University of Utah

50 articles published in JoVE

• Biology

  
  Monitoring On-Target Signaling Responses in Larval Zebrafish - Z-REX Unmasks Precise Mechanisms of Electrophilic Drugs and Metabolites Kuan-Ting Huang¹, Phillippe Ly¹, Jesse R. Poganik², Saba Parvez³, Marcus J. C. Long⁴, Yimon Aye^{1 1}Swiss Federal Institute of Technology Lausanne (EPFL), ²Division of Genetics, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, ³Department of Pharmacology and Toxicology, College of Pharmacy, University of Utah, ⁴University of Lausanne (UNIL) Zebrafish targeting reactive electrophiles and oxidants (Z-REX) is a chemical biology-based method for the investigation of reactive small-molecule signaling. This technique can be applied to live fish of different developmental stages. Here, we couple standard assays in zebrafish with Z-REX for signaling pathway analysis.

• Medicine

  
  Echocardiography Recording in Awake Miniature Pigs Talli Hogen¹, Jing Li^1,2, Pia Balmaceda¹, Thuy Ha¹, Greg W. Brown¹, Robin M. Shaw¹, TingTing Hong^{1,2 1}Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, ²Department of Pharmacology and Toxicology, College of Pharmacy, University of Utah A simple cart construct, built to perform research echocardiography in standing awake minipigs, is described, along with building considerations, training techniques, and representative ultrasound images.

• Medicine

  
  Real-Time Monitoring and Modulation of Blood Pressure in a Rabbit Model of Ischemic Stroke Matthew D. Alexander^1,2, Guillaume Hoareau³, Matthew S. Zabriskie¹, Helen Palatinus³, Nitin Ramanujam Chakravarthula³, Chuanzhuo Wang⁴, M. Austin Johnson^{3 1}Department of Radiology and Imaging Sciences, University of Utah, ²Department of Neurosurgery, University of Utah, ³Department of Emergency Medicine, University of Utah, ⁴Department of Radiology, Shengjing Hospital of China Medical University Continuous arterial blood pressure recording allows the investigation of impacts of various hemodynamic parameters. This report demonstrates the application of continuous arterial blood pressure monitoring in a large animal model of ischemic stroke for determination of stroke pathophysiology, impact of different hemodynamic factors, and the assessment of novel treatment approaches.

• Bioengineering

  
  Noninvasive and Invasive Renal Hypoxia Monitoring in a Porcine Model of Hemorrhagic Shock Lars R. Lofgren¹, Guillaume L. Hoareau^2,3, Kai Kuck^1,4, Natalie A. Silverton^{4,5 1}Department of Biomedical Engineering, University of Utah, ²Department of Emergency Medicine, University of Utah, ³Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, ⁴Department of Anesthesiology, University of Utah, ⁵Geriatric Research, Education, and Clinical Centre, VAMC Presented here is a protocol to measure renal oxygenation in the medulla and noninvasive urine oxygen partial pressure in a hemorrhagic shock porcine model to establish urine oxygen partial pressure as an early indicator of acute kidney injury (AKI) and a novel resuscitative endpoint.

• Biochemistry

  
  Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers Madeline M. Mumbleau*¹, Madeline R. Meyer*¹, Ming C. Hammond^{1 1}Department of Chemistry and Henry Eyring Center for Cell & Genome Science, University of Utah The protocol presents two methods to determine the kinetics of the fluorogenic RNA aptamers Spinach2 and Broccoli. The first method describes how to measure fluorogenic aptamer kinetics in vitro with a plate reader, while the second method details the measurement of fluorogenic aptamer kinetics in cells by flow cytometry.

• Neuroscience

  
  Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes Fatima Abbas¹, Frans Vinberg¹, Silke Becker^{1 1}John A. Moran Eye Center, University of Utah Modification of existing multielectrode array or patch clamp equipment makes the ex vivo electroretinogram more widely accessible. Improved methods to record and maintain ex vivo light responses facilitate the study of photoreceptor and ON-bipolar cell function in the healthy retina, animal models of eye diseases, and human donor retinas.

• Neuroscience

  
  A Model for Epilepsy of Infectious Etiology using Theiler's Murine Encephalomyelitis Virus Gaelle Batot¹, Cameron S. Metcalf¹, Laura A. Bell^1,2, Alberto Pauletti³, Karen S. Wilcox^1,2, Sonja Bröer^{3 1}Department of Pharmacology and Toxicology, University of Utah, ²Interdepartmental Program in Neuroscience, University of Utah, ³Faculty of Veterinary Medicine, Institute of Pharmacology and Toxicology, Freie Universität Berlin Intracerebral infection with the Theiler's murine encephalomyelitis virus (TMEV) in C57BL/6 mice replicates many of the early and chronic clinical symptoms of viral encephalitis and subsequent epilepsy in human patients. This paper describes the virus infection, symptoms, and histopathology of the TMEV model.

• Biology

  
  Removal of an Internal Translational Start Site from mRNA While Retaining Expression of the Full-Length Protein Daisuke Shimura¹, Jennifer Hunter¹, Makoto Katsumata², Robin M. Shaw^{1 1}Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, ²Department of Biomedical Sciences, Cedars-Sinai Medical Center The present protocol describes a single M213L mutation in Gja1 that retains full-length Connexin43 generation but prevents translation of the smaller GJA1-20k internally translated isoform.

• Immunology and Infection

  
  Study of Dendritic Cell Development by Short Hairpin RNA-Mediated Gene Knockdown in a Hematopoietic Stem and Progenitor Cell Line In vitro Yu-Ling Hsiao¹, Hans Häcker², Chien-Kuo Lee^{1 1}Graduate Institute of Immunology, National Taiwan University College of Medicine, ²Department of Pathology, Division of Microbiology and Immunology, University of Utah Here we provide a protocol for screening potential transcription factors involved in the development of dendritic cell (DC) using lentiviral transduction of shRNA to obtain stable knockdown cell lines for in vitro DC differentiation.

• Developmental Biology

  
  Analysis of Transforming Growth Factor ß Family Cleavage Products Secreted Into the Blastocoele of Xenopus laevis Embryos Hyung-Seok Kim¹, Jan L. Christian^{2 1}Department of Neurobiology, University of Utah, ²Departments of Neurobiology and Department of Internal Medicine, Division of Hematology and Hematologic Malignancies, University of Utah, School of Medicine When Transforming Growth Factor ß family precursor proteins are ectopically expressed in Xenopus laevis embryos, they dimerize, get cleaved and are secreted into the blastocoele, which begins at the late blastula to early gastrula stage. We describe a method for aspirating cleavage products from the blastocoele cavity for immunoblot analysis.

• Medicine

  
  In Vivo Quantification of Hip Arthrokinematics during Dynamic Weight-bearing Activities using Dual Fluoroscopy Penny R. Atkins^1,2, Niccolo M. Fiorentino^1,3, Andrew E. Anderson^{1,2,4,5 1}Department of Orthopaedics, University of Utah, ²Scientific Computing and Imaging Institute, University of Utah, ³Department of Mechanical Engineering, University of Vermont, ⁴Department of Biomedical Engineering, University of Utah, ⁵Department of Physical Therapy, University of Utah Dual fluoroscopy accurately captures in vivo dynamic motion of human joints, which can be visualized relative to reconstructed anatomy (e.g., arthrokinematics). Herein, a detailed protocol to quantify hip arthrokinematics during weight-bearing activities of daily living is presented, including the integration of dual fluoroscopy with traditional skin marker motion capture.

• Neuroscience

  
  4-Dimensional Imaging of Zebrafish Optic Cup Morphogenesis Sarah Lusk¹, Macaulie A. Casey¹, Kristen M. Kwan^{1 1}Department of Human Genetics, University of Utah This protocol describes an approach for in toto labeling and multidimensional imaging of zebrafish early eye development. We describe labeling, embedding, and four dimensional (4D) imaging using laser scanning confocal microscopy, and considerations for optimizing acquisition of datasets for dissecting mechanisms of optic cup morphogenesis.

• Neuroscience

  
  Microinjectrode System for Combined Drug Infusion and Electrophysiology M. Isabel Vanegas¹, Kenneth R. Hubbard^1,2, Rahim Esfandyarpour^3,4, Behrad Noudoost^{1 1}Department of Ophthalmology and Visual Sciences, University of Utah, ²Department of Biomedical Engineering, University of Utah, ³Department of Electrical Engineering and Computer Science, University of California, Irvine, ⁴Department of Biomedical Engineering, University of California, Irvine We present a microinjectrode system designed for electrophysiology and assisted delivery of experimental probes (i.e., nanosensors, microelectrodes), with optional drug infusion. Widely available microfluidic components are coupled to a cannula containing the probe. A step-by-step protocol for microinjectrode construction is included, with results during muscimol infusion in macaque cortex.

• Immunology and Infection

  
  Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System Kristi J. Warren^1,3, Todd A. Wyatt^{2,3,4 1}Department of Internal Medicine, University of Utah, ²Research Service, VA Nebraska Iowa Health Care System, ³Department of Internal Medicine, University of Nebraska Medical Center, ⁴Department of Environmental, Agricultural & Occupational Health, University of Nebraska Medical Center In this protocol, lymphocytes are placed in the top chamber of a transmigration system, separated from the bottom chamber by a porous membrane. Chemokine is added to the bottom chamber, which induces active migration along a chemokine gradient. After 48 h, lymphocytes are counted in both chambers to quantitate transmigration.

• Biochemistry

  
  Imaging of Extracellular Vesicles by Atomic Force Microscopy Mikhail Skliar^1,2, Vasiliy S. Chernyshev^{3,4 1}Department of Chemical Engineering, University of Utah, ²The Nano Institute of Utah, University of Utah, ³Center for Photonics and Quantum Materials, Skolkovo Institute of Science and Technology, ⁴Biopharmaceutical Cluster 'Northern', Moscow Institute of Physics and Technology A step-by-step procedure is described for label-free immobilization of exosomes and extracellular vesicles from liquid samples and their imaging by atomic force microscopy (AFM). The AFM images are used to estimate the size of the vesicles in the solution and characterize other biophysical properties. 

• Engineering

  
  Fabrication of Refractive-index-matched Devices for Biomedical Microfluidics Edward R. Polanco¹, Nicholas Western¹, Thomas A. Zangle^{1,2 1}Department of Chemical Engineering, University of Utah, ²Huntsman Cancer Institute, University of Utah This protocol describes the fabrication of microfluidic devices from MY133-V2000 to eliminate artifacts that often arise in microchannels due to the mismatching refractive indices between microchannel structures and an aqueous solution. This protocol uses an acrylic holder to compress the encapsulated device, improving adhesion both chemically and mechanically.

• Bioengineering

  
  Targeting Neuronal Fiber Tracts for Deep Brain Stimulation Therapy Using Interactive, Patient-Specific Models Andrew P. Janson¹, Christopher R. Butson^{1 1}Scientific Computing and Imaging (SCI) Institute, Department of Biomedical Engineering, University of Utah The goal of this project is to develop an interactive, patient-specific modeling pipeline to simulate the effects of deep brain stimulation in near real-time and provide meaningful feedback as to how these devices influence neural activity in the brain.

• Genetics

  
  Dissection of Enhancer Function Using Multiplex CRISPR-based Enhancer Interference in Cell Lines Julia B. Carleton¹, Kristofer C. Berrett¹, Jason Gertz^{1 1}Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah This protocol describes the steps needed to design and perform multiplexed targeting of enhancers with the deactivating fusion protein SID4X-dCas9-KRAB, also known as enhancer interference (Enhancer-i). This protocol enables the identification of enhancers that regulate gene expression and facilitates the dissection of relationships between enhancers regulating a common target gene.

• Genetics

  
  High-throughput Identification of Synergistic Drug Combinations by the Overlap² Method Morgan A. Wambaugh¹, Jessica C. S. Brown^{1 1}Microbiology and Immunology Division, Department of Pathology, University of Utah Synergistic drug combinations are difficult and time-consuming to identify empirically. Here, we describe a method for identifying and validating synergistic small molecules.

• Cancer Research

  
  A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model Andrew K. Fuller^1,2, Benjamin D. Bice^1,2, Ashlee R. Venancio^1,2, Olivia M. Crowley^1,2, Ambur M. Staab^1,2, Stephanie J. Georges^1,2, Julio R. Hidalgo^1,2, Annika V. Warncke^1,2, Melinda L. Angus-Hill^{1,2 1}Division of Gastroenterology, Hepatology, and Nutrition, Department of Internal Medicine, University of Utah, ²Huntsman Cancer Institute, University of Utah Environmental Enrichment (EE) is an animal housing environment that is used to reveal mechanisms that underlie the connections between lifestyle, stress, and disease. This protocol describes a procedure that uses a mouse model of colon tumorigenesis and EE to specifically define alterations in microbiota biodiversity that may impact animal mortality.

• Behavior

  
  Assessing Spatial Memory Impairment in a Mouse Model of Traumatic Brain Injury Using a Radial Water Tread Maze Marcella M. Cline¹, Megan A. Ostlie², Chloe G. Cross³, Gregory G. Garwin², Satoshi Minoshima², Donna J. Cross^{2 1}Department of Radiology, University of Washington, ²Department of Radiology, University of Utah, ³Geriatric Research Education and Clinical Center (GRECC), VA Puget Sound Here we present a protocol for a mouse-specific test of cognition that does not require swimming. This test can be used to successfully distinguish controlled cortical impact-induced traumatic brain injury mice from sham controls.

• Medicine

  
  Ultrasound Assessment of Flow-Mediated Dilation of the Brachial and Superficial Femoral Arteries in Rats Daniel R. Machin¹, Miriam E. Leary², Yuxia He^1,3, Yan-Ting Shiu^1,3, Hirofumi Tanaka², Anthony J. Donato^{1,4,5,6 1}Department of Internal Medicine, University of Utah, ²Department of Kinesiology and Health Education, University of Texas at Austin, ³Division of Nephrology and Hypertension, University of Utah, ⁴Department of Biochemistry, University of Utah, ⁵Department of Exercise and Sport Science, University of Utah, ⁶Geriatric Research Education and Clinical Center, Department of Veterans Affairs Non-invasive assessment of endothelial function in humans can be determined by the flow-mediated dilation technique. Although thousands of studies have used this technique, no study has performed this technique non-invasively in rats. The following article describes non-invasive measurement of flow-mediated dilation in the brachial and superficial femoral arteries of rats.

• Medicine

  
  Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung Ashley E. Van Avermaete¹, Phi T. Trac¹, Theresa W. Gauthier¹, My N. Helms^{2 1}Department of Pediatrics, Neonatology Division, Emory Children's Center, Emory University, ²Department of Internal Medicine, Pulmonary Division, University of Utah We present a protocol to assess the rate of alveolar fluid clearance or pulmonary edema in neonatal mouse lung using X-ray imaging technology.

• Immunology and Infection

  
  Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases Song Buck Tay^1,3, Jeng Yeong Chow², Maybelle Kho Go^1,3, Wen Shan Yew^{1,3 1}Department of Biochemistry, Yong Loo Lin School of Medicine, ²Department of Chemistry, University of Utah, ³Synthetic Biology Research Consortium, National University of Singapore Quorum-quenching enzymes are anti-virulent and anti-bacterial options that can mitigate pathogenesis without risk of incurring resistance, by preventing the expression of virulence factors and genes associated with antibiotic resistance and biofilm formation. In this study, we report a method that demonstrates the efficacy of quorum-quenching enzymes in bacterial biofilm disruption.

• Neuroscience

  
  Long-term Continuous EEG Monitoring in Small Rodent Models of Human Disease Using the Epoch Wireless Transmitter System Andrew Zayachkivsky¹, Mark J. Lehmkuhle², F. Edward Dudek^{2 1}Department of Neurosurgery, Yale University School of Medicine, ²Department of Neurosurgery, University of Utah Here we demonstrate the use of a wireless enabling technology for electroencephalogram (EEG) in neonatal rodent models of human disease. With telemetry, there are no encumbering connections, thus allowing natural behaviors.

• Medicine

  
  In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma Alejandra Bosco¹, Cesar O. Romero¹, Balamurali K. Ambati², Monica L. Vetter^{1 1}Department of Neurobiology & Anatomy, University of Utah, ²Department of Ophthalmology & Visual Sciences, University of Utah Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

• Neuroscience

  
  Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord Jason G. Weinger¹, Milton L. Greenberg², Melanie P. Matheu⁴, Ian Parker³, Craig M. Walsh¹, Thomas E. Lane⁵, Michael D. Cahalan^{2 1}Molecular Biology and Biochemistry, University of California, Irvine, ²Physiology and Biophysics, University of California, Irvine, ³Neurobiology and Behavior, University of California, Irvine, ⁴University of California San Francisco Diabetes Center, University of California, San Francisco, ⁵Pathology, University of Utah A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

• Engineering

  
  Patterning via Optical Saturable Transitions - Fabrication and Characterization Precious Cantu¹, Trisha L. Andrew², Rajesh Menon^{1 1}Department of Electrical and Computer Engineering, The University of Utah, ²Department of Chemistry, The University of Wisconsin-Madison We report that the diffraction limit of conventional optical lithography can be overcome by exploiting the transitions of organic photochromic derivatives induced by their photoisomerization at low light intensities.^1-3 This paper outlines our fabrication technique and two locking mechanisms, namely: dissolution of one photoisomer and electrochemical oxidation.

• Neuroscience

  
  Spinal Cord Transection in the Larval Zebrafish Lisa K. Briona¹, Richard I. Dorsky^{1 1}Department of Neurobiology & Anatomy, University of Utah After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2 days post-injury, and C-bend movement by 3 days post-injury.

• Bioengineering

  
  The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter Sherry N. Davidoff¹, Adam R. Miles¹, Valentin Romanov^1,2, Bruce K. Gale^1,2, Josh W. Eckman¹, Benjamin D. Brooks^{1 1}Wasatch Microfluidics, ²Department of Mechanical Engineering, University of Utah This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

• Medicine

  
  Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography Sandeep Kumar¹, Zachary Berriochoa¹, Alex D. Jones¹, Yingbin Fu^{1,2 1}Department of Ophthalmology & Visual Sciences, University of Utah Health Sciences Center, ²Department of Neurobiology & Anatomy, University of Utah Health Sciences Center Indocyanine Green Angiography (or ICGA) performed by tail vein injection provides high quality ICGA time course images to characterize abnormalities in mouse choroid.

• Bioengineering

  
  Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides Yang Li¹, Catherine A. Foss², Martin G. Pomper^2,3, S. Michael Yu^{1,3 1}Department of Bioengineering, University of Utah, ²Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, ³Institute for NanoBiotechnology, Johns Hopkins University This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

• Biology

  
  Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging Jeffery A Hodges^1,2, Saveez Saffarian^{1,2 1}Department of Physics & Astronomy, University of Utah, ²Center for Cell and Genome, University of Utah The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

• Biology

  
  Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3 Shelly Sorrells¹, Cristhian Toruno¹, Rodney A. Stewart¹, Cicely Jette^{1 1}Department of Oncological Sciences, University of Utah Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

• Biology

  
  Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy Tiffani A Jones¹, Mark M. Metzstein^{1 1}Department of Human Genetics, University of Utah Here, we present a method for light microscopy analysis of tracheal terminal cells in Drosophila larvae. This method allows for quick examination of branch and lumen morphology in whole animals and would be useful for analysis of individual mutants or screens for mutations affecting terminal cell development.

• Biology

  
  Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass Dean Tantin¹, Warren P. Voth¹, Arvind Shakya^{1 1}Department of Pathology, University of Utah School of Medicine We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.

• Engineering

  
  Construction of a High Resolution Microscope with Conventional and Holographic Optical Trapping Capabilities Jacqualine Butterfield¹, Weili Hong¹, Leslie Mershon¹, Michael Vershinin^{1 1}Department of Physics and Astronomy, University of Utah The system described herein employs a traditional optical trap as well as an independent holographic optical trapping line, capable of creating and manipulating multiple traps. This allows for the creation of complex geometric arrangements of refractive particles while also permitting simultaneous high-speed, high-resolution measurements of the activity of biological enzymes.

• Biology

  
  Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle Guangliang Wang¹, H. Joseph Yost², Jeffrey D. Amack^{1 1}Department of Cell and Developmental Biology, State University of New York, Upstate Medical University, ²Department of Neurobiology and Anatomy, Eccles Institute of Human Genetics, University of Utah Cilia-generated fluid flow in Kupffer’s Vesicle (KV) controls left-right patterning of the zebrafish embryo. Here, we describe a technique to modulate gene function specifically in KV cells. In addition, we show how to deliver fluorescent beads into KV to visualize fluid flow.

• Medicine

  
  Quantitative Analysis of Chromatin Proteomes in Disease Emma Monte¹, Haodong Chen¹, Maria Kolmakova¹, Michelle Parvatiyar¹, Thomas M. Vondriska^1,2,3, Sarah Franklin^{1,4 1}Department of Anesthesiology, David Geffen School of Medicine at UCLA, ²Department of Medicine, David Geffen School of Medicine at UCLA, ³Department of Physiology, David Geffen School of Medicine at UCLA, ⁴Department of Internal Medicine, Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

• Biology

  
  Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy Shigeki Watanabe¹, Jackson Richards¹, Gunther Hollopeter¹, Robert J. Hobson¹, Wayne M. Davis¹, Erik M. Jorgensen^{1 1}Department of Biology, Howard Hughes Medical Institute, University of Utah We describe a method to localize fluorescently tagged proteins in electron micrographs. Fluorescence is first localized using photo-activated localization microscopy on ultrathin sections. These images are then aligned to electron micrographs of the same section.

• Biology

  
  Planarian Immobilization, Partial Irradiation, and Tissue Transplantation Otto C. Guedelhoefer IV^1,2, Alejandro Sánchez Alvarado^{3,4 1}Department of Neurobiology and Anatomy, University of Utah School of Medicine, ²Department of Molecular, Cellular and Developmental Biology, UCSB, ³Howard Hughes Medical Institute, ⁴Stowers Institute for Medical Research An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

• Bioengineering

  
  Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization Shannon Meredith¹, Shuai Xu¹, Matthew T. Meredith¹, Shelley D. Minteer^{1 1}Departments of Chemistry and Materials Science and Engineering, University of Utah This article will describe the procedure for synthesizing a hydrophobically modified Nafion enzyme immobilization membrane and how to immobilize proteins and/or enzymes within the membrane and test their specific activity.

• Biology

  
  Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter Gregory M. Dittami¹, Manju Sethi¹, Richard D. Rabbitt², H. Edward Ayliffe^{1 1}Orflo Technologies, ²University of Utah The Moxi Z miniature automated cell counter is a novel instrument that combines the Coulter Principle with patented thin-film sensor technology and a proprietary software algorithm to perform sizing and counting of a broad size range of particles as well as to determine the overall health of monodisperse mammalian cell cultures. This protocol describes the use of this instrument for counting and assessing the health of cell cultures.

• Medicine

  
  Multifocal Electroretinograms Donnell J. Creel^{1 1}John A. Moran Eye Center, University of Utah The development of the multifocal electroretinogram (mfERG) is an important advance in the diagnosis and characterization of retinopathy. Multifocal electroretinograms are a mathematical average of an approximation of a b-wave. Software programs can derive ERGs from more than a hundred retinal areas in a few minutes per eye. Scotomas and retinal dysfunction can be mapped and quantified.

• Bioengineering

  
  Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans Wes Williams¹, Paola Nix¹, Michael Bastiani^{1 1}Department of Biology, University of Utah Laser axotomy followed by time-lapse imaging is a sensitive way to assay the effects of mutations in C. elegans on axon regeneration. A high quality, but inexpensive, laser ablation system can be easily added to most microscopes. Time lapse imaging over 15 hours requires careful immobilization of the worm.

• Biology

  
  Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish George T. Eisenhoffer¹, Jody Rosenblatt^{1 1}Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

• Medicine

  
  Methods for ECG Evaluation of Indicators of Cardiac Risk, and Susceptibility to Aconitine-induced Arrhythmias in Rats Following Status Epilepticus Steven L. Bealer¹, Cameron S. Metcalf¹, Jason G. Little^{1 1}Department of Pharmacology and Toxicology, University of Utah Techniques for measurement of electrical activity of the heart by electrocardiogram (ECG), and analysis of cardiac risk factors and susceptibility to arrhythmias following status epilepticus (SE) in the rat are described.

• Biology

  
  Recapitulation of an Ion Channel IV Curve Using Frequency Components John R. Rigby¹, Steven Poelzing^{1 1}Bioengineering, University of Utah There are technical obstacles to measuring current flux through multiple ion channels simultaneously, and later discerning what portion of the transmembrane current is due to each channel type. To address this need, this method presents a way to generate the IV curve of individual channel types using specific frequency components.

• Biology

  
  Time-lapse Imaging of Mitosis After siRNA Transfection Douglas R. Mackay¹, Katharine S. Ullman¹, Christopher K. Rodesch^{2 1}Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, ²Fluorescence Microscopy Core Facility, University of Utah Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

• Biology

  
  Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees Brad M. Isaacson^1,2, Jeroen G. Stinstra³, Rob S. MacLeod^2,3, Joseph B. Webster^1,4, James P. Beck^1,5, Roy D. Bloebaum^{1,2,5 1}Department of Veteran Affairs, ²Department of Bioengineering, University of Utah, ³Scientific Computing and Imaging Institute , University of Utah, ⁴Department of Physical Medicine and Rehabilitation, University of Utah, ⁵Department of Orthopaedics, University of Utah There is a need to develop alternative prosthesis attachment due to limb loss attributed to vascular occlusive diseases and trauma. The goal of the work is to introduce an osseointegrated intelligent implant design system to increase skeletal fixation and reduce periprosthetic infection rates for patients needing osseointegrated technology.