University of Colorado, Denver View Institution's Website 30 articles published in JoVE Biochemistry Identification of RNA Fragments Resulting from Enzymatic Degradation using MALDI-TOF Mass Spectrometry Shawn W. Schowe1, Conner J. Langeberg2, Erich G. Chapman2, Kitty Brown3, Marino J. E. Resendiz1 1Department of Chemistry, University of Colorado, Denver, 2Department of Chemistry & Biochemistry, University of Denver, 3Analytical Resources Core Bioanalysis & Omics, Colorado State University MALDI-TOF was used to characterize fragments obtained from the reactivity between oxidized RNA and the exoribonuclease Xrn-1. The present protocol describes a methodology that can be applied to other processes involving RNA and/or DNA. Immunology and Infection High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model Howard W. Davidson1, Joseph Ray Cepeda2, Nitin S. Sekhar2, Junying Han2, Ling Gao3, Tomasz Sosinowski1, Li Zhang2 1Barbara Davis Center for Childhood Diabetes, University of Colorado Denver, 2Department of Medicine, Endocrinology, Diabetes & Metabolism, Baylor College of Medicine, 3Scientific Center, Shandong Provincial Hospital affiliated to Shandong University This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo. Medicine Direct Intrabronchial Administration to Improve the Selective Agent Deposition Within the Mouse Lung Sumei Liao1, Oliver Eickelberg1, Eric P. Schmidt1,2, Yimu Yang1 1Department of Medicine, University of Colorado Denver, 2Department of Medicine, Denver Health Medical Center Intratracheal (IT) administration of experimental agents in mice often results in asymmetric delivery to the distal lungs. In this report, we describe a direct intrabronchial (IB) approach to cannulate each lung in living mice non-operatively. This approach can be used to selectively administer agents to one lung or may be adapted to improve the symmetric agent delivery to both lungs. Medicine Generation of Human 3D Lung Tissue Cultures (3D-LTCs) for Disease Modeling Michael Gerckens1,2,3, Hani N. Alsafadi4,5,6, Darcy E. Wagner4,5,6, Michael Lindner2,7, Gerald Burgstaller*1,2,3, Melanie Königshoff*1,2,3,8 1Comprehensive Pneumology Center, Ludwig-Maximilians-Universität and Helmholtz Zentrum Munich, 2German Center of Lung Research (DZL), 3Translational Lung Research and CPC-M bioArchive, Helmholtz Zentrum München, Comprehensive Pneumology Center Munich DZL/CPC-M, 4Department of Experimental Medical Science, Lung Bioengineering and Regeneration, Lund University, 5Wallenberg Center for Molecular Medicine, Lund University, 6Stem Cell Centre, Lund University, 7Asklepios Fachkliniken Munich-Gauting, 8Department of Medicine, Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Here, we present a protocol for the preparation of agarose-filled human precision-cut lung slices from resected patient tissue that are suitable for generating 3D lung tissue cultures to model human lung diseases in biological and biomedical studies. Medicine An Optimized Evans Blue Protocol to Assess Vascular Leak in the Mouse Marilee J. Wick1,2, Julie W. Harral1,2, Zoe L. Loomis1,2, Edward C. Dempsey1,2,3 1Cardiovascular Pulmonary Research Laboratory, University of Colorado Denver, 2Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Denver, 3Denver VA Medical Center In this article, an economical, optimized, and simple protocol is described which uses the Evans blue dye method for assessing plasma extravasation in the organs of FVBN mice that can be adapted for use in other strains, species, and other organs or tissues. Neuroscience Where You Cut Matters: A Dissection and Analysis Guide for the Spatial Orientation of the Mouse Retina from Ocular Landmarks Katelyn B. Sondereker1, Maureen E. Stabio2, Jenna R. Jamil1, Matthew J. Tarchick1, Jordan M. Renna1 1Department of Biology, The University of Akron, 2Department of Cell and Developmental Biology, University of Colorado Denver This protocol provides a comprehensive dissection and analysis guide for the use of deep ocular landmarks, s-opsin immunohistochemistry, Retistruct, and custom code to accurately and reliably orient the isolated mouse retina in anatomical space. Immunology and Infection Electrochemiluminescence Assays for Human Islet Autoantibodies Yong Gu1,2, Zhiyuan Zhao1, Dongmei Miao1, Hilary High1, Tao Yang2, Liping Yu1 1Barbara Davis Center for Childhood Diabetes, University of Colorado Denver, 2Department of Endocrinology, First Affiliated Hospital of Nanjing Medical University Here, we presented the methods to detect human islet autoantibodies using electrochemiluminescence (ECL) assays. The protocol, used to predict type 1 diabetes, can be expanded upon to detect autoantibodies for other autoimmune diseases. Chemistry Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism Andrew J. Francis1, Marino J. E. Resendiz1 1Department of Chemistry, University of Colorado Denver This article provides a detailed procedure on the solid-phase synthesis, purification, and characterization of dodecamers of RNA modified at the C2'-O-position. UV-vis and circular dichroism photometric analyses are used to quantify and characterize structural aspects, i.e., single-strands or double-strands. Neuroscience Spinal Cord Neurons Isolation and Culture from Neonatal Mice Mohamed Eldeiry1, Katsuhiro Yamanaka1, T. Brett Reece1, Muhammad Aftab1 1Department of Surgery, Division of Cardiothoracic Surgery, University of Colorado Anschutz Medical Campus This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage. Chemistry Synthesis of Programmable Main-chain Liquid-crystalline Elastomers Using a Two-stage Thiol-acrylate Reaction Mohand O. Saed1, Amir H. Torbati1,2, Devatha P. Nair2, Christopher M. Yakacki1 1Department of Mechanical Engineering, University of Colorado Denver, 2Department of Ophthalmology, University of Colorado Denver A novel methodology is presented to synthesize and program main-chain liquid-crystalline elastomers using commercially available starting monomers. A wide range of thermomechanical properties was tailored by adjusting the amount of crosslinker, while the actuation performance was dependent on the amount of applied strain during programming. Developmental Biology Forward Genetic Approach to Uncover Stress Resistance Genes in Mice — A High-throughput Screen in ES Cells Michael Ludwig1, David Kitzenberg1, Wallace S. Chick1 1Department of Cell and Developmental Biology, University of Colorado Denver Stress resistance is one of the hallmarks for longevity and is known to be genetically governed. Here, we developed an unbiased high-throughput method to screen for mutations that confer stress resistance in ES cells with which to develop mouse models for longevity studies. Medicine Murine Kidney Transplant Technique Robert Plenter1,2, Swati Jain3, Chelsea M. Ruller4, Trevor L. Nydam4, Alkesh H. Jani3,5 1Colorado Center for Transplantation Care, Research and Education, University of Colorado, Denver, 2Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado, Denver, 3Department of Medicine, Division of Renal Diseases and Hypertension, Medical Center and University of Colorado, Denver, 4Department of Surgery, Division of Transplant Surgery, University of Colorado School of Medicine, University of Colorado-Denver, 5Renal Section, Denver Veterans Affairs Medical Center The goal of this manuscript is to describe the steps required to perform a kidney transplant in a mouse, paying particular attention to the details of the arterial anastomosis. Neuroscience Denver Papillae Protocol for Objective Analysis of Fungiform Papillae Tiffany M. Nuessle1, Nicole L. Garneau1, Meghan M. Sloan1, Stephanie A. Santorico2 1Health Sciences Department, Denver Museum of Nature & Science, 2Department of Mathematical and Statistical Sciences, University of Colorado Denver Here we present a protocol to measure fungiform papilla density from digital photographs. This method builds prioritization and objective characteristic metrics into the original descriptive work on fungiform papillae by Miller & Reedy (1990). Biology Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples Yan Wei Lim1, Matthew Haynes2, Mike Furlan1, Charles E. Robertson3, J. Kirk Harris4, Forest Rohwer1 1Department of Biology, San Diego State University, 2DOE Joint Genome Institute, 3Department of Molecular, Cellular and Developmental Biology, University of Colorado, 4Department of Pediatrics, School of Medicine, University of Colorado Using the cystic fibrosis airway as an example, the manuscript presents a comprehensive workflow comprising a combination of metagenomic and metatranscriptomic approaches to characterize the microbial and viral communities in animal-associated samples. Medicine Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats Gert Blueschke*1, Gabi Hanna*2, Andrew N. Fontanella2, Gregory M. Palmer2, Alina Boico2, Hooney Min2, Mark W. Dewhirst2, David C. Irwin3, Yulin Zhao2, Thies Schroeder4 1Division of Plastic, Maxillofacial, and Oral Surgery, Duke University Medical Center, 2Department of Radiation Oncology, Duke University Medical Center, 3Department of Cardiology, University of Colorado Denver, 4Department of Physical Chemistry, University of Mainz A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents. Biology Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing Jerome E. Lee1, Rui Yi1,2 1Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, 2Linda Crnic Institute for Down Syndrome, University of Colorado, Denver MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs. Bioengineering Multi-step Preparation Technique to Recover Multiple Metabolite Compound Classes for In-depth and Informative Metabolomic Analysis Charmion Cruickshank-Quinn1, Kevin D. Quinn1, Roger Powell1, Yanhui Yang1, Michael Armstrong1, Spencer Mahaffey2, Richard Reisdorph1, Nichole Reisdorph1 1Department of Immunology, National Jewish Health, 2Department of Pharmacology, School of Medicine, University of Colorado Denver The reliability of results in metabolomics experiments depends on the effectiveness and reproducibility of the sample preparation. Described is a rigorous and in-depth method that enables extraction of metabolites from biological fluids with the option of subsequently analyzing up to thousands of compounds, or just the compound classes of interest. Medicine Murine Heterotopic Heart Transplant Technique Robert J. Plenter1, Todd J. Grazia1 1Colorado Center for Transplantation Care, Research and Education and Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Denver The manuscript describes the steps required to perform the heterotopic heart transplant in the mouse. Medicine Assessment of Vascular Function in Patients With Chronic Kidney Disease Kristen L. Jablonski1, Emily Decker1, Loni Perrenoud1, Jessica Kendrick1, Michel Chonchol1, Douglas R. Seals2, Diana Jalal1 1Division of Renal Diseases and Hypertension, University of Colorado, Denver, 2Department of Integrative Physiology, University of Colorado, Boulder The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression. Bioengineering In vitro Cell Culture Model for Toxic Inhaled Chemical Testing Shama Ahmad1, Aftab Ahmad1, Keith B. Neeves2, Tara Hendry-Hofer1, Joan E. Loader1, Carl W. White1, Livia Veress1 1Pediatric Airway Research Center, Department of Pediatrics, University of Colorado, 2Department of Chemical and Biological Engineering, Colorado School of Mines This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents. Environment High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities Colin W. Bell1, Barbara E. Fricks1, Jennifer D. Rocca1, Jessica M. Steinweg2, Shawna K. McMahon3, Matthew D. Wallenstein1 1Natural Resource Ecology Laboratory, Colorado State University, 2Biosciences Division, Oak Ridge National Laboratory, 3Department of Bioengineering, University of Colorado To measure potential rates of soil extracellular enzyme activities, synthetic substrates that are bound to a fluorescent dye are added to soil samples. Enzyme activity is measured as the fluorescent dye is released from the substrate by an enzyme-catalyzed reaction, where higher fluorescence indicates more substrate degradation. Neuroscience Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture Ron Bouchard1,2, Thomas Chong1,2, Subbiah Pugazhenthi1,2 1Section of Endocrinology, Denver VA Medical Center, 2Department of Medicine, University of Colorado Denver School of Medicine Human neuroprogenitor cells (NPCs) were expanded under proliferating conditions. NPCs were differentiated into neuron-rich cultures in the presence of a combination of neurotrophins. Neuronal markers were detected by immunofluorescence staining. To isolate a pure population of neurons, NPCs were differentiated on PEN membrane slides and laser capture microdissection was performed. Biology Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme Hong Li1, Trevor Williams1,2 1Department of Craniofacial Biology, University of Colorado Denver Anschutz Medical Campus, 2Department of Cell and Developmental Biology, University of Colorado Denver Anschutz Medical Campus A protocol for separation of embryo facial ectoderm and mesenchyme is described. We use Dispase II to treat whole embryos first, dissect whole facial prominences out, and then separate the facial ectoderm and mesenchyme. Medicine In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer Yimu Yang1, Gaoqing Yang1, Eric P. Schmidt1 1Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado School of Medicine The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung. Neuroscience Manufacturing and Using Piggy-back Multibarrel Electrodes for In vivo Pharmacological Manipulations of Neural Responses Anna Dondzillo1, Jennifer L. Thornton1, Daniel J. Tollin1, Achim Klug1 1Department of Physiology & Biophysics, University of Colorado Medical Campus Iontophoresis of neural agonists and antagonists during extracellular in vivo recordings is a powerful way to manipulate a neuron’s microenvironment. These manipulations can most easily be done via piggy-back multibarrel electrodes. Here we describe how to manufacture them and use them during auditory recordings. Medicine Use of a Hanging-weight System for Liver Ischemia in Mice Michael Zimmerman1, Eunyoung Tak2, Maria Kaplan1, Mercedes Susan Mandell2, Holger K. Eltzschig2, Almut Grenz2 1UCH Transplant Center, University of Colorado, Denver, 2Department of Anesthesiology, University of Colorado, Denver We established a novel murine model of a hanging weight system for portal triad occlusion. This technique may be useful for future investigations of ischemia in murine hepatic models. Biology Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells Kelsey S. Chow1,2, DuHyun Jun1,2, Karen M. Helm3, David H. Wagner1,2,4, Susan M. Majka1,2,3 1Charles C. Gates Regenerative Medicine and Stem Cell Biology Program, University of Colorado Denver, 2Department of Medicine, University of Colorado Denver, 3Cancer Center, University of Colorado Denver, 4Webb Waring Institute, University of Colorado Denver In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties. Medicine Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury Michael P. Hutchens1, Richard J. Traystman2, Tetsuhiro Fujiyoshi1, Shin Nakayama1, Paco S. Herson1 1Department of Anesthesiology and Perioperative Medicine, Oregon Health & Sciences University, 2Department of Pharmacology, University of Colorado Denver A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI. Immunology and Infection Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry Manira Rayamajhi1, Elizabeth F. Redente2, Tracy V. Condon1, Mercedes Gonzalez-Juarrero3, David W.H. Riches1,2,4, Laurel L. Lenz1,4 1Department of Immunology, University of Colorado School of Medicine, 2Division of Cell Biology, Department of Pediatrics, National Jewish Health, 3Department of Microbiology, Immunology, and Pathology, Colorado State University, 4Department of Immunology, National Jewish Health We illustrate non-surgical delivery of test materials into the lungs of anesthetized mice via the trachea. This method permits lung exposure to bacterial and viral pathogens, cytokines, antibodies, beads, chemicals, or dyes. We further describe harvesting and processing of lungs and lung draining lymph nodes (LDLNs) for flow cytometry. Medicine Use of a Hanging Weight System for Coronary Artery Occlusion in Mice Tobias Eckle1, Michael Koeppen1, Holger Eltzschig1 1Department of Anesthesiology, University of Colorado Denver A murine model for myocardial ischemia and ischemic preconditioning is an important tool study cardioprotective mechanisms in vivo. Here, we report an easy applicable in situ model for cardiac IP using a hanging-weight system for coronary artery occlusion.