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Authors: Louise Lantier
Group: Mouse Metabolic Phenotyping Centers
Summary: The Promethion from Sable Systems (Las Vegas, NV) assesses several key metabolic characteristics of mice such as energy expenditure, preferred metabolic substrate, meal patterns, activity, live body mass. These are measured by individually housing the mice in the Promethion System cages for several days during which numerous parameters are continuously measured (food and water intake, weight, activity, O² and CO²).

Proper citation: Louise Lantier 2019. Vandy – Energy Balance with Promethion. protocols.io https://dx.doi.org/10.17504/protocols.io.yzxfx7n Copy   

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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 https://dx.doi.org/10.17504/protocols.io.yabfsan Copy   

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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Glucose tolerance test measures systemic clearance of glucose following an intraperitoneal bolus injection of 20% dextrose. This experiment measures insulin sensitivity in awake mice assuming that there are no alterations in the animal’s pancreatic -cell function and insulin secretion. Insulin sensitivity is altered in obese mice.

Proper citation: Jason Kim 2019. U Mass - Glucose Tolerance Test. protocols.io https://dx.doi.org/10.17504/protocols.io.xxafpie Copy   

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Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary: Glutathione peroxidase (GPx) catalyzes the reduction of hydroperoxides, including hydrogen peroxides, by reduced glutathione and functions to protect the cell from oxidative damage. With the exception of phospholipid-hydroperoxide GPx, a monomer, all of the GPx enzymes are tetramers of four identical subunits. Each subunit contains a selenocysteine in the active site which participates directly in the two-electron reduction of the peroxide substrate. The enzyme uses glutathione as the ultimate electron donor to regenerate the reduced form of the selenocysteine. The Cayman Chemical Glutathione Peroxidase Assay Kit measures GPx activity indirectly by a coupled reaction with glutathione reductase (GR). Oxidized glutathione (GSSG), produced upon reduction of an organic hydroperoxide by GPx, is recycled to its reduced state by GR and NADPH. The oxidation of NADPH to NADP+ is accompanied by a decrease in absorbance at 340 nm. The rate of decrease in the A340 is directly proportional to the GPx activity in the sample. The Cayman GPx Assay Kit can be used to measure all of the glutathione- dependent peroxidases in plasma, erythrocyte lysates, tissue homogenates, and cell lysates.

Proper citation: Peter Havel 2019. UC Davis - Glutathione Peroxidase. protocols.io https://dx.doi.org/10.17504/protocols.io.ykdfus6 Copy   

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Authors: Jeff Hodgin
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:Collection of urine from experimental animals is a basic requirement to detect albumin/creatinine ratio for glomerular nephropathy. This is a non-invasive, simple, quick, and inexpensive method for collecting spot urine from mouse when a small volume of urine (10-500ul) sample is required.

Proper citation: Jeff Hodgin 2019. U Michigan - Spot urine collection. protocols.io https://dx.doi.org/10.17504/protocols.io.56fg9bn Copy   

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Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary: The enzyme methodology employed in this reagent is based on the reaction first described by Talke and Schubert. To shorten and simplify the assay, the calculations are based on the discovery of Tiffany, et al. that urea concentration is proportional to absorbance change over a fixed time interval. The reaction is monitored by measuring the rate of decrease in absorbance at 340 nm as NADH is converted to NAD.

Proper citation: Peter Havel 2019. UC Davis - Urea Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.yw6fxhe Copy   

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Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: The method employed here is based on an enzymatic colorimetric determination of creatinine which largely eliminates interferences known to the Jaffe method. In the final reaction sequence, the formation of the quinoneimine dye product results in an increase in absorbance at 550 nm (530-570 nm) which is directly proportional to the creatinine concentration in the sample. Potential interference from endogenous creatine and sarcosine are eliminated by the reaction of creatine amidinohydrolase, sarcosine oxidase and catalase before creatinine is determined. Ascorbate oxidase is included in the reagent to eliminate the influence of ascorbate in the sample.

Proper citation: Peter Havel 2019. UC Davis - Creatinine Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.ygjftun Copy   

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Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: Catalase (EC 1.11.1.6; 2H2O2 oxidoreductase) is an ubiquitous antioxidant enzyme that is present in most aerobic cells. Catalase (CAT) is involved in the detoxification of hydrogen peroxide (H2O2), a reactive oxygen species (ROS), which is a toxic product of both normal aerobic metabolism and pathogenic ROS production. This enzyme catalyzes the conversion of two molecules of H2O2 to molecular oxygen and two molecules of water (catalytic activity). CAT also demonstrates peroxidatic activity, in which low molecular weight alcohols can serve as electron donors. While the aliphatic alcohols serve as specific substrates for CAT, other enzymes with peroxidatic activity do not utilize these substrates. In humans, the highest levels of catalase are found in liver, kidney, and erythrocytes, where it is believed to account for the majority of hydrogen peroxide decomposition. The Cayman Chemical Catalase Assay Kit utilizes the peroxidatic function of CAT for determination of enzyme activity. The method is based on the reaction of the enzyme with methanol in the presence of an optimal concentration of H2O2. The formaldehyde produced is measured spectrophotometrically with 4-amino3-hydrazino-5-mercapto-1,2,4-triazole (Purpald) as the chromogen. Purpald specifically forms a bicyclic heterocycle with aldehydes, which upon oxidation changes from colorless to a purple color. The assay can be used to measure CAT activity in plasma, serum, erythrocyte lysates, tissue homogenates, and cell lysates.

Proper citation: Peter Havel 2019. UC Davis - Catalase. protocols.io https://dx.doi.org/10.17504/protocols.io.ygbftsn 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. Serum levels of HDL-cholesterol reflect cholesterol metabolism and are associated with cardiovascular disease.

Proper citation: Jason Kim 2019. U Mass - Cholesterol (HDL). protocols.io https://dx.doi.org/10.17504/protocols.io.xt6fnre Copy   

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Authors: Jennifer Rutkowsky
Group: Mouse Metabolic Phenotyping Centers
Summary: Factor VIII is a glycoprotein present in human plasma, human endothelial cells, megakaryocytes and platelets. Immunohistochemical staining for factor VIII related antigen could be used to determine if the benign and malignant neoplastic lesions are of endothelial origin. Furthermore, it can be utilized to determine vessel density in a tissue of interestModified from: IHC Methods and Materials VMTH - Anatomic Pathology, UC-Davis.

Proper citation: Jennifer Rutkowsky 2019. UC Davis - Immunohistochemistry Factor VIII. protocols.io https://dx.doi.org/10.17504/protocols.io.56qg9dw Copy   

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Authors: Jennifer Rutkowsky
Group: Mouse Metabolic Phenotyping Centers
Summary: SummaryIn multiple studies, access to the circulatory system in laboratory studies is necessary and femoral vein is one of the more common. Implanted femoral catheters permit a researcher to effectively administer substances and perform repeated blood sampling from an anesthetized mouse ( for instance administration of gadolinium for MRI BBB permeability studies).

Proper citation: Jennifer Rutkowsky 2019. UC Davis - Femoral Vein Cannulation. protocols.io https://dx.doi.org/10.17504/protocols.io.zavf2e6 Copy   

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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. Serum glucagon levels reflect glucagon secretion and pancreatic α-cell function. Serum glucagon levels regulate hepatic glucose production and glucose homeostasis. Serum glucagon levels are altered in obesity, insulin resistance, and counterregulatory stress response.

Proper citation: Jason Kim 2019. U Mass - Glucagon. protocols.io https://dx.doi.org/10.17504/protocols.io.xw3fpgn Copy   

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Authors: Saivageethi Nuthikattu, Jennifer Rutkowsky
Group: Mouse Metabolic Phenotyping Centers
Summary: To quantify regions or cell type specific alterations in RNA expression. Laser capture microscopy is utilized to isolate region or cell-specific regions in the brain and qualitative real time-PCR (qRT-PCR) is used to assess relative or semi-quantitative RNA expression of genes of interest.Modified from: Laser Capture Microscopy Protocol- Van Winkle Lab

Proper citation: Saivageethi Nuthikattu, Jennifer Rutkowsky 2019. UC Davis - Laser Capture Microscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.zawf2fe 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. 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 https://dx.doi.org/10.17504/protocols.io.yxefxje Copy   

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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 https://dx.doi.org/10.17504/protocols.io.56eg9be Copy   

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Authors: Patrick Tso, Dana Lee
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary: Food Intake Monitoring - Oxymax/CLAMS monitors the cumulative amount of food eaten as well as the amount eaten in each bout with the use of a Mettler Toledo balance with a resolution of 0.01g. Feeders are designed to account for spillage of food and to prevent foraging, a combination which provides the most accurate food intake monitor on the market. Food Access Control - Access to food can be placed under automatic computerized control. A pneumatic driven shield prevents access to the food, and can be controlled by times intervals and/or by mass of food. Additionally, this option also includes paradigms for Yolked or Paired feeding. Center Feeder Cages: The Center Feeder Cage maintains the same sealed environment but adds a feeder to the center of the cage to allow food intake measurements. The Center Feeder is the most popular and universal in application, supporting all subject body types from lean to obese. An adjustable food guard allows mechanical fine tuning of food access to minimize foraging.

Proper citation: Patrick Tso, Dana Lee 2019. U Cinn - Meal Pattern Analysis Food Intake Procedure. protocols.io https://dx.doi.org/10.17504/protocols.io.xjqfkmw Copy   

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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 https://dx.doi.org/10.17504/protocols.io.x96fr9e Copy   

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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 https://dx.doi.org/10.17504/protocols.io.56mg9c6 Copy   

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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 https://dx.doi.org/10.17504/protocols.io.xr8fm9w Copy   

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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 https://dx.doi.org/10.17504/protocols.io.yaafsae Copy   

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