.doi.org/10.17504/protocols.io.68ehhte Copy   

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Authors: Henri Brunengraber
Group: Mouse Metabolic Phenotyping Centers
Summary: This is the standard protocol for most routine glucose tolerance testing. It is performed on awake mice, fasted for 18 hr (overnight) or 6 hours.

Proper citation: Henri Brunengraber 2019. Case - Intraperitoneal Glucose Tolerance Test. protocols.io https://dx.doi.org/10.17504/protocols.io.ydtfs6n Copy   

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Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: The RIA (RadioImmuno Assay) is an assay method used for the quantification of various proteins. A standardized concentration of antibody specific to the analyte of interest is pipetted into test tubes. A standardized concentration of the analyte of interest that is labeled with a radioisotope (usually 125I) is added to the tubes. Standards and samples are pipetted into the tubes and the tubes are incubated. During the incubation period the standardized concentration of labeled analyte and the unknown concentration of analyte in the samples will compete for binding sites on the antibody. After the incubation period a precipitating reagent is added to the tubes and the bound antibodies are precipitated out using the double antibody-polyethyleneglycol precipitation technique. The tubes are centrifuged and the supernatant is aspirated and the precipitate is counted (using a gamma counter if 125I is used). If there is a low concentration of the analyte of interest in the samples, the labeled analyte will have a higher probability for binding to the antibody and thus there will be a higher count in the precipitate. If there is a high concentration of the analyte of interest in the samples there will be a lower count of labeled analyte in the precipitate. The counts and known concentrations of the standards are used to generate a standard curve, and the counts of the samples are used to interpolate quantitative concentrations for the analyte of interest from the standard curve.

Proper citation: Peter Havel 2019. UC Davis - RadioImmuno Assay (RIA) Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.yv8fw9w Copy   

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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer. Serum levels of total cholesterol reflect cholesterol metabolism and are associated with cardiovascular disease.

Proper citation: Jason Kim 2019. U Mass - Cholesterol (Total). protocols.io https://dx.doi.org/10.17504/protocols.io.xucfnsw Copy   

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Authors: John Rutledge
Group: Mouse Metabolic Phenotyping Centers
Summary: One of the three indices of arterial function that are compromised to a varying degree in individuals with cardiovascular disease is vascular permeability. This assay measures vascular permeability (as flux of labeled large molecular weight molecules: i.e. albumin or dextran) and lipid permeability (as flux of labeled lipid) in coronary or carotid arteries.Modified from: Walsh et. al. Arterioscler Thromb Vasc Biol. 1999 Apr;19(4):840-6.

Proper citation: John Rutledge 2019. UC Davis - Macrovascular Permeability and Lipoprotein Flux. protocols.io https://dx.doi.org/10.17504/protocols.io.yrrfv56 Copy   

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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer. Serum levels of aspartate transferase reflect liver function.

Proper citation: Jason Kim 2019. U Mass - Aspartate Transferase. protocols.io https://dx.doi.org/10.17504/protocols.io.xr5fm86 Copy   

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Authors: Lori Haapanen
Group: Mouse Metabolic Phenotyping Centers
Summary: This core will provide services for quantification and comparison in animal models for hormone measurements; kidney function analytes; lipid metabolism analytes; liver function analytes; miscellaneous protein analytes; and immune function including cytokines/chemokines. The Luminex assay enables the quantification of all or any combination of analytes for which there are assay beads in tissue/cell lysate, tissue culture supernatant samples and plasma/serum. For example, cytokines and chemokines are peptides, which act as regulators in normal and pathological conditions, effecting interactions between cells as well as regulating processes occurring in the extracellular environment.

Proper citation: Lori Haapanen 2019. UC Davis - Luminex/Multiplex. protocols.io https://dx.doi.org/10.17504/protocols.io.56wg9fe Copy   

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Authors: Gary Cline, John Stack
Group: Mouse Metabolic Phenotyping Centers
Summary: Procedure followed to detect the concentration of creatinine in serum, plasma, and urine.

Proper citation: Gary Cline, John Stack 2019. Yale - Blood and Urine Creatinine. protocols.io https://dx.doi.org/10.17504/protocols.io.y2dfya6 Copy   

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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer. Serum levels of LDL-cholesterol reflect cholesterol metabolism and are associated with cardiovascular disease.

Proper citation: Jason Kim 2019. U Mass - Cholesterol (LDL). protocols.io https://dx.doi.org/10.17504/protocols.io.xt9fnr6 Copy   

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Authors: Mari Golub
Group: Mouse Metabolic Phenotyping Centers
Summary: The y-maze is a modification of the T- maze which evaluates memory and special learning in rodents though quantification of spontaneous alternation (a measure of spatial cognition). This measure has previously been shown to be affected in mouse models of Alzheimer’s. References: Ohno et al. Neuron 41:27-33, 2004. Oakley et al. J Neuroscience 26(40):10129-10140, 2006. Ohno et al. Neurobiol Dis 26:134-145, 2007. Holcomb et al. Nat Med 4:97-100, 1998.

Proper citation: Mari Golub 2019. UC Davis - Y-Maze. protocols.io https://dx.doi.org/10.17504/protocols.io.yw7fxhn Copy   

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Authors: Kristin Evans
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary: Vertical sleeve Gastrectomy (VSG) is a common surgical treatment of obesity, type-2 diabetes, and other comorbidities of obesity. The VSG mouse surgical model provides researchers with a mechanism to evaluate mechanistic studies to explore the methods by which VSG bariatric surgery produces beneficial effects on obesity. The surgical procedure is performed under sterile conditions in accordance with AAALAC standards. Surgical animals will be able to ship 2 weeks after the post-operative monitoring period is completed. Cohorts of Vertical Sleeve gastrectomy (VSG), a pair fed SHAM, and an ad lib SHAM will be provided with each cohort ordered.

Proper citation: Kristin Evans 2019. UC Davis - Vertical Sleeve Gastrectomy. protocols.io https://dx.doi.org/10.17504/protocols.io.za3f2gn Copy   

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Authors: David A. Antonetti
Group: Mouse Metabolic Phenotyping Centers
Summary: A virtual optometry system is used to quantify the spatial vision of laboratory animal.

Proper citation: David A. Antonetti 2019. U Michigan - Optokinetic Measurements of Visual Acuity and Contrast Sensitivity. protocols.io https://dx.doi.org/10.17504/protocols.io.x99fr96 Copy   

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Authors: Trina Knotts
Group: Mouse Metabolic Phenotyping Centers
Summary: The work of Gordon and colleagues (i.e., Nature. 2006 Dec 21;444(7122):1027-31) has shown that obesity can result in marked shifts in the gut microbiome in mice and other models including humans. While the role of the microbiome remains to be fully elucidated, the gut microbiota can no longer be ignored as a potentially important factor when assessing metabolic phenotype. This service involves 16S gene variable region (V1-V3 or V3-V4 directed primers) sequencing by Titanium 454 (10,000 avg seq read depth) of feces, cecal, or other GI contents. Sequences can be processed through a bioinformatics pipeline (Qiime) to taxonomically classify them and to assess alpha and beta diversity of the community. In addition, Principal Components Analysis (PCA) or partial least squares- discriminant analysis (PLS-DA) can leverage variances in the relative microbial abundances to better understand how specific microbes contribute to separation by group. Correlational analyses can identify which variables of host metadata associate with specific microbes. The Core’s gut microbiome assay will employ this approach to uncover unique microbiota fingerprints in test mice. One caveat is that with fecal samples, patterns are only a surrogate for actual gut microbiota patterns, and may not exactly reflect the intestinal populations.

Proper citation: Trina Knotts 2019. UC Davis - Gut Microbiome Analysis (454-10K reads). protocols.io https://dx.doi.org/10.17504/protocols.io.ykjfuun Copy   

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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:Hepatic gluconeogensis is estimated using pyruvate tolerance test that measures systemic elevation of glucose partly derived from pyruvate and hepatic gluconeogensis following an intraperitoneal bolus injection of pyruvate in awake mice. Hepatic gluconeogenesis is affected by obesity and regulates glucose homeostasis.

Proper citation: Jason Kim 2019. U Mass - Hepatic gluconeogenesis. protocols.io https://dx.doi.org/10.17504/protocols.io.xy7fpzn Copy   

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Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary: The lipid extraction method is based on work by Folch 1957. Tissue is homogenized and put into a 2:1 chloroform and methanol mix. The mix is separated into two phases by adding a salt solution. The upper phase contains any non-lipid substances and the lower phase contains the chloroform with the lipids. The upper phase is removed and a sample is taken from the bottom phase and evaporated. The sample is reconstituted with 2-propanol with a volume to bring it to the desired concentration to assay. Triglycerides are enzymatically hydrolyzed by lipase to free fatty acids and glycerol. The glycerol is phosphorylated by adenosine triphosphate (ATP) with glycerol kinase (GK) to produce glycerol-3- phosphate and adenosine diphosphate. Glycerol-3-phosphate is oxidized by dihydroxyacetone phosphate (DAP) by glycerolphosphate oxidase producing hydrogen peroxide (H²O²). In a Trinder5 type color reaction catalyzed by peroxidase, the H²O² reacts with 4-aminoantipyrine (4-AAP) and 3,5-dichloro-2- hydroxybenzene sulfonate (DHBS) to produce a red colored dye. The absorbance of this dye is proportional to the concentration of triglycerides present in the sample. Cholesterol esters are enzymatically hydrolysed by cholesterol esterase to cholesterol and free fatty acids. Free cholesterol, including that originally present, is then oxidized by cholesterol oxidase to cholest-4-en-3- one and hydrogen peroxide. The hydrogen peroxide combines with HBA and 4-aminoantipyrine to form a chromophore (quinoneimine dye) which may be quantitated at 500-550nm.

Proper citation: Peter Havel 2019. UC Davis - Tissue TG & TC Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.62thgen Copy   

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Authors: Kristin Evans
Group: Mouse Metabolic Phenotyping Centers
Summary: SummaryA high-fat diet of varying composition and percent fat is administered to induce obesity in mice. High-fat diet induced obesity is causally associated with insulin resistance and type 2 diabetes. Diets will be provided in consultation with the investigator needs. This service applies to specialized feeding for vendor supplied animals being supplied out of the UC Davis vendor approved barrier facility.

Proper citation: Kristin Evans 2019. UC Davis - High fat diet feeding. protocols.io https://dx.doi.org/10.17504/protocols.io.xfmfjk6 Copy   

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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: This experiment provides the quantification of multiple cytokines and chemokines using multiplexed-Luminex technology based on beads containing specific antibodies. Serum cytokine levels reflect chronic or acute inflammation, and circulating cytokines and chemokines are altered in obesity. Cytokines Panel I include IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, KC (IL-8 homologue), IL-9, IL-10 (interleukin-10), IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, TNFα (tumor necrosis factor alpha), IFNγ (interferon gamma), IP-10 (interferon gamma-induced protein 10; CXCL-10), Eotaxin (CCL-11), G-CSF (granulocyte colony stimulating factor), GM-CSF (granulocyte macrophage colony stimulating factor), LIF (leukemia inhibitory factor), LIX (LPSinduced CXC chemokine), MCP-1 (monocyte chemotactic protein-1; CCL-2), M-CSF (macrophage colony stimulating factor), MIG (monokine induced by gamma interferon; CXCL9), MIP-1α (macrophage inflammatory protein alpha; CCL-3), MIP-1β (macrophage inflammatory protein beta; CCL-4), and MIP-2/RANTES (macrophage inflammatory protein 2alpha; CXCL-2). A service can be requested for all or any combination of listed cytokines/chemokines for customized multiplexed Luminex assay.

Proper citation: Jason Kim 2019. U Mass - Gamma-glutamyl Transferase. protocols.io https://dx.doi.org/10.17504/protocols.io.xwzfpf6 Copy   

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