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

•   Source: Protocols.io

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 dx.doi.org/10.17504/protocols.io.yymfxu6 Copy   

•   Source: Protocols.io

Authors: Amy Ehrlich, Trina Knotts
Group: Mouse Metabolic Phenotyping Centers
Summary: Gut tissue (by region- e.g ileum & colon) will be opened along the mesenteric border and mounted in Ussing chambers (Physiologic Instruments, San Diego, CA, USA), exposing 0.3 cm² of tissue surface area to 2.5ml of oxygenated Krebs-glucose (10mM) and Krebs-mannitol (10mM) at 37°C on the serosal and luminal sides, respectively. The paracellular pathway and transcellular pathway will be measured as the flux of FITC-Dextran 4000 (FD-4, Sigma –Aldrich) and horseradish peroxidase (HRP Type VI, Sigma Aldrich), respectively. FD-4 (400µg/ml) and HRP (200µg/ml) will be added to the mucosal chamber and samples will be collected from the serosal chamber every 30 min for 2 hours. Concentration of FD-4 is measured via fluorescence at excitation 485 nm, emission 538 nm. O-dianisidine substrate is used to detect HRP at absorbance 450 nm.

Proper citation: Amy Ehrlich, Trina Knotts 2019. UC Davis - Ex vivo assessment of barrier function-gut permeability. protocols.io dx.doi.org/10.17504/protocols.io.yipfudn Copy   

•   Source: Protocols.io

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 dx.doi.org/10.17504/protocols.io.7bwhipe Copy   

•   Source: Protocols.io

Authors: Patrick Tso
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary: Oxygen consumption and carbon dioxide production is measured using indirect calorimetry. The Columbus Instruments Oxymax Equal Flow System is an indirect open circuit calorimeter designed to simultaneously measure metabolic performance of multiple subjects that have similar ventilation needs. uses an open circuit calorimetry technique. This system allows sixteen animal cages to be simultaneously monitored. Variables provided by this measurement include VO2, VCO2, RQ, and HEAT.

Proper citation: Patrick Tso 2019. U Cinn - Energy Expenditure Measurements. protocols.io dx.doi.org/10.17504/protocols.io.xi9fkh6 Copy   

•   Source: Protocols.io

Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary: Glucose is oxidized by glucose oxidase to gluconic acid and hydrogen peroxide. The hydrogen peroxide reacts in the presence of peroxidase with HBA and 4-aminoantipyrine forming a red quinoneimine dye. The intensity of the color formed is proportional to the glucose concentration and can be measured photometrically between 460 and 560 nm.

Proper citation: Peter Havel 2019. UC Davis - Glucose Protocol. protocols.io dx.doi.org/10.17504/protocols.io.yj9fur6 Copy   

•   Source: Protocols.io

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 dx.doi.org/10.17504/protocols.io.ydsfs6e Copy   

•   Source: Protocols.io

Authors: Henri Brunengraber
Group: Mouse Metabolic Phenotyping Centers
Summary: SummaryA known quantity of tissue / plasma is hydrolyzed and extracted after adding known amounts of internal standards: eg. heptadecanoic acid and cholesterol-d7. Fatty acids / cholesterol are analyzed as their trimethylsilyl derivatives using gas chromatography-electron impact ionization mass spectrometry (GCMS) (note: this protocol outlines the processing for palmitate and cholesterol; other fatty acids and sterols can be assayed using this preparation, see refs 1,2).References: 1. 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. 2009, 284(10):6101-8. 2. Influence of diet on the modeling of adipose tissue triglycerides during growth. Brunengraber DZ, McCabe BJ, Kasumov T, Alexander JC, Chandramouli V, Previs SF. Am J Physiol Endocrinol Metab. 2003, 285(4):E917-25.

Proper citation: Henri Brunengraber 2019. Case - Lipid Analysis Assay by GC-mass spectrometry. protocols.io dx.doi.org/10.17504/protocols.io.yehftb6 Copy   

•   Source: Protocols.io

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 dx.doi.org/10.17504/protocols.io.xy3fpyn Copy   

•   Source: Protocols.io

Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Lipid metabolism is estimated by measuring systemic clearance of [1-14C] palmitate following a bolus injection in awake mice. Lipid metabolism is altered in obese mice.

Proper citation: Jason Kim 2019. U Mass - Lipid metabolism. protocols.io dx.doi.org/10.17504/protocols.io.x3gfqjw Copy   

•   Source: Protocols.io

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 dx.doi.org/10.17504/protocols.io.y3sfyne Copy   

•   Source: Protocols.io

Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary: Insulin clearance test measure systemic clearance of insulin following a bolus injection. Liver accounts for the majority of systemic insulin clearance following secretion from pancreatic β- cells into portal circulation. Hepatic clearance of insulin may be affected by obesity and in other mouse models of altered metabolism. Alterations in insulin clearance may also affect glucose and lipid metabolism.

Proper citation: Jason Kim 2019. U Mass - Insulin clearance. protocols.io dx.doi.org/10.17504/protocols.io.x2wfqfe Copy   

•   Source: Protocols.io

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

•   Source: Protocols.io

Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: 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. Serum C-peptide levels reflect insulin secretion and pancreatic -cell function. Serum C-peptide levels are altered in obesity, insulin resistance, and type 1 and type 2 diabetes.

Proper citation: Jason Kim 2019. U Mass - C-Peptide. protocols.io dx.doi.org/10.17504/protocols.io.xttfnnn 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 creatinine reflect systemic protein metabolism.

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

•   Source: Protocols.io

Authors: Patrick Tso, Dana Lee
Group: Mouse Metabolic Phenotyping Centers
Summary: Determinations of triglycerides in plasma/serum/lymph will be made using a Randox Triglycerides colorimetric kit. The triglycerides are determined after enzymatic hydrolysis with lipases.

Proper citation: Patrick Tso, Dana Lee 2019. U Cinn - Triglyceride Assay. protocols.io dx.doi.org/10.17504/protocols.io.xm4fk8w Copy   

•   Source: Protocols.io

Authors: John Stack, Gary Cline
Group: Mouse Metabolic Phenotyping Centers
Summary: Procedure used to determine the concentration of calcium in blood, serum, and plasma. Calcium is measured as the complex with arsenazo III and monitored at 600nm.

Proper citation: John Stack, Gary Cline 2019. Yale - Blood or Urine Calcium. protocols.io dx.doi.org/10.17504/protocols.io.y3nfyme Copy   

•   Source: Protocols.io

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. The hyperglycemic clamp is used to assess insulin secretory capacity in conscious mice in response to hyperglycemia. Plasma insulin and C-Pedtide concentrations are measured at various times during the 2-hr clamp period.

Proper citation: Li Kang 2019. Vandy - Hyperglycemic clamp. protocols.io dx.doi.org/10.17504/protocols.io.yxefxje Copy   

•   Source: Protocols.io

Authors: Teri Stevenson, Vance L. Albaugh
Group: Mouse Metabolic Phenotyping Centers
Summary: This is the protocol for the vertical sleeve gastrectomy in the mouse. This procedure eliminates the greater curvature of the stomach leaving approximately 30% of the preoperative stomach capacity. Specifically, this procedure eliminates the greater curvature of the stomach where the ghrelin-producing cells are located.

Proper citation: Teri Stevenson, Vance L. Albaugh 2019. Vandy - Vertical Sleeve Gastrectomy in Mice. protocols.io dx.doi.org/10.17504/protocols.io.zcjf2un Copy   

•   Source: Protocols.io

Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: The ELISA (Enzyme Linked ImmunoSorbent Assay) is an assay method used for the quantification of various proteins. A plate is coated with a capture antibody specific to the analyte of interest. Standards and samples are pipetted into the plate. After an incubation period the wells are washed and any unbound analyte is removed. An enzyme-linked antibody that is specific to the analyte of interest is added to the wells. After an incubation period the wells are washed and any unbound antibody is removed. The analyte of interest is now sandwiched between the capture antibody and the enzyme-linked antibody. A substrate (normally 3,3’,5,5’-tetramethylbenzidine) is added which reacts with the enzyme (normally horseradish peroxidase) conjugated to the secondary antibody. A blue color develops in proportion to the amount of analyte sandwiched between the two antibodies. After an incubation period the reaction is stopped with the addition of an acid which turns the blue color to yellow. The intensity of the color development is measured using a spectrophotometer. The absorbance readings and known concentrations of the standards are used to generate a standard curve, and the absorbance readings 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 - ELISA Protocol. protocols.io dx.doi.org/10.17504/protocols.io.yibfuan Copy   

•   Source: Protocols.io