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Washington University School of Medicine

63 ARTICLES PUBLISHED IN JoVE

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Biology

Presynaptically Silent Synapses Studied with Light Microscopy
Krista L. Moulder 1, Xiaoping Jiang 1, Amanda A. Taylor 1, Ann M. Benz 1, Steven Mennerick 1,2,3
1Department of Psychiatry, Washington University School of Medicine, 2Department of Anatomy, Washington University School of Medicine, 3Department of Neurobiology, Washington University School of Medicine

Glutamatergic synapses can switch from an active mode to a silent mode. We demonstrate that presynaptic activity status in dissociated culture of rodent neurons is visualized using a fixable form of the FM1-43 dye to visualize active synapses and immunostaining with vGluT-1 antibody to visualize all glutamate synapses.

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Neuroscience

Multielectrode Array Recordings of the Vomeronasal Epithelium
Hannah A. Arnson 1, Xiaoyan Fu 1, Timothy E. Holy 1
1Anatomy and Neurobiology, Washington University School of Medicine

Multielectrode array (MEA) recordings provide a method for studying the electrical activity of large populations of neurons. Here, we present the details of a MEA preparation to record from the mouse vomeronasal epithelium while simultaneously stimulating the tissue.

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Immunology and Infection

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host
Colin J. Thalhofer 1, Joel W. Graff 2, Laurie Love-Homan 3, Suzanne M. Hickerson 4, Noah Craft 5, Stephen M. Beverley 4, Mary E. Wilson 6,7
1Interdisciplinary Immunology Program, University of Iowa, and the VA Medical Center, 2Department of Biochemistry, University of Iowa, and the VA Medical Center, 3Department of Internal Medicine, University of Iowa, 4Department of Molecular Microbiology, Washington University School of Medicine, 5Division of Dermatology, Harbor-UCLA Medical Center, Hanley-Hardison Research Center, 6Interdisciplinary Immunology Program, Iowa City VA Medical Center, 7Departments of Internal Medicine, Microbiology and Epidemiology, University of Iowa

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

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Biology

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation
Enrique Amaya 1, Kristen Kroll 2
1The Healing Foundation Centre, Faculty of Life Sciences, University of Manchester, 2Department of Developmental Biology, Washington University School of Medicine

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

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Immunology and Infection

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells
Nayan J. Sarma 1, Akiko Takeda 1, Nabeel R. Yaseen 1
1Department of Pathology and Immunology, Washington University School of Medicine

The colony forming cell (CFC) assay is an in vitro assay in which hematopoietic progenitors form colonies in a semi-solid medium. A combination of colony morphology, cell morphology, and flow cytometry are used to assess the ability of the progenitors to proliferate and differentiate along the different hematopoietic lineages.

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Neuroscience

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation
Cynthia L. Montana 1, Connie A. Myers 1, Joseph C. Corbo 1
1Department of Pathology and Immunology, Washington University School of Medicine

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

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Neuroscience

Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses
Alexander V. Kolesnikov 1, Vladimir J. Kefalov 1
1Department of Ophthalmology and Visual Sciences, Washington University School of Medicine

We describe a relatively simple method of transretinal electroretinogram (ERG) recordings for obtaining rod and cone photoresponses from intact mouse retina. This approach takes advantage of the block of synaptic transmission from photoreceptors to isolate their light responses and record them using field electrodes placed across the isolated flat-mounted retina.

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Bioengineering

Measuring Left Ventricular Pressure in Late Embryonic and Neonatal Mice
Victoria P. Le 1, Attila Kovacs 2, Jessica E. Wagenseil 1
1Department of Biomedical Engineering, Saint Louis University, 2Department of Internal Medicine, Washington University School of Medicine

Measuring left ventricular pressure (LV) in embryonic and neonatal mice is described. Pressure is measured by inserting a needle connected to a fluid-filled transducer into the LV under ultrasound guidance. Care must be taken to maintain normal cardiac function during the experimental protocol.

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Neuroscience

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo
Ariel M. Lyons-Warren 1, Tsunehiko Kohashi 1,2, Steven Mennerick 3, Bruce A. Carlson 1
1Department of Biology, Washington University in St. Louis , 2Division of Biological Science, Graduate School of Science, Nagoya University, 3Department of Psychiatry, Washington University in St. Louis

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

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Biology

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER
Francesco Vallania 1, Enrique Ramos 1, Sharon Cresci 2, Robi D. Mitra 1, Todd E. Druley 1,3
1Center for Genome Sciences and Systems Biology, Department of Genetics, Washington University School of Medicine, 2Department of Internal Medicine, Washington University School of Medicine, 3Department of Pediatrics, Washington University School of Medicine

Pooled DNA sequencing is a fast and cost-effective strategy to detect rare variants associated with complex phenotypes in large cohorts. Here we describe the computational analysis of pooled, next-generation sequencing of 32 cancer-related genes using the SPLINTER software package. This method is scalable, and applicable to any phenotype of interest.

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Neuroscience

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System
Mingjie Li 1, Nada Husic 1, Ying Lin 1, B. Joy Snider 1
1Department of Neurology and Hope Center for Neurological Disorders, Washington University School of Medicine

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

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Medicine

Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)
Ameet I. Thaker 1, Anisa Shaker 1, M. Suprada Rao 1, Matthew A. Ciorba 1
1Division of Gastroenterology, Washington University School of Medicine

We demonstrate a protocol in which administration of the genotoxic agent azoxymethane (AOM) followed by three cycles of the pro-inflammatory agent dextran sulfate sodium (DSS) rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis-associated cancer.

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Immunology and Infection

Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium
Megan E. Lau 1, David A. Hunstad 1,2
1Department of Pediatrics, Washington University School of Medicine, 2Department of Molecular Microbiology, Washington University School of Medicine

We developed an in vitro model that mimics an important component of the acute inflammatory response during infection of the bladder with uropathogenic Escherichia coli. The transuroepithelial neutrophil migration assay enables quantitative assessment of human neutrophil migration across bladder epithelia, cultured on permeable supports, in response to bacterial infection or chemoattractant substances.

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Neuroscience

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging
Richard S. Stewart 1, Ilona M. Kiss 1, Robert S. Wilkinson 1
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.

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Biology

Isolation of Myofibroblasts from Mouse and Human Esophagus
Matthew Gargus 1, Chao Niu 1, Anisa Shaker 1
1Department of Medicine, Keck School of Medicine, University of Southern California

We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express α-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions.

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Medicine

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model
Russell K. Pachynski 1, Alexander Scholz 3, Justin Monnier 2,3, Eugene C. Butcher 3, Brian A. Zabel 2
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.

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Medicine

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice
Katherine Poinsatte 1, Uma Maheswari Selvaraj 1, Sterling B. Ortega 1, Erik J. Plautz 1, Xiangmei Kong 1, Jeffrey M. Gidday 2, Ann M. Stowe 1
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.

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Medicine

Establishment and Characterization of UTI and CAUTI in a Mouse Model
Matt S. Conover 1, Ana L. Flores-Mireles 1, Michael E. Hibbing 1, Karen Dodson 1, Scott J. Hultgren 1
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.

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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. Brett 2,3,5,6,7
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.

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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. Hegab 1, Naofumi Kameyama 1, Aoi Kuroda 1, Shizuko Kagawa 1, Yongjun Yin 2, David Ornitz 2, Tomoko Betsuyaku 1
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.

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Chemistry

Laboratory Production of Biofuels and Biochemicals from a Rapeseed Oil through Catalytic Cracking Conversion
Siauw H. Ng 1, Yu Shi 1, Nicole E. Heshka 1, Yi Zhang 1, Edward Little 1
1CanmetENERGY, Natural Resources Canada

This paper presents an experimental method to produce biofuels and biochemicals from canola oil mixed with a fossil-based feed in the presence of a catalyst at mild temperatures. Gaseous, liquid, and solid products from a reaction unit are quantified and characterized. Conversion and individual product yields are calculated and reported.

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Neuroscience

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains
Antonio J. Tito 1, Shebna Cheema 2, Mian Jiang 2, Sheng Zhang 1,3
1Center for Metabolic and Degenerative Diseases, The Brown Foundation Institute of Molecular Medicine, Programs in Human and Molecular Genetics and Neuroscience, The Graduate School of Biomedical Sciences at Houston, The University of Texas Health Science Center at Houston, 2Department of Natural Sciences, University of Houston - Downtown, 3Department of Neurobiology and Anatomy, McGovern Medical School, The University of Texas Health Science Center at Houston

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

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Medicine

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers
Catherine E. Lang 1,2,3, Kimberly J. Waddell 1, Joseph W. Klaesner 1,4,5, Marghuretta D. Bland 1,2,3
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.

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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 Rusch 1, Abul Usmani 1, Alexandra M. Lopes 3,4, Donald F. Conrad 1
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.

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Medicine

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation
Wyatt E. Lanik 1, Lily Xu 2, Cliff J. Luke 1, Elise Z. Hu 2, Pranjal Agrawal 2, Victoria S. Liu 2, Rajesh Kumar 1, Alexa M. Bolock 1, Congrong Ma 3, Misty Good 1
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.

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Behavior

Use of a Video Scoring Anchor for Rapid Serial Assessment of Social Communication in Toddlers
Natasha Marrus 1, Stefanie Kennon-McGill 1, Brooke Harris 2, Yi Zhang 1, Anne L. Glowinski 1, John N. Constantino 1
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.

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JoVE Core

Measuring the Carotid to Femoral Pulse Wave Velocity (Cf-PWV) to Evaluate Arterial Stiffness
Hongwei Ji 1, Jing Xiong 1, Shikai Yu 1, Chen Chi 1, Bin Bai 1, Jiadela Teliewubai 1, Yuyan Lu 1, Yi Zhang 1, Yawei Xu 1
1Department of Cardiology, Shanghai Tenth People's Hospital, Tongji University School of Medicine

This protocol describes a method to standardize the measurements of carotid-femoral pulse wave velocity to evaluate arterial stiffness.

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Genetics

Rare Event Detection Using Error-corrected DNA and RNA Sequencing
Wing H. Wong *1,2, R. Spencer Tong *1,2, Andrew L. Young 1,2, Todd E. Druley 1,2
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.

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Developmental Biology

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization
Stephanie A. Michalski *1, Sangappa B. Chadchan *1, Emily S. Jungheim 1, Ramakrishna Kommagani 1
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.

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Genetics

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)
Erin A. Brettmann 1, Inez Y. Oh 1, Cristina de Guzman Strong 1
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.

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Cancer Research

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad
Gan-Lin Zhang 1, Yi Zhang 1, Ke-Xin Cao 1, Xiao-Min Wang 1
1Oncology Department, Beijing Hospital of Traditional Chinese Medicine, Capital Medical University

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

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Biology

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis
Jeffrey R. Millman 1,2, Teresa Doggett 3, Christina Thebeau 3, Sheng Zhang 3, Clay F. Semenkovich 1, Rithwick Rajagopal 3
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.

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Medicine

Multi-Modal Home Sleep Monitoring in Older Adults
Cristina D. Toedebusch *1, Jennifer S. McLeland *1, Claire M. Schaibley 1, Ian R. Banks 1, Jill Boyd 1, John C. Morris 1,2,3, David M. Holtzman 1,2,3, Brendan P. Lucey 1,2
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.

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Chemistry

Reactive Inkjet Printing and Propulsion Analysis of Silk-based Self-propelled Micro-stirrers
David A. Gregory 1, Piyush Kumar 1, Ana Jimenez-Franco 1, Yi Zhang 1, Yu Zhang  2, Stephen J. Ebbens 1, Xiubo Zhao 1
1Department of Chemical and Biological Engineering, University of Sheffield, 2School of Engineering, University of Liverpool

This protocol demonstrates the ability to utilize reactive inkjet printing to print self-motile biocompatible and environmentally friendly micro-stirrers for use in biomedical and environmental applications.

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Chemistry

Breath Collection from Children for Disease Biomarker Discovery
Amalia Z. Berna 1, Brian DeBosch 1,2, Janis Stoll 3, Audrey R. Odom John 1,4
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.

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Medicine

Inverse Probability of Treatment Weighting (Propensity Score) using the Military Health System Data Repository and National Death Index
Joshua D. Mitchell 1, Brian F. Gage 2, Nicole Fergestrom 3, Eric Novak 1, Todd C. Villines 4
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.

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Immunology and Infection

Isolation of Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens
Geetika Bajpai 1, Kory J. Lavine 1,2,3
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.

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JoVE Journal

Measuring Diurnal Rhythms in Autophagic and Proteasomal Flux
Mikhail Ryzhikov 1, Anna Eubanks 1, Jeffrey A. Haspel 1
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.

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Bioengineering

Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence Microscopy
Avani V. Pisapati 1, Yi Wang *2, Megan E. Blauch *3, Nathan J. Wittenberg 3, Xuanhong Cheng 1,2, X. Frank Zhang 1,4
1Department of Bioengineering, Lehigh University, 2Department of Materials Science and Engineering, Lehigh University, 3Department of Chemistry, Lehigh University, 4Department of Mechanical Engineering and Mechanics, Lehigh University

We present a protocol for immobilizing single macromolecules in microfluidic devices and quantifying changes in their conformations under shear flow. This protocol is useful for characterizing the biomechanical and functional properties of biomolecules such as proteins and DNA in a flow environment.

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Chemistry

A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules
Yi Zhang 1, Kanako Kurosawa 1, Daisuke Nishiura 2, Mika Tei 1, Mikiko Tsudome 3
1SUGAR Program, X-star, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2Center for Mathematical Science and Advanced Technology, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 3Deep-Sea Nanoscience Research Group, Japan Agency for Marine-Earth Science and Technology (JAMSTEC)

The overall goal of the protocol is to prepare over one million ordered, uniform, stable, and biocompatible femtoliter droplets on a 1 cm2 planar substrate that can be used for cell-free protein synthesis.

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Modeling Intestinal Development and Injury-Repair In Vitro Using Mouse Colonic Stem Cell Monolayer Culture Under Air-Liquid Interface
Brian D. Muegge *1, Yi Wang *2, Thaddeus S. Stappenbeck 2,3
1Division of Endocrinology, Diabetes, and Lipid Metabolism Research, Department of Medicine, Washington University School of Medicine, 2Department of Pathology and Immunobiology, Washington University School of Medicine, 3Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic

This protocol describes the generation of a long-lived self-renewing monolayer culture system for mouse colonic stem cells that contains all major epithelial cell types. This culture system can be used to study epithelial biology, intestinal wound repair, and host-pathogen interactions.

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Medicine

Lymphatic and Blood Network Analysis During Obesity
Rafael S Czepielewski *1, Alexandre Gallerand *2,3, Jérôme Gilleron 2,4, Narges Khedher 2,3, Gwendalyn J. Randolph 1, Stoyan Ivanov 2,3
1Department of Pathology and Immunology, Washington University School of Medicine, 2Université Côte d'Azur, 3Mediterranean Center of Molecular Medicine C3M, Team 13, Institut National de la Santé et de la Recherche Médicale (Inserm) UMR1065, 4Mediterranean Center of Molecular Medicine C3M, Team 7, Institut National de la Santé et de la Recherche Médicale (Inserm) UMR1065

Obesity is a growing global public health issue. It has been previously associated with lymphatic dysfunction, suggesting a vital crosstalk between the adipose tissue and the lymphatic system. Here, we propose an accessible methodology allowing the distinct labeling of blood and lymphatic vasculatures within the subcutaneous adipose tissue.

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Immunology and Infection

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice
Valerie P. O'Brien 1,2,6, Matthew S. Joens 3,7, Amanda L. Lewis 1,2,4,8, Nicole M. Gilbert 2,5
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.

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Neuroscience

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina
Zelun Wang 1,2, Sean McCracken 1,2, Philip R. Williams 1,3,4
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.

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Neuroscience

The Sleep Nullifying Apparatus: A Highly Efficient Method of Sleep Depriving Drosophila
Krishna Melnattur 1, Ellen Morgan 1, Vincent Duong 1, Arjan Kalra 1, Paul J. Shaw 1
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.

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Medicine

Biological Preparation and Mechanical Technique for Determining Viscoelastic Properties of Zonular Fibers
Juan Rodriguez 1,2, Matthew Reilly 3, Robert P. Mecham 4, Steven Bassnett 2,4
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.

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Neuroscience

Assessment of Swim Endurance and Swim Behavior in Adult Zebrafish
Brooke Burris 1, Nicholas Jensen 1, Mayssa H. Mokalled 1,2
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.

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Immunology and Infection

Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria
Sydney R. Morrill 1,2,3, Kavita Agarwal 2,3, Sudeshna Saha 2,3, Warren G. Lewis 2,3, Nicole M. Gilbert 4, Amanda L. Lewis 2,3
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.

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Immunology and Infection

Mouse Heterotopic Cervical Cardiac Transplantation Utilizing Vascular Cuffs
Wenjun Li 1, Hailey M. Shepherd 1, Alexander S. Krupnick 2, Andrew E. Gelman 1,3, Kory J. Lavine 4, Daniel Kreisel 1,3
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.

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Neuroscience

A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants
Jalin A. Stelzer 1, Jason J. Yi 1
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.

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Neuroscience

Real-Time Dynamic Collection of Hippocampal Extracellular Fluid from Conscious Rats Using a Microdialysis System
Xiaobo Wang 1,2, Na Xie 3, Yi Zhang 4, Xianli Meng 1,2,3, Ya Hou 3, Sanyin Zhang 1,2
1Innovative Institute of Chinese Medicine and Pharmacy, Chengdu University of Traditional Chinese Medicine, 2Research Institute of Integrated TCM & Western Medicine, Chengdu University of Traditional Chinese Medicine, 3School of Pharmacy, Chengdu University of Traditional Chinese Medicine, 4School of Ethnic Medicine, Chengdu University of Traditional Chinese Medicine

The protocol here provides a detailed real-time dynamic sampling of extracellular fluid from the hippocampus of awake rats using a microdialysis system.

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Medicine

Dynamic Continuous Blood Extraction from Rat Heart via Noninvasive Microdialysis Technique
Ya Hou 1, Jinrong Bai 1,2, Yi Zhang 3, Xianli Meng 1,2,4, Sanyin Zhang 4, Xiaobo Wang 2
1School of Pharmacy, Chengdu University of Traditional Chinese Medicine, 2Research Institute of Integrated TCM & Western Medicine, Chengdu University of Traditional Chinese Medicine, 3School of Ethnic Medicine, Chengdu University of Traditional Chinese Medicine, 4Innovative Institute of Chinese Medicine and Pharmacy, Chengdu University of Traditional Chinese Medicine

The present protocol describes a simple and efficient method for the real-time and dynamic collection of rat heart blood using the microdialysis technique.

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JoVE Core

An In Vitro Dissolution Determination of Multi-Index Components in Tibetan Medicine Rhodiola Granules
Qinyun Du 1, Qingxiu He 2, Fengming Zhang 3, Jiao Mi 3, Yuying Li 3, Shaohui Wang 1, Yi Zhang 1
1School of Pharmacy, Chengdu University of Traditional Chinese Medicine, 2School of Ethnic Medicine, Chengdu University of Traditional Chinese Medicine, 3Tibet Rhodiola Pharmaceutical Holding Co. Ltd

Here, we test the dissolution of Rhodiola granules (RG) in vitro, draw dissolution curves of salidroside, gallic acid, and ethyl gallate in ultrapure water, and fit the curves to different mathematical models. This protocol provides information and guidance for in vivo bioequivalence and in vivo-in vitro correlation studies of RG.

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Bioengineering

Synthesis of Strong Adhesive Hydrogel, Gelatin O-Nitrosobenzaldehyde
Yuelong Liang *1,2, Zhengze Huang *1, Yi Zhang *3,4, Yi Hong 3,4, Qijiang Mao 1,2,5, Xu Feng 1
1Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, 2Zhejiang Province Medical Research Center of Minimally Invasive Diagnosis and Treatment of Abdominal Diseases, 3Dr. Li Dak Sum & Yip Yio Chin Center for Stem Cells and Regenerative Medicine, Department of Orthopedic Surgery of the Second Affiliated Hospital, Zhejiang University School of Medicine, 4Zhejiang University-University of Edinburgh Institute, Zhejiang University School of Medicine, Key Laboratory of Tissue Engineering and Regenerative Medicine of Zhejiang Province, Zhejiang University School of Medicine, 5Zhejiang Provincial Key Laboratory of Laparoscopic Technology

The protocol presented here shows the synthesis of a strong adhesive hydrogel gelatin o-nitrosobenzaldehyde (gelatin-NB). Gelatin-NB has rapid and efficient tissue adhesion ability, which can form a strong physical barrier to protect wound surfaces, so it is expected to be applied to the field of injury repair biotechnology.

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Medicine

Voltage-Dependent Potassium Current Recording on H9c2 Cardiomyocytes via the Whole-Cell Patch-Clamp Technique
Hong Jiang *1, Yating Zhang *1, Ya Hou 1, Lijun Li 2, Sanyin Zhang 3,4, Yi Zhang 5, Xianli Meng 1,3,4, Xiaobo Wang 4
1State Key Laboratory of Southwestern Chinese Medicine Resources, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, 2Chengdu Techman Instrument Co., Ltd., 3Innovative Institute of Chinese Medicine and Pharmacy, Chengdu University of Traditional Chinese Medicine, 4Research Institute of Integrated TCM & Western Medicine, Chengdu University of Traditional Chinese Medicine, 5School of Ethnic Medicine, Chengdu University of Traditional Chinese Medicine

The present protocol describes an efficient method for the real-time and dynamic acquisition of voltage-gated potassium (Kv) channel currents in H9c2 cardiomyocytes using the whole-cell patch-clamp technique.

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JoVE Core

A Coregistered Ultrasound and Photoacoustic Imaging Protocol for the Transvaginal Imaging of Ovarian Lesions
Haolin Nie 1, Hongbo Luo 2, Lin Chen 1, Quing Zhu 1,3
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.

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Cancer Research

Quantifying Replication Stress in Ovarian Cancer Cells Using Single-Stranded DNA Immunofluorescence
Natasha Ramakrishnan 1, Ena Haseljic 1, Priyanka Verma 1
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.

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Bioengineering

Electromyometrial Imaging of Uterine Contractions in Pregnant Women
Hui Wang *1,2,3, Zichao Wen *2,3, Wenjie Wu 2,3,4, Zhexian Sun 2,3,4, Qing Wang 5, Alan L. Schwartz 6, Phillip Cuculich 7, Alison G. Cahill 8, George A. Macones 8, Yong Wang 2,3,4,5
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.

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Neuroscience

Multi-Tracer Studies of Brain Oxygen and Glucose Metabolism Using a Time-of-Flight Positron Emission Tomography-Computed Tomography Scanner
John J. Lee 1, Nicholas Metcalf 1, Tony A. Durbin 1, Jennifer Byers 1, Kim Casey 1, Hussain Jafri 1, Manu S. Goyal *1, Andrei G. Vlassenko *1
1Mallinckrodt Institute of Radiology, Washington University School of Medicine

Quantitative measurements of oxygen and glucose metabolism by PET are established technologies, but details of practical protocols are sparsely described in the literature. This paper presents a practical protocol successfully implemented on a state-of-the-art positron emission tomography-computed tomography scanner.

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Chemistry

Histone Modification Screening using Liquid Chromatography, Trapped Ion Mobility Spectrometry, and Time-Of-Flight Mass Spectrometry
Meiby Fernandez-Rojas 1, Cassandra N. Fuller 1, Lilian Valadares Tose 1, Matthew Willetts 2, Melvin A. Park 2, Natarajan V. Bhanu 3, Benjamin A. Garcia 3, Francisco Fernandez-Lima 1,4
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).

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Medicine

Microfluidic Model of Necrotizing Enterocolitis Incorporating Human Neonatal Intestinal Enteroids and a Dysbiotic Microbiome
Lauren C. Frazer 1, Yukihiro Yamaguchi 1, Corey M. Jania 1, Wyatt E. Lanik 2, Qingqing Gong 3, Dhirendra K. Singh 1, Stephen Mackay 1, Natalia S. Akopyants 1, Misty Good 1
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.

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Medicine

Surgical Perfusion and Isolation of the Porcine Pancreas for Islet Isolation
Sophia H. Roberts 1,2, Louai Alrata 1,2, Dahlia Abdulsattar 1,2, Karan Joseph 1,2, Wahid Abu-Amer 1,2, Mohamed Zaghloul 1,2, Batool Arif 1,2, Tarek Alhamad 3, Maria S. Remedi 4, Yiing Lin 5, Mohamed A. Zayed 1,2,6,7
1Department of Surgery, Section of Vascular Surgery, Washington University School of Medicine, 2CardioVascular Research Innovation in Surgery and Engineering Center, Washington University School of Medicine, 3Department of Medicine, Division of Nephrology, Washington University School of Medicine, 4Department of Medicine, Division of Endocrinology, Washington University School of Medicine, 5Department of Surgery, Section of Transplant Surgery, Washington University School of Medicine, 6Division of Molecular Cell Biology, Washington University School of Medicine, 7Department of Biomedical Engineering, Washington University McKelvey School of Engineering

This protocol provides a step-by-step guide for the procurement of a porcine pancreas for islet isolation and purification.

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Measurement of Mitochondrial Respiration in Human and Mouse Skeletal Muscle Fibers by High-Resolution Respirometry

Measurement of Mitochondrial Respiration in Human and Mouse Skeletal Muscle Fibers by High-Resolution Respirometry
Terri A. Pietka 1,2, Rita T. Brookheart 1,3
1John T. Milliken Department of Medicine, Division of Nutritional Sciences and Obesity Medicine, Washington University School of Medicine, 2Nutrition and Obesity Research Center, Cellular and Molecular Biology Core, Washington University School of Medicine, 3Nutrition and Obesity Research Center, Animal Model Research Core, Washington University School of Medicine

Here, we describe a comprehensive method for measuring mitochondrial oxidative phosphorylation in fresh permeabilized skeletal muscle fibers from either human or mouse muscle. This method allows for the real-time quantification of mitochondrial respiration and the assessment of fuel preference and metabolic flexibility while preserving existing mitochondrial networks and membrane integrity.

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