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Authors: John Stack, Gary Cline
Group: Mouse Metabolic Phenotyping Centers
Summary: Procedure used to determine the concentration of magnesium in blood, serum, and plasma. Magnesium is measured as the chlorophosphonazo III complex (addition of EGTA prevents calcium interference) and monitored at 600 nm.

Proper citation: John Stack, Gary Cline 2019. Yale - Magnesium. protocols.io https://dx.doi.org/10.17504/protocols.io.y3zfyp6 Copy   

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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Streptozotocin can selectively destroy the pancreatic β-cells with rapid and irreversible necrosis and can be used to generate a chronic model of hyperglycemia and type 1 diabetes.

Proper citation: Jason Kim 2019. U Mass - STZ-induced type 1 diabetes model. protocols.io https://dx.doi.org/10.17504/protocols.io.xh8fj9w Copy   

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Authors: Oliver Fiehn
Group: Mouse Metabolic Phenotyping Centers, Metabolomics Protocols & Workflows
Summary: Summary:This SOP describes sample extraction and sample preparation for lipid profiling by liquid chromatography / quadrupole time of flight mass spectrometry (LC-QTOF) or nanoelectrospray ion trap-FTICR MS.

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

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Authors: Henri Brunengraber
Group: Mouse Metabolic Phenotyping Centers, Metabolomics Protocols & Workflows
Summary: Heavy water can be used as a tracer for estimating metabolic rates (in vivo, in vitro) such as total energy expenditure (TEE) and fractional synthesis rates (FSR; eg. protein, lipids, triglycerides). When using both ²H²O and H²18O (DLW), TEE is estimated from the total production of CO² as measured by the differences in decay rates of labeled the 18O and ²H in body water over time following a single bolus of DLW (1). ²H²O can be used to estimate fractional synthesis rates of metabolic reactions such as those associated with proteins, lipids, triglycerides, and cholesterol (2,4).References: 1. Gas chromatography-mass spectrometry assay of the (18) O enrichment of water as trimethyl phosphate. Brunengraber DZ, McCabe BJ, Katanik J, and Previs SF. Anal Biochem 306: 278– 282 (2002). 2. Increased plasma membrane cholesterol in cystic fibrosis cells correlates with CFTR genotype and depends on de novo cholesterol synthesis. Fang D, West RH, Manson ME, Ruddy J, Jiang D, Previs SF, Sonawane ND, Burgess JD, Kelley TJ.Respir Res.; 11:61 (2010). 3. Triglyceride synthesis in epididymal adipose tissue: contribution of glucose and non-glucose carbon sources. Bederman IR, Foy S, Chandramouli V, Alexander JC, Previs SF. J Biol Chem.; 284(10):6101-8 (2009). 4. Novel application of the "doubly labeled" water method: measuring CO2 production and the tissue-specific dynamics of lipid and protein in vivo. Bederman IR, Dufner DA, Alexander JC, Previs SF. Am J Physiol Endocrinol Metab.; 290(5):E1048-56 (2006).

Proper citation: Henri Brunengraber 2019. Case - Heavy Water Assays by GC-mass spectrometry. protocols.io https://dx.doi.org/10.17504/protocols.io.ydsfs6e Copy   

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Authors: John Stack, Gary Cline
Group: Mouse Metabolic Phenotyping Centers
Summary: Procedure used to determine the creatine kinase activity in blood , serum, and plasma. Creatine kinase activity is measured by the enzymatically coupled reactions of creatine kinase, hexokinase, and glucose-6-P dehydrogenase. The rate of NADPH formation is monitored by the change in absorbance at 340 nm.

Proper citation: John Stack, Gary Cline 2019. Yale - Creatine Kinase Activity. protocols.io https://dx.doi.org/10.17504/protocols.io.y3sfyne Copy   

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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 (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 - Hemoglobin A1c. protocols.io https://dx.doi.org/10.17504/protocols.io.xy3fpyn Copy   

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

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

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Authors: Louise Lantier
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary: The exercise stress test is a maximal exercise test used to calculate VO2 max. Mice are run to complete exhaustion as the treadmill speed is increased every three minutes (See table 1). During the test, direct measurements of the oxygen consumption and carbon dioxide output by the mouse are made, which are then used to calculate VO2 max, VCO2, and RER. VO2 max refers to the maximum amount of oxygen that the mouse can utilize during maximal exercise.

Proper citation: Louise Lantier 2019. Vandy - Exercise Stress Test. protocols.io https://dx.doi.org/10.17504/protocols.io.yxcfxiw 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.

Proper citation: John Rutledge 2019. UC Davis - Microvascular Permeability and Lipoprotien Flux. protocols.io https://dx.doi.org/10.17504/protocols.io.56vg9e6 Copy   

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Authors: Henri Brunengraber
Group: Mouse Metabolic Phenotyping Centers
Summary: During the course of development or during a diet study the amount of food and body weights are measured 1-2 times per week.

Proper citation: Henri Brunengraber 2019. Case - Measurement of Food Consumption and Body Weight. protocols.io https://dx.doi.org/10.17504/protocols.io.yeqftdw Copy   

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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:In-cage free running wheel is used to voluntarily induce exercise in awake mice. Exercise affects insulin sensitivity and glucose metabolism.

Proper citation: Jason Kim 2019. U Mass - Exercise model. protocols.io https://dx.doi.org/10.17504/protocols.io.xwyfpfw Copy   

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Authors: Amparo Villablanca
Group: Mouse Metabolic Phenotyping Centers
Summary: Increased lipid accumulation in the artery wall and atherosclerotic lesion development are two hallmarks of atherosclerotic cardiovascular disease. Atheroma quantification and characterization assesses neutral lipid accumulation in the vessel wall, percentage of lesions per aortic segment (quantitative) and lesion morphology, complexity, and severity (qualitative).Modified from: Villablanca AC, et.al. J Cardiovasc Transl Res. 2009 Sep;2(3):289-99.

Proper citation: Amparo Villablanca 2019. UC Davis - Atheroma Quantification. protocols.io https://dx.doi.org/10.17504/protocols.io.7bwhipe Copy   

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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: The adiponectin enzyme-linked immunosorbent assay (ELISA) is a standard immunoassay for the detection of total adiponectin levels in serum, adipocyte extracts, and cell culture media samples. This experiment uses a 96-well plate coated with anti-adiponectin antibodies and developed with a secondary anti-adiponectin antibody linked to horseradish peroxidase. The ELISA is developed with 3,3',5,5'-Tetramethylbenzidine (TMB), a chromogenic substrate and read on an automated plate spectrophotometer at 450 nm. Adiponectin is an adipocyte-derived hormone (adipokine) that regulates insulin sensitivity and glucose metabolism.

Proper citation: Jason Kim 2019. U Mass - Adiponectin. protocols.io https://dx.doi.org/10.17504/protocols.io.xm9fk96 Copy   

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Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary: Hydrogen peroxide (H2O2) is a ubiquitous, toxic, metabolic by-product of aerobic respiration, oxidative stress, and oxidative injury. Cayman's Hydrogen Peroxide Assay Kit utilizes the well established xylenol orange detection method of quantifying the oxidation of ferrous ions (Fe2+) to ferric ions (Fe3+) by hydrogen peroxide. A unique feature of Cayman's assay is the inclusion of catalase as an H2O2 scavenger for the purpose of confirming the specificity of the reaction for H2O2. The sensitivity and the specificity of the assay make it well suited to accurately measure urinary levels H2O2 in a 96 well plate format. Each kit contains hydrogen peroxide, reagent 1, reagent 2, catalase, a 96 well plate, plate cover, and complete instructions.

Proper citation: Peter Havel 2019. UC Davis - Hydrogen Peroxide. protocols.io https://dx.doi.org/10.17504/protocols.io.ypqfvmw Copy   

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Authors: Chee Lim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:The tail-cuff blood pressure measurement is a non-invasive test and the principle is the same as that of the inflatable blood pressure cuff used in the standard clinic visit. The Visitech system uses an LED light source to detect the pulse signal wave. Since the measurement is sensitive to motion artifact, the procedure requires training sessions to acclimate the mouse to the process.

Proper citation: Chee Lim 2019. Vandy - Tail-Cuff Blood Pressure. protocols.io https://dx.doi.org/10.17504/protocols.io.yymfxu6 Copy   

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Authors: Brandon Willis
Group: Mouse Metabolic Phenotyping Centers
Summary: Standard PCR conditions for genotyping of mutant mouse DNA extracted from somatic (e.g., tail) tissue.

Proper citation: Brandon Willis 2019. UC Davis - Genotyping. protocols.io https://dx.doi.org/10.17504/protocols.io.yjifuke 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 creatinine reflect systemic protein metabolism.

Proper citation: Jason Kim 2019. U Mass - Creatinine. protocols.io https://dx.doi.org/10.17504/protocols.io.xvjfn4n Copy   

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Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary: Albumin blue dye is a stain for the specific and sensitive spectrofluorometric determination of albumin in natural matrices. AB 580 binds to the albumin present in urine samples and the fluorescence can be quantified using a fluorimeter.

Proper citation: Peter Havel 2019. UC Davis - Uninary Albumin Excretion (UAE) Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.yw5fxg6 Copy   

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Authors: Jennifer Rutkowsky
Group: Mouse Metabolic Phenotyping Centers
Summary: Ionized calcium-binding adapter molecule 1 (IBA1) is specifically expressed in macrophages / microglia and is upregulated during the activation of these cells. Iba1 expression is up-regulated in microglia following nerve injury,[4] central nervous system ischemia, and several other brain diseases. Furthermore it has been found in athlesclerotic plaques and at sigts of vascular injury. Modified from: IHC Methods and Materials VMTH - Anatomic Pathology, UC-Davis

Proper citation: Jennifer Rutkowsky 2019. UC Davis - Immunohistochemistry IBA1. protocols.io https://dx.doi.org/10.17504/protocols.io.56pg9dn Copy   

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Authors: Thomas Huser
Group: Mouse Metabolic Phenotyping Centers
Summary: Raman spectroscopy analyzes non-elastically scattered light from chemical bonds (Fig. 1). Photons from a laser source are scattered by interaction with molecular bond vibrations and either deposit energy into a particular bond or receive additional energy from an already excited bond vibration. Different molecular bonds have different vibrational frequencies and lead to specific peaks in the Raman spectrum. For instance, a C-C bond can be easily distinguished from a C=C bond, allowing us to distinguish saturated from unsaturated lipids. All biomacromolecules, including DNA, RNA, proteins, and lipids, have characteristic Raman modes, which enable the nondestructive determination of their chemical composition and structure. Raman spectroscopy provides complementary information to mass spectrometry or magnetic resonance imaging (MRI) without destroying cells in the process of the analysis, with high spatial resolution akin to confocal optical microscopy, and without the need for contrast agents - enabling live imaging at the cellular level. In our LTRS system we have further increased the sensitivity of Raman spectroscopy at the cellular and sub cellular level by combining laser trapping and Raman spectroscopy. Here, a tightly focused laser beam, such as the one obtained by sending the beam through a high resolution immersion oil microscope objective, forms an optical laser trap which captures cells or lipoproteins and confines them to the laser focus until the laser is turned off or another particle collides with the one captured. Modified from: Chan JW, et.al. Anal. Chem. 2005 Sep 15;77(18):5870-76.

Proper citation: Thomas Huser 2019. UC Davis - Lipoprotein analysis by LTRS. protocols.io https://dx.doi.org/10.17504/protocols.io.56yg9fw Copy   

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