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Authors: Kent Lloyd, Kristin Grimsrud
Group: Mouse Metabolic Phenotyping Centers
Summary: The intravenous glucose tolerance test is used to assess insulin sensitivity, glucose disposal and islet function in vivo by measuring glucose and insulin responses following intravenous glucose administration.
Proper citation: Kent Lloyd, Kristin Grimsrud 2019. UC Davis - Intravenous Glucose Tolerance Test. protocols.io dx.doi.org/10.17504/protocols.io.8i7huhn Copy
Authors: Jeff Hodgin
Group: Mouse Metabolic Phenotyping Centers
Summary: Massons Trichrome staining is used for detection of collagen fibers for tubulointerstitial fibrosis on formalin-fixed paraffin-embedded kidney tissue section. The collagen fibers will be stained blue, the nuclei black, and the background is stained red.
Proper citation: Jeff Hodgin 2019. U Michigan - Massons Trichrome staining. protocols.io dx.doi.org/10.17504/protocols.io.56ug9ew 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 aspartate transferase reflect liver function.
Proper citation: Jason Kim 2019. U Mass - Aspartate Transferase. protocols.io dx.doi.org/10.17504/protocols.io.xr5fm86 Copy
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. Leptin is an adipocyte-derived hormone (adipokine) that regulates feeding behavior (hunger/satiety). Serum leptin levels are altered in obesity, insulin resistance, and type 2 diabetes.
Proper citation: Jason Kim 2019. U Mass - Leptin. protocols.io dx.doi.org/10.17504/protocols.io.x23fqgn Copy
Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:Glucose tolerance test with insulin secretion measures systemic clearance of glucose and systemic appearance of insulin following an intraperitoneal bolus injection of 20% dextrose. This experiment measures insulin sensitivity and insulin secretion (pancreatic -cell function) in awake mice assuming that there are no alterations in systemic insulin clearance. Insulin sensitivity and pancreatic -cell function are altered in obese mice.
Proper citation: Jason Kim 2019. U Mass - Glucose Tolerance Test with insulin secretion. protocols.io dx.doi.org/10.17504/protocols.io.xxbfpin Copy
Authors: John Stack, Gary Cline
Group: Mouse Metabolic Phenotyping Centers
Summary: Procedure used to determine the concentration of HDL cholesterol in blood, serum, and plasma. HDL Cholesterol is determined in a two-step procedure. First chylomicrons, VLDL, and LDL are selectively reacted with cholesterol esterase and eliminated from the reaction. In the second step, the remaining HDL-cholesterol is assayed as described for total cholesterol.
Proper citation: John Stack, Gary Cline 2019. Yale - HDL Cholesterol. protocols.io dx.doi.org/10.17504/protocols.io.y3ufynw Copy
Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary:
This experiment measures serum and plasma concentrations of non-esterified fatty acids using a 96-well kit. The experiment involves a coupled reaction to measure non-esterified fatty acids (NEFA) which ultimately forms a purple product that absorbs light at 550nm. This allows the concentration of NEFA to be determined from the optical density measured at 540~550nm. Serum fatty acids levels reflect systemic lipid metabolism, lipid digestion/absorption, and lipid clearance. Serum fatty acids levels are altered in obesity, insulin resistance, and type 2 diabetes.
Proper citation: Jason Kim 2019. U Mass - Non-esterified fatty acids. protocols.io dx.doi.org/10.17504/protocols.io.x3hfqj6 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 total protein reflect systemic protein metabolism.
Proper citation: Jason Kim 2019. U Mass - Total protein. protocols.io dx.doi.org/10.17504/protocols.io.x5gfq3w Copy
Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary:
The EchoMRI 3-in-1 uses ¹H- magnetic resonance spectroscopy to noninvasively measure fat mass, lean mass, and water mass in individual organs. Fat, lean, and water mass composition in specific organs is altered in obesity.
Proper citation: Jason Kim 2019. U Mass - Body composition (organs). protocols.io dx.doi.org/10.17504/protocols.io.xsnfnde Copy
Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers, Metabolomics Protocols & Workflows
Summary: This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer. Triglyceride levels may be measured in serum, plasma, and tissue samples. Serum and tissue triglyceride levels are affected by alterations in systemic lipid metabolism, lipid digestion/absorption, and lipid clearance. Serum and tissue triglyceride levels are altered in obesity, insulin resistance, type 2 diabetes, alcoholic steatohepatitis, non-alcoholic fatty liver disease, and non-alcoholic steatohepatitis.
Proper citation: Jason Kim 2019. U Mass - Triglyceride. protocols.io dx.doi.org/10.17504/protocols.io.x5pfq5n Copy
Authors: K.C. Kent Lloyd
Group: Mouse Metabolic Phenotyping Centers
Summary: Basic Adipocyte Culture Protocol
Proper citation: K.C. Kent Lloyd 2019. UC Davis - Basic Adipocyte Culture Protocol. protocols.io dx.doi.org/10.17504/protocols.io.7bvhin6 Copy
Authors: Eva Feldman
Group: Mouse Metabolic Phenotyping Centers
Summary: This protocol measures and quantifies the pain in rodent hind paw. These measurements can be used to evaluate the pathology present in the nervous system.
Proper citation: Eva Feldman 2019. U Michigan - Hind Paw Withdrawal for Rodents. protocols.io dx.doi.org/10.17504/protocols.io.x67frhn Copy
Authors: Henri Brunengraber
Group: Mouse Metabolic Phenotyping Centers
Summary: This is the standard protocol for most routine glucose tolerance testing. It is performed on awake mice, fasted for 18 hr (overnight) or 6 hours.
Proper citation: Henri Brunengraber 2019. Case - Intraperitoneal Glucose Tolerance Test. protocols.io dx.doi.org/10.17504/protocols.io.ydtfs6n Copy
Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:
Glutathione reductase (GR, EC 1.6.4.2) is a flavoprotein that catalyzes the NADPH- dependent reduction of oxidized glutathione (GSSG) to glutathione (GSH). This enzyme is essential for the GSH redox cycle which maintains adequate levels of reduced cellular GSH. A high GSH/GSSG ratio is essential for protection against oxidative stress. The Cayman Chemical Glutathione Reductase Assay Kit measures GR activity by measuring the rate of NADPH oxidation. The oxidation of NADPH to NADP+ is accompanied by a decrease in absorbance at 340 nm. Since GR is present at rate limiting concentrations, the rate of decrease in the A340 is directly proportional to the GR activity in the sample. The Cayman GR Assay Kit is provided in a convenient 96 well plate format and can be used to measure GR activity inplasma, erythrocyte lysates, tissue homogenates, and cell lysates.
Proper citation: Peter Havel 2019. UC Davis - Glutathione Reductase. protocols.io dx.doi.org/10.17504/protocols.io.68ehhte Copy
Authors: Lin Zhong, Chee Lim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:Imlantable radiotelemetry devices allow for long-term continous monitoring of blood pressure and heart rate in conscious freely moving caged animals. This protocol describes the surgical implantation of a radio transmitter device in the mouse.
Proper citation: Lin Zhong, Chee Lim 2019. Vandy - Mouse Blood Pressure Telemetry. protocols.io dx.doi.org/10.17504/protocols.io.6xnhfme Copy
Authors: Fawaz G. Haj
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:
An intraperitoneal Glucose tolerance test or ipGTT is designed to determine clearance of an intraperitoneally injected glucose load from the body. Animals are fasted for approximately 16 hours, a solution of glucose is administered by intraperitoneal (IP) injection and blood glucose is measured at different time points during 2 hours post-injection.
Proper citation: Fawaz G. Haj 2019. UC Davis - Intraperitoneal Glucose Tolerance Test. protocols.io dx.doi.org/10.17504/protocols.io.yp4fvqw Copy
Authors: Vincent Young
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:
This protocol is for the submission of DNA to generate libraries for 16S rRNA sequencing, which can be used for bacterial community analysis and detect variations in the microbiota under differing conditions.DNA is aliquoted into 96 well plates, which are properly packaged and shipped to the MMPC and libraries are prepared for community analysis as previously described by Seekatz et al. (1). Briefly, barcoded dual-index primers specific to the V4 region of the 16S rRNA gene amplify the DNA (2). PCR reactions are composed of 5 μL of 4 μM equimolar primer set, 0.15 μL of AccuPrime Taq DNA High Fidelity Polymerase, 2 μL of 10x AccuPrime PCR Buffer II (Thermo Fisher Scientific, catalog no. 12346094), 11.85 μL of PCR-grade water, and 1 μL of DNA template. The PCR conditions used consisted of 2 min at 95°C, followed by 30 cycles of 95°C for 20 s, 55°C for 15 s, and 72°C for 5 min, followed by 72°C for 10 min. Each PCR reaction is normalized using the SequalPrep Normalization Plate Kit (Thermo Fisher Scientific, catalog no. A1051001). The normalized reactions are pooled and quantified using the Kapa Biosystems Library qPCR MasterMix (ROX Low) Quantification kit for Illumina platforms (catalog no. KK4873). The Agilent Bioanalyzer is used to confirm the size of the amplicon library (~399 bp) using a high-sensitive DNA analysis kit (catalog no. 5067-4626). Pooled amplicon library is then sequenced on the Illumina MiSeq platform using the 500 cycle MiSeq V2 Reagent kit (catalog no. MS-102-2003) according to the manufacturer's instructions with modifications of the primer set with custom read 1/read 2 and index primers added to the reagent cartridge. The“Preparing Libraries for Sequencing on the MiSeq” (part 15039740, Rev. D) protocol was used to prepare libraries with a final load concentration of 5.5 pM, spiked with 15% PhiX to create diversity within the run. FASTQ files are distributed to the client when the 2 x 250 bp sequencing completes.References:1. Seekatz AM, Theriot CM, Molloy CT, Wozniak KL, Bergin IL, Young VB. 2015. Fecal Microbiota Transplantation Eliminates Clostridium difficile in a Murine Model of Relapsing Disease. Infect Immun 83:3838-3846. 10.1128/IAI.00459-15.
2. Kozich JJ, Westcott SL, Baxter NT, Highlander SK, Schloss PD. 2013. Development of a dual- index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Appl Environ Microbiol 79:5112–5120. 10.1128/AEM.01043-13.
Proper citation: Vincent Young 2019. U Michigan - Illumina 16S rRNA gene sequencing using DNA. protocols.io dx.doi.org/10.17504/protocols.io.7bzhip6 Copy
Authors: Patrick Tso, Dana Lee
Group: Mouse Metabolic Phenotyping Centers
Summary: Quantitative determinations of non-esterified fatty acids in plasma/serum/lymph will be made using the NEFA-HR enzymatic colorimetric method assay.
Proper citation: Patrick Tso, Dana Lee 2019. U Cinn - NEFA Concentration. protocols.io dx.doi.org/10.17504/protocols.io.xmnfk5e Copy
Authors: Jennifer Rutkowsky
Group: Mouse Metabolic Phenotyping Centers
Summary: This technique has been used routinely for the non-invasive measurement of blood pressure in rats, and more recently in mice. The technique provides a good estimate of actual systolic pressure. Hypertension major risk factor for stroke myocardial infarction, heart failure, aneurysm of the arteries, peripheral artery disease and is a cause of chronic kidney disease and erectile dysfunction (ED)Modified from: UC Davis MBP SOP-CODA Blood Pressure, Todd Tolentino 11/02/2012
Proper citation: Jennifer Rutkowsky 2019. UC Davis - Blood Pressure by Tail Cuff. protocols.io dx.doi.org/10.17504/protocols.io.yetften Copy
Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:Phloridzin is a potent inhibitor of renal glucose reabsorption and may be used to lower serum glucose levels. Phloridzin may be administered chronically to induce glycosuria and lower hyperglycemia in diabetic mice. Phloridzin may be administered acutely to reduce serum glucose levels for experiments in diabetic mice.
Proper citation: Jason Kim 2019. U Mass - Chronic/acute phloridzin treatment. protocols.io dx.doi.org/10.17504/protocols.io.xujfnun Copy
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