Washington University School of Medicine 36 articles published in JoVE Chemistry Histone Modification Screening using Liquid Chromatography, Trapped Ion Mobility Spectrometry, and Time-Of-Flight Mass Spectrometry Meiby Fernandez-Rojas1, Cassandra N. Fuller1, Lilian Valadares Tose1, Matthew Willetts2, Melvin A. Park2, Natarajan V. Bhanu3, Benjamin A. Garcia3, Francisco Fernandez-Lima1,4 1Department of Chemistry and Biochemistry, Florida International University, 2Bruker Daltonics Inc., 3Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 4Biomolecular Sciences Institute, Florida International University An analytical workflow based on liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) for high confidence and highly reproducible "bottom-up" analysis of histone modifications and identification based on principal parameters (retention time [RT], collision cross section [CCS], and accurate mass-to-charge [m/z] ratio). Medicine Microfluidic Model of Necrotizing Enterocolitis Incorporating Human Neonatal Intestinal Enteroids and a Dysbiotic Microbiome Lauren C. Frazer1, Yukihiro Yamaguchi1, Corey M. Jania1, Wyatt E. Lanik2, Qingqing Gong3, Dhirendra K. Singh1, Stephen Mackay1, Natalia S. Akopyants1, Misty Good1 1Division of Neonatal-Perinatal Medicine, Department of Pediatrics, University of North Carolina at Chapel Hill, 2University of Nebraska College of Medicine, 3Department of Surgery, Washington University School of Medicine This protocol describes an in vitro model of necrotizing enterocolitis (NEC), which can be used for mechanistic studies into disease pathogenesis. It features a microfluidic chip seeded with intestinal enteroids derived from the human neonatal intestine, endothelial cells, and the intestinal microbiome of a neonate with severe NEC. Bioengineering Electromyometrial Imaging of Uterine Contractions in Pregnant Women Hui Wang*1,2,3, Zichao Wen*2,3, Wenjie Wu2,3,4, Zhexian Sun2,3,4, Qing Wang5, Alan L. Schwartz6, Phillip Cuculich7, Alison G. Cahill8, George A. Macones8, Yong Wang2,3,4,5 1Department of Physics, Washington University, 2Center for Reproductive Health Sciences, Washington University School of Medicine, 3Department of Obstetrics and Gynecology, Washington University School of Medicine, 4Department of Biomedical Engineering, Washington University, 5Mallinckrodt Institute of Radiology, Washington University School of Medicine, 6Department of Pediatrics, Washington University School of Medicine, 7Department of Cardiology, Washington University School of Medicine, 8Department of Women's Health, University of Texas at Austin, Dell Medical School We present a protocol for conducting electromyometrial imaging (EMMI), including the following procedures: multiple electromyography electrode sensor recordings from the body surface, magnetic resonance imaging, and uterine electrical signal reconstruction. Engineering A Coregistered Ultrasound and Photoacoustic Imaging Protocol for the Transvaginal Imaging of Ovarian Lesions Haolin Nie1, Hongbo Luo2, Lin Chen1, Quing Zhu1,3 1Department of Biomedical Engineering, Washington University, 2Department of Electrical & Systems Engineering, Washington University, 3Department of Radiology, Washington University School of Medicine We report a coregistered ultrasound and photoacoustic imaging protocol for the transvaginal imaging of ovarian/adnexal lesions. The protocol may be valuable to other translational photoacoustic imaging studies, especially those using commercial ultrasound arrays for the detection of photoacoustic signals and standard delay-and-sum beamforming algorithms for imaging. Cancer Research Quantifying Replication Stress in Ovarian Cancer Cells Using Single-Stranded DNA Immunofluorescence Natasha Ramakrishnan1, Ena Haseljic1, Priyanka Verma1 1Division of Oncology, Department of Medicine, Siteman Cancer Center, Washington University School of Medicine Here, we describe an immunofluorescence-based method to quantify the levels of single-stranded DNA in cells. This efficient and reproducible method can be utilized to examine replication stress, a common feature in several ovarian cancers. Additionally, this assay is compatible with an automated analysis pipeline, which further increases its efficiency. Neuroscience A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants Jalin A. Stelzer1, Jason J. Yi1 1Department of Neuroscience, Washington University School of Medicine A simple and scalable method was developed to assess the functional significance of missense variants in Ube3a, a gene whose loss and gain of function are linked to both Angelman syndrome and autism spectrum disorder. Immunology and Infection Mouse Heterotopic Cervical Cardiac Transplantation Utilizing Vascular Cuffs Wenjun Li1, Hailey M. Shepherd1, Alexander S. Krupnick2, Andrew E. Gelman1,3, Kory J. Lavine4, Daniel Kreisel1,3 1Department of Surgery, Washington University School of Medicine, 2Department of Surgery, The University of Maryland, 3Department of Pathology & Immunology, Washington University School of Medicine, 4Department of Medicine, Washington University School of Medicine Mouse cardiac transplantation models represent valuable research tools for studying transplantation immunology. The present protocol details mouse heterotopic cervical cardiac transplantation that involves the placement of cuffs on the recipient's common carotid artery and the donor's pulmonary artery trunk to allow for laminar blood flow. Immunology and Infection Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria Sydney R. Morrill1,2,3, Kavita Agarwal2,3, Sudeshna Saha2,3, Warren G. Lewis2,3, Nicole M. Gilbert4, Amanda L. Lewis2,3 1Division of Biology and Biomedical Sciences, Washington University School of Medicine, 2Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, 3Glycobiology Research and Training Center, UCSD, 4Department of Pediatrics, Washington University School of Medicine The protocol presents a mouse model of vaginal colonization with anaerobically cultured human vaginal bacteria. We focus on Gardnerella vaginalis, while including suggestions for Prevotella bivia and Fusobacterium nucleatum. This protocol can also be used as a guide for vaginal inoculations and viable recovery of other anaerobically grown bacteria. Medicine Biological Preparation and Mechanical Technique for Determining Viscoelastic Properties of Zonular Fibers Juan Rodriguez1,2, Matthew Reilly3, Robert P. Mecham4, Steven Bassnett2,4 1Department of Basic Sciences, University of Health Sciences and Pharmacy, 2Department of Ophthalmology & Visual Sciences, Washington University School of Medicine, 3Department of Biomedical Engineering, The Ohio State University, 4Department of Cell Biology & Physiology, Washington University School of Medicine The protocol describes a method for the study of extracellular matrix viscoelasticity and its dependence on protein composition or environmental factors. The matrix system targeted is the mouse zonule. The performance of the method is demonstrated by comparing the viscoelastic behavior of wild-type zonular fibers with those lacking microfibril-associated glycoprotein-1. Neuroscience Assessment of Swim Endurance and Swim Behavior in Adult Zebrafish Brooke Burris1, Nicholas Jensen1, Mayssa H. Mokalled1,2 1Department of Developmental Biology, Washington University School of Medicine, 2Center of Regenerative Medicine, Washington University School of Medicine Capable of functional recovery after spinal cord injury, adult zebrafish is a premier model system to elucidate innate mechanisms of neural regeneration. Here, we describe swim endurance and swim behavior assays as functional readouts of spinal cord regeneration. Neuroscience Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina Zelun Wang1,2, Sean McCracken1,2, Philip R. Williams1,3,4 1John F. Hardesty, MD Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, 2Graduate Program in Neuroscience, Washington University School of Medicine, 3Department of Neuroscience, Washington University School of Medicine, 4Hope Center for Neurological Disorders, Washington University School of Medicine In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina. Neuroscience The Sleep Nullifying Apparatus: A Highly Efficient Method of Sleep Depriving Drosophila Krishna Melnattur1, Ellen Morgan1, Vincent Duong1, Arjan Kalra1, Paul J. Shaw1 1Department of Neuroscience, Washington University School of Medicine Sleep deprivation is a powerful tool to investigate sleep function and regulation. We describe a protocol to sleep deprive Drosophila using the Sleep Nullifying Apparatus, and to determine the extent of rebound sleep induced by deprivation. Immunology and Infection Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice Valerie P. O'Brien1,2,6, Matthew S. Joens3,7, Amanda L. Lewis1,2,4,8, Nicole M. Gilbert2,5 1Department of Molecular Microbiology, Washington University School of Medicine in Saint Louis, 2Center for Women’s Infectious Disease Research, Washington University School of Medicine in Saint Louis, 3Center for Cellular Imaging, Washington University School of Medicine in Saint Louis, 4Department of Obstetrics and Gynecology, Washington University School of Medicine in Saint Louis, 5Department of Pediatrics, Washington University School of Medicine in Saint Louis, 6Fred Hutchinson Cancer Research Center, 7TESCAN USA, Inc., 8University of California San Diego A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy. Neuroscience A Revised Surgical Approach to Induce Endolymphatic Hydrops in the Guinea Pig Carla V. Valenzuela1, Choongheon Lee1, Craig A. Buchman1, Jeffery T. Lichtenhan1 1Department of Otolaryngology, Head and Neck Surgery, Washington University School of Medicine in St. Louis This article demonstrates an extradural approach to obliterate the guinea pig endolymphatic sac and injure the endolymphatic duct with a fine pick in order to induce experimental endolymphatic hydrops. Medicine Inverse Probability of Treatment Weighting (Propensity Score) using the Military Health System Data Repository and National Death Index Joshua D. Mitchell1, Brian F. Gage2, Nicole Fergestrom3, Eric Novak1, Todd C. Villines4 1Cardiovascular Division, Washington University School of Medicine, 2General Medical Sciences, Washington University School of Medicine, 3Center for Advancing Population Science, Medical College of Wisconsin, 4Cardiology Service, Department of Medicine, Walter Reed National Military Medical Center When randomized controlled trials are not feasible, a comprehensive health care data source like the Military Health System Data Repository provides an attractive alternative for retrospective analyses. Incorporating mortality data from the national death index and balancing differences between groups using propensity weighting helps reduce biases inherent in retrospective designs. Immunology and Infection Isolation of Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens Geetika Bajpai1, Kory J. Lavine1,2,3 1Department of Medicine, Washington University School of Medicine, 2Department of Immunology and Pathology, Washington University School of Medicine, 3Department of Developmental Biology, Washington University School of Medicine Presented here is a protocol to isolate various subsets of macrophages and other non-immune cells from human and mouse myocardium by preparing a single cell suspension through enzymatic digestion. Gating schemes for flow cytometry based identification and characterization of isolated macrophages are also presented. Biology Measuring Diurnal Rhythms in Autophagic and Proteasomal Flux Mikhail Ryzhikov1, Anna Eubanks1, Jeffrey A. Haspel1 1Division of Pulmonary and Critical Care Medicine, Washington University School of Medicine We describe our protocol for measuring biological rhythms in protein catabolism via autophagy and the proteasome in mouse liver. Genetics A Complete Pipeline for Isolating and Sequencing MicroRNAs, and Analyzing Them Using Open Source Tools Meredith M. Course1, Kathryn Gudsnuk1, Paul N. Valdmanis1 1Division of Medical Genetics, University of Washington School of Medicine Here, we describe a step-by-step strategy for isolating small RNAs, enriching for microRNAs, and preparing samples for high-throughput sequencing. We then describe how to process sequence reads and align them to microRNAs, using open source tools. Chemistry Breath Collection from Children for Disease Biomarker Discovery Amalia Z. Berna1, Brian DeBosch1,2, Janis Stoll3, Audrey R. Odom John1,4 1Department of Pediatrics, Washington University School of Medicine, 2Cell Biology & Physiology, Washington University School of Medicine, 3Division of Gastroenterology, Hepatology and Nutrition, Washington University School of Medicine, 4Department of Molecular Microbiology, Washington University School of Medicine This protocol describes a simple method for the acquisition of breath samples from children. Briefly, samples of mixed air are pre-concentrated in sorbent tubes prior to gas chromatography-mass spectrometry analysis. Breath biomarkers of infectious and non-infectious diseases can be identified using this breath collection method. Medicine Multi-Modal Home Sleep Monitoring in Older Adults Cristina D. Toedebusch*1, Jennifer S. McLeland*1, Claire M. Schaibley1, Ian R. Banks1, Jill Boyd1, John C. Morris1,2,3, David M. Holtzman1,2,3, Brendan P. Lucey1,2 1Department of Neurology, Washington University School of Medicine, 2Hope Center for Neurological Disorders, Washington University School of Medicine, 3Knight Alzheimer's Disease Research Center, Washington University School of Medicine Here, we present a protocol to improve the quality of data from home sleep testing by providing a method to enhance instructions through a structured participant visit. This protocol includes the implementation of a step-by-step educational manual with photos to ensure proper placement of equipment. Biology Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis Jeffrey R. Millman1,2, Teresa Doggett3, Christina Thebeau3, Sheng Zhang3, Clay F. Semenkovich1, Rithwick Rajagopal3 1Division of Metabolism, Endocrinology and Lipid Research, Department of Medicine, Washington University School of Medicine, 2Department of Biomedical Engineering, Washington University in Saint Louis, 3Department of Ophthalmology and Visual Science, Washington University School of Medicine This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer. Genetics High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq) Erin A. Brettmann1, Inez Y. Oh1, Cristina de Guzman Strong1 1Division of Dermatology, Center for Pharmacogenomics, Center for the Study of Itch, Department of Medicine, Washington University School of Medicine The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step. Medicine A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts Lianxiang Bi*1, Bradley K. Wacker*1, David A. Dichek1 1Department of Medicine, University of Washington School of Medicine This method describes the placement of interposition vein grafts in rabbits, the transduction of the grafts, and the achievement of durable transgene expression. This allows the investigation of physiological and pathological roles of transgenes and their protein products in grafted veins, and testing of gene therapies for vein graft disease. Developmental Biology Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization Stephanie A. Michalski*1, Sangappa B. Chadchan*1, Emily S. Jungheim1, Ramakrishna Kommagani1 1Department of Obstetrics and Gynecology, Center for Reproductive Health Sciences, Washington University School of Medicine This study presents a validated and optimized procedure for the isolation and culture of human endometrial stromal cells to conduct in vitro decidualization assay. Further, this study provides a detailed method to efficiently knockdown a specific gene using siRNAs in human endometrial stromal cells. Genetics Rare Event Detection Using Error-corrected DNA and RNA Sequencing Wing H. Wong*1,2, R. Spencer Tong*1,2, Andrew L. Young1,2, Todd E. Druley1,2 1Department of Pediatrics, Division of Hematology and Oncology, Washington University School of Medicine, 2Center for Genome Sciences and Systems Biology, Washington University School of Medicine Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5–2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001. Behavior Use of a Video Scoring Anchor for Rapid Serial Assessment of Social Communication in Toddlers Natasha Marrus1, Stefanie Kennon-McGill1, Brooke Harris2, Yi Zhang1, Anne L. Glowinski1, John N. Constantino1 1Department of Psychiatry, Washington University School of Medicine, 2Veteran's Administration Palo Alto Health Care System Here we present a protocol for administration of the video-referenced rating of reciprocal social behavior (vrRSB) using a paper or online version. This survey quantifies RSB, a prerequisite for social competence, in toddlers through "video-referenced" items whereby a subject is compared to a reference child shown in a three-minute video. Medicine Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation Wyatt E. Lanik1, Lily Xu2, Cliff J. Luke1, Elise Z. Hu2, Pranjal Agrawal2, Victoria S. Liu2, Rajesh Kumar1, Alexa M. Bolock1, Congrong Ma3, Misty Good1 1Division of Newborn Medicine, Department of Pediatrics, Washington University School of Medicine, 2Washington University, 3Division of Newborn Medicine, Department of Pediatrics, University of Pittsburgh School of Medicine This protocol describes how to establish an enteroid culture system from neonatal mouse or premature human intestine as well as an efficient method to collect milk from mice. Developmental Biology A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry Ana C. Lima*1,2,3,4, Min Jung*1, Jannette Rusch1, Abul Usmani1, Alexandra M. Lopes3,4, Donald F. Conrad1 1Department of Genetics, Washington University School of Medicine, 2Graduate Program in Areas of Basic and Applied Biology (GABBA), Abel Salazar Institute of Biomedical Sciences, University of Porto, 3Instituto de Investigação e Inovação em Saúde, University of Porto, 4IPATIMUP - Instituto de Patologia e Imunologia Molecular da Universidade do Porto, 5Department of Pathology & Immunology, Washington University School of Medicine This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology. Medicine A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers Catherine E. Lang1,2,3, Kimberly J. Waddell1, Joseph W. Klaesner1,4,5, Marghuretta D. Bland1,2,3 1Program in Physical Therapy, Washington University School of Medicine, 2Program in Occupational Therapy, Washington University School of Medicine, 3Department of Neurology, Washington University School of Medicine, 4Mallinckrodt Institute of Radiology, Washington University School of Medicine, 5Department of Biomedical Engineering, Washington University This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers. Medicine Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer Ahmed E. Hegab1, Naofumi Kameyama1, Aoi Kuroda1, Shizuko Kagawa1, Yongjun Yin2, David Ornitz2, Tomoko Betsuyaku1 1Division of Pulmonary Medicine, Keio University School of Medicine, 2Department of Developmental Biology, Washington University School of Medicine We describe a method for the detection of tumor nodule development in the lungs of an adenocarcinoma mouse model using micro-computed tomography and its use for monitoring changes in nodule size over time and in response to treatment. The accuracy of the assessment was confirmed with end-point histological quantification. Biology Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization Daniel L. Kober*1,2,3, Zeynep Yurtsever*2,3,4,5, Thomas J. Brett2,3,5,6,7 1Molecular Microbiology and Microbial Pathogenesis Program, Washington University School of Medicine, 2Department of Internal Medicine, Washington University School of Medicine, 3Drug Discovery Program in Pulmonary and Critical Care Medicine, Washington University School of Medicine, 4Biochemistry Program, Washington University School of Medicine, 5Center for the Investigation of Membrane Excitability Diseases, Washington University School of Medicine, 6Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 7Department of Cell Biology and Physiology, Washington University School of Medicine This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies. Developmental Biology Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles Paola L. Carvajal Monroy1, Zipora Yablonka-Reuveni2, Sander Grefte3, Anne Marie Kuijpers-Jagtman1, Frank A.D.T.G. Wagener1, Johannes W. Von den Hoff1 1Department of Orthodontics and Craniofacial Biology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, 2Department of Biological Structure, University of Washington School of Medicine, 3Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells. Medicine Establishment and Characterization of UTI and CAUTI in a Mouse Model Matt S. Conover1, Ana L. Flores-Mireles1, Michael E. Hibbing1, Karen Dodson1, Scott J. Hultgren1 1Department of Molecular Microbiology and Center for Women's Infectious Disease Research, Washington University School of Medicine The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work’s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans. Medicine Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice Katherine Poinsatte1, Uma Maheswari Selvaraj1, Sterling B. Ortega1, Erik J. Plautz1, Xiangmei Kong1, Jeffrey M. Gidday2, Ann M. Stowe1 1Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center, 2Department of Neurological Surgery, Washington University School of Medicine This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection. Medicine Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model Russell K. Pachynski1, Alexander Scholz3, Justin Monnier2,3, Eugene C. Butcher3, Brian A. Zabel2 1Division of Oncology, Department of Medicine, Washington University School of Medicine, 2Palo Alto Institute for Research and Education, Veterans Affairs Palo Alto Health Care System, 3Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses. Neuroscience Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging Richard S. Stewart1, Ilona M. Kiss1, Robert S. Wilkinson1 1Department of Cell Biology and Physiology, Washington University School of Medicine Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals. Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.
