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University of Colorado Anschutz Medical Campus

31 ARTICLES PUBLISHED IN JoVE

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Behavior

Method for Simultaneous fMRI/EEG Data Collection during a Focused Attention Suggestion for Differential Thermal Sensation
Pamela K. Douglas 1,2, Maureen Pisani 2, Rory Reid 1, Austin Head 2, Edward Lau 2, Ebrahim Mirakhor 3, Jennifer Bramen 2, Billi Gordon 2, Ariana Anderson 2, Wesley T. Kerr 2, Chajoon Cheong 4, Mark S. Cohen 1,2
1Neuropsychiatric Institute, University of California, Los Angeles, 2Laboratory of Neuroimaging Technology, University of California, Los Angeles, 3Yale School of Medicine, 4Korean Basic Science Institute

We present a protocol for concurrent collection of EEG/fMRI data, and synchronized MR clock signal recording. We demonstrate this method using a unique paradigm whereby subjects receive ‘cold glove’ instructions during scanning, and EEG/fMRI data are recorded along with hand temperature measurements both before and after hypnotic induction.

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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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JoVE Journal

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia
Sandra Christoph 1,2, Alisa B. Lee-Sherick 1, Susan Sather 1, Deborah DeRyckere 1, Douglas K. Graham 1
1Department of Pediatrics, University of Colorado Anschutz Medical Campus, 2Department of Bone Marrow Transplantation, University Hospital of Essen

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

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Medicine

Reproducable Paraplegia by Thoracic Aortic Occlusion in a Murine Model of Spinal Cord Ischemia-reperfusion
Marshall T. Bell 1, T. Brett Reece 1, Phillip D. Smith 1, Joshua Mares 1, Michael J. Weyant 1, Joseph C. Cleveland Jr. 1, Kirsten A. Freeman 1, David A. Fullerton 1, Ferenc Puskas 2
1Department of Surgery, Division of Cardiothoracic Surgery, University of Colorado, 2Department of Anesthesiology, University of Colorado

The lack of mechanistic understanding of spinal cord ischemia-reperfusion injury has hindered further adjuncts to prevent paraplegia following high risk aortic operations. Thus, the development of animal models is imperative. This manuscript demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion in a murine model.

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Medicine

A Preclinical Murine Model of Hepatic Metastases
Kevin C. Soares 1, Kelly Foley 2, Kelly Olino 1, Ashley Leubner 2, Skye C. Mayo 1, Ajay Jain 1, Elizabeth Jaffee 2, Richard D. Schulick 3, Kiyoshi Yoshimura 1,2, Barish Edil 1,2, Lei Zheng 1,2
1Department of Surgery, The Johns Hopkins University School of Medicine, 2Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, 3Department of Surgery, University of Colorado Anschutz Medical Campus

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

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Neuroscience

Isolation and Cannulation of Cerebral Parenchymal Arterioles
Paulo W. Pires 1, Fabrice Dabertrand 2, Scott Earley 1
1Department of Pharmacology, University of Nevada School of Medicine, 2Department of Pharmacology, University of Vermont College of Medicine

This manuscript describes a simple and reproducible protocol for isolation of intracerebral arterioles (a group of blood vessels encompassing parenchymal arterioles, penetrating arterioles and pre-capillary arterioles) from mice, to be used in pressure myography, immunofluorescence, biochemistry, and molecular studies.

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Biology

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice
Emily J. Sharpe *1, Joshua R. St. Clair *1,2, Catherine Proenza 1,3
1Department of Physiology and Biophysics, University of Colorado Anschutz Medical Campus, 2Department of Bioengineering, University of Colorado Anschutz Medical Campus, 3Department of Medicine, Division of Cardiology, University of Colorado Anschutz Medical Campus

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

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Biology

A Buoyancy-based Method of Determining Fat Levels in Drosophila
Kelsey E. Hazegh 1, Tânia Reis 1
1Department of Medicine, Division of Endocrinology, Metabolism, and Diabetes, University of Colorado Anschutz Medical Campus

Here we present a method to measure organismal fat levels in the third instar (L3) larval stage of Drosophila melanogaster. This method exploits the comparatively low density of fat tissue to differentiate between larvae with altered fat stores. Buoyancy-based analysis is a valuable tool for rapid, reproducible, and economical screening.

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Cancer Research

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies
Colton Hanna 1, Letty Kwok 1, Jessica Finlay-Schultz 1, Carol A. Sartorius 1, Diana M. Cittelly 1
1Department of Pathology, School of Medicine, University of Colorado Anschutz Medical Campus

We describe a method for stable labeling of patient-derived xenografts (PDXs) with lentiviral particles expressing green-fluorescent protein and luciferase reporters. This method allows for tracking the growth of PDXs at the primary site, as well as detecting spontaneous and experimental metastases using in vivo imaging systems.

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Medicine

A Rat Tibial Growth Plate Injury Model to Characterize Repair Mechanisms and Evaluate Growth Plate Regeneration Strategies
Christopher B. Erickson 1, Nichole Shaw 2, Nancy Hadley-Miller 2, Michael S. Riederer 3, Melissa D. Krebs 3, Karin A. Payne 4
1Department of Bioengineering, Department of Orthopedics, University of Colorado Anschutz Medical Campus, 2Department of Orthopedics, University of Colorado Anschutz Medical Campus, 3Department of Chemical & Biological Engineering, Colorado School of Mines, 4Department of Orthopedics, Gates Center for Regenerative Medicine, University of Colorado Anschutz Medical Campus

The growth plate is a cartilaginous region in children's long bones where longitudinal growth occurs. When injured, bony tissue can form and impair growth. We describe a rat model of growth plate injury that leads to bony repair tissue, allowing the study of repair mechanisms and growth plate regeneration strategies.

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Neuroscience

Spinal Cord Neurons Isolation and Culture from Neonatal Mice
Mohamed Eldeiry 1, Katsuhiro Yamanaka 1, T. Brett Reece 1, Muhammad Aftab 1
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.

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JoVE Journal

Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing
Elliott P. Brooks 1, James T. Nichols 1
1Department of Craniofacial Biology, University of Colorado Anschutz Medical Campus

The goal of this protocol is to alter the penetrance of lethal skeletal mutant phenotypes in zebrafish by selective breeding. Lethal mutants cannot be grown to adulthood and bred themselves, therefore this protocol describes a method for tracking and selecting penetrance through multiple generations by progeny testing.

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Chemistry

Phthalic Acid Ester-Binding DNA Aptamer Selection, Characterization, and Application to an Electrochemical Aptasensor
Xiao Wu *1, Donglin Diao *1, Zhangwei Lu *1, Yu Han 1, Shi Xu 2, Xinhui Lou 1
1Department of Chemistry, Capital Normal University, 2College of Life Sciences, Capital Normal University

A protocol for the in vitro selection and characterization of group-specific phthalic acid ester- binding DNA aptamers is presented. The application of the selected aptamer in an electrochemical aptasensor is also included.

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Developmental Biology

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes
Andrew S. Riching 1, Yuanbiao Zhao 1, Yingqiong Cao 1, Pilar Londono 1, Hongyan Xu 2, Kunhua Song 1
1Division of Cardiology, Department of Medicine, University of Colorado Anschutz Medical Campus, 2Department of Population Health Sciences, Medical College of Georgia, Augusta University

Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-β signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

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JoVE Core

Use of Electron Paramagnetic Resonance in Biological Samples at Ambient Temperature and 77 K
Hanan B. Elajaili 1, Laura Hernandez-Lagunas 1, Kalina Ranguelova 2, Sergey Dikalov 3, Eva Nozik-Grayck 1
1Cardiovascular Pulmonary Research Laboratories and Pediatric Critical Care Medicine, Department of Pediatrics, University of Colorado Anschutz Medical Campus, 2Bruker BioSpin Corp, 3Department of Medicine, Division of Clinical Pharmacology, Vanderbilt University Medical Center

Electron paramagnetic resonance (EPR) spectroscopy is an unambiguous method to measure free radicals. The use of selective spin probes allows for detection of free radicals in different cellular compartments. We present a practical, efficient method to collect biological samples that facilitate treating, storing, and transferring samples for EPR measurements.

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Immunology and Infection

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells
Jeffrey M. Finlon 1, Matthew A. Burchill 1, Beth A. Jirón Tamburini 1,2
1Division of Gastroenterology and Hepatology, Department of Medicine, University of Colorado Anschutz Medical Campus, School of Medicine, 2Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

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Developmental Biology

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry
Josiah Fernandez 1, Enrique C Torchia 1
1Department of Dermatology and Charles C. Gates Center for Regenerative Medicine, University of Colorado Anschutz Medical Campus

This protocol describes how to isolate skin keratinocytes from mouse models, to stain with metal-tagged antibodies, and to analyze stained cells by mass cytometry in order to profile the expression pattern of proteins of interest in the different cell cycle phases.

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Neuroscience

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset (Callithrix jacchus)
Samuel Alexander Budoff *1,2, José Firmino Rodrigues Neto *1, Valéria Arboés 1, Manuela Sales Lima Nascimento 1, Carolina Bione Kunicki 1, Mariana Ferreira Pereira de Araújo 1,3
1Edmond and Lily Safra International Institute of Neuroscience, Santos Dumont Institute, 2Neuroscience Program, University of Colorado Anschutz Medical Campus, 3Department of Physiological Sciences, Health Sciences Center, Federal University of Espírito Santo

This work presents a protocol to perform a stereotaxic, neurosurgical implantation of microelectrode arrays in the common marmoset. This method specifically enables electrophysiological recordings in freely behaving animals but can be easily adapted to any other similar neurosurgical intervention in this species (e.g., cannula for drug administration or electrodes for brain stimulation).

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JoVE Journal

NF-κB-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells
Jonathan M. Mendez 1, A. Marijke Keestra-Gounder 1
1Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus

Here, we present a protocol to quickly and easily measure nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation in cell lines expressing NF-κB::luciferase reporter constructs, via measurements of luminescence in the cell lysate. Additionally, gene expression is determined via RT-qPCR isolated from cells infected with Salmonella Typhimurium.

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Neuroscience

Ex Vivo Pressurized Hippocampal Capillary-Parenchymal Arteriole Preparation for Functional Study
Amanda C. Rosehart 1, Abbie C. Johnson 2, Fabrice Dabertrand 1,3
1Department of Anesthesiology, University of Colorado Anschutz Medical Campus, 2Department of Neurological Sciences, University of Vermont Larner College of Medicine, 3Department of Pharmacology, University of Colorado Anschutz Medical Campus

The present manuscript details how to isolate hippocampal arterioles and capillaries from the mouse brain and how to pressurize them for pressure myography, immunofluorescence, biochemistry, and molecular studies.

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Medicine

Establishing In Situ Closed Circuit Perfusion of Lower Abdominal Organs and Hind Limbs in Mice
Ping Ren 1,2,3, Chunyan Yang 2,4, Laren A. Lofchy 2, Guankui Wang 2, Fangfang Chen *2,4,5, Dmitri Simberg *2
1Department of Thoracic Surgery, The First Hospital of Jilin University, 2The Skaggs School of Pharmacy and Pharmaceutical Sciences, Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, 3Key Laboratory of Zoonoses Research, Ministry of Education, Jilin University, 4Department of Cardiology, China-Japan Union Hospital of Jilin University, 5Department of Gastrointestinal, Colorectal and Anal Surgery, China-Japan Union Hospital of Jilin University

A protocol is described for in situ perfusion of the mouse lower body, including the bladder, the prostate, sex organs, bone, muscle and foot skin.

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Biochemistry

In vitro Monitoring of Extracellular pH in Real-Time
Ian M. Cartwright 1,2,3, Sean P. Colgan 1,2,3
1Mucosal Inflammation Program, University of Colorado Anschutz Medical Campus, 2Department of Medicine, University of Colorado Anschutz Medical Campus, 3Rocky Mountain Regional Veterans Affairs Medical Center

This article represents a useful in vitro assay to measure changes in extracellular pH during neutrophil (PMN) transepithelial migration (TEM)

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Developmental Biology

Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells
Lauren A. Shechtman *1, Christina M. Piarowski *1, Jennifer K. Scott 1, Erin J. Golden 1, Dany Gaillard 1, Linda A. Barlow 1
1Department of Cell and Developmental Biology and the Rocky Mountain Taste and Smell Center, University of Colorado Anschutz Medical Campus

The protocol presents a method for culturing and processing lingual organoids derived from taste stem cells isolated from the posterior taste papilla of adult mice.

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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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Biochemistry

Shotgun Proteomics Sample Processing Automated by an Open-Source Lab Robot
Yu Han 1,3, Cody T. Thomas 1,3, Sara A. Wennersten 1,3, Edward Lau 1,3, Maggie P. Y. Lam 1,2,3
1Department of Medicine-Cardiology, University of Colorado Anschutz Medical Campus, 2Department of Biochemistry & Molecular Genetics, University of Colorado Anschutz Medical Campus, 3Consortium for Fibrosis Research & Translation, School of Medicine, University of Colorado Anschutz Medical Campus

Detailed protocol and three Python scripts are provided for operating an open-source robotic liquid handling system to perform semi-automated protein sample preparation for mass spectrometry experiments, covering detergent removal, protein digestion, and peptide desalting steps.

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Developmental Biology

Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis
Madison A. Rogers *1, Brenna J. C. Dennison *1, Katherine A. Fantauzzo 1
1Department of Craniofacial Biology, School of Dental Medicine, University of Colorado Anschutz Medical Campus

The protocol presents a method for isolating whole cell protein lysates from dissected mouse embryo facial processes or cultured mouse embryonic palatal mesenchyme cells and performing subsequent western blotting to assess phosphorylated protein levels.

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Bioengineering

Fabrication of Size-Controlled and Emulsion-Free Chitosan-Genipin Microgels for Tissue Engineering Applications
Michael A. Stager 1, Christopher B. Erickson 2, Karin A. Payne 2, Melissa D. Krebs 1
1Department of Chemical and Biological Engineering, Colorado School of Mines, 2Department of Orthopedics, University of Colorado Anschutz Medical Campus

The present protocol describes a non-emulsion-based method for the fabrication of chitosan-genipin microgels. The size of these microgels can be precisely controlled, and they can display pH-dependent swelling, degrade in vivo, and be loaded with therapeutic molecules that release over time in a sustained manner, making them highly relevant for tissue engineering applications.

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Cancer Research

Positron Emission Tomography Imaging of Cell Trafficking: A Method of Cell Radiolabeling
Aditya Bansal 1, Timothy R. DeGrado 2, Mukesh K. Pandey 1
1Division of Nuclear Medicine, Department of Radiology, Mayo Clinic, 2Department of Radiology, University of Colorado Anschutz Medical Campus

Presented here is a protocol to radiolabel cells with a positron emission tomography (PET) radioisotope, 89Zr (t1/2 78.4 h), using a ready-to-use radiolabeling synthon, [89Zr]Zr-p-isothiocyanatobenzyl-desferrioxamine ([89Zr]Zr-DBN). Radiolabeling cells with [89Zr]Zr-DBN allows noninvasive tracking and imaging of administered radiolabeled cells in the body with PET for up to 7 days post-administration.

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Biology

Isolation and Characterization of the Murine Uterosacral Ligaments and Pelvic Floor Organs
Catalina S. Bastías 1, Lea M. Savard 2, Kevin N. Eckstein 2, Kathleen Connell 3, Callan M. Luetkemeyer 2,5, Virginia L. Ferguson 1,2,4, Sarah Calve 1,2,4
1Biomedical Engineering Program, University of Colorado Boulder, 2Paul M. Rady Department of Mechanical Engineering, University of Colorado Boulder, 3Department of Obstetrics and Gynecology, University of Colorado Anschutz Medical Campus, 4BioFrontiers Institute, University of Colorado Boulder, 5Department of Mechanical Science and Engineering, University of Illinois Urbana-Champaign

This article presents a detailed protocol for dissecting uterosacral ligaments and other pelvic floor tissues, including the cervix, rectum, and bladder in mice, to expand the study of female reproductive tissues.

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Bioengineering

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation
Alicia E. Tanneberger 1, Layla Blair 1, Duncan Davis-Hall 1, Chelsea M. Magin 1,2,3
1Department of Bioengineering, University of Colorado Denver | Anschutz Medical Campus, 2Department of Pediatrics, University of Colorado Anschutz Medical Campus, 3Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado Anschutz Medical Campus

This article describes how to 3D bioprint phototunable hydrogels to study extracellular matrix stiffening and fibroblast activation.

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Neuroscience

Miniscope Recording Calcium Signals at Hippocampus of Mice Navigating an Odor Plume
Fabio M. Simoes de Souza 1, Ryan Williamson 2, Connor McCullough 3, Alec Teel 1, Gregory Futia 3, Ming Ma 1, Aaron True 4, John P. Crimaldi 4, Emily Gibson 3,5, Diego Restrepo 1,5
1Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Campus, 2Neurotechnology Center, University of Colorado Anschutz Medical Campus, 3Department of Bioengineering, University of Colorado Anschutz Medical Campus, 4Civil, Environmental & Architectural Engineering, University of Colorado, 5Neuroscience Graduate Program, University of Colorado Anschutz Medical Campus

This protocol investigates the brain-behavior relationship in hippocampal CA1 in mice navigating an odor plume. We provide a step-by-step protocol, including surgery to access imaging of the hippocampus, behavioral training, miniscope GCaMP6f recording and processing of the brain, and behavioral data to decode the mouse position from ROI neural activity.

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