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Authors: Patrick Tso, Dana Lee
Group: Mouse Metabolic Phenotyping Centers
Summary: In vitro quantification of cholesterol in serum or plasma is determined using an Infinity Total Cholesterol Assay kit. This assay enzymatically hydrolyzes the cholesterol esters in the sample to cholesterol and free fatty acids. The free cholesterol is further oxidized and combined with HBA to allow it to be quantitatively measured in the serum or plasma.
Proper citation: Patrick Tso, Dana Lee 2019. U Cinn - Cholesterol Concentration. protocols.io dx.doi.org/10.17504/protocols.io.xiifkce Copy
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 dx.doi.org/10.17504/protocols.io.zawf2fe Copy
Authors: Jeff Hodgin, Jharna Saha
Group: Mouse Metabolic Phenotyping Centers
Summary: This protocol is used to stain and count podocyte nuclei in mouse glomeruli for diabetic nephropathy study.
Proper citation: Jeff Hodgin, Jharna Saha 2019. U Michigan - Podocyte Counting And Density Analysis. protocols.io dx.doi.org/10.17504/protocols.io.565g9g6 Copy
Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer. Serum uric acid levels are affected by alterations in systemic protein and nitrogen metabolism. Serum uric acid levels are altered in kidney failure and renal complications of diabetes.
Proper citation: Jason Kim 2019. U Mass - Uric Acid. protocols.io dx.doi.org/10.17504/protocols.io.x5sfq6e Copy
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 dx.doi.org/10.17504/protocols.io.xmvfk66 Copy
Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:
Triglycerides are enzymatically hydrolyzed by lipase to free fatty acids and glycerol. The glycerol is phosphorylated by adenosine triphosphate (ATP) with glycerol kinase (GK) to produce glycerol-3- phosphate and adenosine diphosphate. Glycerol-3-phosphate is oxidized by dihydroxyacetone phosphate (DAP) by glycerolphosphate oxidase producing hydrogen peroxide (H2O2). In a Trinder5 type color reaction catalyzed by peroxidase, the H2O2 reacts with 4-aminoantipyrine (4-AAP) and 3,5-dichloro-2- hydroxybenzene sulfonate (DHBS) to produce a red colored dye. The absorbance of this dye is proportional to the concentration of triglycerides present in the sample.
Proper citation: Peter Havel 2019. UC Davis - Triglyceride Protocol. protocols.io dx.doi.org/10.17504/protocols.io.yw3fxgn Copy
Authors: Bingle Bracy
Group: Mouse Metabolic Phenotyping Centers
Summary: Study of the unstressed mouse requires prior implantation of catheters in the carotid artery and jugular vein. The arterial catheter is used to sample blood and the venous catheter is used as an infusion port. The surgical procedure is performed under sterile conditions in accordance with AAALAC standards.
Proper citation: Bingle Bracy 2019. Vandy – Chronic Catheterization of carotid artery and jugular vein. protocols.io dx.doi.org/10.17504/protocols.io.zddf226 Copy
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 dx.doi.org/10.17504/protocols.io.yevfte6 Copy
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 dx.doi.org/10.17504/protocols.io.ywafxae Copy
Authors: Jennifer Rutkowsky
Group: Mouse Metabolic Phenotyping Centers
Summary: BODIPY® 493/503 can be used as a stain for neutral lipids and as a tracer for oil and other nonpolar lipids. It has uses for identifying and quantifying lipid droplets in various cell and tissue type. Abnormal lipid droplet accumulation can be associated with various metabolic disorders such as Diabetes, cardiovascular disease and cognitive impairment.
Proper citation: Jennifer Rutkowsky 2019. UC Davis - Immunohistochemistry BODIPY. protocols.io dx.doi.org/10.17504/protocols.io.56rg9d6 Copy
Authors: Fawaz G. Haj
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:
An in-vivo glucose-stimulates insulin secretion test is designated to determine alterations in insulin secretion by the pancreas upon a bolus IP Glucose injection.
Proper citation: Fawaz G. Haj 2019. UC Davis - IN-VIVO Glucose-stimulates Insulin Secretion Test. protocols.io dx.doi.org/10.17504/protocols.io.yptfvnn Copy
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 dx.doi.org/10.17504/protocols.io.xprfmm6 Copy
Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: The RIA (RadioImmuno Assay) is an assay method used for the quantification of various proteins. A standardized concentration of antibody specific to the analyte of interest is pipetted into test tubes. A standardized concentration of the analyte of interest that is labeled with a radioisotope (usually 125I) is added to the tubes. Standards and samples are pipetted into the tubes and the tubes are incubated. During the incubation period the standardized concentration of labeled analyte and the unknown concentration of analyte in the samples will compete for binding sites on the antibody. After the incubation period a precipitating reagent is added to the tubes and the bound antibodies are precipitated out using the double antibody-polyethyleneglycol precipitation technique. The tubes are centrifuged and the supernatant is aspirated and the precipitate is counted (using a gamma counter if 125I is used). If there is a low concentration of the analyte of interest in the samples, the labeled analyte will have a higher probability for binding to the antibody and thus there will be a higher count in the precipitate. If there is a high concentration of the analyte of interest in the samples there will be a lower count of labeled analyte in the precipitate. The counts and known concentrations of the standards are used to generate a standard curve, and the counts 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 - Corticosterone RIA. protocols.io dx.doi.org/10.17504/protocols.io.ygiftue Copy
Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: A subcutaneous or intraperitoneal implantation of Alzet osmotic pump is used to chronically administer selected drug in mice. Chronic drug delivery may be used to examine intermediate to long-term effects of selected drug on obesity, insulin resistance, and metabolism.
Proper citation: Jason Kim 2019. U Mass - Chronic drug delivery. protocols.io dx.doi.org/10.17504/protocols.io.xuefnte Copy
Authors: Teri Stevenson, Vance L. Albaugh
Group: Mouse Metabolic Phenotyping Centers
Summary: This is the protocol for the modified gastric bypass in the mouse. In this procedure the stomach is bypassed and the food flows through the bypass arm directly into the jejunum.
Proper citation: Teri Stevenson, Vance L. Albaugh 2019. Vandy - Modified Roux-en-Y Gastric Bypass in Mice. protocols.io dx.doi.org/10.17504/protocols.io.zchf2t6 Copy
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 dx.doi.org/10.17504/protocols.io.56gg9bw Copy
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 dx.doi.org/10.17504/protocols.io.ygkftuw Copy
Authors: Lin Zhong, Jeffrey Rottman, Chee Lim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:The most common cause of cardiovascular mortality in man is the outcome from myocardial infarction. Accordingly, it is necesarry to study the corresponding process of heart injury recovery in many mouse models relevant to human cardiovascular disease. This protocol describes the surgical induction of myocardial injury via 1) permanent ligation of a coronary artery in the heart (myocardial infarction), 2) cryoinjury, and 3) transient occlusion of a coronary artery followed by reperfusion (ischemia-reperfusion injury).
Proper citation: Lin Zhong, Jeffrey Rottman, Chee Lim 2019. Vandy - Mouse Myocardial Infarction. protocols.io dx.doi.org/10.17504/protocols.io.6xghfjw Copy
Authors: John Stack, Gary Cline
Group: Mouse Metabolic Phenotyping Centers
Summary: Procedure used to measure the concentration of Blood Urea Nitrogen(BUN) in blood, plasma, and serum. Urea is determined by the enzymatically coupled reactions of urease (to form ammonia) and glutamate dehydrogenase (conversion of ammonia and glutamate to glutamine with oxidation of NADH to NAD). The rate of NAD formation is monitored by the change in absorbance at 340 nm.
Proper citation: John Stack, Gary Cline 2019. Yale - Blood Urea Nitrogen. protocols.io dx.doi.org/10.17504/protocols.io.y3qfymw Copy
Authors: Gary Cline, John Stack
Group: Mouse Metabolic Phenotyping Centers
Summary: Procedure used to determine the concentration of β-Hydroxybutyrate in blood, serum, and plasma. β-Hydroxybutyrate is measured by the oxidation to acetoacetate, followed by reduction of an indicator dye (monitored at 505 nm) by NADH.
Proper citation: Gary Cline, John Stack 2019. Yale - Beta hydroxybutyrate (Cobas). protocols.io dx.doi.org/10.17504/protocols.io.y2bfyan Copy
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