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62 ARTICLES PUBLISHED IN JoVE

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Biology

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time
Yi-Ping Ho 1,2, Hunter H. Chen 2,3, Kam W. Leong 2, Tza-Huei Wang 1,3
1Mechanical Engineering, Johns Hopkins University, 2Biomedical Engineering, Duke University, 3Biomedical Engineering, Johns Hopkins University

We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.

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Biology

Assembly, Tuning and Use of an Apertureless Near Field Infrared Microscope for Protein Imaging
Melissa Paulite 1, Zahra Fakhraai 2, Boris B. Akhremitchev 3, Kerstin Mueller 1, Gilbert C. Walker 1
1Department of Chemistry, University of Toronto, 2Department of Chemistry, University of Wisconsin, 3Department of Chemistry, Duke University

The assembly of a nearfield infrared microscope for imaging protein aggregates is described.

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Neuroscience

Human Fear Conditioning Conducted in Full Immersion 3-Dimensional Virtual Reality
Nicole C. Huff 1, David J. Zielinski 2, Matthew E. Fecteau 1, Rachael Brady 2, Kevin S. LaBar 1
1Center for Cognitive Neuroscience, Duke University, 2Pratt School of Engineering, Duke University

Classical fear conditioning paradigm was adapted for human participants in a fully immersive virtual reality setting. Using a discrimination paradigm, conditioned fear, cue and context memory retention, and extinction was measured with skin conductance response to dynamic virtual snakes and spiders (the conditioned stimuli) in two distinct virtual contexts.

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Neuroscience

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number
Dominic M. Ippolito 1, Cagla Eroglu 2
1Department of Neurobiology, Duke University, 2Department of Cell Biology, Duke University

This protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers.

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Neuroscience

Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells
Sandeepa Dey 1, Senmiao Zhan 2, Hiroaki Matsunami 1
1Department of Molecular Genetics and Molecular Biology , Duke University, 2Department of Chemistry, Duke University

Here we demonstrate a protocol to carry out live cell staining that can be used to detect odorant receptors on the surface of HEK293T cells conveniently. In addition, it may also be used to assay for surface expression of other chemosensory receptors or GPCRs.

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Neuroscience

Brain Imaging Investigation of the Memory-Enhancing Effect of Emotion
Andrea Shafer 1, Alexandru Iordan 2, Roberto Cabeza 3, Florin Dolcos 1,4
1Centre for Neuroscience, University of Alberta, 2Neuroscience Program, University of Illinois, Urbana-Champaign, 3Center for Cognitive Neuroscience, Duke University, 4Psychology Department, Neuroscience Program, & Beckman Institute, University of Illinois, Urbana-Champaign

We present a protocol that uses functional magnetic resonance imaging to investigate the neural correlates of the memory-enhancing effect of emotion. This protocol allows identification of brain activity specifically linked to memory-related processing, contrary to more general perceptual processing, and can be used with healthy and clinical populations.

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Bioengineering

Procedure for Lung Engineering
Elizabeth A. Calle *1, Thomas H. Petersen *2, Laura E. Niklason 1,3
1Department of Biomedical Engineering, Yale University, 2Department of Biomedical Engineering, School of Medicine, Duke University, 3Department of Anesthesia, Yale University

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

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Immunology and Infection

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay
Philip M. Armstrong 1, Theodore G. Andreadis 1, Shannon L. Finan 1, John J. Shepard 1, Michael C. Thomas 1
1Center for Vector Biology and Zoonotic Diseases, Department of Environmental Sciences, The Connecticut Agricultural Experiment Station

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

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Biology

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue
Chun-Chun Chen 1, Kazuhiro Wada 2, Erich D. Jarvis 1
1Howard Hughes Medical Institute, Department of Neurobiology, Duke University , 2Department of Biological Sciences, Hokkaido University

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

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Biology

In Vivo Modeling of the Morbid Human Genome using Danio rerio
Adrienne R. Niederriter 1,2, Erica E. Davis 1,3, Christelle Golzio 1, Edwin C. Oh 1, I-Chun Tsai 1, Nicholas Katsanis 1
1Center for Human Disease Modeling, Department of Cell Biology, Duke University Medical Center, 2Department of Evolutionary Anthropology, Duke University, 3Department of Pediatrics, Duke University Medical Center

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

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Neuroscience

Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling
Gustavo Arriaga 1, Joshua J. Macopson 1, Erich D. Jarvis 1,2
1Department of Neurobiology, Duke University Medical Center, 2Howard Hughes Medical Institute

Transsynaptic tracing has become a powerful tool for analyzing central efferents regulating peripheral targets through multi-synaptic circuits. Here we present a protocol that exploits the transsynaptic pseudorabies virus to identify and localize a functional brain circuit, followed by classical tract tracing techniques to validate specific connections in the circuit between identified groups of neurons.

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Medicine

Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage
Beilei Lei 1, Huaxin Sheng 1, Haichen Wang 2, Christopher D. Lascola 3, David S. Warner 1,4, Daniel T. Laskowitz 1,2,4, Michael L. James 1,2
1Multidisciplinary Neuroprotection Laboratories, Department of Anesthesiology, Duke University, 2Department of Neurology, Duke University, 3Department of Radiology, Duke University, 4Department of Neurobiology, Duke University

Preclinical models of intracerebral hemorrhage are utilized to mimic certain aspects of clinical disease. Thus, mechanisms of injury and potential therapeutic strategies may be explored. In this protocol, two models of intracerebral hemorrhage are described, intrastriatal (basal ganglia) injections of autologous blood or collagenase.

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Biology

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli
Sarah R. MacEwan *1,2, Wafa Hassouneh *1,2, Ashutosh Chilkoti 1,2
1Department of Biomedical Engineering, Duke University, 2Research Triangle MRSEC, Duke University

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

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Medicine

An Isolated Working Heart System for Large Animal Models
Matthew A. Schechter 1, Kevin W. Southerland 1, Bryan J. Feger 1, Dean Linder Jr. 1, Ayyaz A. Ali 2, Linda Njoroge 1, Carmelo A. Milano 1, Dawn E. Bowles 1
1Department of Surgery, Duke University Medical Center, 2Department of Cardiothoracic Surgery, University Hospital of South Manchester

Most studies involving the Langendorff apparatus use small animal models due to the increased complexity of systems for larger mammals. We describe a Langendorff system for large animal models that allows for use across a range of species, including humans, and relatively easy data acquisition.

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Immunology and Infection

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care
Katherine Riccione 1,2, Carter M. Suryadevara 1,3, David Snyder 1, Xiuyu Cui 1, John H. Sampson 1,2,3, Luis Sanchez-Perez 1
1Duke Brain Tumor Immunotherapy Program, Department of Neurosurgery, Duke University, 2Department of Biomedical Engineering, Duke University, 3Department of Pathology, Duke University

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

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Medicine

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo
Cara C. Rada 1, Stephanie L. Pierce 2, Chad A. Grotegut 2, Sarah K. England 1
1Department of Obstetrics and Gynecology, Washington University in St. Louis, 2Department of Obstetrics and Gynecology, Duke University

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

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Neuroscience

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae
Jared D. Chrispell 1, Tatiana I. Rebrik 2, Ellen R. Weiss 1
1Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, 2Department of Ophthalmology, Duke University

We present a method for the electroretinographic (ERG) analysis of zebrafish larvae utilizing micromanipulation and electroretinography techniques. This is a simple and straightforward method for assaying visual function of zebrafish larvae in vivo.

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Neuroscience

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster
Bin Gou *1, Edward Zhu *2, Ruo He 1, Ulrich Stern 1, Chung-Hui Yang 1
1Department of Neurobiology, Duke University, 2Department of Pharmacology, Duke University

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

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Engineering

Fabrication and Operation of Acoustofluidic Devices Supporting Bulk Acoustic Standing Waves for Sheathless Focusing of Particles
C. Wyatt Shields IV 1,2, Daniela F. Cruz 1,2, Korine A. Ohiri 1,3, Benjamin B. Yellen 1,2,3, Gabriel P. Lopez 1,2,3
1NSF Research Triangle Materials Research Science and Engineering Center, Duke University, 2Department of Biomedical Engineering, Duke University, 3Department of Mechanical Engineering and Materials Science, Duke University

Acoustofluidic devices use ultrasonic waves within microfluidic channels to manipulate, concentrate and isolate suspended micro and nanoscopic entities. This protocol describes the fabrication and operation of such a device supporting bulk acoustic standing waves to focus particles in a central streamline without the aid of sheath fluids.

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Engineering

Colloidal Synthesis of Nanopatch Antennas for Applications in Plasmonics and Nanophotonics
Thang B. Hoang 1,2, Jiani Huang 1,2, Maiken H. Mikkelsen 1,2,3
1Department of Physics, Duke University, 2Center for Metamaterials and Integrated Plasmonics, Duke University, 3Department of Electrical and Computer Engineering, Duke University

A protocol for the colloidal synthesis of silver nanocubes and fabrication of plasmonic nanoscale patch antennas with sub-10 nm gaps is presented.

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Immunology and Infection

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids
Daniel E. Rothschild 1, Tara Srinivasan 2, Linette A. Aponte-Santiago 1, Xiling Shen 2,3,4, Irving C. Allen 1
1Department of Biomedical Sciences and Pathobiology, Virginia Maryland College of Veterinary Medicine, Virginia Tech, 2Department of Biomedical Engineering, Cornell University, 3School of Electrical and Computer Engineering, Cornell University, 4Department of Biomedical Engineering, Duke University

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

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Environment

Spotting Cheetahs: Identifying Individuals by Their Footprints
Zoe C. Jewell 1, Sky K. Alibhai 1, Florian Weise 2,3, Stuart Munro 2, Marlice Van Vuuren 4, Rudie Van Vuuren 4
1WildTrack and Nicholas School of the Environment, Duke University, 2N∕a′an ku sê Research Programme, 3Division of Biology and Conservation Ecology, School of Science and the Environment, Manchester Metropolitan University, 4N∕a′an ku sê Foundation

The cheetah (Acinonyx jubatus) is an iconic, endangered species, but conservation efforts are challenged by habitat shrinkage and conflict with commercial farmers. The footprint identification technique, a robust, accurate and cost-effective image classification system, is a new approach to monitoring cheetahs.

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Behavior

Eliciting and Analyzing Male Mouse Ultrasonic Vocalization (USV) Songs
Jonathan Chabout 1,2, Joshua Jones-Macopson 1, Erich D. Jarvis 1,2,3
1Department of Neurobiology, Duke University, 2Howard Hughes Medical Institute, 3The Rockefeller University

Mice produce a complex multisyllabic repertoire of ultrasonic vocalizations (USVs). These USVs are widely used as readouts for neuropsychiatric disorders. This protocol describes some of the practices we learned and developed to consistently induce, collect, and analyze the acoustic features and syntax of mouse songs.

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Bioengineering

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior
Kai Wang 1, Kam W. Leong 2, Yong Yang 1
1Department of Chemical and Biomedical Engineering, West Virginia University, 2Department of Biomedical Engineering, Columbia University

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

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Bioengineering

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)–Cell Interaction and the Resultant Bioeffects at the Single-cell Level
Fenfang Li 1, Fang Yuan 2, Georgy Sankin 1, Chen Yang 1, Pei Zhong 1
1Mechanical Engineering and Materials Science, Duke University, 2Huacells Corp

A microfluidic chip was fabricated to produce pairs of gold dots for tandem bubble generation and fibronectin-coated islands for single-cell patterning nearby. The resultant flow field was characterized by particle image velocimetry and was employed to study various bioeffects, including cell membrane poration, membrane deformation, and intracellular calcium response.

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Biology

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus
Hae-Na Chung 1, Stacy D. Rodriguez 1, Victoria K. Carpenter 2, Julia Vulcan 1, C. Donovan Bailey 1, Madhugiri Nageswara-Rao 1, Yiyi Li 3, Geoffrey M. Attardo 4, Immo A. Hansen 1,5
1Department of Biology, New Mexico State University, 2Department of Molecular Genetics and Microbiology, Duke University, 3Department of Computer Sciences, New Mexico State University, 4Department of Epidemiology of Microbial Diseases, Yale School of Public Health, 5Institute of Applied Biosciences, New Mexico State University

The fat body is the central metabolic organ in insects. We present a live organ culture system that enables the user to study the responses of isolated fat body tissue to various stimuli.

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Developmental Biology

Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells
Kathryn E. Ware 1, Shivee Gilja 1, Shenghan Xu 1, Samantha Shetler 1, Mohit K. Jolly 2, Xueyang Wang 3, Suzanne Bartholf Dewitt 4, Alexander J. Hish 1, Sarah Jordan 1, William Eward 5, Herbert Levine 2, Andrew J. Armstrong 4, Jason A. Somarelli 1
1Department of Medicine, Duke University, 2Department of Bioengineering, Rice University, 3Department of Molecular Genetics and Microbiology, Duke University, 4Solid Tumor Program and the Duke Prostate Center, Duke University Medical Center, 5Duke University Medical Center

We present here a cell culture method for inducing mesenchymal-epithelial transitions (MET) in sarcoma cells based on combined ectopic expression of microRNA-200 family members and grainyhead-like 2 (GRHL2). This method is suitable for better understanding the biological impact of phenotypic plasticity on cancer aggressiveness and treatments.

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Neuroscience

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay
Yuetian Zhang *1, Yi Pan *1, Hiroaki Matsunami 2,3, Hanyi Zhuang 1,4
1Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao Tong University School of Medicine, 2Department of Molecular Genetics and Microbiology, Duke University Medical Center, 3Department of Neurobiology, Duke Institute for Brain Sciences, Duke University Medical Center, 4Institute of Health Science, Chinese Academy of Science/Shanghai Jiao Tong University School of Medicine

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

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Medicine

Use of Two Intracorporeal Ventricular Assist Devices As a Total Artificial Heart
Muath Bishawi 1, Jun-Neng Roan 1,2, Jordan Richards 1, Zachary Brown 1, Laura Blue 1, Mani A. Daneshmand 1, Jacob N. Schroder 1, Dawn E. Bowles 3, Carmelo A. Milano 1
1Division of Cardiothoracic Surgery, Department of Surgery, Duke University, 2Division of Cardiovascular Surgery, Department of Surgery, National Cheng Kung University Hospital and College of Medicine, 3Division of Surgical Sciences, Department of Surgery, Duke University

Here, we present a protocol using two centrifugal pumps as a total artificial heart replacement.

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JoVE Journal

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope
Zackary N. Scholl 1, Qing Li 1, Eric Josephs 1, Dimitra Apostolidou 1, Piotr E. Marszalek 1
1Mechanical Engineering and Materials Science, Duke University

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

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Bioengineering

A Protocol for Real-time 3D Single Particle Tracking
Shangguo Hou 1, Kevin Welsher 1
1Department of Chemistry, Duke University

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

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Cancer Research

A GPC3-targeting Bispecific Antibody, GPC3-S-Fab, with Potent Cytotoxicity
Yanlan Wang 1,2, Jiayu Liu 1,2, Haitao Pan 1,2, Jieyu Xing 1,2, Xiaoqiong Wu 1,2, Qing Li 1,2, Zhong Wang 1,2
1School of Pharmaceutical Sciences, Sun Yat-Sen University, 2Center for Cellular & Structural Biology, Sun Yat-Sen University

Here, we present a protocol to produce a bispecific antibody GPC3-S-Fab in Escherichia coli. The purified GPC3-S-Fab has potent cytotoxicity against GPC3 positive liver cancer cells.

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Developmental Biology

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila
Scott Barish 1, Pelin C. Volkan 1
1Department of Biology, Duke University

Here, we present a protocol to stage and dissect developing olfactory tissue from Drosophila species. The dissected tissue can later be used for molecular analyses, such as quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) or RNA sequencing (RNAseq), as well as in vivo analyses such as immunohistochemistry or in situ hybridization.

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Medicine

Ferric Chloride-induced Canine Carotid Artery Thrombosis: A Large Animal Model of Vascular Injury
Allyson L. Huttinger 1, Debra G. Wheeler 1, Surya Gnyawali 2, David Dornbos III 1, Juliana M. Layzer 3, Nicholas Venetos 1, Spencer Talentino 1, Nicholas J. Musgrave 1, Cheyenne Jones 1, Camille Bratton 1, Matthew E. Joseph 4, Chandan Sen 2, Bruce A. Sullenger 3, Shahid M. Nimjee 1
1Department of Neurological Surgery, Ohio State University, 2Department of Surgery, Ohio State University, 3Department of Surgery, Duke University, 4Department of Internal Medicine, Ohio State University

Here, we present the modifications necessary to a well characterized and commonly used small animal ferric chloride-induced (FeCl3) carotid artery injury model for use in a large animal vascular injury model. The resulting model can be utilized for pre-clinical trial assessment of both prophylactic and thrombolytic pharmacological and mechanical interventions.

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Biology

Reverse Dissection and DiceCT Reveal Otherwise Hidden Data in the Evolution of the Primate Face
Anne M. Burrows 1,2, Kailey M Omstead 1, Ashley R Deutsch 3,5, Justin T. Gladman 4, Adam Hartstone-Rose 3
1Department of Physical Therapy, Duquesne University, 2Department of Anthropology, University of Pittsburgh, 3College of Sciences, North Carolina State University, 4Shared Materials Instrumentation Facility, Duke University, 5Department of Anthropology, University of Florida

Facial expressions are a mode of visual communication produced by mimetic muscles. Here, we present protocols for the novel techniques of reverse dissection and DiceCT to fully visualize and assess mimetic muscles. These combined techniques can examine both morphological and physiological aspects of mimetic musculature to determine functional aspects.

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JoVE Journal

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques
Katheryn E. Rothenberg 1, Ishaan Puranam 1, Brenton D. Hoffman 1
1Department of Biomedical Engineering, Duke University

Here, we present a protocol for the simultaneous use of Förster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

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Bioengineering

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells
Emma Pipó-Ollé 1, Prasad Walke 2, Martine K. Notabi 1, Rime B. El-Houri 1, Morten Østergaard Andersen 1, David Needham 2,3,4, Eva C. Arnspang 1
1Department of Chemical Engineering, Biotechnology and Environmental Technology, University of Southern Denmark, 2Center for Single Particle Science and Engineering (SPSE), Institute for Molecular Medicine, Health Sciences, University of Southern Denmark, 3Department of Mechanical Engineering and Material Science, Duke University, 4School of Pharmacy, University of Nottingham

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

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Neuroscience

Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase
Claire A. de March 1, Yosuke Fukutani 1,2, Aashutosh Vihani 1,3, Hitoshi Kida 1,4, Hiroaki Matsunami 1,3,5,6
1Department of Molecular Genetics and Microbiology, Duke University Medical Center, 2Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 3Department of Neurobiology, Duke University Medical Center, 4Department of Mechanical Systems, Engineering, Tokyo University of Agriculture and Technology, 5Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, 6Duke Institute for Brain Sciences, Duke University

Physiologically, odorant receptors are activated by odorant molecules inhaled in the vapor phase. However, most in vitro systems utilize liquid phase odorant stimulation. Here, we present a method that allows real-time in vitro monitoring of odorant receptor activation upon odorant stimulation in vapor phase.

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Biochemistry

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling
Stefan Ilic 1, Shira Cohen 1, Ariel Afek 2, Raluca Gordan 2, David B. Lukatsky 1, Barak Akabayov 1
1Department of Chemistry, Ben-Gurion University of the Negev, 2Center for Genomic and Computational Biology, Department of Biostatistics and Bioinformatics, Duke University

Protein binding microarray (PBM) experiments combined with biochemical assays link the binding and catalytic properties of DNA primase, an enzyme that synthesizes RNA primers on template DNA. This method, designated as high-throughput primase profiling (HTPP), can be used to reveal DNA-binding patterns of a variety of enzymes.

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Behavior

Evaluating Skilled Prehension in Mice Using an Auto-Trainer
Robert Hubbard 1, Jason Dunthorn 2, Richard J. O’Brien 3, Dan Tasch 2, Uri Tasch 2, Steven R. Zeiler 1
1Department of Neurology, Johns Hopkins University, 2Step Analysis, LLC, 3Department of Neurology, Duke University

Method to assess the impact of training on motor skills is a useful tool. Unfortunately, most behavioral assessments can be labor intensive and/or expensive.We describe here a robotic method of assessing prehension (reach-to-grasp) skill in mice.

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Engineering

Evaluation of an Exclusive Spur Dike U-Turn Design with Radar-Collected Data and Simulation
Yang Shao 1, Hongtao Yu 1, Huan Wu 2, Xueyan Han 1, Xizhen Zhou 1, Christian G. Claudel 3, Hualing Zhang 4, Chen Yang 5
1Highway Academy, Chang'an University, 2Human Resource Department, Xi'an Shiyou University, 3Cockrell School of Engineering, University of Texas at Austin, 4Department of Earth and Atmospheric Science, University of Houston, 5School of Foreign Languages, Xi'an Shiyou University

This protocol describes the process of solving a microscopic traffic problem with simulation. The whole process contains a detailed description of data collection, data analysis, simulation model build, simulation calibration, and sensitive analysis. Modifications and troubleshooting of the method are also discussed.

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Immunology and Infection

Optimization of the Cuff Technique for Murine Heart Transplantation
Yunhan Ma *1,2, Baiyi Xie *1,2, Helong Dai *3,4,5, Chenxi Wang 6, Shujiao Liu 6, Tianshu Lan 7, Shuangyue Xu 1,2, Guoliang Yan 1,2,6, Zhongquan Qi 1,2,8
1Organ Transplantation Institute, School of Medicine, Xiamen University, 2Fujian Key Laboratory of Organ and Tissue Regeneration, 3Department of Kidney Transplantation, Center of Organ Transplantation, The Second Xiangya Hospital of Central South University, 4Clinical Research Center for Organ Transplantation in Hunan Province, 5Clinical Immunology Center, Central South University, 6School of Medicine, Xiamen University, 7Xiamen Medical College, 8Medical College, Guangxi University

We introduce an inner tube approach to the cuff technique for mouse cervical heterotopic heart transplantation to help evert the vessel over the cuff. We found that cooperation between two experienced surgeons markedly shortens the operation time.

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Bioengineering

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions
Morgan Burt 1, Rohan Bhattachaya 1, Arinze E. Okafor 1, Samira Musah 1,2
1Department of Biomedical Engineering, Duke University, 2Department of Medicine, Division of Nephrology, Duke University School of Medicine

Presented here is a chemically defined protocol for the derivation of human kidney podocytes from induced pluripotent stem cells with high efficiency (>90%) and independent of genetic manipulations or subpopulation selection. This protocol produces the desired cell type within 26 days and could be useful for nephrotoxicity testing and disease modeling.

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Neuroscience

Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments
Rui Yang 1,2,3, Vann Bennett 2,3
1School of Basic Medical Sciences, Xi'an Jiaotong University, 2Department of Cell Biology, Duke University, 3Department of Biochemistry, Duke University

Here, we described a protocol to quantitatively study the assembly and structure of the axon initial segments (AIS) of hippocampal neurons that lack pre-assembled AIS due to the absence of a giant ankyrin-G.

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Biology

Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke
Katrina L. Wilson 1, S. Thomas Carmichael 2, Tatiana Segura 1,3,4
1Department of Biomedical Engineering, Duke University, 2Department of Neurology, University of California Los Angeles, 3Department of Neurology, Duke University, 4Department of Dermatology, Duke University

Stroke is a global issue with minimal treatment options and no current clinical therapy for regenerating the lost brain tissue. Here we describe methods for creating precise photothrombotic stroke in the motor cortex of rodents and subsequent injection of hydrogel biomaterials to study their effects on tissue regeneration after stroke.

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Bioengineering

Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection
Gabriella Shull *1,2, Christiane Haffner *3, Wieland B. Huttner 3, Elena Taverna 3,4, Suhasa B. Kodandaramaiah 1,5,6
1Department of Biomedical Engineering, University of Minnesota, 2Department of Biomedical Engineering, Duke University, 3Max Planck Institute of Molecular Cell Biology and Genetics, 4Max Planck Institute for Evolutionary Anthropology, 5Department of Mechanical Engineering, University of Minnesota, 6Graduate Program in Neuroscience, University of Minnesota

This protocol demonstrates the use of a robotic platform for microinjection into single neural stem cells and neurons in brain slices. This technique is versatile and offers a method of tracking cells in tissue with high spatial resolution.

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Developmental Biology

Clonal Analysis of the Neonatal Mouse Heart using Nearest Neighbor Modeling
Melanie Bakovic *1, Devang Thakkar *2, Paige DeBenedittis 1, Diana C. Chong 1, Michael C. Thomas 1, Edwin S. Iversen 3, Ravi Karra 1,4
1Department of Medicine, Duke University, 2Center for Genomic and Computational Biology, Duke University, 3Department of Statistical Science, Duke University, 4Regeneration Next, Duke University

Presented here is a protocol for using multicolor lineage tracing and nearest-neighbor modeling to identify clonally derived cardiomyocytes during growth and regeneration in mice. This approach is objective, works across different labeling conditions, and can be adapted to incorporate a variety of image analysis pipelines.

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Bioengineering

Building a Simple and Versatile Illumination System for Optogenetic Experiments
Phillip Kyriakakis 1, Lourdes Fernandez de Cossio 2, Patrick Wade Howard 1, Sivleng Kouv 1, Marianne Catanho 1, Vincent J. Hu 3, Robert Kyriakakis 1, Molly E. Allen 1, Yunhan Ma 4, Marcelo Aguilar-Rivera 1, Todd P. Coleman 1
1Department of Bioengineering, University of California, San Diego, 2University of California, San Diego, 3Department of Mathematical Computational, and Systems biology, University of California, Irvine, 4Department of Biomedical Engineering, Duke University

This protocol describes how to perform optogenetic experiments for controlling gene expression with red and far-red light using PhyB and PIF3. Included are step-by-step instructions for building a simple and flexible illumination system, which enables the control of gene expression or other optogenetics with a computer.

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Neuroscience

Assessment of Sensory Thresholds in Dogs Using Mechanical and Hot Thermal Quantitative Sensory Testing
Rachael M. Cunningham 1,2, Rachel M. Park 1,2, David Knazovicky 3, B. Duncan X. Lascelles 2,4,5,6, Margaret E. Gruen 1,2,4
1Comparative Behavioral Research, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 2Translational Research in Pain (TRiP) Program, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 3Small Animal Orthopedic Surgery, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 4Comparative Pain Research and Education Center, College of Veterinary Medicine, North Carolina State University, 5Thurston Arthritis Center, UNC School of Medicine, University of North Carolina - Chapel Hill, 6Center for Translational Pain Research, Department of Anesthesiology, Duke University

This work describes a standard protocol for mechanical and hot thermal quantitative sensory testing to evaluate the somatosensory system in dogs. Sensory thresholds are measured using an electronic von Frey anesthesiometer, pressure algometer, and hot contact thermode.

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Neuroscience

Quantifying Levels of Dopaminergic Neuron Morphological Alteration and Degeneration in Caenorhabditis elegans
Shefali R. Bijwadia 1, Katherine Morton 1, Joel N. Meyer 1
1Nicholas School of the Environment, Duke University

In this article, we showcase how to use a seven-point scoring system to consistently quantify changes to dopaminergic neuron dendrite morphology in C. elegans. This system is intended for analyses of dopaminergic neurodegeneration assays utilizing genetic, chemical, and age-based models of neurodegenerative disorders.

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Biology

Assessment of Cellular Bioenergetics in Mouse Hematopoietic Stem and Primitive Progenitor Cells using the Extracellular Flux Analyzer
Surinder Kumar 1, Morgan Jones 2, Qing Li 2,3,4, David B. Lombard 1,4,5
1Department of Pathology, University of Michigan, Ann Arbor, 2Department of Internal Medicine, University of Michigan, Ann Arbor, 3Department of Cellular and Developmental Biology, University of Michigan, Ann Arbor, 4Rogel Cancer Center, University of Michigan, Ann Arbor, 5Institute of Gerontology, University of Michigan, Ann Arbor

The method presented here summarizes optimized protocols for assessing cellular bioenergetics in non-adherent mouse hematopoietic stem and primitive progenitor cells (HSPCs) using the extracellular flux analyzer to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of HSPCs in real time.

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Biology

Inherent Dynamics Visualizer, an Interactive Application for Evaluating and Visualizing Outputs from a Gene Regulatory Network Inference Pipeline
Robert C. Moseley 1, Sophia Campione 1, Bree Cummins 2, Francis Motta 3, Steven B. Haase 1
1Department of Biology, Duke University, 2Department of Mathematical Sciences, Montana State University, 3Department of Mathematical Sciences, Florida Atlantic University

The Inherent Dynamics Visualizer is an interactive visualization package that connects to a gene regulatory network inference tool for enhanced, streamlined generation of functional network models. The visualizer can be used to make more informed decisions for parameterizing the inference tool, thus increasing confidence in the resulting models.

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Medicine

A Porcine Heterotopic Heart Transplantation Protocol for Delivery of Therapeutics to a Cardiac Allograft
Michelle Mendiola Pla 1, Amy Evans 2, Franklin H. Lee 3, Yuting Chiang 1, Muath Bishawi 1, Andrew Vekstein 1, Lillian Kang 1, Diego Zapata 4, Ryan Gross 3, Alexis Carnes 2, Lynden E. Gault 5, Julie A. Balko 6, Desiree Bonadonna 2, Sam Ho 5, Paul Lezberg 7, Benjamin S. Bryner 1, Jacob N. Schroder 1, Carmelo A. Milano 1, Dawn E. Bowles 3
1Division of Cardiothoracic Surgery, Department of Surgery, Duke University, 2Perfusion Services, Duke University, 3Division of Surgical Sciences, Department of Surgery, Duke University, 4Division of Laboratory Animal Resources, Duke University, 5Gift of Hope Organ and Tissue Donor Network, 6College of Veterinary Medicine, North Carolina State University, 7TransMedics, Inc

We present a protocol for utilizing a normothermic ex vivo sanguinous perfusion system for the delivery of therapeutics to an entire cardiac allograft in a porcine heterotopic heart transplant model.

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Methods to Visualize Structural and Functional Changes at Synapses
Thomas M. Newpher 1
1Department of Psychology and Neuroscience, Duke Institute for Brain Sciences, Duke University

Methods to Visualize Structural and Functional Changes at Synapses

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Bioengineering

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells
Yasmin Roye 1, Samira Musah 1,2,3,4
1Department of Biomedical Engineering, Pratt School of Engineering, Duke University, 2Department of Medicine, Division of Nephrology, Duke University School of Medicine, 3Department of Cell Biology, Duke University, 4Center for Biomolecular and Tissue Engineering, Duke University

Presented here is a protocol to engineer a personalized organ-on-a-chip system that recapitulates the structure and function of the kidney glomerular filtration barrier by integrating genetically matched epithelial and vascular endothelial cells differentiated from human induced pluripotent stem cells. This bioengineered system can advance kidney precision medicine and related applications.

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Bioengineering

Controlling Particle Fraction in Microporous Annealed Particle Scaffolds for 3D Cell Culture
Alexa R. Anderson 1, Tatiana Segura 2
1Department of Biomedical Engineering, Duke University, 2Departments of Biomedical Engineering, Neurology, and Dermatology, Duke University

Minimizing the variability in the particle fraction within granular scaffolds facilitates reproducible experimentation. This work describes methods for generating granular scaffolds with controlled particle fractions for in vitro tissue engineering applications.

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Bioengineering

An Intra-Tissue Radiometry Microprobe for Measuring Radiance In Situ in Living Tissue
Amanda L. Holt 1,3, Yakir Luc Gagnon 2,4, Alison M. Sweeney 1,3
1Department of Physics, Yale University, 2Lund Vision Group, Department of Biology, Lund University, 3Department of Physics and Astronomy, University of Pennsylvania, 4Department of Biology, Duke University

In this paper, a method for measuring radiance in situ in living tissue is described. This work includes details of the construction of micro-scale probes for different measurements of radiance and irradiance, provides guidance for mounting tissue for the characterization of radiance, and outlines computational methods for analyzing the resulting data.

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Genetics

A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration
Michael Landowski 1,2, Samuel Grindel 1, Ying Hao 3, Sakae Ikeda 1,2, Catherine Bowes Rickman 3,4, Akihiro Ikeda 1,2
1Department of Medical Genetics, University of Wisconsin-Madison, 2McPherson Eye Research Institute, University of Wisconsin-Madison, 3Department of Ophthalmology, Duke University, 4Department of Cell Biology, Duke University

Mouse models can be useful tools for investigating the biology of the retinal pigmented epithelium (RPE). It has been established that mice can develop an array of RPE pathologies. Here, we describe a phenotyping protocol to elucidate and quantify RPE pathologies in mice using light, transmission electron, and confocal microscopy.

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Neuroscience

Placement of Extracranial Stimulating Electrodes and Measurement of Cerebral Blood Flow and Intracranial Electrical Fields in Anesthetized Mice
Simone Degan 1, Yu Feng 1, Ulrike Hoffmann 2, Dennis A. Turner 1,3,4,5
1Department of Neurosurgery, Duke University Medical Center, 2Department of Anesthesiology and Pain Management, University Texas Southwestern Medical School, 3Department of Neurobiology, Duke University Medical Center, 4Department of Biomedical Engineering, Duke University, 5Research and Surgery Services, Durham VA Medical Center

We describe a protocol for assessing dose-response curves for extracranial stimulation in terms of brain electrical field measurements and a relevant biomarker-cerebral blood flow. Since this protocol involves invasive electrode placement into the brain, general anesthesia is needed, with spontaneous breathing preferred rather than controlled respirations.

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Neuroscience

Integrated Photoacoustic, Ultrasound, and Angiographic Tomography (PAUSAT) for NonInvasive Whole-Brain Imaging of Ischemic Stroke
Luca Menozzi *1, Ángela del Águila *2, Tri Vu 1, Chenshuo Ma 1, Wei Yang 2, Junjie Yao 1
1Department of Biomedical Engineering, Duke University, 2Multidisciplinary Brain Protection Program, Department of Anesthesiology, Duke University School of Medicine

This work demonstrates the use of a multimodal ultrasound-based imaging platform for noninvasive imaging of ischemic stroke. This system allows for the quantification of blood oxygenation through photoacoustic imaging and impaired perfusion in the brain through acoustic angiography.

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Neuroscience

Mouse Cardiac Arrest Model for Brain Imaging and Brain Physiology Monitoring During Ischemia and Resuscitation
Ran Li 1, Weina Duan 1, Dong Zhang 2, Ulrike Hoffmann 3, Junjie Yao 2, Wei Yang 1, Huaxin Sheng 1
1Multidisciplinary Brain Protection Program, Department of Anesthesiology, Duke University Medical Center, 2Department of Biomedical Engineering, Duke University, 3Department of Anesthesiology and Pain Management, UT Southwestern University Medical Center

This protocol demonstrates a unique mouse model of asphyxia cardiac arrest that does not require chest compression for resuscitation. This model is useful for monitoring and imaging the dynamics of brain physiology during cardiac arrest and resuscitation.

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Biology

Alignment of Synchronized Time-Series Data Using the Characterizing Loss of Cell Cycle Synchrony Model for Cross-Experiment Comparisons
Sophia A. Campione 1, Christina M. Kelliher 2, David A. Orlando 3, Trung Q. Tran 4, Steven B. Haase 1
1Department of Biology, Duke University, 2Department of Biology, University of Massachusetts, 3Orlando Data Science LLC, 4Department of Computer Science, Duke University

One challenge of analyzing synchronized time-series experiments is that the experiments often differ in the length of recovery from synchrony and the cell-cycle period. Thus, the measurements from different experiments cannot be analyzed in aggregate or readily compared. Here, we describe a method for aligning experiments to allow for phase-specific comparisons.

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