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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: 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 (LPS- induced CXC chemokine), MCP-1 (monocyte chemotactic protein-1; CCL-2), M-CSF (macrophage colony stimulating factor), MIG (monokine induced by gamma interferon; CXCL- 9), MIP-1α (macrophage inflammatory protein alpha; CCL-3), MIP-1 (macrophage inflammatory protein beta; CCL-4), and MIP-2/RANTES (macrophage inflammatory protein 2- alpha; 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 - Cytokines Panel I - multiplex. protocols.io https://dx.doi.org/10.17504/protocols.io.xvnfn5e Copy   

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Authors: Patrick Tso, Dana Lee
Group: Mouse Metabolic Phenotyping Centers
Summary: Quantitative determinations of non-esterified fatty acids in plasma/serum/lymph will be made using the NEFA-HR enzymatic colorimetric method assay.

Proper citation: Patrick Tso, Dana Lee 2019. U Cinn - NEFA Concentration. protocols.io https://dx.doi.org/10.17504/protocols.io.xmnfk5e Copy   

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Authors: Mark Kelly, Timothy P. Fitzgibbons
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary: This is a Mouse model of Transverse aortic constriction (TAC). A commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart failure. Initially leading to compensated hypertrophy of the heart, which often is associated with a temporary enhancement of cardiac contractility. Resulting in cardiac dilatation and heart failure.

Proper citation: Mark Kelly, Timothy P. Fitzgibbons 2019. U Mass - Transverse Aortic Constriction. protocols.io https://dx.doi.org/10.17504/protocols.io.56ig9ce Copy   

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Authors: John Stack, Gary Cline
Group: Mouse Metabolic Phenotyping Centers
Summary: Procedure for determining the glycogen content within liver and muscle tissue.

Proper citation: John Stack, Gary Cline 2019. Yale - Tissue Glycogen. protocols.io https://dx.doi.org/10.17504/protocols.io.y39fyr6 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. Albumin is a major circulating protein in body.

Proper citation: Jason Kim 2019. U Mass - Albumin. protocols.io https://dx.doi.org/10.17504/protocols.io.xprfmm6 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 alkaline phosphatase reflect liver function.

Proper citation: Jason Kim 2019. U Mass - Alkaline Phosphatase. protocols.io https://dx.doi.org/10.17504/protocols.io.xptfmnn Copy   

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Authors: Oliver Fiehn
Group: Mouse Metabolic Phenotyping Centers, Metabolomics Protocols & Workflows
Summary: This SOP describes sample extraction and sample preparation for primary metabolism profiling by gas chromatography / time of flight mass spectrometry (GCTOF)References: Fiehn O, Kind T (2006) Metabolite profiling in blood plasma. In: Metabolomics: Methods and Protocols. Weckwerth W (ed.), Humana Press, Totowa NJ

Proper citation: Oliver Fiehn 2019. UC Davis - Metabolomics: Sample preparation for GCTOF analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.ytjfwkn Copy   

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Authors: Trina Knotts
Group: Mouse Metabolic Phenotyping Centers
Summary: Changes in fecal energy loss can result in differences in net energy balance and feed efficiency, and may result from, e.g., alterations in bile acid production, secretion or re-uptake, and changes in activities of enzymes involved in macronutrient digestion or uptake (e.g., pancreatic lipase). This assay will be coupled to food intake determinations and will involve fecal collection and feces weight measures for 48h, which can be coordinated with the meal pattern studies (catalog item D4003) or indirect calorimetry (catalog item D4007). Diet and fecal energy content will be measured by bomb calorimetry and the following reported: fecal energy density, total fecal energy loss/day, and fecal energy loss as a percent of energy intake. Alternatively, fecal samples and diet samples can be examined without food intake measurements. In this case, only diet and fecal energy density will be reported. Additional tests can be done through proximate analysis (D4005- Digestible Energy by Proximate Analysis) to determine protein or fat absorption.

Proper citation: Trina Knotts 2019. UC Davis - Digestible Energy by Bomb Calorimetry. protocols.io https://dx.doi.org/10.17504/protocols.io.ygkftuw Copy   

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Authors: Kent Lloyd
Group: Mouse Metabolic Phenotyping Centers
Summary: This is a generic protocol for making genetically-altered mouse models for research. Investigators who do not have a mutant mouse to send for testing at the MMPC @ UC Davis can ask for the mutant mouse to be derived by the MMPC @ UC Davis, through the UC Davis Mouse Biology Program. Genetically altered mice can be made on most genetic backgrounds, such as C57BL/6, FVB/N, B6D2F1 and other hybrids, and others. Transgenic (random genomic integration of expression-competent transgenes) and gene targeted (specific insertion and/or replacement of an endogenous genetic locus) mutant mice can generated. “Knockout” mice can be made so as to delete endogenous gene expression ubiquitously and conditionally in either a specific cell or tissue type (spatial conditional) and/or at a specific age (temporal conditional). Numerous technologies are available for the derivation of mutant mice, including molecular construct design, gene targeted in embryonic stem (ES) cells, pronuclear microinjection of transgenic constructs into zygotes, ES cell microinjection into blastocysts, genome editing in ES cells and zygotes (e.g., TALENs, CRISPR/Cas9). Mutant mice can be genotype confirmed (see MMPC item D2004) and custom bred to generate cohorts of male and/or female mice for phenotyping at the MMPC @ UC Davis.

Proper citation: Kent Lloyd 2019. UC Davis - Mouse Model Creation. protocols.io https://dx.doi.org/10.17504/protocols.io.56gg9bw Copy   

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Authors: John Stack, Gary Cline
Group: Mouse Metabolic Phenotyping Centers
Summary: Procedure used to determine the concentration of triglycerides in blood, serum, and plasma. Triglycerides are determined by coupling lipase, glucokinase, glycerol phosphate oxidase, and peroxidase to form a quinonemine dye which is measured at 500 nm.

Proper citation: John Stack, Gary Cline 2019. Yale - Triglycerides. protocols.io https://dx.doi.org/10.17504/protocols.io.y4bfysn Copy   

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Authors: Mark Kelly LAT, Timothy P. Fitzgibbons
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary: This is a mouse model of acute myocardial infarction. This protocol is suitable for studying mouse models thought to be relevant to coronary ischemia or the development of congestive heart failure.

Proper citation: Mark Kelly LAT, Timothy P. Fitzgibbons 2019. U Mass - Coronary Artery Ligation. protocols.io https://dx.doi.org/10.17504/protocols.io.56kg9cw Copy   

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Authors: Li Kang
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:Mice with catheters implanted in the jugular vein (infusions) and carotid artery (sampling) are used for this procedure (V3002). The hyperinsulinemic, euglycemic clamp or “insulin clamp” has been used in a variety of species to assess insulin action. In an insulin clamp, the rate of glucose infused to maintain euglycemia is an index of whole body insulin sensitivity. Isotopes can be used during an insulin clamp to distinguish between insulin’s effects on endogenous glucose production and glucose utilization, or to examine insulin’s effects on specific tissues and metabolic pathways. The insulin infusion rate used in a study depends on a number of factors such as whether the experiment requires a sensitive analysis of endogenous glucose production, or whether the mouse is known to be unusually insulin resistant (e.g. ob/ob). Finally the insulin clamp technique, especially when isotopes are involved, is most quantitative under steady state conditions. Therefore, clamps should be of a duration (~2 h) that is sufficient for the actions of insulin to obtain steady state. The section that follows describes, a) the technical procedures used on the day of a clamp experiment; b) preparation of insulin and tracer infusates; and c) analyses of plasma and tissue radioactivity. Familiarity with these procedures combined with laboratory demonstration provides the investigator with the necessary background to clamp the conscious mouse.

Proper citation: Li Kang 2019. Vandy - Hyperinsulinemic-euglycemic Clamp. protocols.io https://dx.doi.org/10.17504/protocols.io.yyafxse Copy   

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Authors: K.C. Kent Lloyd
Group: Mouse Metabolic Phenotyping Centers
Summary: SummaryThe purpose of this standard operating procedure (SOP) is to provide steps for the acquisition, acclimation and quarantine of animals obtained from other institutions or laboratories and introduced to the MBP Vivarium with minimal risk to the current mouse colony population. This procedure applies to the import/export staff, veterinary staff and any principal investigator requesting animals.

Proper citation: K.C. Kent Lloyd 2019. UC Davis - Acquisition/Acclimation/Quarantine. protocols.io https://dx.doi.org/10.17504/protocols.io.xfifjke Copy   

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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: The EchoMRI 3-in-1 uses ¹H- magnetic resonance spectroscopy to noninvasively measure fat mass, lean mass, and water mass in individual organs. Fat, lean, and water mass composition in specific organs is altered in obesity.

Proper citation: Jason Kim 2019. U Mass - Body composition (organs). protocols.io https://dx.doi.org/10.17504/protocols.io.xsnfnde Copy   

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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer.

Proper citation: Jason Kim 2019. U Mass - Ammonia. protocols.io https://dx.doi.org/10.17504/protocols.io.xq3fmyn Copy   

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Authors: Patrick Tso, Dana Lee
Group: Mouse Metabolic Phenotyping Centers
Summary: Determinations of phospholipids in plasma/serum/lymph will be made using the Wako Phospholipids C enzymatic assay. In this assay, phospholipids in the sample are hydrolyzed ultimately producing a blue pigment. The amount of phospholipids in the sample is determined by measuring the absorbance of the blue color.

Proper citation: Patrick Tso, Dana Lee 2019. U Cinn - Phospholipids Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.xmvfk66 Copy   

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Authors: John Stack, Gary Cline
Group: Mouse Metabolic Phenotyping Centers
Summary: Procedure used to determine the concentration of LDL cholesterol in blood, serum, and plasma. LDL-cholesterol is determined by an elimination method to selectively remove HDL, VLDL, and IDL subfractions prior to assay of LDL-cholesterol as described for total cholesterol.

Proper citation: John Stack, Gary Cline 2019. Yale - LDL Cholesterol. protocols.io https://dx.doi.org/10.17504/protocols.io.y3vfyn6 Copy   

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Authors: Jeff Hodgin, Jharna Saha
Group: Mouse Metabolic Phenotyping Centers
Summary: Periodic acid-Schiff (PAS) is a staining method used to detect glycogen on formalin-fixed, paraffin-embedded kidney tissue sections. PAS staining highlights basement membranes and is frequently used to diagnose glomerular mesangial matrix expansion.

Proper citation: Jeff Hodgin, Jharna Saha 2019. U Michigan - Mesangial matrix evalutaion. protocols.io https://dx.doi.org/10.17504/protocols.io.562g9ge Copy   

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Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: When a sample is mixed with R1, AcAc in the sample is broken down to acetone by AADC. Upon addition of R2, 3-HB in the sample is oxidized in the presence of 3-HBDH and Thio-NAD. This oxidation triggers the cyclic reactions. Since the original AcAc in the sample has been removed, only 3-HB is assayed by measureing the rate of Thio-NADH production spectrophotometrically.

Proper citation: Peter Havel 2019. UC Davis - ß hydroxy butyrate Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.ywafxae Copy   

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Authors: Lynette Bower
Group: Mouse Metabolic Phenotyping Centers
Summary: Utilizing this telemetry we have the ability to simultaneously measure a pressure signal and biopotential signal (ECG, EEG, EMG) in a single mouse. This can provide a more comprehensive physiological assessment of cardiovascular function, the presence of any damage to the heart, the effects of drugs, specific genes or interventions used to regulate the heart.Modified from: Butz et al. Physiol Genomics. 2001 Mar 8;5(2):89-97.

Proper citation: Lynette Bower 2019. UC Davis - Blood Pressure/Heart Rate by Telemetry . protocols.io https://dx.doi.org/10.17504/protocols.io.yevfte6 Copy   

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