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University of Colorado School of Medicine

25 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry
Manira Rayamajhi 1, Elizabeth F. Redente 2, Tracy V. Condon 1, Mercedes Gonzalez-Juarrero 3, David W.H. Riches 1,2,4, Laurel L. Lenz 1,4
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.

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Medicine

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury
Michael P. Hutchens 1, Richard J. Traystman 2, Tetsuhiro Fujiyoshi 1, Shin Nakayama 1, Paco S. Herson 1
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.

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Neuroscience

Manufacturing and Using Piggy-back Multibarrel Electrodes for In vivo Pharmacological Manipulations of Neural Responses
Anna Dondzillo 1, Jennifer L. Thornton 1, Daniel J. Tollin 1, Achim Klug 1
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.

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Behavior

P50 Sensory Gating in Infants
Anne Spencer Ross 1,2, Sharon Kay Hunter 1, Mark A Groth 1, Randal Glenn Ross 1
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.

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Medicine

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer
Yimu Yang 1, Gaoqing Yang 1, Eric P. Schmidt 1
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.

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Neuroscience

Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways
Stacy L. Fairbanks *1, Rebekah Vest *1, Saurabh Verma 2, Richard J. Traystman 1,3, Paco S. Herson 1,3
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.

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Medicine

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors
Angela M. Pierce 1, Amy K. Keating 1
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.

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Neuroscience

Laser-guided Neuronal Tracing In Brain Explants
Otto Albrecht 1, Achim Klug 1
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.

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Immunology and Infection

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function
Mia J. Smith 1,2, Thomas A. Packard 1, Shannon K. O'Neill 1, Rochelle M. Hinman 1, Marynette Rihanek 3, Peter A. Gottlieb 3, John C. Cambier 1,4
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.

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Biology

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast
Colby P. Fees 1, Cassi Estrem 1, Jeffrey K. Moore 1
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.

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Medicine

Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition
Sidharth Tyagi 1, Donald Beqollari 1, Chang Seok Lee 2, Lori A. Walker 1, Roger A. Bannister 1
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.

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Immunology and Infection

Transurethral Instillation Procedure in Adult Male Mouse
Sanghee Lee *1, Alonso Carrasco Jr. *2, Randall B. Meacham 1, Anna P. Malykhina 1
1Division of Urology, Department of Surgery, University of Colorado School of Medicine, 2Pediatric Urology, Children's Hospital Colorado

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.

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Immunology and Infection

Electrochemiluminescence Assays for Human Islet Autoantibodies
Yong Gu 1,2, Zhiyuan Zhao 1, Dongmei Miao 1, Hilary High 1, Tao Yang 2, Liping Yu 1
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.

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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-Perez 1, William E. O'Gorman 2, Elena W.Y. Hsieh 1,3
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.

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Medicine

Direct Intrabronchial Administration to Improve the Selective Agent Deposition Within the Mouse Lung
Sumei Liao 1, Oliver Eickelberg 1, Eric P. Schmidt 1,2, Yimu Yang 1
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.

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Cancer Research

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts
Sanghee Lee 1,2, Danielle N. Burner 1,2, Theresa R. Mendoza 1,2, Michelle T. Muldong 1,2, Catalina Arreola 1,2, Christina N. Wu 2,3, Nicholas A. Cacalano 4, Anna A. Kulidjian 2,5, Christopher J. Kane 1,2, Christina A. M. Jamieson 1,2
1Department of Urology, University of California, San Diego, 2Moores Cancer Center, University of California, San Diego, 3Department of Medicine, University of California, San Diego, 4Department of Radiation Oncology, University of California, Los Angeles, 5Department of Orthopedic Surgery, University of California, San Diego

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

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Biochemistry

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling
Steven E. Guard 1, Christopher C. Ebmeier 1, William M. Old 1,2
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.

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Developmental Biology

Imaging Dpp Release from a Drosophila Wing Disc
Laura Faith George 1, Emily Anne Bates 1
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.

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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 Gu 1, Hilary High 1, Liping Yu 1
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.

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Biology

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining
Wells B. LaRiviere 1,2, Xiaorui Han 3,4, Kaori Oshima 1, Sarah A. McMurtry 1, Robert J. Linhardt 3, Eric P. Schmidt 1,5
1Department of Medicine, University of Colorado Anschutz Medical Campus, 2Medical Scientist Training Program, University of Colorado Anschutz Medical Campus, 3Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, 4Department of Health Sciences, Curtin University, 5Department of Medicine, Denver Health Medical Center

This report describes techniques to isolate and purify sulfated glycosaminoglycans (GAGs) from biological samples and a polyacrylamide gel electrophoresis approach to approximate their size. GAGs contribute to tissue structure and influence signaling processes via electrostatic interaction with proteins. GAG polymer length contributes to their binding affinity for cognate ligands.

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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 Miao 1, Caiguo Zhang 1, Marian Rewers 1, Liping Yu 1
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.

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Neuroscience

Coherent Anti-Stokes Raman Spectroscopy (CARS) Application for Imaging Myelination in Brain Slices
Elizabeth A. McCullagh 1, Shani Poleg 2, Dominik Stich 3, Radu Moldovan *3, Achim Klug *2
1Department of Integrative Biology, Oklahoma State University, 2Department of Physiology and Biophysics, University of Colorado Anschutz, 3Advanced Light Microscopy Core, University of Colorado Anschutz

Visualizing myelination is an important goal for many researchers studying the nervous system. CARS is a technique that is compatible with immunofluorescence that can natively image lipids within tissue such as the brain illuminating specialized structures such as myelin.

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Biology

Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria
Rihab Bouchareb 1, Liping Yu 1, Emelie Lassen 1, Ilse S. Daehn 1
1Department of Medicine, Division of Nephrology, The Icahn School of Medicine at Mount Sinai

The article describes the method for isolating conditionally immortalized glomerular endothelial cells from the kidneys of transgenic mice expressing the thermolabile simian virus 40 and photo-activatable mitochondria, PhAMexcised. We describe the procedure for glomeruli isolation from whole kidneys using beads, digestion steps, seeding, and culturing of GECs-CD31 positive.

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Neuroscience

Functional Magnetic Resonance Imaging (fMRI) of the Visual Cortex with Wide-View Retinotopic Stimulation
Andre Galenchik-Chan *1, Diane Chernoff *1, Wei Zhou 2, Timothy Q. Duong 3, Eric R. Muir 4
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.

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Neuroscience

Modeling Highly Repetitive Low-level Blast Exposure in Mice
Adam Crabtree *1, Cory McEvoy *1,2, Peter Muench 1, Rebecca A. Ivory 3,4,5, Josh Rodriguez 5, Mohamed Omer 5, Trinity Charles 5, James S. Meabon 5,6
1United States Army Special Operations Command, 2CU Anschutz Center for COMBAT Research, Department of Emergency Medicine, University of Colorado School of Medicine, 3University of Washington School of Nursing, 4University of Delaware School of Nursing, 5Veterans Affairs Northwest Mental Illness Research, Education, and Clinical Center (MIRECC), VA Puget Sound Health Care System, 6Department of Psychiatry and Behavioral Sciences, University of Washington School of Medicine

Presented here are methods for producing repeated low-intensity blast exposures using mice.

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