Duke University View Institution's Website 64 articles published in JoVE Biology Alignment of Synchronized Time-Series Data Using the Characterizing Loss of Cell Cycle Synchrony Model for Cross-Experiment Comparisons Sophia A. Campione1, Christina M. Kelliher2, David A. Orlando3, Trung Q. Tran4, Steven B. Haase1 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. Bioengineering An Intra-Tissue Radiometry Microprobe for Measuring Radiance In Situ in Living Tissue Amanda L. Holt1,3, Yakir Luc Gagnon2,4, Alison M. Sweeney1,3 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. Neuroscience Placement of Extracranial Stimulating Electrodes and Measurement of Cerebral Blood Flow and Intracranial Electrical Fields in Anesthetized Mice Simone Degan1, Yu Feng1, Ulrike Hoffmann2, Dennis A. Turner1,3,4,5 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. Neuroscience Integrated Photoacoustic, Ultrasound, and Angiographic Tomography (PAUSAT) for NonInvasive Whole-Brain Imaging of Ischemic Stroke Luca Menozzi*1, Ángela del Águila*2, Tri Vu1, Chenshuo Ma1, Wei Yang2, Junjie Yao1 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. Neuroscience Mouse Cardiac Arrest Model for Brain Imaging and Brain Physiology Monitoring During Ischemia and Resuscitation Ran Li1, Weina Duan1, Dong Zhang2, Ulrike Hoffmann3, Junjie Yao2, Wei Yang1, Huaxin Sheng1 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. Genetics A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration Michael Landowski1,2, Samuel Grindel1, Ying Hao3, Sakae Ikeda1,2, Catherine Bowes Rickman3,4, Akihiro Ikeda1,2 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. Bioengineering Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells Yasmin Roye1, Samira Musah1,2,3,4 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. Bioengineering Controlling Particle Fraction in Microporous Annealed Particle Scaffolds for 3D Cell Culture Alexa R. Anderson1, Tatiana Segura2 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. Medicine A Porcine Heterotopic Heart Transplantation Protocol for Delivery of Therapeutics to a Cardiac Allograft Michelle Mendiola Pla1, Amy Evans2, Franklin H. Lee3, Yuting Chiang1, Muath Bishawi1, Andrew Vekstein1, Lillian Kang1, Diego Zapata4, Ryan Gross3, Alexis Carnes2, Lynden E. Gault5, Julie A. Balko6, Desiree Bonadonna2, Sam Ho5, Paul Lezberg7, Benjamin S. Bryner1, Jacob N. Schroder1, Carmelo A. Milano1, Dawn E. Bowles3 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. Biology Inherent Dynamics Visualizer, an Interactive Application for Evaluating and Visualizing Outputs from a Gene Regulatory Network Inference Pipeline Robert C. Moseley1, Sophia Campione1, Bree Cummins2, Francis Motta3, Steven B. Haase1 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. Neuroscience Quantifying Levels of Dopaminergic Neuron Morphological Alteration and Degeneration in Caenorhabditis elegans Shefali R. Bijwadia1, Katherine Morton1, Joel N. Meyer1 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. Neuroscience Assessment of Sensory Thresholds in Dogs Using Mechanical and Hot Thermal Quantitative Sensory Testing Rachael M. Cunningham1,2, Rachel M. Park1,2, David Knazovicky3, B. Duncan X. Lascelles2,4,5,6, Margaret E. Gruen1,2,4 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. Immunology and Infection Intranasal Immunization and Milk Collection in Studies of Maternal Immunization in New Zealand White Rabbits (Oryctolagus cuniculus) Chelsea D. Landon1,2, Gabriella Dancourt1, Vivian Shing2, Herman F. Staats2,3,4 1Division of Laboratory Animal Resources, Duke University Medical Center, 2Department of Pathology, Duke University Medical Center, 3Department of Immunology, Duke University Medical Center, 4Duke Human Vaccine Institute, Duke University Medical Center This article describes and demonstrates the administration of intranasal vaccines and the collection of milk from lactating rabbits (Oryctolagus cuniculus) as a means to assess mucosal immunity in a translationally appropriate model of maternal immunization. Neuroscience Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments Rui Yang1,2,3, Vann Bennett2,3 1 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. Bioengineering Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection Gabriella Shull*1,2, Christiane Haffner*3, Wieland B. Huttner3, Elena Taverna3,4, Suhasa B. Kodandaramaiah1,5,6 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. Bioengineering Building a Simple and Versatile Illumination System for Optogenetic Experiments Phillip Kyriakakis1, Lourdes Fernandez de Cossio2, Patrick Wade Howard1, Sivleng Kouv1, Marianne Catanho1, Vincent J. Hu3, Robert Kyriakakis1, Molly E. Allen1, Yunhan Ma4, Marcelo Aguilar-Rivera1, Todd P. Coleman1 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. Biology Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke Katrina L. Wilson1, S. Thomas Carmichael2, Tatiana Segura1,3,4 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. Bioengineering Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions Morgan Burt1, Rohan Bhattachaya1, Arinze E. Okafor1, Samira Musah1,2 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. Biochemistry DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling Stefan Ilic1, Shira Cohen1, Ariel Afek2, Raluca Gordan2, David B. Lukatsky1, Barak Akabayov1 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. Behavior Evaluating Skilled Prehension in Mice Using an Auto-Trainer Robert Hubbard1, Jason Dunthorn2, Richard J. O’Brien3, Dan Tasch2, Uri Tasch2, Steven R. Zeiler1 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. Neuroscience Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase Claire A. de March1, Yosuke Fukutani1,2, Aashutosh Vihani1,3, Hitoshi Kida1,4, Hiroaki Matsunami1,3,5,6 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. Biochemistry Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope Zackary N. Scholl1, Qing Li1, Eric Josephs1, Dimitra Apostolidou1, Piotr E. Marszalek1 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. Bioengineering Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells Emma Pipó-Ollé1, Prasad Walke2, Martine K. Notabi1, Rime B. El-Houri1, Morten Østergaard Andersen1, David Needham2,3,4, Eva C. Arnspang1 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. Biology Reverse Dissection and DiceCT Reveal Otherwise Hidden Data in the Evolution of the Primate Face Anne M. Burrows1,2, Kailey M Omstead1, Ashley R Deutsch3,5, Justin T. Gladman4, Adam Hartstone-Rose3 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. Bioengineering Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques Katheryn E. Rothenberg1, Ishaan Puranam1, Brenton D. Hoffman1 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. Medicine Ferric Chloride-induced Canine Carotid Artery Thrombosis: A Large Animal Model of Vascular Injury Allyson L. Huttinger1, Debra G. Wheeler1, Surya Gnyawali2, David Dornbos III1, Juliana M. Layzer3, Nicholas Venetos1, Spencer Talentino1, Nicholas J. Musgrave1, Cheyenne Jones1, Camille Bratton1, Matthew E. Joseph4, Chandan Sen2, Bruce A. Sullenger3, Shahid M. Nimjee1 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. Developmental Biology Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila Scott Barish1, Pelin C. Volkan1 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. Medicine Use of Two Intracorporeal Ventricular Assist Devices As a Total Artificial Heart Muath Bishawi1, Jun-Neng Roan1,2, Jordan Richards1, Zachary Brown1, Laura Blue1, Mani A. Daneshmand1, Jacob N. Schroder1, Dawn E. Bowles3, Carmelo A. Milano1 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. Bioengineering A Protocol for Real-time 3D Single Particle Tracking Shangguo Hou1, Kevin Welsher1 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. Biology Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus Hae-Na Chung1, Stacy D. Rodriguez1, Victoria K. Carpenter2, Julia Vulcan1, C. Donovan Bailey1, Madhugiri Nageswara-Rao1, Yiyi Li3, Geoffrey M. Attardo4, Immo A. Hansen1,5 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. Behavior Eliciting and Analyzing Male Mouse Ultrasonic Vocalization (USV) Songs Jonathan Chabout1,2, Joshua Jones-Macopson1, Erich D. Jarvis1,2,3 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. Developmental Biology Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells Kathryn E. Ware1, Shivee Gilja1, Shenghan Xu1, Samantha Shetler1, Mohit K. Jolly2, Xueyang Wang3, Suzanne Bartholf Dewitt4, Alexander J. Hish1, Sarah Jordan1, William Eward5, Herbert Levine2, Andrew J. Armstrong4, Jason A. Somarelli1 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. Bioengineering A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)–Cell Interaction and the Resultant Bioeffects at the Single-cell Level Fenfang Li1, Fang Yuan2, Georgy Sankin1, Chen Yang1, Pei Zhong1 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. Engineering Colloidal Synthesis of Nanopatch Antennas for Applications in Plasmonics and Nanophotonics Thang B. Hoang1,2, Jiani Huang1,2, Maiken H. Mikkelsen1,2,3 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. Immunology and Infection The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids Daniel E. Rothschild1, Tara Srinivasan2, Linette A. Aponte-Santiago1, Xiling Shen2,3,4, Irving C. Allen1 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. Environment Spotting Cheetahs: Identifying Individuals by Their Footprints Zoe C. Jewell1, Sky K. Alibhai1, Florian Weise2,3, Stuart Munro2, Marlice Van Vuuren4, Rudie Van Vuuren4 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. Neuroscience High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster Bin Gou*1, Edward Zhu*2, Ruo He1, Ulrich Stern1, Chung-Hui Yang1 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. Engineering Fabrication and Operation of Acoustofluidic Devices Supporting Bulk Acoustic Standing Waves for Sheathless Focusing of Particles C. Wyatt Shields IV1,2, Daniela F. Cruz1,2, Korine A. Ohiri1,3, Benjamin B. Yellen1,2,3, Gabriel P. Lopez1,2,3 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. Medicine Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo Cara C. Rada1, Stephanie L. Pierce2, Chad A. Grotegut2, Sarah K. England1 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. Neuroscience Electroretinogram Analysis of the Visual Response in Zebrafish Larvae Jared D. Chrispell1, Tatiana I. Rebrik2, Ellen R. Weiss1 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. Immunology and Infection Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care Katherine Riccione1,2, Carter M. Suryadevara1,3, David Snyder1, Xiuyu Cui1, John H. Sampson1,2,3, Luis Sanchez-Perez1 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. Medicine Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage Beilei Lei1, Huaxin Sheng1, Haichen Wang2, Christopher D. Lascola3, David S. Warner1,4, Daniel T. Laskowitz1,2,4, Michael L. James1,2 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. 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 Chilkoti1,2 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. Biology In Vivo Modeling of the Morbid Human Genome using Danio rerio Adrienne R. Niederriter1,2, Erica E. Davis1,3, Christelle Golzio1, Edwin C. Oh1, I-Chun Tsai1, Nicholas Katsanis1 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. Medicine Intraoperative Detection of Subtle Endometriosis: A Novel Paradigm for Detection and Treatment of Pelvic Pain Associated with the Loss of Peritoneal Integrity Bruce A. Lessey1, H. Lee Higdon III1, Sara E. Miller2, Thomas A. Price3 1Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Greenville Hospital System, 2Department of Pathology, Duke University Health System, 3Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Duke University Loss of peritoneal integrity provides a new paradigm to understand and treat chronic pelvic pain in women with mild forms of endometriosis and can be easily detected using intraoperative instillation of dye at the time of laparoscopy. Medicine Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate David D. Lo*1,2, Jeong S. Hyun*1,3, Michael T. Chung1, Daniel T. Montoro1, Andrew Zimmermann1, Monica M. Grova1,4, Min Lee5, Derrick C. Wan1, Michael T. Longaker1 1Department of Surgery, Stanford University, 2Department of Surgery, Duke University, 3Department of Surgery, Saint Joseph Mercy Hospital, 4School of Medicine, University of California, San Francisco, 5School of Dentistry, University of California, Los Angeles This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration. Medicine 3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System Teagan J. Walter1,2, Erin E. Sparks3, Stacey S. Huppert2 1Department of Cell and Developmental Biology, Center for Stem Cell Biology, Vanderbilt University, 2Division of Gastroenterology, Hepatology, and Nutrition, Cincinnati Children's Hospital, 3Department of Biology, Duke University A method of visualizing and quantifying the 3-dimensional structure of mouse hepatic portal vein or intrahepatic bile duct is described. This resin cast technique can also be applied to other ductal or vascular systems and allows for in situ visualization or quantification of a system's intact communicating architecture. Neuroscience Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI Gabriel P. Howles1, Yi Qi2, Stephen J. Rosenzweig3, Kathryn R. Nightingale3, G. Allan Johnson2 1Department of Radiology, Stanford University, 2Center for In Vivo Microscopy, Duke University Medical Center, 3Department of Biomedical Engineering, Duke University A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity. Biology Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation Yufen Goh*1, Melissa J. Fullwood*1,2,3, Huay Mei Poh1, Su Qin Peh1, Chin Thing Ong1, Jingyao Zhang1, Xiaoan Ruan1, Yijun Ruan1,3 1Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore, 2A*STAR-Duke-NUS Neuroscience Research Partnership, Singapore, 3Department of Biochemistry, National University of Singapore, Singapore Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control. Biology Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue Chun-Chun Chen1, Kazuhiro Wada2, Erich D. Jarvis1 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. Neuroscience Brain Imaging Investigation of the Impairing Effect of Emotion on Cognition Gloria Wong1,2, Sanda Dolcos1,3, Ekaterina Denkova1, Rajendra Morey4,5,6, Lihong Wang4,5, Gregory McCarthy6,7, Florin Dolcos1,2,3,8,9 1Department of Psychiatry, University of Alberta, 2Centre for Neuroscience, University of Alberta, 3Department of Psychology, University of Illinois, 4Brain Imaging and Analysis Center, Duke University, 5Department of Psychiatry and Behavioral Sciences, Duke University, 6Mid-Atlantic Mental Illness Research Education and Clinical Center, VA Medical Center, 7Department of Psychology, Yale University, 8Neuroscience Program, University of Illinois, 9Beckman Institute for Advanced Science & Technology, University of Illinois We present a protocol that allows investigation of the neural mechanisms mediating the detrimental impact of emotion on cognition, using functional magnetic resonance imaging. This protocol can be used with both healthy and clinical participants. Bioengineering Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress Whitney O. Lane1, Alexandra E. Jantzen2, Tim A. Carlon2, Ryan M. Jamiolkowski3, Justin E. Grenet1, Melissa M. Ley1, Justin M. Haseltine2, Lauren J. Galinat2, Fu-Hsiung Lin1, Jason D. Allen4, George A. Truskey2, Hardean E. Achneck1 1Department of Surgery, Duke University Medical Center, 2Department of Biomedical Engineering, Duke University, 3School of Medicine, University of Pennsylvania, 4Department of Medicine, Division of Cardiology, Duke University Medical Center We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture. Bioengineering Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium Carleen M. Bowers1, Eric J. Toone1, Robert L. Clark2, Alexander A. Shestopalov3 1Department of Chemistry, Duke University, 2Hajim School of Engineering and Applied Sciences, University of Rochester, 3Department of Chemical Engineering, University of Rochester Here we describe a simple method for patterning oxide-free silicon and germanium with reactive organic monolayers and demonstrate functionalization of the patterned substrates with small molecules and proteins. The approach completely protects surfaces from chemical oxidation, provides precise control over feature morphology, and provides ready access to chemically discriminated patterns. Immunology and Infection Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging Joel R. Meyerson1,2, Tommi A. White1, Donald Bliss3, Amy Moran3, Alberto Bartesaghi1, Mario J. Borgnia1, M. Jason V. de la Cruz1, David Schauder1, Lisa M. Hartnell1, Rachna Nandwani1,4, Moez Dawood5, Brianna Kim6, Jun Hong Kim7, John Sununu8, Lisa Yang9, Siddhant Bhatia10, Carolyn Subramaniam1, Darrell E. Hurt11, Laurent Gaudreault12, Sriram Subramaniam1 1Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 2The Medical Research Council Mitochondrial Biology Unit, University of Cambridge, 3National Library of Medicine, National Institutes of Health, 4Massachusetts Institute of Technology, 5William Fremd High School, 6University of Virginia, 7Duke University, 8Yale University, 9University of Notre Dame, 10Washington University in St. Louis, 11Bioinformatics and Computational Biosciences Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12Thomas Jefferson High School for Science and Technology The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis. Immunology and Infection Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes Dana C. Upton1, Shyam Unniraman1 1Department of Immunology, Duke University We describe how to perform retroviral or lentiviral infections of overexpression or shRNA-containing constructs in the human Ramos B-cell line and how to measure somatic hypermutation in these cells. Bioengineering Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model Alexandra E. Jantzen1, Whitney O. Lane2, Shawn M. Gage3, Justin M. Haseltine1, Lauren J. Galinat1, Ryan M. Jamiolkowski4, Fu-Hsiung Lin3, George A. Truskey1, Hardean E. Achneck3 1Department of Biomedical Engineering, Duke University, 2School of Medicine, Duke University, 3Department of Surgery, Duke University Medical Center, 4School of Medicine, University of Pennsylvania A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices. Immunology and Infection Visualizing Dengue Virus through Alexa Fluor Labeling Summer Zhang1, Hwee Cheng Tan2, Eng Eong Ooi3 1Defence Medical and Environmental Research Institute, DSO National Laboratories, 2Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, 3Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell. Neuroscience Brain Imaging Investigation of the Memory-Enhancing Effect of Emotion Andrea Shafer1, Alexandru Iordan2, Roberto Cabeza3, Florin Dolcos1,4 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. Bioengineering Procedure for Lung Engineering Elizabeth A. Calle*1, Thomas H. Petersen*2, Laura E. Niklason1,3 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. Neuroscience Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells Sandeepa Dey1, Senmiao Zhan2, Hiroaki Matsunami1 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. Neuroscience Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number Dominic M. Ippolito1, Cagla Eroglu2 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. Neuroscience Human Fear Conditioning Conducted in Full Immersion 3-Dimensional Virtual Reality Nicole C. Huff1, David J. Zielinski2, Matthew E. Fecteau1, Rachael Brady2, Kevin S. LaBar1 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. Biology Assembly, Tuning and Use of an Apertureless Near Field Infrared Microscope for Protein Imaging Melissa Paulite1, Zahra Fakhraai2, Boris B. Akhremitchev3, Kerstin Mueller1, Gilbert C. Walker1 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. Biology Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time Yi-Ping Ho1,2, Hunter H. Chen2,3, Kam W. Leong2, Tza-Huei Wang1,3 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.