University of Colorado School of Medicine 16 articles published in JoVE Neuroscience Functional Magnetic Resonance Imaging (fMRI) of the Visual Cortex with Wide-View Retinotopic Stimulation Andre Galenchik-Chan*1, Diane Chernoff*1, Wei Zhou2, Timothy Q. Duong3, Eric R. Muir4 1Renaissance School of Medicine at Stony Brook University, 2Department of Radiology, University of Colorado School of Medicine, 3Department of Radiology, Montefiore-Einstein, 4Department of Radiology, School of Medicine, Stony Brook University We have developed techniques for mapping the visual cortex function utilizing more of the visual field than is commonly used. This approach has the potential to enhance the evaluation of vision disorders and eye diseases. Immunology and Infection A High-Throughput Multiplexed Screening for Type 1 Diabetes, Celiac Diseases, and COVID-19 Xiaofan Jia*1, Ling He*1,2, Dongmei Miao1, Caiguo Zhang1, Marian Rewers1, Liping Yu1 1Barbara Davis Center for Diabetes, University of Colorado School of Medicine, 2Department of Endocrinology, Guangzhou First People’s Hospital, School of Medicine, South China University of Technology In line with the urgent need for screening for type 1 diabetes, celiac disease, and coronavirus disease 2019, we developed a high throughput 6-Plex electrochemiluminescence assay to simultaneously detect all four islet autoantibodies, tissue transglutaminase autoantibodies, and antibodies to the receptor binding domain of severe acute respiratory syndrome coronavirus 2. Medicine A High-Throughput Electrochemiluminescence 7-Plex Assay Simultaneously Screening for Type 1 Diabetes and Multiple Autoimmune Diseases Xiaofan Jia*1,2, Ling He*1,3, Yong Gu1, Hilary High1, Liping Yu1 1Barbara Davis Center for Diabetes, University of Colorado School of Medicine, 2Department of Endocrinology, Beijing Hospital, National Center of Gerontology; Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, 3Department of endocrinology, Guangzhou First People's Hospital, School of Medicine, South China University of Technology We model a simple multiplexed ECL assay that combines 7 autoantibody assays together. The assay is capable of screening for T1D and multiple other autoimmune diseases, simultaneously, including celiac disease, autoimmune thyroid disease, and autoimmune polyglandular syndrome 1. Biochemistry Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling Steven E. Guard1, Christopher C. Ebmeier1, William M. Old1,2 1Department of Molecular, Cellular and Developmental Biology, University of Colorado, 2Linda Crnic Institute for Down Syndrome, University of Colorado School of Medicine Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline. Developmental Biology Imaging Dpp Release from a Drosophila Wing Disc Laura Faith George1, Emily Anne Bates1 1Department of Pediatrics, University of Colorado School of Medicine The timing of exposure to ligands may impact their developmental consequences. Here we show how to image release of a Drosophila bone morphogenetic protein (BMP) called Dpp from cells of the wing disc. Developmental Biology RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells Patrick S. McGrath1,2,3, Nicole Diette1,2, Igor Kogut1,2,3, Ganna Bilousova1,2,3 1Department of Dermatology, University of Colorado School of Medicine, Anschutz Medical Campus, 2Charles C. Gates Center for Regenerative Medicine, University of Colorado School of Medicine, Anschutz Medical Campus, 3Stem Cell Biobank and Disease Modeling Core, University of Colorado School of Medicine, Anschutz Medical Campus Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60. Immunology and Infection Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry Ryan M. Baxter*1, Daniel S. Kong*1, Josselyn E. Garcia-Perez1, William E. O'Gorman2, Elena W.Y. Hsieh1,3 1Department of Immunology and Microbiology, University of Colorado School of Medicine, 2OMNI Biomarkers, Development Sciences, Genentech, 3Department of Pediatrics, Division of Allergy and Immunology, University of Colorado School of Medicine Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry. Immunology and Infection Transurethral Instillation Procedure in Adult Male Mouse Sanghee Lee*1, Alonso Carrasco Jr.*2, Randall B. Meacham1, Anna P. Malykhina1 1Division of Urology, Department of Surgery, University of Colorado School of Medicine, 2 Transurethral instillation is a challenging procedure and has not been well described in the literature. The aim of this manuscript is to describe a technique for transurethral insertion of a catheter for intravesical delivery of liquids with active substances into the urinary bladder and/or prostate in adult male mice. Medicine Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition Sidharth Tyagi1, Donald Beqollari1, Chang Seok Lee2, Lori A. Walker1, Roger A. Bannister1 1Department of Medicine-Cardiology Division, University of Colorado School of Medicine, 2Department of Molecular Physiology and Biophysics, Baylor College of Medicine Immunohistochemical staining of myosin heavy chain isoforms has emerged as the state-of-the-art discriminator of skeletal muscle fiber-type (i.e., type I, type IIA, type IIX, type IIB). Here, we present a staining protocol along with a novel semi-automated algorithm that facilitates rapid assessment of fiber-type and fiber morphology. Biology High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast Colby P. Fees1, Cassi Estrem1, Jeffrey K. Moore1 1Department of Cell and Developmental Biology, University of Colorado School of Medicine Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells. Immunology and Infection Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function Mia J. Smith1,2, Thomas A. Packard1, Shannon K. O'Neill1, Rochelle M. Hinman1, Marynette Rihanek3, Peter A. Gottlieb3, John C. Cambier1,4 1Department of Immunology and Microbiology, University of Colorado School of Medicine, 2Department of Microbiology, Immunology, and Pathology, Colorado State University, 3Barbara Davis Center for Childhood Diabetes, University of Colorado School of Medicine, 4Department of Biomedical Research, National Jewish Health A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described. Neuroscience Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors Andrea Linares1, Leena E. Couling2, Eduardo J. Carrera3, Robert C. Speth2 1Farquhar College of Arts and Sciences, Nova Southeastern University, 2Department of Pharmaceutical Sciences, College of Pharmacy, Nova Southeastern University, 3School of Medicine, University of Colorado Here we present a protocol to describe the localization of angiotensin II Type 1 receptors in the rat brain by quantitative, densitometric, in vitro receptor autoradiography using an iodine-125 labeled analog of angiotensin II. Neuroscience Laser-guided Neuronal Tracing In Brain Explants Otto Albrecht1, Achim Klug1 1Department of Physiology and Biophysics, University of Colorado School of Medicine We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy. Medicine Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors Angela M. Pierce1, Amy K. Keating1 1Department of Pediatrics, University of Colorado School of Medicine The brain is a unique site with qualities that are not well represented by in vitro or ectopic analyses. Orthotopic mouse models with reproducible location and growth characteristics can be reliably created with intracranial injections using a stereotaxic fixation instrument and a low pressure syringe pump. Behavior P50 Sensory Gating in Infants Anne Spencer Ross1,2, Sharon Kay Hunter1, Mark A Groth1, Randal Glenn Ross1 1Department of Psychiatry, University of Colorado School of Medicine, 2Colorado State University Methods to record auditory P50 sensory gating, a physiological marker of cerebral inhibition which reflects early stages of attention, are described. Neuroscience Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways Stacy L. Fairbanks*1, Rebekah Vest*1, Saurabh Verma2, Richard J. Traystman1,3, Paco S. Herson1,3 1Department of Anesthesiology, University of Colorado School of Medicine, 2Oregon National Primate Research Center, Oregon Health & Science University, 3Department of Pharmacology, University of Colorado School of Medicine Primary disassociated embryonic hippocampal neuronal cultures are useful for investigating the signaling mechanisms involved in neuron death. Sexing the embryos before the isolation and dissociation of the hippocampus allows the preparation of separate male and female cultures, which enables the researcher to identify and investigate sex-specific cell signaling.
