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Authors: Trina Knotts
Group: Mouse Metabolic Phenotyping Centers
Summary: Rapid and accurate measures of fat and lean mass will be carried out in isoflurane-anesthetized mice, using a PixiMus dual energy X-ray absorptiometry (DEXA) device. This approach is especially attractive when it is desirable to utilize the same mice for several phenotypic measurements (e.g., when relatively few mice of a particular genotype are available), and measurements may be taken within ~5 min/mouse. Total bone mineral density (BMD, in gm/cm²) and bone mineral content (BMC, in.mg.) values are obtained automatically from the DEXA scan and will be provided to MMPC clients as part of the dataset. Regional determinations of BMD and BMC can also be provided for a nominal data processing fee by utilizing the region-of-interest (ROI) capability of the PixiMus software. When terminal studies are conducted, whole-animal DEXA data can be complemented by determinations of adiposity (catalog item D4002) by dissection and measurement of adipose depot-specific weights (summed weights of gonadal, retroperitoneal, and femoral subcutaneous fat depots divided by live weight.)

Proper citation: Trina Knotts 2019. UC Davis - Gross Body Composition (DEXA). protocols.io https://dx.doi.org/10.17504/protocols.io.68ahhse Copy   

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Authors: Malcolm Low
Group: Mouse Metabolic Phenotyping Centers

Proper citation: Malcolm Low 2019. U Michigan - Cryoembedding. protocols.io https://dx.doi.org/10.17504/protocols.io.xidfka6 Copy   

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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 https://dx.doi.org/10.17504/protocols.io.8i7huhn Copy   

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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 https://dx.doi.org/10.17504/protocols.io.56ug9ew 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. 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 https://dx.doi.org/10.17504/protocols.io.x23fqgn Copy   

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Authors: Louise Lantier
Group: Mouse Metabolic Phenotyping Centers
Summary: After the last sample is taken for the clamp, the mouse is anesthetized in order to harvest tissues. It is very important that the mouse remain alive during tissue harvesting for future signaling or metabolite analysis. The mouse is therefore given a sub-lethal dose of pentobarbital to induce anesthesia (~70mg/kg pi or 35mg/kg iv).

Proper citation: Louise Lantier 2019. Vandy – Post Clamp Anesthesia. protocols.io https://dx.doi.org/10.17504/protocols.io.zdnf25e Copy   

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Authors: Louise Lantier
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:The minispec Body Composition Analyzer is based on Time Domain NMR. It acquires and analyzes TD-NMR signals from all protons in the entire sample volume and can provide 3 components of interest: Fat, Free Body Fluid and Lean Tissue values. Bruker's minispec Body Composition Analyzer provides a precise method for measurement of lean tissue, fat and fluid in living mice. TD-NMR uses NMR technology, providing analysis of fat tissue, lean tissue and free fluid by the same physical selection rules that give rise to contrast in MRI. Tissue contrast is high between fat and muscle based on relative relaxation times. This Magnetic Resonance technology provides our users with the opportunity to make multiple measurements during the life of the animal with the following benefits: ♦ Rapid analysis: measurement takes less than 2 minutes, no sample preparation ♦ Economical procedures: no consumables, retain expensive lab animals for entire study ♦ Reduced animal stress: no need for anesthetics; animals are measured "as-is" ♦ Allows more frequent testing, due to reduction of risks to animal health ♦ Better accuracy and precision compared to DEXA (X-ray) method

Proper citation: Louise Lantier 2019. Vandy – Body Composition in conscious mice. protocols.io https://dx.doi.org/10.17504/protocols.io.yypfxvn Copy   

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Authors: John Rutledge
Group: Mouse Metabolic Phenotyping Centers
Summary: SummaryUtilizing the TA-F10 (DSI) telemeter we can simultaneously collect temperature and activity measures in a single mouse.  This can provide a comprehensive assessment of circadian patterns, activity levels, and core body temperature in multiple interventions (drug, altered fed access, etc.) or genetic mouse models.  The following protocol describes the implantation and measurement parameters and intervals.Modified from: Butz et al. Physiol Genomics. 2001 Mar 8;5(2):89-97.

Proper citation: John Rutledge 2019. UC Davis - Temperature and activity by Telemetry. protocols.io https://dx.doi.org/10.17504/protocols.io.ywtfxen Copy   

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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 https://dx.doi.org/10.17504/protocols.io.6xnhfme Copy   

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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 https://dx.doi.org/10.17504/protocols.io.7bzhip6 Copy   

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Authors: Jennifer Rutkowsky
Group: Mouse Metabolic Phenotyping Centers
Summary: Mitochondria are the power house of the cell. They are distinct organelles with two membranes. Usually they are rod shaped, however they can be round. Several diseases and disorders including schizophrenia, bipolar disorder, dementia, Alzheimer's disease, Parkinson's disease, epilepsy, stroke, cardiovascular disease, retinitis pigmentosa, and diabetes mellitus have all shown association to mitochondrial dysfunction and disorder.

Proper citation: Jennifer Rutkowsky 2019. UC Davis - Immunohistochemistry Mitochondrial marker. protocols.io https://dx.doi.org/10.17504/protocols.io.56ng9de Copy   

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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 https://dx.doi.org/10.17504/protocols.io.yp4fvqw Copy   

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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Glucose uptake in individual organs can be measured using a bolus injection of 2-deoxy-D-[1-14C] glucose, a non-metabolizable glucose analog, and by determining labeled metabolite levels in select tissues. Insulin resistance is characterized by reduced glucose metabolism and develops in obese mice.

Proper citation: Jason Kim 2019. U Mass - Organ-specific glucose uptake. protocols.io https://dx.doi.org/10.17504/protocols.io.x4cfqsw Copy   

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Authors: Louise Lantier
Group: Mouse Metabolic Phenotyping Centers
Summary: The glucose tolerance test measures the clearance of an oral glucose load from the body. It is used to detect disturbances in glucose metabolism that can be linked to diabetes or metabolic syndrome. Animals are fasted for 5 hours, fasted blood glucose levels are determined before a solution of glucose is administered by oral gavage. Subsequently, the blood glucose and plasma insulin levels are measured at different time points during the following 120 minutes.

Proper citation: Louise Lantier 2019. Vandy – Oral glucose tolerance test. protocols.io https://dx.doi.org/10.17504/protocols.io.yz2fx8e Copy   

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Authors: Li-Ming Jin
Group: Mouse Metabolic Phenotyping Centers
Summary: Erectile function is well regulated by vasorelaxant and vasoconstrictive mechanisms, which can be perturbed by various disease states including cardiovascular disease, diabetes, and hormonal insufficiencies. This protocol enables us to asses erectile function by measuring the mean arterial blood pressure (MAP) and intracavernosal pressure (ICP) upon electrical stimulation of the cavernous nerve.Modified from: Burnett et al Science. 1992 Jul 17;257(5068):401-3.; Quinlan et al J Urol. 1989 Mar;141(3):656-61.; and Jin et al J Sex Med. 2008 Mar;5(3):544-51.

Proper citation: Li-Ming Jin 2019. UC Davis - Erectile Dysfunction. protocols.io https://dx.doi.org/10.17504/protocols.io.yikfucw Copy   

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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:Chronic indwelling catheter is placed in the jugular vein for intravenous infusion and injection of drug, hormones, and metabolites. Surival surgery is performed in anesthetized mice, and mice recover from surgery after 4~5 days.

Proper citation: Jason Kim 2019. U Mass - Surgery – jugular vein cannulation. protocols.io https://dx.doi.org/10.17504/protocols.io.x5cfq2w Copy   

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Authors: Fawaz G. Haj
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary: This test is designated to determine defects in the insulin signaling pathway, through evaluation of the activation state of the insulin receptor (IR) and its substrate (IRS1/2), as well as downstream target, mainly Akt and MAP kinases.

Proper citation: Fawaz G. Haj 2019. UC Davis - Insulin signaling pathway. protocols.io https://dx.doi.org/10.17504/protocols.io.yp2fvqe Copy   

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Authors: Jon Ramsey
Group: Mouse Metabolic Phenotyping Centers
Summary: The major fat pads of mice will be dissected and weighed. This approach provides a way to determine if an intervention has an impact on specific depots of fat.

Proper citation: Jon Ramsey 2019. UC Davis - Adiposity. protocols.io https://dx.doi.org/10.17504/protocols.io.ybsfsne Copy   

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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 https://dx.doi.org/10.17504/protocols.io.56sg9ee 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 C-reactive peptide reflect systemic inflammation and may be altered in obesity.

Proper citation: Jason Kim 2019. U Mass - C-reactive Peptide. protocols.io https://dx.doi.org/10.17504/protocols.io.xt5fnq6 Copy   

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