University of Utah View Institution's Website 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 Aye1 1Swiss 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 Li1,2, Pia Balmaceda1, Thuy Ha1, Greg W. Brown1, Robin M. Shaw1, TingTing Hong1,2 1Nora 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. Alexander1,2, Guillaume Hoareau3, Matthew S. Zabriskie1, Helen Palatinus3, Nitin Ramanujam Chakravarthula3, Chuanzhuo Wang4, M. Austin Johnson3 1Department 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. Hoareau2,3, Kai Kuck1,4, Natalie A. Silverton4,5 1Department 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. Hammond1 1Department 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 Becker1 1John 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. Bell1,2, Alberto Pauletti3, Karen S. Wilcox1,2, Sonja Bröer3 1Department 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. Shaw1 1Nora 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 Lee1 1Graduate 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. Christian2 1Department 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. Atkins1,2, Niccolo M. Fiorentino1,3, Andrew E. Anderson1,2,4,5 1Department 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. Kwan1 1Department 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. Hubbard1,2, Rahim Esfandyarpour3,4, Behrad Noudoost1 1Department 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. Warren1,3, Todd A. Wyatt2,3,4 1Department 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 Skliar1,2, Vasiliy S. Chernyshev3,4 1Department 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. Zangle1,2 1Department 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. Butson1 1Scientific 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 Gertz1 1Department 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. Brown1 1Microbiology 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. Fuller1,2, Benjamin D. Bice1,2, Ashlee R. Venancio1,2, Olivia M. Crowley1,2, Ambur M. Staab1,2, Stephanie J. Georges1,2, Julio R. Hidalgo1,2, Annika V. Warncke1,2, Melinda L. Angus-Hill1,2 1Division 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. Cross2 1Department 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 He1,3, Yan-Ting Shiu1,3, Hirofumi Tanaka2, Anthony J. Donato1,4,5,6 1Department 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. Helms2 1Department 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 Tay1,3, Jeng Yeong Chow2, Maybelle Kho Go1,3, Wen Shan Yew1,3 1Department 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 Dudek2 1Department 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. Vetter1 1Department 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. Cahalan2 1Molecular 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 Menon1 1Department 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. Dorsky1 1Department 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 Romanov1,2, Bruce K. Gale1,2, Josh W. Eckman1, Benjamin D. Brooks1 1Wasatch 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 Fu1,2 1Department 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. Pomper2,3, S. Michael Yu1,3 1Department 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 Hodges1,2, Saveez Saffarian1,2 1Department 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 Jette1 1Department 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. Metzstein1 1Department 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 Shakya1 1Department 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 Vershinin1 1Department 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. Amack1 1Department 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. Vondriska1,2,3, Sarah Franklin1,4 1Department 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. Jorgensen1 1Department 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 IV1,2, Alejandro Sánchez Alvarado3,4 1Department 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. Minteer1 1Departments 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 Ayliffe1 1Orflo 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. Creel1 1John 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 Bastiani1 1Department 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 Rosenblatt1 1Department 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. Little1 1Department 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 Poelzing1 1Bioengineering, 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. Rodesch2 1Department 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. Isaacson1,2, Jeroen G. Stinstra3, Rob S. MacLeod2,3, Joseph B. Webster1,4, James P. Beck1,5, Roy D. Bloebaum1,2,5 1Department 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.