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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 dx.doi.org/10.17504/protocols.io.xvnfn5e Copy
Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:
Whole body glucose turnover and hepatic glucose production rates are measured at basal state using an intravenous infusion of labeled glucose in awake mice. Whole body glucose turnover and hepatic glucose production regulate basal glucose levels and are altered in obesity.
Proper citation: Jason Kim 2019. U Mass - Basal glucose metabolism. protocols.io dx.doi.org/10.17504/protocols.io.xr8fm9w Copy
Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Hyperinsulinemic-euglycemic clamp is the gold-standard method to assess insulin sensitivity. The hyperinsulinemic-euglycemic clamp is widely used in clinics and laboratories to measure insulin action on glucose utilization in humans and animals for clinical and basic science research. Incorporation of radioactive-labeled glucose during hyperinsulinemic-euglycemic clamps makes it possible to measure glucose metabolism in individual organs in awake mice. Impaired insulin sensitivity (insulin resistance) is a major characteristic of obesity and an early requisite event in the development of type 2 diabetes.
Proper citation: Jason Kim 2019. U Mass - Protein metabolism. protocols.io dx.doi.org/10.17504/protocols.io.x4ffqtn Copy
Authors: Fawaz G. Haj
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:
An intraperitoneal insulin tolerance test or ipITT is designed to determine the sensitivity of insulin-responsive tissues in the rodent. This is determined by measurement of glucose remaining in the circulation over time after a bolus ip insulin injection.
Proper citation: Fawaz G. Haj 2019. UC Davis - Intraperitoneal Insulin Tolerance Test. protocols.io dx.doi.org/10.17504/protocols.io.yp5fvq6 Copy
Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: A 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.
Proper citation: Jason Kim 2019. U Mass - Chronic high-fat feeding. protocols.io dx.doi.org/10.17504/protocols.io.xugfntw Copy
Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:
The Wako enzymatic method relies upon the acylation of coenzyme A (CoA) by the fatty acids in the presence of added acyl-CoA synthetase (ACS). The acyl-CoA thus produced is oxidized by added acyl- CoA oxidase (ACOD) with generation of hydrogen peroxide, in the presence of peroxidase (POD) permits the oxidative condensation of 3-methy-N-ethyl-N(β-hydroxyethyl)-aniline (MEFA) with 4-aminoantipyrine to form a purple colored adduct which can be measured colorimetrically at 550 nm.
Proper citation: Peter Havel 2019. UC Davis - Non-Esterified Fatty Acids Protocol. protocols.io dx.doi.org/10.17504/protocols.io.yutfwwn Copy
Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer. Serum levels of bilirubin reflect bile acid metabolism.
Proper citation: Jason Kim 2019. U Mass - Bilirubin. protocols.io dx.doi.org/10.17504/protocols.io.xscfnaw Copy
Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary:
The EchoMRI 3-in-1 uses ¹H- magnetic resonance spectroscopy to noninvasively measure whole body fat mass, lean mass, and water mass in awake mice. Whole body fat mass is a measure of adiposity that is altered in obesity. Whole body lean mass may be altered by exercise and in mouse models of altered metabolism.
Proper citation: Jason Kim 2019. U Mass - Body composition (whole body). protocols.io dx.doi.org/10.17504/protocols.io.xsqfndw Copy
Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:
Alanine transaminase (ALT) also known as alanine aminotransferase or (sGPT) is a metabolic enzyme expressed primarily in the liver. Elevation of ALT levels is an indication of liver damage and has been associated with liver injury. ALT levels are monitored routinely in patients with liver diseases. ALT is also a very useful tool for preclinical investigation of experimental drug formulations and ALT levels are commonly used to monitor and attenuate the hepatotoxic effects of experimental drugs in rodents.
Proper citation: Peter Havel 2019. UC Davis - Alanine transaminase. protocols.io dx.doi.org/10.17504/protocols.io.ybtfsnn Copy
Authors: Patrick Tso, Dana Lee
Group: Mouse Metabolic Phenotyping Centers
Summary: Dietary fat containing 5% sucrose polybehenate, a non-absorbable food additive, is fed in a semi-synthetic diet containing absorbable fat to rodents. The animals eat the diet ad lib for 3 days. Two or more fecal pellets are collected on days 3 and 4. Fat absorption is calculated from the ratios of behenic acid to other fatty acids in the diet and in the feces as analyzed by gas chromatography of fatty acid methyl esters.
Proper citation: Patrick Tso, Dana Lee 2019. U Cinn - Non-invasive Measurement of Intestinal Fat Absorption. protocols.io dx.doi.org/10.17504/protocols.io.xmtfk6n Copy
Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:The TSE PhenoMaster/LabMaster Metabolic Cage system is used to measure indirect calorimetry, food/water intake, energy expenditure, and physical activity in individually housed awake mice. The experiment noninvasively measures VO² consumption and VCO² production rates in individual mice using metabolic chambers and calculates the respiratory exchange ratio (respiratory quotient) to reflect energy expenditure. Metabolic cage measurement is conducted continuously for 72 hours (3 days) to account for acclimation of mice housed in home cages during the study. With our high-speed Siemens O²/CO² sensing unit, indirect calorimetry measurements may be performed at a 20-min interval for a full 12-cage study.
Proper citation: Jason Kim 2019. U Mass - Energy balance – food intake, energy expenditure, physical activity. protocols.io dx.doi.org/10.17504/protocols.io.xwwfpfe Copy
Authors: David A. Antonetti
Group: Mouse Metabolic Phenotyping Centers
Summary: A spectral domain ophthalmic imaging system based on the technology of optical coherence tomography (OCT) will be used to acquire the microstructure image of retina in live animals. Like the ultrasound, but uses light waves instead of sound waves, the light backscattered from within a sample is processed to develop a high-resolution, depth-resolved image suitable for analyzing internal microstructure in vivo.
Proper citation: David A. Antonetti 2019. U Michigan - Retinal Microstructure Imaging OCT. protocols.io dx.doi.org/10.17504/protocols.io.yabfsan Copy
Authors: Oliver Fiehn
Group: Mouse Metabolic Phenotyping Centers, Metabolomics Protocols & Workflows
Summary: Summary:Lipidomic analysis by UPLC-QTOF mass spectrometry
Proper citation: Oliver Fiehn 2019. UC Davis - Metabolomics: Lipidomics analysis. protocols.io dx.doi.org/10.17504/protocols.io.ys7fwhn Copy
Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Chronic indwelling catheter is placed in the carotid artery for blood sampling during experiments. Surival surgery is performed in anesthetized mice, and mice recover from surgery after 4~5 days.
Proper citation: Jason Kim 2019. U Mass - Surgery – carotid artery cannulation. protocols.io dx.doi.org/10.17504/protocols.io.x4pfqvn Copy
Authors: Jeff Hodgin
Group: Mouse Metabolic Phenotyping Centers
Summary: This protocol provides information for the non-invasive measurement of blood pressure--tail cuff blood pressure determination operated by CODA tail-cuff blood pressure system in mice.
Proper citation: Jeff Hodgin 2019. U Michigan - Tail Cuff Blood Pressure Determination. protocols.io dx.doi.org/10.17504/protocols.io.56eg9be Copy
Authors: Eva Feldman
Group: Mouse Metabolic Phenotyping Centers
Summary: To confirm the presence of diabetic neuropathy, nerve conduction velocity (NCV) studies are performed. The animals are anesthetized with isoflurane to prevent discomfort. Body temperature is monitered with a dermal temperature probe and maintained at 32ºC with a warming lamp during NCV. Body temperature is maintained at 37ºC after NCV using a warming pad to ease animal stress from anesthetic. The nerve studies last less than 30 min per rat or mouse. The electrodes are cleaned with 70% alcohol between animals to maintain pathogen-free status.
Proper citation: Eva Feldman 2019. U Michigan - Nerve Conduction Velocity. protocols.io dx.doi.org/10.17504/protocols.io.x96fr9e Copy
Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: Direct Enzymatic HbA1c test is an enzymatic assay in which lysed whole blood samples are subjected to extensive protease digestion with Bacillus sp protease. This process releases amino acids including glycated valines from the hemoglobin beta chains. Glycated valines then serve as substrates for specific recombinant fructosyl valine oxidase (FVO) enzyme, produced in E. coli. The recombinant FVO specifically cleaves N-terminal valines and produces hydrogen peroxide. This, in turn, is measured using a horseradish per-oxidase (POD) catalyzed reaction and a suitable chromagen. The HbA1c concentration is expressed directly as %HbA1c by use of a suit-able calibration curve in which the calibrators have values for each level in %HbA1c.
Proper citation: Peter Havel 2019. UC Davis - HbA1c Protocol. protocols.io dx.doi.org/10.17504/protocols.io.ynrfvd6 Copy
Authors: Jennifer Rutkowsky
Group: Mouse Metabolic Phenotyping Centers
Summary: Massons trichrome staining is used for detection of collagen fibers in tissues such as skin, heart, etc. on formalin-fixed, paraffin-embedded sections, and may be used for frozen sections as well. The collagen fibers will be stained blue and the nuclei will be stained black and the background is stained red.Modified from: Massons Trichrome UC Davis Clinical Pathology lab
Proper citation: Jennifer Rutkowsky 2019. UC Davis - Massons Trichrome. protocols.io dx.doi.org/10.17504/protocols.io.56mg9c6 Copy
Authors: Gary Cline, John Stack
Group: Mouse Metabolic Phenotyping Centers
Summary: Procedure used to measure the Alanine Amino activity in blood, plasma, and serum. Alanine Amino (ALT) activity is measured by the enzymatically coupled reactions of ALT (to form pyruvate from alanine and α-ketoglutarate) and Lactate dehydrogenase (conversion of pyruvate to lactate with oxdiation of NADH to NAD). The rate of NAD formation is monitored by the change in absorbance at 340 nm.
Proper citation: Gary Cline, John Stack 2019. Yale - Alanine Aminotransferase. protocols.io dx.doi.org/10.17504/protocols.io.yz4fx8w Copy
Authors: Jennifer Rutkowsky
Group: Mouse Metabolic Phenotyping Centers
Summary: Staining tissues on a slide with Heatoxylin and Eosin.Modified from: H &E protocol, UC Davis Clinical Pathology lab
Proper citation: Jennifer Rutkowsky 2019. UC Davis - Hematoxylin and Eosin (H&E). protocols.io dx.doi.org/10.17504/protocols.io.56sg9ee Copy
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