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Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: LDL and VLDL are separated from HDL using a precipitation reagent. Then the HDL fraction is measured for either TC or TG using the same reagents for total cholesterol or triglyceride.
Proper citation: Peter Havel 2019. UC Davis - LDL Protocol. protocols.io dx.doi.org/10.17504/protocols.io.yrcfv2w Copy
Authors: Oliver Fiehn
Group: Mouse Metabolic Phenotyping Centers, Metabolomics Protocols & Workflows
Summary: This SOP describes sample extraction and sample preparation for primary metabolism profiling by gas chromatography / time of flight mass spectrometry (GCTOF)References: Fiehn O, Kind T (2006) Metabolite profiling in blood plasma. In: Metabolomics: Methods and Protocols. Weckwerth W (ed.), Humana Press, Totowa NJ
Proper citation: Oliver Fiehn 2019. UC Davis - Metabolomics: Sample preparation for GCTOF analysis. protocols.io dx.doi.org/10.17504/protocols.io.ytjfwkn Copy
Authors: John Stack, Gary Cline
Group: Mouse Metabolic Phenotyping Centers
Summary: Procedure used to determine the concentration of triglycerides in blood, serum, and plasma. Triglycerides are determined by coupling lipase, glucokinase, glycerol phosphate oxidase, and peroxidase to form a quinonemine dye which is measured at 500 nm.
Proper citation: John Stack, Gary Cline 2019. Yale - Triglycerides. protocols.io dx.doi.org/10.17504/protocols.io.y4bfysn Copy
Authors: Jeff Hodgin
Group: Mouse Metabolic Phenotyping Centers
Summary: Immunoblotting (also called Western Blotting) is a technique to separate and identify individual proteins in a protein mixture (e.g. a cell lysate). In Immunoblotting (Western blotting) the protein mixture is applied to a gel electrophoresis (SDS-PAGE gel electrophoresis) to sort the proteins by its molecular weight size in individual protein bands. The separated protein bands are then transferred to a nitrocellulose transfer membrane. The proteins adhere to the membrane in the same pattern as they have been separated due to interactions of charges. To visualize the protein of interest the membrane is commonly first probed using a primary protein- specific antibody followed by a labeled secondary antibody. The specificity of the antibody-antigen interaction enables a target protein to be identified in the midst of a complex protein mixture. The bound antibodies are then detected by western blotting substrate (e.g. Pierce ECL Western Blotting Substrate), and then visualized by using adeveloping film (HyBlot CL Autoradiography Film) in the darkroom. The thickness of the band corresponds to the amount of protein present.
Proper citation: Jeff Hodgin 2019. U Michigan - Western Blot. protocols.io dx.doi.org/10.17504/protocols.io.56hg9b6 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 alkaline phosphatase reflect liver function.
Proper citation: Jason Kim 2019. U Mass - Alkaline Phosphatase. protocols.io dx.doi.org/10.17504/protocols.io.xptfmnn Copy
Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:
Cayman Chemical's Protein Carbonyl Colorimetric Assay Kit is a convenient colorimetric assay for the measurement of oxidized proteins. Protein samples are derivatized by making use of the reaction between 2,4-dinitrophenylhydrazine (DNPH) and protein carbonyls. Formation of a Schiff base produces the corresponding hydrazone which can be analyzed spectrophotometrically at 360-385 nm. This assay can be used to measure oxidized protein in plasma, serum, cell lysates, and tissue homogenates.
Proper citation: Peter Havel 2019. UC Davis - Protein Carbonyl. protocols.io dx.doi.org/10.17504/protocols.io.63hhgj6 Copy
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 (V3002). The hyperinsulinemic, euglycemic clamp or “insulin clamp” has been used in a variety of species to assess insulin action. In an insulin clamp, the rate of glucose infused to maintain euglycemia is an index of whole body insulin sensitivity. Isotopes can be used during an insulin clamp to distinguish between insulin’s effects on endogenous glucose production and glucose utilization, or to examine insulin’s effects on specific tissues and metabolic pathways. The insulin infusion rate used in a study depends on a number of factors such as whether the experiment requires a sensitive analysis of endogenous glucose production, or whether the mouse is known to be unusually insulin resistant (e.g. ob/ob). Finally the insulin clamp technique, especially when isotopes are involved, is most quantitative under steady state conditions. Therefore, clamps should be of a duration (~2 h) that is sufficient for the actions of insulin to obtain steady state. The section that follows describes, a) the technical procedures used on the day of a clamp experiment; b) preparation of insulin and tracer infusates; and c) analyses of plasma and tissue radioactivity. Familiarity with these procedures combined with laboratory demonstration provides the investigator with the necessary background to clamp the conscious mouse.
Proper citation: Li Kang 2019. Vandy - Hyperinsulinemic-euglycemic Clamp. protocols.io dx.doi.org/10.17504/protocols.io.yyafxse Copy
Authors: Mari Golub
Group: Mouse Metabolic Phenotyping Centers
Summary: The radial arm water maze (RAWM) measures spatial learning by combining both a water maze and radial arm maze (RAM). This test measures both working and reference memory through a reward system, in this case escape from water. This method is advantageous to other RAM which use food as a reward in that it may be utilized with mice on specialized diets. Reference: Behavioral consequences of ovarian atrophy and estrogen replacement in the APPsswe mouse. Golub et al. Neurobiol Aging (2008) 29(10): 1512-1523.
Proper citation: Mari Golub 2019. UC Davis - Radial Arm Water Maze. protocols.io dx.doi.org/10.17504/protocols.io.yv2fw8e Copy
Authors: Mark Kelly LAT, Timothy P. Fitzgibbons
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:
This is a mouse model of acute myocardial infarction. This protocol is suitable for studying mouse models thought to be relevant to coronary ischemia or the development of congestive heart failure.
Proper citation: Mark Kelly LAT, Timothy P. Fitzgibbons 2019. U Mass - Coronary Artery Ligation. protocols.io dx.doi.org/10.17504/protocols.io.56kg9cw Copy
Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:
Aspartate transaminase (AST) also known as aspartate aminotransferase or (sGOT) is a metabolic enzyme expressed primarily in the liver. Elevation of AST levels is an indication of liver damage and has been associated with liver injury. AST levels are monitored routinely in patients with liver diseases. AST is also a very useful tool for preclinical investigation of experimental drug formulations and AST levels are commonly used to monitor and attenuate the hepatotoxic effects of experimental drugs in rodents.
Proper citation: Peter Havel 2019. UC Davis - Aspartate Aminotransferase. protocols.io dx.doi.org/10.17504/protocols.io.ybvfsn6 Copy
Authors: K.C. Kent Lloyd
Group: Mouse Metabolic Phenotyping Centers
Summary: SummaryThe purpose of this standard operating procedure (SOP) is to provide steps for the acquisition, acclimation and quarantine of animals obtained from other institutions or laboratories and introduced to the MBP Vivarium with minimal risk to the current mouse colony population. This procedure applies to the import/export staff, veterinary staff and any principal investigator requesting animals.
Proper citation: K.C. Kent Lloyd 2019. UC Davis - Acquisition/Acclimation/Quarantine. protocols.io dx.doi.org/10.17504/protocols.io.xfifjke Copy
Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer.
Proper citation: Jason Kim 2019. U Mass - Ammonia. protocols.io dx.doi.org/10.17504/protocols.io.xq3fmyn Copy
Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer.
Proper citation: Jason Kim 2019. U Mass - Amylase. protocols.io dx.doi.org/10.17504/protocols.io.xq4fmyw Copy
Authors: Trina Knotts
Group: Mouse Metabolic Phenotyping Centers, Microbiome Centers
Summary: The work of Gordon and colleagues (i.e., Nature. 2006 Dec 21;444(7122):1027-31) has shown that obesity can result in marked shifts in the gut microbiome in mice and other models including humans. While the role of the microbiome remains to be fully elucidated, the gut microbiota can no longer be ignored as a potentially important factor when assessing metabolic phenotype. This service involves 16S gene variable region (V3-V4 using primer pair 515F-806R) pair end sequencing (2x300bp) by Illumina (20,000 avg seq read depth) of feces, cecal, or other GI contents. Sequences can be processed through a bioinformatics pipeline (Qiime) to taxonomically classify them and to assess alpha and beta diversity of the community. In addition, Principal Components Analysis (PCA) or partial least squares- discriminant analysis (PLS-DA) can leverage variances in the relative microbial abundances to better understand how specific microbes contribute to separation by group. Correlational analyses can identify which variables of host metadata associate with specific microbes. The Core’s gut microbiome assay will employ this approach to uncover unique microbiota fingerprints in test mice. One caveat is that with fecal samples, patterns are only a surrogate for actual gut microbiota patterns, and may not exactly reflect the intestinal populations.
Proper citation: Trina Knotts 2019. UC Davis - Gut Microbiome Analysis (Illumina-20K reads, 2X300bp). protocols.io dx.doi.org/10.17504/protocols.io.ynpfvdn Copy
Authors: Jeff Hodgin, Jharna Saha
Group: Mouse Metabolic Phenotyping Centers
Summary: Periodic acid-Schiff (PAS) is a staining method used to detect glycogen on formalin-fixed, paraffin-embedded kidney tissue sections. PAS staining highlights basement membranes and is frequently used to diagnose glomerular mesangial matrix expansion.
Proper citation: Jeff Hodgin, Jharna Saha 2019. U Michigan - Mesangial matrix evalutaion. protocols.io dx.doi.org/10.17504/protocols.io.562g9ge Copy
Authors: John Stack, Gary Cline
Group: Mouse Metabolic Phenotyping Centers
Summary: Procedure used to determine the concentration of LDL cholesterol in blood, serum, and plasma. LDL-cholesterol is determined by an elimination method to selectively remove HDL, VLDL, and IDL subfractions prior to assay of LDL-cholesterol as described for total cholesterol.
Proper citation: John Stack, Gary Cline 2019. Yale - LDL Cholesterol. protocols.io dx.doi.org/10.17504/protocols.io.y3vfyn6 Copy
Authors: Stephen M. Griffey
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:
Gross necropsy with documentation of changes present. Collection and histologic processing (paraffin blocks/H&E slides) and interpretation of kidneys, liver, spleen, pancreas, heart, lungs, esophagus, trachea, thymus, mesenteric lymph nodes, GI Tract, cerebrum, cerebellum, urinary bladder, and reproductive tract and additional tissues with gross changes. This includes photodocumentation of significant findings. This assay can be customized to include other target organs based on findings from other MMPC assays.
This procedure is to be followed by trained Comparative Pathology Laboratory (CPL) staff responsible for performing necrospy diagnostics procedures at CPL.
Proper citation: Stephen M. Griffey 2019. UC Davis - Mouse Gross Necropsy with Histology. protocols.io dx.doi.org/10.17504/protocols.io.yusfwwe Copy
Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary:
This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer. Serum alanine transferase levels reflect liver function.
Proper citation: Jason Kim 2019. U Mass - Alanine Transferase. protocols.io dx.doi.org/10.17504/protocols.io.xpmfmk6 Copy
Authors: Lin Zhong, Jeffrey Rottman, Chee Lim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:Hypertension in man can be modeled in the mouse by introducing a constriction in the major artery (aorta) thereby obstructing outflow and increasing the afterload to the heart. This protocol describes the procedure for aortic banding aka transverse aortic constriction (TAC).
Proper citation: Lin Zhong, Jeffrey Rottman, Chee Lim 2019. Vandy - Transverse Aortic Constriction. protocols.io dx.doi.org/10.17504/protocols.io.6xdhfi6 Copy
Authors: Gary Cline, John Stack
Group: Mouse Metabolic Phenotyping Centers
Summary: Procedure used to determine the concentration of albumin in blood, plasma, and serum. Albumin is measured as its conjugate with bromocresol green monitored at 600 nm.
Proper citation: Gary Cline, John Stack 2019. Yale - Blood Albumin. protocols.io dx.doi.org/10.17504/protocols.io.y2cfyaw Copy
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