University of Colorado Anschutz Medical Campus 28 articles published in JoVE Cancer Research Positron Emission Tomography Imaging of Cell Trafficking: A Method of Cell Radiolabeling Aditya Bansal1, Timothy R. DeGrado2, Mukesh K. Pandey1 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. Bioengineering 3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation Alicia E. Tanneberger1, Layla Blair1, Duncan Davis-Hall1, Chelsea M. Magin1,2,3 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. Biology Isolation and Characterization of the Murine Uterosacral Ligaments and Pelvic Floor Organs Catalina S. Bastías1, Lea M. Savard2, Kevin N. Eckstein2, Kathleen Connell3, Callan M. Luetkemeyer2,5, Virginia L. Ferguson1,2,4, Sarah Calve1,2,4 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. Bioengineering Fabrication of Size-Controlled and Emulsion-Free Chitosan-Genipin Microgels for Tissue Engineering Applications Michael A. Stager1, Christopher B. Erickson2, Karin A. Payne2, Melissa D. Krebs1 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. 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. Fantauzzo1 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. Biochemistry Shotgun Proteomics Sample Processing Automated by an Open-Source Lab Robot Yu Han1,3, Cody T. Thomas1,3, Sara A. Wennersten1,3, Edward Lau1,3, Maggie P. Y. Lam1,2,3 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. 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. Scott1, Erin J. Golden1, Dany Gaillard1, Linda A. Barlow1 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. Biology Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining Wells B. LaRiviere1,2, Xiaorui Han3,4, Kaori Oshima1, Sarah A. McMurtry1, Robert J. Linhardt3, Eric P. Schmidt1,5 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. Medicine Establishing In Situ Closed Circuit Perfusion of Lower Abdominal Organs and Hind Limbs in Mice Ping Ren1,2,3, Chunyan Yang2,4, Laren A. Lofchy2, Guankui Wang2, 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. Immunology and Infection NF-κB-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells Jonathan M. Mendez1, A. Marijke Keestra-Gounder1 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. Neuroscience Ex Vivo Pressurized Hippocampal Capillary-Parenchymal Arteriole Preparation for Functional Study Amanda C. Rosehart1, Abbie C. Johnson2, Fabrice Dabertrand1,3 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. 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 Arboés1, Manuela Sales Lima Nascimento1, Carolina Bione Kunicki1, Mariana Ferreira Pereira de Araújo1,3 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). Cancer Research Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing Michael Seager1, Dara L. Aisner1, Kurtis D. Davies1 1Department of Pathology, University of Colorado - Anschutz Medical Campus This article details the use of an anchored multiplex polymerase chain reaction-based library preparation kit followed by next-generation sequencing to assess for oncogenic gene fusions in clinical solid tumor samples. Both wet-bench and data analysis steps are described. Developmental Biology Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry Josiah Fernandez1, Enrique C Torchia1 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. Cancer Research Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment Ayman J. Oweida1, Shilpa Bhatia1, Benjamin Van Court1, Laurel Darragh1, Natalie Serkova1,2,3, Sana D. Karam1 1Department of Radiation Oncology, University of Colorado Denver - Anschutz Medical Campus, 2Department of Anesthesiology, University of Colorado Denver - Anschutz Medical Campus, 3Division of Radiology, University of Colorado Denver - Anschutz Medical Campus Here we present a reproducible method for developing an orthotopic murine model of head and neck squamous cell carcinoma. Tumors demonstrate clinically relevant histopathological features of the disease, including necrosis, poor differentiation, nodal metastases, and immune infiltration. Tumor-bearing mice develop clinically relevant symptoms including dysphagia, jaw displacement, and weight loss. Biochemistry Use of Electron Paramagnetic Resonance in Biological Samples at Ambient Temperature and 77 K Hanan B. Elajaili1, Laura Hernandez-Lagunas1, Kalina Ranguelova2, Sergey Dikalov3, Eva Nozik-Grayck1 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. Immunology and Infection Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells Jeffrey M. Finlon1, Matthew A. Burchill1, Beth A. Jirón Tamburini1,2 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. 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. Developmental Biology Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes Andrew S. Riching1, Yuanbiao Zhao1, Yingqiong Cao1, Pilar Londono1, Hongyan Xu2, Kunhua Song1 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. Developmental Biology Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing Elliott P. Brooks1, James T. Nichols1 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. Medicine A Rat Tibial Growth Plate Injury Model to Characterize Repair Mechanisms and Evaluate Growth Plate Regeneration Strategies Christopher B. Erickson1, Nichole Shaw2, Nancy Hadley-Miller2, Michael S. Riederer3, Melissa D. Krebs3, Karin A. Payne4 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. Neuroscience Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons Rosa L. Moreno1, Megan Josey2, Angeles B. Ribera1,2 1Department of Physiology and Biophysics, University of Colorado Anschutz Medical Campus (UCAMC), 2Neuroscience Graduate Program, University of Colorado Anschutz Medical Campus (UCAMC) This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages. Cancer Research Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies Colton Hanna1, Letty Kwok1, Jessica Finlay-Schultz1, Carol A. Sartorius1, Diana M. Cittelly1 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. Biology A Buoyancy-based Method of Determining Fat Levels in Drosophila Kelsey E. Hazegh1, Tânia Reis1 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. 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 Proenza1,3 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. Medicine Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS Touraj Shokati1, Nicholas Bodenberger1, Holly Gadpaille1, Björn Schniedewind1, Alexander A. Vinks2, Wenlei Jiang3, Rita R. Alloway4, Uwe Christians1 1iC42 Clinical Research and Development, University of Colorado, Anschutz Medical Campus, 2 Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction. Medicine A Preclinical Murine Model of Hepatic Metastases Kevin C. Soares1, Kelly Foley2, Kelly Olino1, Ashley Leubner2, Skye C. Mayo1, Ajay Jain1, Elizabeth Jaffee2, Richard D. Schulick3, Kiyoshi Yoshimura1,2, Barish Edil1,2, Lei Zheng1,2 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. Medicine Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia Sandra Christoph1,2, Alisa B. Lee-Sherick1, Susan Sather1, Deborah DeRyckere1, Douglas K. Graham1 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.