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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 dx.doi.org/10.17504/protocols.io.x4kfquw Copy   

•   Source: Protocols.io

Authors: John Stack, Gary Cline
Group: Mouse Metabolic Phenotyping Centers
Summary: Procedure used to determine the concentration of NEFA in blood, serum, and plasma. NEFA; Free fatty acids are measured in a multistep reaction to form an colored adduct of 3-methyl-Nethyl-N-(b-hydroxy-ethyl)-analine and 4-aminoantipyridine monitored at 560 nm.

Proper citation: John Stack, Gary Cline 2019. Yale - Non-Esterified Fatty Acids. protocols.io dx.doi.org/10.17504/protocols.io.y38fyrw Copy   

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Authors: Trina Knotts
Group: Mouse Metabolic Phenotyping Centers
Summary: Plasma samples will be assayed for lipopolysaccharide binding protein (LBP) as surrogate for bacterial LPS/measure of endotoxemia via ELISA.

Proper citation: Trina Knotts 2019. UC Davis - Lipoprotein Binding Protein (LBP)-Endotoxemia Assay. protocols.io dx.doi.org/10.17504/protocols.io.yrhfv36 Copy   

•   Source: Protocols.io

Authors: John Stack, Gary Cline
Group: Mouse Metabolic Phenotyping Centers
Summary: Procedure used to determine the concentration of inorganic phosphorus in blood, serum, and plasma. Phosphorus (Inorganic) is measured as the complex with molybdate and monitored at 340nm.

Proper citation: John Stack, Gary Cline 2019. Yale - Blood and Urine Inorganic Phosphorous. protocols.io dx.doi.org/10.17504/protocols.io.y2gfybw Copy   

•   Source: Protocols.io

Authors: Henri Brunengraber
Group: Mouse Metabolic Phenotyping Centers
Summary: Analysis of the citric acid cycle (CAC) and related intermediates (such as glutamate, glutamine, GABA, and aspartate) using gas chromatography mass spectrometry (GC-MS) is an analytical approach to identifying unexpected correlations between apparently related and unrelated pathways of energy metabolism. Intermediates can be as expressed as their absolute concentrations or relative ratios by using known amounts of added reference standards to the sample. GC-MS can also distinguish between heavy labelled molecules (eg. 2H- or 13C-labelled) and the naturally occurring most abundant molecules. The use of metabolic tracers (eg. 2H- or 13C-labelled) can offer additional information that can lead to the understanding of interrelationships between the pools of intermediary metabolism, such as with the CAC, and specific metabolic pathways. Applications using tracers can also assess the turnover of specific metabolic pools under various physiological and pathological conditions. The following SOP presents a relatively simple method that is sensitive for simultaneously measuring concentrations and heavy labelled molecules of CAC intermediates (relative and absolute) and other related intermediates of energy metabolism using GC-MS technology. Note: the following extraction procedure can also be used parallel analysis for acyl-CoA’s and lipids (for details on isolating and mass spectrometry assay procedures see, refs 4-6).References: 1. Yang, L., Kasumov, T., Kombu, R. S., Zhu, S. H., Cendrowski, A. V., David, F., Anderson, V. E., Kelleher, J. K., and Brunengraber, H. Metabolomic and mass isotopomer analysis of liver gluconeogenesis and citric acid cycle: II. Heterogeneity of metabolite labeling pattern. J. Biol. Chem. (2008) 283, 21988-21996 2. Yang, L., Kombu, R. S., Kasumov, T., Zhu, S. H., Cendrowski, A. V., David, F., Anderson, V. E., Kelleher, J. K., and Brunengraber, H. Metabolomic and mass isotopomer analysis of liver gluconeogenesis and citric acid cycle. I. Interrelation between gluconeogenesis and cataplerosis; formation of methoxamates from aminooxyacetate and ketoacids. J. Biol. Chem. (2008) 283, 21978-21987 3. Kombu RS, Brunengraber H, and Puchowicz MA. Analysis of the Citric Acid Cycle Intermediates using Gas Chromatography-Mass Spectrometry In: Methods Molec Biol, “Metabolic Profiling: Methods and Protocols” (ed. Metz,TO), Humana Press, (2011) 708:14757 4. Zhang Y, Zhang S, Marin-Valencia I, Puchowicz MA. Decreased Carbon Shunting From Glucose Towards Oxidative Metabolism In Diet-Induced Ketotic Rat Brain. J Neurochem. (2015) 132(3): 301-12 5. Harris SR, Zhang GF, Sadhukhan S, Wang H, Shi C, Puchowicz MA, Anderson VE, Salomon RG, Tochtrop GP, Brunengraber H. Metabolomics and Mass Isotopomer Analysis as a Strategy for Pathway Discovery: Pyrrolyl and Cyclopentenyl Derivatives of the Pro-Drug of Abuse, Levulinate. Chem Res Toxicol. (2012) 26(2): 213-20 6. Zhang, G. F., Kombu, R. S., Kasumov, T., Han, Y., Sadhukhan, S., Zhang, J., Sayre, L. M., Ray, D., Gibson, K. M., Anderson, V. A., Tochtrop, G. P., and Brunengraber, H. Catabolism of 4-hydroxyacids and 4-hydroxynonenal via 4-hydroxy-4-phosphoacyl-CoAs. J. Biol. Chem. (2009) 284, 33521-33534

Proper citation: Henri Brunengraber 2019. Case - Citric Acid Cycle and Related Intermediates. protocols.io dx.doi.org/10.17504/protocols.io.ydbfs2n Copy   

•   Source: Protocols.io

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 dx.doi.org/10.17504/protocols.io.yv8fw9w Copy   

•   Source: Protocols.io

Authors: Trina Knotts
Group: Mouse Metabolic Phenotyping Centers
Summary: Rapid and accurate measures of fat and lean mass will be carried out in isoflurane-anesthetized mice, using a PixiMus dual energy X-ray absorptiometry (DEXA) device. This approach is especially attractive when it is desirable to utilize the same mice for several phenotypic measurements (e.g., when relatively few mice of a particular genotype are available), and measurements may be taken within ~5 min/mouse. Total bone mineral density (BMD, in gm/cm²) and bone mineral content (BMC, in.mg.) values are obtained automatically from the DEXA scan and will be provided to MMPC clients as part of the dataset. Regional determinations of BMD and BMC can also be provided for a nominal data processing fee by utilizing the region-of-interest (ROI) capability of the PixiMus software. When terminal studies are conducted, whole-animal DEXA data can be complemented by determinations of adiposity (catalog item D4002) by dissection and measurement of adipose depot-specific weights (summed weights of gonadal, retroperitoneal, and femoral subcutaneous fat depots divided by live weight.)

Proper citation: Trina Knotts 2019. UC Davis - Gross Body Composition (DEXA). protocols.io dx.doi.org/10.17504/protocols.io.68ahhse Copy   

•   Source: Protocols.io

Authors: Malcolm Low
Group: Mouse Metabolic Phenotyping Centers

Proper citation: Malcolm Low 2019. U Michigan - Cryoembedding. protocols.io dx.doi.org/10.17504/protocols.io.xidfka6 Copy   

•   Source: Protocols.io

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 dx.doi.org/10.17504/protocols.io.xucfnsw Copy   

•   Source: Protocols.io

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 dx.doi.org/10.17504/protocols.io.yrrfv56 Copy   

•   Source: Protocols.io

Authors: John Rutledge
Group: Mouse Metabolic Phenotyping Centers
Summary: SummaryUtilizing the TA-F10 (DSI) telemeter we can simultaneously collect temperature and activity measures in a single mouse.  This can provide a comprehensive assessment of circadian patterns, activity levels, and core body temperature in multiple interventions (drug, altered fed access, etc.) or genetic mouse models.  The following protocol describes the implantation and measurement parameters and intervals.Modified from: Butz et al. Physiol Genomics. 2001 Mar 8;5(2):89-97.

Proper citation: John Rutledge 2019. UC Davis - Temperature and activity by Telemetry. protocols.io dx.doi.org/10.17504/protocols.io.ywtfxen Copy   

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Authors: Louise Lantier
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:The minispec Body Composition Analyzer is based on Time Domain NMR. It acquires and analyzes TD-NMR signals from all protons in the entire sample volume and can provide 3 components of interest: Fat, Free Body Fluid and Lean Tissue values. Bruker's minispec Body Composition Analyzer provides a precise method for measurement of lean tissue, fat and fluid in living mice. TD-NMR uses NMR technology, providing analysis of fat tissue, lean tissue and free fluid by the same physical selection rules that give rise to contrast in MRI. Tissue contrast is high between fat and muscle based on relative relaxation times. This Magnetic Resonance technology provides our users with the opportunity to make multiple measurements during the life of the animal with the following benefits: ♦ Rapid analysis: measurement takes less than 2 minutes, no sample preparation ♦ Economical procedures: no consumables, retain expensive lab animals for entire study ♦ Reduced animal stress: no need for anesthetics; animals are measured "as-is" ♦ Allows more frequent testing, due to reduction of risks to animal health ♦ Better accuracy and precision compared to DEXA (X-ray) method

Proper citation: Louise Lantier 2019. Vandy – Body Composition in conscious mice. protocols.io dx.doi.org/10.17504/protocols.io.yypfxvn Copy   

•   Source: Protocols.io

Authors: Louise Lantier
Group: Mouse Metabolic Phenotyping Centers
Summary: After the last sample is taken for the clamp, the mouse is anesthetized in order to harvest tissues. It is very important that the mouse remain alive during tissue harvesting for future signaling or metabolite analysis. The mouse is therefore given a sub-lethal dose of pentobarbital to induce anesthesia (~70mg/kg pi or 35mg/kg iv).

Proper citation: Louise Lantier 2019. Vandy – Post Clamp Anesthesia. protocols.io dx.doi.org/10.17504/protocols.io.zdnf25e Copy   

•   Source: Protocols.io

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 dx.doi.org/10.17504/protocols.io.56wg9fe Copy   

•   Source: Protocols.io

Authors: David A. Antonetti
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:Capture nucleosomes from the cytoplasm of apoptotic cells on a micro titer plate and detect the associated histones with HRP-conjugated antibody. Designed for use with cell culture but adapted for use with homogenized mouse retinas.

Proper citation: David A. Antonetti 2019. U Michigan - Retinal Cell Death. protocols.io dx.doi.org/10.17504/protocols.io.yaafsae Copy   

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Authors: K.C. Kent Lloyd
Group: Mouse Metabolic Phenotyping Centers
Summary: The purpose of this document is to describe the procedure for accepting commercial vendor and courier-delivered (“Imports”) shipments of mice to the 2nd Street facility of the UC Davis Mouse Biology Program. This procedure is to be followed by administrative personnel responsible for accepting commercial or courier-delivered shipments of mice and by Vivarium personnel responsible for unpacking and caging mice. Transportation of mice is stressful and potentially dangerous to their health, especially in warm summer months. Mice need to be unpacked, have a health inspection, and housed in appropriate caging in appropriate numbers as soon as possible after arrival. This insures they are acclimated and in the best physical health prior to their use by the Mouse Biology Program.

Proper citation: K.C. Kent Lloyd 2019. UC Davis - Receipt of Vendor and Courier Mice. protocols.io dx.doi.org/10.17504/protocols.io.xh4fj8w Copy   

•   Source: Protocols.io

Authors: Vincent Young
Group: Mouse Metabolic Phenotyping Centers
Summary: This protocol is for the submission of tissue/fecal samples for DNA extraction and subsequent processing to generate libraries for 16S rRNA sequencing, which can be used for bacterial community analysis and detect variations in the microbiota under differing conditions. Up to 250 uL or 0.25 g of sample is added to each well of the Bead plate provided by the MMPC. Plates are properly packaged and shipped to the MMPC and processed using the Qiagen MagAttract PowerMicrobiome kit DNA/RNA kit (Qiagen, catalog no. 27500-4-EP) on the EpMotion 5075 (Eppendorf) liquid handler. DNA is lysed using mechanical bead beating and extracted using magnetic bead technology. Extracted DNA is then used to generate 16S rRNA libraries for community analysis. The process used for library generation has been previously described by Seekatz et al. (1). Briefly, barcoded dual-index primers specific to the V4 region of the 16S rRNA gene amplify the DNA (2). PCR reactions are composed of 5 µL of 4 µM equimolar primer set, 0.15 µL of AccuPrime Taq DNA High Fidelity Polymerase, 2 µL of 10x AccuPrime PCR Buffer II (Thermo Fisher Scientific, catalog no. 12346094), 11.85 µL of PCR-grade water, and 1 µL of DNA template. The PCR conditions used consisted of 2 min at 95°C, followed by 30 cycles of 95°C for 20 s, 55°C for 15 s, and 72°C for 5 min, followed by 72°C for 10 min. Each PCR reaction is normalized using the SequalPrep Normalization Plate Kit (Thermo Fisher Scientific, catalog no. A1051001). The normalized reactions are pooled and quantified using the Kapa Biosystems Library qPCR MasterMix (ROX Low) Quantification kit for Illumina platforms (catalog no. KK4873). The Agilent Bioanalyzer is used to confirm the size of the amplicon library (~399 bp) using a high-sensitive DNA analysis kit (catalog no. 5067-4626). Pooled amplicon library is then sequenced on the Illumina MiSeq platform using the 500 cycle MiSeq V2 Reagent kit (catalog no. MS-102-2003) according to the manufacturer's instructions with modifications of the primer set with custom read 1/read 2 and index primers added to the reagent cartridge. The “Preparing Libraries for Sequencing on the MiSeq” (part 15039740, Rev. D) protocol was used to prepare libraries with a final load concentration of 5.5 pM, spiked with 15% PhiX to create diversity within the run. FASTQ files are distributed to the client when the 2 x 250 bp sequencing completes.References:1. Seekatz AM, Theriot CM, Molloy CT, Wozniak KL, Bergin IL, Young VB. 2015. Fecal Microbiota Transplantation Eliminates Clostridium difficile in a Murine Model of Relapsing Disease. Infect Immun 83:3838-3846. 10.1128/IAI.00459-15.2. Kozich JJ, Westcott SL, Baxter NT, Highlander SK, Schloss PD. 2013. Development of a dual- index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Appl Environ Microbiol 79:5112–5120. 10.1128/AEM.01043-13.

Proper citation: Vincent Young 2019. U Michigan - DNA Extraction for Illumina 16S rRNA Extraction. protocols.io dx.doi.org/10.17504/protocols.io.7kxhkxn Copy   

•   Source: Protocols.io

Authors: Fawaz G. Haj
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary: This test is designated to determine if rodents exhibit inflammatory disorders, through evaluation of the activation state of key proteins such as JNK and p38 MAP kinases. We will examine the inflammatory state of adipose and liver tissues.

Proper citation: Fawaz G. Haj 2019. UC Davis - Inflammation pathway. protocols.io dx.doi.org/10.17504/protocols.io.ypyfvpw Copy   

•   Source: Protocols.io