University of Michigan, Ann Arbor View Institution's Website 122 articles published in JoVE Cancer Research Cell-Free DNA Extraction of Vitreous and Aqueous Humor Specimens for Diagnosis and Monitoring of Vitreoretinal Lymphoma Noah A. Brown1, Rajesh C. Rao1,2,3,4,5,6,7,8, Bryan L. Betz1 1Department of Pathology, University of Michigan, Ann Arbor, 2Department of Ophthalmology and Visual Sciences, Kellogg Eye Center, University of Michigan, Ann Arbor, 3Department of Human Genetics, University of Michigan, Ann Arbor, 4Rogel Cancer Center, University of Michigan, Ann Arbor, 5Center of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, 6Center for RNA Biomedicine, University of Michigan, Ann Arbor, 7A. Alfred Taubman Medical Research Institute, University of Michigan, Ann Arbor, 8Section of Ophthalmology, Surgery Service, Veterans Administration Ann Arbor Healthcare System A procedure to extract cell-free DNA from vitreous and aqueous humor to perform molecular studies for diagnosing vitreoretinal lymphoma is established here. The method offers the ability to concurrently extract DNA from the cellular component of the sample or to reserve it for ancillary testing. Biology CorrelationCalculator and Filigree: Tools for Data-Driven Network Analysis of Metabolomics Data Gayatri Iyer1, Marci Brandenburg1,2, Christopher Patsalis1, George Michailidis3, Alla Karnovsky1 1Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, 2Taubman Health Sciences Library, University of Michigan, Ann Arbor, 3Department of Statistics, University of Florida We present CorrelationCalculator and Filigree, two tools for data-driven network construction and analysis of metabolomics data. CorrelationCalculator supports building a single interaction network of metabolites based on expression data, while Filigree allows building a differential network, followed by network clustering and enrichment analysis. Biology Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic β-Cells Elena Levi-D’Ancona1,2, Vaibhav Sidarala1, Scott A. Soleimanpour1,3,4 1Department of Internal Medicine, Division of Metabolism, Endocrinology and Diabetes, University of Michigan, Ann Arbor, 2Graduate Program in Immunology, University of Michigan Medical School, 3Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, 4VA Ann Arbor Healthcare System This protocol outlines two methods for the quantitative analysis of mitophagy in pancreatic β-cells: first, a combination of cell-permeable mitochondria-specific dyes, and second, a genetically encoded mitophagy reporter. These two techniques are complementary and can be deployed based on specific needs, allowing for flexibility and precision in quantitatively addressing mitochondrial quality control. Neuroscience P300-Based Brain-Computer Interface Speller Performance Estimation with Classifier-Based Latency Estimation Nazmun N. Khan1, Taylor Sweet1, Chase A. Harvey1, Seth Warschausky2, Jane E. Huggins3,4, David E. Thompson1 1Brain and Body Sensing Lab, Mike Wiegers Department of Electrical & Computer Engineering, Kansas State University, 2Adaptive Cognitive Assessment Laboratory, Department of Physical Medicine and Rehabilitation, University of Michigan, Ann Arbor, 3Direct Brain Interface Laboratory, Department of Physical Medicine and Rehabilitation, University of Michigan, Ann Arbor, 4Direct Brain Interface Laboratory, Department of Biomedical Engineering, University of Michigan, Ann Arbor This article presents a method for estimating same-day P300 speller Brain-Computer Interface (BCI) accuracy using a small testing dataset. Neuroscience Remote Neuronal Activation Coupled with Automated Blood Sampling to Induce and Measure Circulating Luteinizing Hormone in Mice Cristina Sáenz de Miera1, Jiane Feng1,2, Carol F. Elias1,2, Nathan Qi1,2 1Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, 2Mouse Metabolic Phenotyping Center-Live, University of Michigan, Ann Arbor Luteinizing hormone (LH) pulsatility is a hallmark of reproductive function. We describe a protocol for remote activation of specific neuronal populations linked to serial automated blood collection. This technique allows timed hormonal modulation, multiplexing, and minimizing manipulation effects on LH levels in conscious freely moving, and undisturbed animals. Cancer Research Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor Brittany Turner-Ivey1, Jody Fromm Longo2, Dorea P. Jenkins2, Stephen T. Guest3, Steven L. Carroll1,2 1Hollings Cancer Center, Medical University of South Carolina, 2Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 3Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor We have developed a cross-species comparative oncogenomics approach utilizing genomic analyses and functional genomic screens to identify and compare therapeutic targets in tumors arising in genetically engineered mouse models and the corresponding human tumor type. Biology Methodology to Metabolically Inactivate Bacteria for Caenorhabditis elegans Research Safa Beydoun*1, Elizabeth S. Kitto*1, Emily Wang1, Shijiao Huang1, Scott F. Leiser1,2 1Molecular and Integrative Physiology Department, University of Michigan, Ann Arbor, 2Department of Internal Medicine, University of Michigan, Ann Arbor The food source for Caenorhabditis elegans in the lab is live Escherichia coli. Since bacteria are metabolically active, they present a confounding variable in metabolic and drug studies in C. elegans. A detailed protocol to metabolically inactivate bacteria using paraformaldehyde is described here. Biology Improved Lipofuscin Models and Quantification of Outer Segment Phagocytosis Capacity in Highly Polarized Human Retinal Pigment Epithelial Cultures Qitao Zhang1, Gillian Autterson1, Jason M. L. Miller1,2 1Kellogg Eye Center, University of Michigan, Ann Arbor, 2Cellular and Molecular Biology Program, University of Michigan, Ann Arbor This protocol describes a lipofuscin accumulation model in highly differentiated and polarized human retinal pigment epithelial (RPE) cultures and an improved outer segment (OS) phagocytosis assay to detect the total OS consumption/degradation capacity of the RPE. These methods overcome the limitations of previous lipofuscin models and classical pulse-chase outer segment phagocytosis assays. Bioengineering High-Throughput Cardiotoxicity Screening Using Mature Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Monolayers Andre Monteiro da Rocha1, Andrew Allan2, Travis Block3, Jeffery Creech1, Todd J. Herron1 1Frankel Cardiovascular Regeneration Core Laboratory, Cardiovascular Medicine-Internal Medicine, University of Michigan, Ann Arbor, 2CAIRN Research, 3StemBioSys, Inc. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an alternative to using animals for preclinical cardiotoxicity screening. A limitation to the widespread adoption of hiPSC-CMs in preclinical toxicity screening is the immature, fetal-like phenotype of the cells. Presented here are protocols for robust and rapid maturation of hiPSC-CMs. Medicine Troubleshooting FoCUS Image Acquisition: Patient Positioning, Transducer Manipulation, and Image Optimization Adam L. Gottula1,2,3, Suhas Devangam1, Jessica L. Koehler2, Matthew J. Sigakis1 1Department of Anesthesiology, Division of Critical Care, University of Michigan, Ann Arbor, 2Department of Emergency Medicine, University of Michigan, Ann Arbor, 3Max Harry Weil Institute for Critical Care Research and Innovation, University of Michigan, Ann Arbor Here, we present a protocol to allow providers to perform focused cardiac ultrasound (FoCUS) in the clinical environment. We describe methods of transducer manipulation, review common pitfalls of transducer movements, and suggest tips to optimize phased array transducer use. Medicine Assessment of Ex Vivo Murine Biventricular Function in a Langendorff Model Pierre-Emmanuel Noly1,2, Wei Huang1, Suyash Naik1, Paul Tang1, Ienglam Lei1 1University of Michigan, Ann Arbor, 2University of Montreal Presented here is a protocol to reliably quantify the right and left ventricular function of donor hearts after cold preservation using an ex vivo perfusion system. Biology Determination of the Mating Efficiency of Haploids in Saccharomyces cerevisiae Anjali Mahilkar*1,2, Prachitha Nagendra*1, Supreet Saini1 1Department of Chemical Engineering, Indian Institute of Technology Bombay, 2Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor In this work, a robust method for the quantification of mating efficiency in the yeast Saccharomyces cerevisiae is described. This method is particularly useful for the quantification of pre-zygotic barriers in speciation studies. Medicine Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes Emily A. Lavey1, Margaret V. Westfall1 1Department of Cardiac Surgery, Michigan Medicine, University of Michigan, Ann Arbor A set of protocols are presented that describe the measurement of contractile function via sarcomere length detection along with calcium (Ca2+) transient measurement in isolated rat myocytes. The application of this approach for studies in animal models of heart failure is also included. Medicine In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter Zhaokai Li*1,2, Liu Liu*1, Zhong Wang1 1Department of Cardiac Surgery, Cardiovascular Center, The University of Michigan, Ann Arbor, 2Department of Cardiovascular Medicine, Xiangya Hospital, Central South University We describe here, the establishment and application of an Tg(Myh6-cre)1Jmk/J /Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as αMHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 below) mouse reporter line for cardiac reprogramming assessment. Neonatal cardiac fibroblasts (NCFs) isolated from the mouse strain are converted into induced cardiomyocytes (iCMs), allowing for convenient and efficient evaluation of reprogramming efficiency and functional maturation of iCMs via calcium (Ca2+) flux. Biology Mouse Abdominal Aortic Aneurysm Model Induced by Perivascular Application of Elastase Chao Xue1, Guizhen Zhao1, Yang Zhao1, Y Eugene Chen1, Jifeng Zhang1 1Department of Internal Medicine, Frankel Cardiovascular Center, University of Michigan, Ann Arbor The present protocol describes a standardized surgical method for the elastase-induced AAA model through the direct application of elastase to the adventitia of infrarenal abdominal aorta in mice. Bioengineering The Muscle Cuff Regenerative Peripheral Nerve Interface for the Amplification of Intact Peripheral Nerve Signals Shelby R. Svientek1, Justin P. Wisely1, Amir Dehdashtian1, Jarred V. Bratley1, Paul S. Cederna1,2, Stephen W. P. Kemp1,2 1Department of Surgery, Section of Plastic Surgery, The University of Michigan Health System, 2Department of Biomedical Engineering, The University of Michigan, Ann Arbor This manuscript provides an innovative method for developing a biologic peripheral nerve interface termed the Muscle Cuff Regenerative Peripheral Nerve Interface (MC-RPNI). This surgical construct can amplify its associated peripheral nerve's motor efferent signals to facilitate accurate detection of motor intent and the potential control of exoskeleton devices. Cancer Research Longitudinal Intravital Imaging Through Clear Silicone Windows Laura Maiorino*1,2,3, Margaret Shevik*1,4,5, José M. Adrover1, Xiao Han1,6, Elias Georgas7,8, John Erby Wilkinson9, Harrison Seidner1, Leonie Foerschner1, David A. Tuveson1, Yi-Xian Qin8, Mikala Egeblad1 1Cold Spring Harbor Laboratory, 2Cold Spring Harbor Laboratory School of Biological Sciences, 3Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 4Medical Scientist Training Program, School of Medicine, Stony Brook University, 5Graduate Program in Pharmacology, Stony Brook University, 6Graduate Program in Genetics, Stony Brook University, 7Graduate Program in Biomedical Engineering, Stony Brook University, 8Department of Biomedical Engineering, Stony Brook University, 9Department of Pathology, University of Michigan An approach is here presented for long-term intravital imaging using optically clear, silicone windows that can be glued directly to the tissue/organ of interest and the skin. These windows are cheaper and more versatile than others currently used in the field, and the surgical insertion causes limited inflammation and distress to the animals. Bioengineering Rapid Encapsulation of Reconstituted Cytoskeleton Inside Giant Unilamellar Vesicles Yashar Bashirzadeh*1, Nadab Wubshet*1, Thomas Litschel2, Petra Schwille3, Allen P. Liu1,4,5,6 1Department of Mechanical Engineering, University of Michigan, Ann Arbor, 2John A. Paulson School of Engineering and Applied Sciences, Harvard University, 3Department of Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, 4Department of Biomedical Engineering, University of Michigan, Ann Arbor, 5Department of Biophysics, University of Michigan, Ann Arbor, 6Cellular and Molecular Biology Program, University of Michigan, Ann Arbor This article introduces a simple method for expeditious production of giant unilamellar vesicles with encapsulated cytoskeletal proteins. The method proves to be useful for bottom-up reconstitution of cytoskeletal structures in confinement and cytoskeleton-membrane interactions. Bioengineering Open-source Toolkit: Benchtop Carbon Fiber Microelectrode Array for Nerve Recording Julianna M. Richie1, Paras R. Patel1, Elissa J. Welle1, Tianshu Dong2, Lei Chen3, Albert J. Shih2, Cynthia A. Chestek1,4,5,6 1Department of Biomedical Engineering, University of Michigan, Ann Arbor, 2Department of Mechanical Engineering, University of Michigan, Ann Arbor, 3Department of Mechanical Engineering, University of Massachusetts Lowell, 4Department of Electrical Engineering and Computer Science, University of Michigan, Ann Arbor, 5Neuroscience Graduate Program, University of Michigan, Ann Arbor, 6Robotics Graduate Program, University of Michigan, Ann Arbor Here, we describe fabrication methodology for customizable carbon fiber electrode arrays for recording in vivo in nerve and brain. Biology Assessment of Cellular Bioenergetics in Mouse Hematopoietic Stem and Primitive Progenitor Cells using the Extracellular Flux Analyzer Surinder Kumar1, Morgan Jones2, Qing Li2,3,4, David B. Lombard1,4,5 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. Engineering Performing In Situ Closed-Cell Gas Reactions in the Transmission Electron Microscope Kinga A. Unocic1, Dale K. Hensley1, Franklin S. Walden2, Wilbur C. Bigelow3, Michael B. Griffin4, Susan E. Habas4, Raymond R. Unocic1, Lawrence F. Allard5 1Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, 2Protochips Inc., 3Department of Materials Science & Engineering, University of Michigan, 4Catalytic Carbon Transformation & Scale-up, National Renewable Energy Laboratory, 5Materials Science & Technology Division, Oak Ridge National Laboratory Here, we present a protocol for performing in situ TEM closed-cell gas reaction experiments while detailing several commonly used sample preparation methods. Immunology and Infection Systematic Scoring Analysis for Intestinal Inflammation in a Murine Dextran Sodium Sulfate-Induced Colitis Model Vicky Garcia-Hernandez1, Philipp-Alexander Neumann2, Stefan Koch3,4, Renae Lyons1, Asma Nusrat1, Charles A. Parkos1 1Department of Pathology, University of Michigan, 2Department of Surgery, Klinikum Rechts der Isar, Technische Universität München, 3Wallenberg Centre for Molecular Medicine, Linköping University, 4Department of Biomedical and Clinical Sciences (BKV), Linköping University Systematic scoring of intestinal inflammation using a free computer-assisted system is a powerful tool to quantitatively compare histopathological changes in colitis models characterized by the presence of ulcers and inflammatory changes. Histological colitis score evaluation strengthens clinical observations and facilitates data interpretation. Immunology and Infection Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model Kevin Boerner1, Anny-Claude Luissint1, Charles A. Parkos1 1Department of Pathology, University of Michigan, Ann Arbor Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo. Biology Generation of Murine Primary Colon Epithelial Monolayers from Intestinal Crypts Chithra K Muraleedharan1, Jay Mierzwiak1, Darius Feier1, Asma Nusrat1, Miguel Quiros1 1Department of Pathology, School of Medicine, University of Michigan In this protocol, we describe how to generate murine primary epithelial colon monolayers directly from intestinal crypts. We provide experimental approaches to generate confluent monolayers on permeable filters, confluent monolayers for scratch wound healing and biochemical studies, and sparse and confluent monolayers for immunofluorescence analysis. Bioengineering Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels Ariel A. Szklanny1, Dylan B. Neale2, Joerg Lahann2, Shulamit Levenberg1 1Faculty of Biomedical Engineering, Technion, 2Department of Chemical Engineering and Biointerfaces Institute, University of Michigan, Ann Arbor Engineered tissues heavily rely on proper vascular networks to provide vital nutrients and gases and remove metabolic waste. In this work, a stepwise seeding protocol of endothelial cells and support cells creates highly organized vascular networks in a high-throughput platform for studying developing vessel behavior in a controlled 3D environment. Cancer Research Quantifying the Brain Metastatic Tumor Micro-Environment using an Organ-On-A Chip 3D Model, Machine Learning, and Confocal Tomography C. Ryan Oliver*1,2, Trisha M. Westerhof*1,2, Maria G. Castro2,3,4, Sofia D. Merajver1,2 1Department of Internal Medicine, University of Michigan Ann Arbor, 2Rogel Cancer Center, University of Michigan Ann Arbor, 3Department of Neurosurgery, University of Michigan Ann Arbor, 4Department of Cell and Developmental Biology, University of Michigan Ann Arbor Here, we present a protocol for preparing and culturing a blood brain barrier metastatic tumor micro-environment and then quantifying its state using confocal imaging and artificial intelligence (machine learning). Engineering Simulating Imaging of Large Scale Radio Arrays on the Lunar Surface Alexander M. Hegedus1 1Department of Climate and Space Sciences and Engineering, University of Michigan A simulation framework for testing the imaging capabilities of large-scale radio arrays on the lunar surface is presented. Major noise components are discussed, and a software pipeline is walked through with details on how to customize it for novel scientific uses. Bioengineering Fabrication of the Composite Regenerative Peripheral Nerve Interface (C-RPNI) in the Adult Rat Shelby R. Svientek1, Dan C. Ursu1, Paul S. Cederna1,2, Stephen W. P. Kemp1,2 1Department of Surgery, Division of Plastic Surgery, University of Michigan, Ann Arbor, 2Department of Biomedical Engineering, University of Michigan, Ann Arbor The following manuscript describes a novel method for developing a biologic, closed loop neural feedback system termed the composite regenerative peripheral nerve interface (C-RPNI). This construct has the ability to integrate with peripheral nerves to amplify efferent motor signals while simultaneously providing afferent sensory feedback. Neuroscience Measuring and Manipulating Functionally Specific Neural Pathways in the Human Motor System with Transcranial Magnetic Stimulation Elana R. Goldenkoff1, Amir Mashni1, Katherine J. Michon1, Hannah Lavis1, Michael Vesia1 1School of Kinesiology, Brain Behavior Laboratory, University of Michigan This article describes new approaches to measure and strengthen functionally specific neural pathways with transcranial magnetic stimulation. These advanced noninvasive brain stimulation methodologies can provide new opportunities for the understanding of brain-behavior relations and development of new therapies to treat brain disorders. Neuroscience Long-range Channelrhodopsin-assisted Circuit Mapping of Inferior Colliculus Neurons with Blue and Red-shifted Channelrhodopsins David Goyer1, Michael T. Roberts1 1Kresge Hearing Research Institute, Department of Otolaryngology - Head and Neck Surgery, University of Michigan Channelrhodopsin-assisted circuit mapping (CRACM) is a precision technique for functional mapping of long-range neuronal projections between anatomically and/or genetically identified groups of neurons. Here, we describe how to utilize CRACM to map auditory brainstem connections, including the use of a red-shifted opsin, ChrimsonR. Chemistry Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly Samuel C. Leguizamon1, Abdulla F. Alqubati1, Timothy F. Scott2,3 1Department of Chemical Engineering, University of Michigan, 2Department of Chemical Engineering, Monash University, 3Department of Materials Science and Engineering, Monash University A protocol is presented for the synthesis of information-encoded peptoid oligomers and for the sequence-directed self-assembly of these peptoids into molecular ladders using amines and aldehydes as dynamic covalent reactant pairs and Lewis acidic rare-earth metal triflates as multi-role reagents. Bioengineering Focused Ion Beam Lithography to Etch Nano-architectures into Microelectrodes Shreya Mahajan1, Jonah A. Sharkins2,4, Allen H. Hunter5, Amir Avishai6, Evon S. Ereifej2,3,4 1Department of Electrical Engineering and Computer Science, University of Michigan, 2Veteran Affairs Ann Arbor Healthcare System, 3Department of Neurology, School of Medicine, University of Michigan, 4Department of Biomedical Engineering, University of Michigan, 5Michigan Center for Materials Characterization, University of Michigan, 6Carl Zeiss SMT, Inc. We have shown that the etching of nano-architecture into intracortical microelectrode devices may reduce the inflammatory response and has the potential to improve electrophysiological recordings. The methods described herein outline an approach to etch nano-architectures into the surface of non-functional and functional single shank silicon intracortical microelectrodes. Bioengineering A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk Carmen G. Flesher1, Nicki A. Baker1, Clarissa Strieder-Barboza1,2, Dominic Polsinelli5, Phillip J. Webster1,2, Oliver A. Varban1, Carey N. Lumeng2,3,4, Robert W. O'Rourke1,6 1Department of Surgery, University of Michigan Medical School, 2Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, 3Graduate Program in Immunology, University of Michigan Medical School, 4Graduate Program in Cellular and Molecular Biology, University of Michigan Medical School, 5Undergraduate Research Opportunity Program, University of Michigan, 6Department of Surgery, Ann Arbor Veterans Affairs Healthcare System We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype. Cancer Research Optimization, Design and Avoiding Pitfalls in Manual Multiplex Fluorescent Immunohistochemistry Jenny Lazarus1, Yagiz Akiska1, Mirna Perusina Lanfranca1, Lawrence Delrosario1, Lei Sun1, Daniel Long2, Jiaqi Shi3, Howard Crawford2, Marina P. Di Magliano1, Weiping Zou1, Timothy Frankel1 1Department of Surgery, University of Michigan, 2Department of Molecular and Cellular Physiology, University of Michigan, 3Department of Pathology, University of Michigan Multiplex fluorescent immunohistochemistry is an emerging technology that enables the visualization of multiple cell types within intact formalin-fixed, paraffin embedded (FFPE) tissue. Presented are guidelines for ensuring a successful 7-color multiplex with instructions for optimizing antibodies and reagents, preparing slides, design and tips for avoiding common problems. Behavior Automated Rat Single-Pellet Reaching with 3-Dimensional Reconstruction of Paw and Digit Trajectories Alexandra Bova1, Krista Kernodle1, Kaitlyn Mulligan2, Daniel Leventhal2,3,4 1Neuroscience Graduate Program, University of Michigan, 2Department of Neurology, University of Michigan, 3Department of Biomedical Engineering, University of Michigan, 4Department of Neurology, VA Ann Arbor Health System Rodent skilled reaching is commonly used to study dexterous skills, but requires significant time and effort to implement the task and analyze the behavior. We describe an automated version of skilled reaching with motion tracking and three-dimensional reconstruction of reach trajectories. Cancer Research Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells Michael E. Bregenzer*1, Ciara Davis*1, Eric N. Horst*1, Pooja Mehta*2, Caymen M. Novak*1, Shreya Raghavan*2, Catherine S. Snyder*2, Geeta Mehta1,2,3,4 1Department of Biomedical Engineering, University of Michigan, 2Department of Materials Science and Engineering, University of Michigan, 3Rogel Cancer Center, School of Medicine, University of Michigan, 4Macromolecular Science and Engineering, University of Michigan This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates’ potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease. Developmental Biology Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone Jingwen Yang*1,2, Haichun Pan*2, Yuji Mishina2 1The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory for Oral Biomedicine of Ministry of Education, School and Hospital of Stomatology, Wuhan University, 2Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan Here, we present a detailed protocol to detect and quantify protein levels during craniofacial morphogenesis/pathogenesis by immunostaining using mouse craniofacial tissues as examples. In addition, we describe a method for preparation and cryosectioning of undecalcified hard tissues from young mice for immunostaining. Cancer Research Droplet Digital TRAP (ddTRAP): Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction Mohammed E. Sayed1, Aaron L. Slusher1, Andrew T. Ludlow1 1School of Kinesiology, University of Michigan We successfully converted the standard telomere repeat amplification protocol (TRAP) assay to be employed in droplet digital polymerase chain reactions. This new assay, called ddTRAP, is more sensitive and quantitative, allowing for better detection and statistical analysis of telomerase activity within various human cells. Medicine Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay Gemma Pearson1, Scott A. Soleimanpour1,2 1Department of Internal Medicine, Division of Metabolism, Endocrinology and Diabetes, University of Michigan, Ann Arbor, 2VA Ann Arbor Healthcare System This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples. Developmental Biology Generation of Organoids from Mouse Extrahepatic Bile Ducts Junya Shiota1, Nureen H. Mohamad Zaki1, Juanita L. Merchant1,2, Linda C. Samuelson1,2, Nataliya Razumilava1 1Departments of Internal Medicine, The University of Michigan, 2Molecular & Integrative Physiology, The University of Michigan This protocol describes the production of a mouse extrahepatic bile duct 3-dimensional organoid system. These biliary organoids can be maintained in culture to study cholangiocyte biology. Biliary organoids express markers of both progenitor and biliary cells and are composed of polarized epithelial cells. Behavior Experimental Paradigm for Measuring the Effects of Self-distancing in Young Children Amanda Grenell1, Rachel E. White2, Emily O. Prager1, Catherine Schaefer1, Ethan Kross3, Angela L. Duckworth4, Stephanie M. Carlson1 1Insitute of Child Development, University of Minnesota-Twin Cities, 2Department of Psychology, Hamilton College, 3Department of Psychology, University of Michigan, 4Department of Psychology, University of Pennsylvania An experimental paradigm was created to measure the effects of self-distancing in young children (4-6-year-olds). Self-distancing is a process through which individuals adopt a less egocentric perspective. This paradigm has been used to examine the effects of self-distancing on young children's self-regulation. Cancer Research Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures Felipe J. Núñez1,2, Flor M. Mendez2, Maria B. Garcia-Fabiani1,2, Joaquín Pardo1,3, Marta Edwards1,2, Pedro R. Lowenstein1,2, Maria G. Castro1,2 1Department of Neurosurgery, University of Michigan Medical School, 2Department of Cell & Developmental Biology, University of Michigan, 3INIBIOLP, Histology B-Pathology B, School of Medicine, UNLP Neurospheres grown as 3D cultures constitute a powerful tool to study glioma biology. Here we present a protocol to perform immunohistochemistry while maintaining the 3D structure of glioma neurospheres through paraffin embedding. This method enables the characterization of glioma neurosphere properties such as stemness and neural differentiation. Biochemistry An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity Antonio E. Chambers*1, Adam E. Richardson*1, David F. Read2, Thomas J. Waller3, Douglas A. Bernstein1, Philip J. Smaldino1 1Department of Biology, Ball State University, 2Department of Genome Sciences, University of Washington, 3Department of Molecular, Cellular, and Developmental Biology, University of Michigan Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes. Biochemistry Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts Ye Guan1,2, Shiyuan Wang1, Minjun Jin2, Haotian Xu3, Qiong Yang1,4 1Department of Biophysics, University of Michigan, Ann Arbor, 2Department of Chemistry, University of Michigan, Ann Arbor, 3Department of Computer Science, Wayne State University, 4Department of Physics, University of Michigan, Ann Arbor We present a method for the generation of in vitro self-sustained mitotic oscillations at the single-cell level by encapsulating egg extracts of Xenopus laevis in water-in-oil microemulsions. Medicine Complete and Partial Aortic Occlusion for the Treatment of Hemorrhagic Shock in Swine Aaron M. Williams*1, Umar F. Bhatti*1, Isabel S. Dennahy1, Kiril Chtraklin1, Panpan Chang1, Nathan J. Graham1, Basil M. Baccouche1, Shalini Roy1, Mohammed Harajli1, Jing Zhou1, Vahagn C. Nikolian1, Qiufang Deng1, Yuzi Tian1, Baoling Liu1, Yongqing Li1, Gregory L. Hays1,2, Julia L. Hays1,2, Hasan B. Alam1 1Department of Surgery, University of Michigan, 2Hays Innovations Here, we present a protocol demonstrating a hemorrhagic shock model in swine that uses aortic occlusion as a bridge to definitive care in trauma. This model has application in testing a wide range of surgical and pharmacological therapeutic strategies. Biology Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse Zhen Wang1,4, Ramon Ocadiz-Ruiz1, Sinju Sundaresan1, Lin Ding1, Michael Hayes1, Nirakar Sahoo3, Haoxing Xu1,2, Juanita L. Merchant1,2,5 1Department of Internal Medicine-Gastroenterology, University of Michigan, 2Department of Molecular and Integrative Physiology, University of Michigan, 3Department of Molecular, Cellular and Developmental Biology, University of Michigan, 4Department of Gastrointestinal Surgery, The First Affiliated Hospital of Guangxi Medical University, 5Division of Gastroenterology, University of Arizona College of Medicine Here, we describe the isolation of enteric-glial cells from the intestinal-submucosa using sequential EDTA incubations to chelate divalent cations and then incubation in non-enzymatic cell recovery solution. Plating the resultant cell suspension on poly-D-lysine and laminin results in a highly enriched culture of submucosal glial cells for functional analysis. Engineering Bulk and Thin Film Synthesis of Compositionally Variant Entropy-stabilized Oxides Sai Sivakumar*1, Elizabeth Zwier*1, Peter Benjamin Meisenheimer*1, John T. Heron1 1Department of Materials Science and Engineering, University of Michigan The synthesis of high quality bulk and thin film (Mg0.25(1-x)CoxNi0.25(1-x)Cu0.25(1-x)Zn0.25(1-x))O and (Mg0.25(1-x)Co0.25(1-x)Ni0.25(1-x)CuxZn0.25(1-x))O entropy-stabilized oxides is presented. Immunology and Infection Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration Emma Teal1, Nina G. Steele2, Jayati Chakrabarti1, Loryn Holokai3, Yana Zavros1 1Department of Pharmacology and Systems Physiology, University of Cincinnati, 2Department of Cell & Developmental Biology, University of Michigan, 3Department of Molecular Genetics, Biochemistry & Microbiology, University of Cincinnati Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described. Genetics A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins Harriet Prior*1, Lauren MacConnachie*1, Jose L. Martinez1, Georgina C.B. Nicholl1, Asim A. Beg1 1Department of Pharmacology, University of Michigan Here, we present a method to engineer the genome of C. elegans using CRISPR-Cas9 ribonucleoproteins and homology dependent repair templates. Behavior Methods of Pairing and Pair Maintenance of New Zealand White Rabbits (Oryctolagus Cuniculus) Via Behavioral Ethogram, Monitoring, and Interventions Sarah Thurston1,2, Lisa Burlingame1,2, Patrick A. Lester1,2, Jennifer Lofgren1,2 1Unit for Laboratory Animal Medicine, University of Michigan, 2Refinement and Enrichment Advancements Laboratory, University of Michigan Though European rabbits are a social species, socially housing them can be challenging. Therefore, there must be a thorough understanding of behaviors and social structures of pair-housed laboratory rabbits. Here we present a protocol to identify pairing methods, species-typical hierarchy establishment behaviors and behaviors that warrant appropriate intervention. Engineering Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes Chao Tian1, Wei Zhang2,3, Van Phuc Nguyen1, Xueding Wang2,4, Yannis M. Paulus1,2 1Kellogg Eye Center, Department of Ophthalmology and Visual Sciences, University of Michigan, 2Department of Biomedical Engineering, University of Michigan, 3Institute of Biomedical Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College, 4Department of Radiology, University of Michigan This manuscript describes the novel setup and operating procedure of a photoacoustic microscopy and optical coherence tomography dual-modality system for noninvasive, label-free chorioretinal imaging of larger animals, such as rabbits. Genetics Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing Ahmed Elaswad*1,2, Karim Khalil*1,3, David Cline1, Patrick Page-McCaw4, Wenbiao Chen4, Maximilian Michel5, Roger Cone5, Rex Dunham1 1School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, 2Department of Animal Wealth Development, Faculty of Veterinary Medicine, Suez Canal University, 3Anatomy and Embryology Department, Faculty of Veterinary Medicine, Cairo University, 4Department of Molecular Physiology and Biophysics, Vanderbilt University, 5Life Science Institute, University of Michigan A simple and efficient microinjection protocol for gene editing in channel catfish embryos using the CRISPR/Cas9 system is presented. In this protocol, guide RNAs and Cas9 protein were microinjected into the yolk of one-cell embryos. This protocol has been validated by knocking out two channel catfish immune-related genes. Developmental Biology Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids David R. Hill1, Sha Huang1, Yu-Hwai Tsai1, Jason R. Spence1,2, Vincent B. Young1,3 1Department of Internal Medicine, Division of Gastroenterology, University of Michigan, 2Department of Cell and Developmental Biology, University of Michigan, 3Department of Internal Medicine, Division of Infectious Disease, University of Michigan This protocol describes the measurement of epithelial barrier permeability in real-time following pharmacologic treatment in human intestinal organoids using fluorescent microscopy and live cell microscopy. Neuroscience Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers Hussein S. Hamid1, John M. Hayes2, Eva L. Feldman2, Stephen I. Lentz3 1University of Michigan Medical School, 2Department of Neurology, University of Michigan, 3Department of Internal Medicine, University of Michigan This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal. Developmental Biology Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos Antionette L. Williams1, Brenda L. Bohnsack1 1Department of Ophthalmology and Visual Sciences, Kellogg Eye Center, University of Michigan A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos. Engineering Scalable Solution-processed Fabrication Strategy for High-performance, Flexible, Transparent Electrodes with Embedded Metal Mesh Arshad Khan1, Sangeon Lee2, Taehee Jang3, Ze Xiong4, Cuiping Zhang1,5, Jinyao Tang4, L. Jay Guo2,3, Wen-Di Li1,5 1Department of Mechanical Engineering, University of Hong Kong, 2Department of Mechanical Engineering, University of Michigan, 3Department of Electrical Engineering and Computer Science, University of Michigan, 4Department of Chemistry, University of Hong Kong, 5HKU-Shenzhen Institute of Research and Innovation This protocol describes a solution-based fabrication strategy for high-performance, flexible, transparent electrodes with fully-embedded, thick metal mesh. Flexible transparent electrodes fabricated by this process demonstrate among the highest reported performances, including ultra-low sheet resistance, high optical transmittance, mechanical stability under bending, strong substrate adhesion, surface smoothness, and environmental stability. Genetics Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform Nicolaas H. Fourie1, Ralph M. Peace1,2, Sarah K. Abey1, LeeAnne B. Sherwin1, John W. Wiley3, Wendy A. Henderson1 1Digestive Disorders Unit, National Institute of Nursing Research, National Institutes of Health, DHHS, 2National Institutes of Health Research Scholar, Howard Hughes Medical Institute, 3Internal Medicine, Medical School, University of Michigan We describe the use of a multiplexed high-throughput gene expression platform that quantitates gene expression by barcoding and counting molecules in biological substrates without the need for amplification. We used the platform to quantitate microRNA (miRNA) expression in whole blood in subjects with and without irritable bowel syndrome. Developmental Biology Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes Hidehito Saito1,2, Kumiko Iwabuchi3, Noemi Fusaki4,5, Fumito Ito3,6 1Department of Surgery, University of Michigan, 2Department of Biochemistry II, Kanazawa Medical University, 3Center for Immunotherapy, Roswell Park Cancer Institute, 4DNAVEC Corporation, 5Department of Ophthalmology, Keio University School of Medicine, 6Department of Surgical Oncology, Roswell Park Cancer Institute The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells. Biochemistry Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo Jan-Ulrik Dahl1, Philipp Koldewey1,2, James C. A. Bardwell1,2, Ursula Jakob1 1Department of Molecular, Cellular, and Developmental Biology, University of Michigan, 2Howard Hughes Medical Institute, University of Michigan This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions. Biology G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay Ansley Semack1, Rabia U. Malik2, Sivaraj Sivaramakrishnan1 1Genetics, Cell Biology, and Development, University of Minnesota, 2Department of Cell and Developmental Biology, University of Michigan Simple methods to detect the selective activation of G proteins by G protein-coupled receptors remain an outstanding challenge in cell signaling. Here, Fӧrster resonance energy transfer (FRET) biosensors have been developed by pairwise tethering a GPCR to G protein peptides to probe conformational changes at controlled concentrations in live cells. Biology Measuring In Vitro ATPase Activity for Enzymatic Characterization Chelsea S. Rule1, Marcella Patrick1, Maria Sandkvist1 1Department of Microbiology and Immunology, University of Michigan We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase. Immunology and Infection Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle (GUV) Membranes Balaji Olety1, Sarah L. Veatch2, Akira Ono1 1Department of Microbiology and Immunology, University of Michigan Medical School, 2Department of Biophysics, University of Michigan We illustrate here an in vitro membrane binding assay in which interactions between HIV-1 Gag and lipid membranes are visually analyzed using YFP-tagged Gag synthesized in a wheat germ-based in vitro translation system and GUVs prepared by an electroformation technique. Medicine Cutaneous Surgical Denervation: A Method for Testing the Requirement for Nerves in Mouse Models of Skin Disease Shelby C. Peterson1, Isaac Brownell*2, Sunny Y. Wong*1 1Dermatology, Cell and Developmental Biology, University of Michigan, 2Dermatology Branch, National Cancer Institute, National Institutes of Health This article includes detailed protocols for genetic labeling of mouse skin, surgical denervation, skin biopsy and visualizing labeled epithelia by whole-mount β-galactosidase staining. These methods can be used to test the requirement for nerves in mouse models of normal and pathological skin. Chemistry Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability Hui Gao1, Eshita Khera2, Jung-Kul Lee1, Fei Wen2 1Department of Chemical Engineering, Konkuk University, 2Department of Chemical Engineering, University of Michigan Presented is the protocol for co-immobilizing whole-cell biocatalysts for cofactor regeneration and improved reusability, using the production of L-xylulose as an example. The cofactor regeneration is achieved by coupling two Escherichia coli strains expressing functionally complementary enzymes; the whole-cell biocatalyst immobilization is achieved by cell encapsulation in calcium alginate beads. Behavior Pavlovian Conditioned Approach Training in Rats Christopher J. Fitzpatrick1, Jonathan D. Morrow2 1Neuroscience Graduate Program, University of Michigan, 2Department of Psychiatry, University of Michigan Here, we present a protocol to elicit Pavlovian conditioned approach behavior in rats. This procedure can be used to measure individual differences in the tendency to approach and attribute incentive salience to reward-related cues and investigate addiction vulnerability. Developmental Biology Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye Sarah J. Smith*1,2, Eric J. Horstick*3,4, Ann E. Davidson1,2, James Dowling1,2,4 1Program in Genetics & Genome Biology, The Hospital for Sick Children, 2Department of Molecular Genetics, The University of Toronto, 3Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, 4Departments of Pediatrics and Neurology, University of Michigan In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics. Immunology and Infection Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells Gregory J. Baker1,2, Maria G. Castro1,2, Pedro R. Lowenstein1,2 1Department of Neurosurgery, University of Michigan, 2Department of Cell and Developmental Biology, University of Michigan Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment. Neuroscience Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain Alzahra J. Al Omran1, Hannah C. Saternos1, Tongyu Liu2, Surya M. Nauli3, Wissam A. AbouAlaiwi1 1Department of Pharmacology and Experimental Therapeutics, University of Toledo, College of Pharmacy and Pharmaceutical Sciences, 2Life Sciences Institute, University of Michigan, 3Department of Biomedical & Pharmaceutical Sciences, Chapman University, School of Pharmacy, Rinker Health Science campus Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties. Immunology and Infection Isolation of Leukocytes from the Human Maternal-fetal Interface Yi Xu1, Olesya Plazyo1, Roberto Romero1,2,3,4, Sonia S. Hassan1,5, Nardhy Gomez-Lopez1,5,6 1Perinatology Research Branch, NICHD/NIH/DHHS, 2Department of Obstetrics and Gynecology, University of Michigan, 3Department of Epidemiology and Biostatistics, Michigan State University, 4Department of Molecular Obstetrics and Genetics, Wayne State University, 5Department of Obstetrics and Gynecology, Wayne State University School of Medicine, 6Department of Immunology and Microbiology, Wayne State University School of Medicine Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis. Immunology and Infection Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination Lukasz J. Ochyl1,2, James J Moon1,2,3 1Department of Pharmaceutical Sciences, University of Michigan, 2Biointerfaces Institute, University of Michigan, 3Department of Biomedical Engineering, University of Michigan We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination. Biology Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution Francine E. Garrett-Bakelman*1, Caroline K. Sheridan*1, Thadeous J. Kacmarczyk1, Jennifer Ishii1, Doron Betel1,2, Alicia Alonso1, Christopher E. Mason3, Maria E. Figueroa4, Ari M. Melnick1 1Department of Medicine, Weill Cornell Medical College, 2Institute for Computational Biomedicine, Weill Cornell Medical College, 3Department of Physiology and Biophysics, Weill Cornell Medical College, 4Department of Pathology, University of Michigan Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions. Medicine Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma Anda-Alexandra Calinescu1, Felipe Javier Núñez1, Carl Koschmann2, Bradley L. Kolb1, Pedro R. Lowenstein1,3, Maria G. Castro1,3 1Department of Neurosurgery, University of Michigan School of Medicine, 2Department of Pediatrics, Division of Hematology-Oncology, University of Michigan School of Medicine, 3Department of Cell and Developmental Biology, University of Michigan Here we describe an efficient and versatile protocol to induce, monitor and analyze novel glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate and generate tumors with histo-pathological characteristics of human GBM. Developmental Biology Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells Ieng Lam Lei1, Lei Bu2, Zhong Wang1 1Cardiac Surgery, University of Michigan, 2Leon H Charney Division of Cardiology, New York University School of Medicine In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines. Medicine A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys Liwei Huang1, An Xiao1, Andrea Wecker1, Daniel A. McBride1, Soo Young Choi2, Weibin Zhou3, Joshua H. Lipschutz2 1Department of Medicine, Eastern Virginia Medical School, 2Department of Medicine, Medical University of South Carolina, 3Department of Pediatrics, University of Michigan We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish. Bioengineering Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms Derek S. Samarian1, Nicholas S. Jakubovics2, Ting L. Luo1, Alexander H. Rickard1 1Department of Epidemiology, School of Public Health, The University of Michigan, 2Centre for Oral Health Research, School of Dental Sciences, Newcastle University The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque. Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted. Immunology and Infection Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins Natalie Maricic1, Suzanne Dawid1,2 1Microbiology and Immunology, University of Michigan, 2Pediatrics and Communicable Diseases, University of Michigan Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species. Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. Medicine Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty Hassan N. Tausif1, Lauren Johnson2, Michael Titus2, Kyle Mavin2, Navasuja Chandrasekaran1, Maria A. Woodward1, Roni M. Shtein1, Shahzad I. Mian1 1Department of Ophthalmology, University of Michigan, 2MidWest Eye Banks Endothelial keratoplasty is a surgical technique continuously evolving as advancements result in transplantation of thinner grafts with each succession1. Here we present a technique for controlled manual tissue dissection to allow safe and repeatable separation of endothelium and Descemet's membranes from donor corneoscleral buttons for transplantation2. Biology Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development Katherine D. Walton1, Åsa Kolterud2 1Cell and Developmental Biology, University of Michigan, 2Department of Biosciences and Nutrition, Karolinska Instituet Novum Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine. Biology SIVQ-LCM Protocol for the ArcturusXT Instrument Jason D. Hipp*1, Jerome Cheng*2, Jeffrey C. Hanson*1, Avi Z. Rosenberg1, Michael R. Emmert-Buck1, Michael A. Tangrea1, Ulysses J. Balis2 1Laboratory of Pathology, National Cancer Institute, National Institutes of Health, 2Department of Pathology, University of Michigan SIVQ-LCM is an innovative approach that harnesses a computer algorithm, Spatially Invariant Vector Quantization (SIVQ), to drive the laser capture microdissection (LCM) process. The SIVQ-LCM workflow greatly improves the speed and accuracy of microdissection, with applications in both the research and clinical settings. Medicine 3D-Neuronavigation In Vivo Through a Patient's Brain During a Spontaneous Migraine Headache Alexandre F. DaSilva*1,2,3, Thiago D. Nascimento*1, Tiffany Love*3, Marcos F. DosSantos1, Ilkka K. Martikainen1,3, Chelsea M. Cummiford3, Misty DeBoer1, Sarah R. Lucas1, MaryCatherine A. Bender1, Robert A. Koeppe4, Theodore Hall5, Sean Petty5, Eric Maslowski5, Yolanda R. Smith6, Jon-Kar Zubieta3 1Headache & Orofacial Pain Effort (H.O.P.E.), Biological & Materials Sciences Department, University of Michigan School of Dentistry, 2Michigan Center for Oral Health Research (MCOHR), University of Michigan School of Dentistry, 3Translational Neuroimaging Laboratory, Molecular & Behavioral Neuroscience Institute, University of Michigan, 4PET Physics Section, Division of Nuclear Medicine, Radiology Department, University of Michigan, 53DLab, University of Michigan, 6Department of Obstetrics and Gynecology, University of Michigan In this study, the authors report for the first time a novel 3D-Immersive & Interactive Neuronavigation (3D-IIN) through the impact of a spontaneous migraine headache attack in the μ-opioid system of a patient's brain in vivo. Engineering Fabrication and Testing of Microfluidic Optomechanical Oscillators Kewen Han1, Kyu Hyun Kim2, Junhwan Kim1, Wonsuk Lee2,3, Jing Liu3, Xudong Fan3, Tal Carmon2, Gaurav Bahl1 1Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, 2Electrical Engineering and Computer Science, University of Michigan, 3Biomedical Engineering, University of Michigan Parametric optomechanical excitations have recently been experimentally demonstrated in microfluidic optomechanical resonators by means of optical radiation pressure and stimulated Brillouin scattering. This paper describes the fabrication of these microfluidic resonators along with methodologies for generating and verifying optomechanical oscillations. Medicine Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells Anupama Pal1, Celina G. Kleer2 1Department of Internal Medicine, University of Michigan Comprehensive Cancer Center, 2Department of Pathology, University of Michigan Comprehensive Cancer Center Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues. Bioengineering Lipid Bilayer Vesicle Generation Using Microfluidic Jetting Christopher W. Coyne1, Karan Patel1, Johanna Heureaux1, Jeanne Stachowiak3, Daniel A. Fletcher4,5, Allen P. Liu1,2 1Department of Mechanical Engineering, University of Michigan, 2Department of Biomedical Engineering, University of Michigan, 3Department of Biomedical Engineering, Institute for Cellular and Molecular Biology, The University of Texas at Austin, 4Department of Bioengineering, University of California, Berkeley, 5Physical Biosciences Division, Lawrence Berkeley National Laboratory Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired. Bioengineering Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae Bibhudatta Mishra1, Mostafa Ghannad-Rezaie2, Jiaxing Li1, Xin Wang 1, Yan Hao1, Bing Ye3,4, Nikos Chronis2,5, Catherine A. Collins1 1Department of Molecular, Cellular and Developmental Biology, University of Michigan, 2Department of Biomedical Engineering, University of Michigan, 3Life Sciences Institute, University of Michigan, 4Department of Cell and Developmental Biology, University of Michigan, 5Department of Mechanical Engineering, University of Michigan Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the 'larva chip' for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae. Immunology and Infection Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells Qiao Li1, Lin Lu1, Huimin Tao1, Carolyn Xue1, Seagal Teitz-Tennenbaum2, John H. Owen3, Jeffrey S Moyer3, Mark E.P. Prince3, Alfred E. Chang1, Max S. Wicha2 1Department of Surgery, University of Michigan, 2Department of Internal Medicine, University of Michigan, 3Department of Otolaryngology, University of Michigan Evaluated in syngeneic immunocompetent hosts, cancer stem cell (CSC) based dendritic cell (DC) vaccine demonstrated significantly higher antitumor immunity than traditional DC vaccines pulsed with heterogeneous bulk tumor cells. Medicine Production of Apolipoprotein C-III Knockout Rabbits using Zinc Finger Nucleases Dongshan Yang1, Jifeng Zhang1, Jie Xu1, Tianqing Zhu1, Yanbo Fan1, Jianglin Fan2, Y. Eugene Chen1 1Center for Advanced Models for Translational Sciences and Therapeutics, Department of Internal Medicine, University of Michigan Medical Center, 2Department of Molecular Pathology, University of Yamanashi Recent development in gene targeting tools makes production of knockout (KO) rabbits possible. In the present work, we generated five Apolipoprotein (Apo) C-III KO rabbits using Zinc Finger Nucleases (ZFN). This work demonstrated that ZFN is a highly efficient method to produce KO rabbits. Bioengineering Quantitative and Temporal Control of Oxygen Microenvironment at the Single Islet Level Joe Fu-Jiou Lo1, Yong Wang2,3, Zidong Li1, Zhengtuo Zhao1, Di Hu1, David T. Eddington3, Jose Oberholzer2,3 1Department of Mechanical Engineering, University of Michigan-Dearborn, 2Department of Surgery/Transplant, University of Illinois at Chicago, 3Department of Bioengineering, University of Illinois at Chicago Microfluidic oxygen control confers more than just convenience and speed over hypoxic chambers for biological experiments. Especially when implemented via diffusion through a membrane, microfluidic oxygen can provide simultaneous liquid and gas phase modulations at the microscale-level. This technique enables dynamic multi-parametric experiments critical for studying islet pathophysiology. Biology Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations Eric J. Horstick1, Elizabeth M. Gibbs1, Xingli Li1, Ann E. Davidson1, James J. Dowling1 1Departments of Pediatrics and Neurology, University of Michigan Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology. Biology Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae Darcée D. Sloboda1, Dennis R. Claflin1,2, James J. Dowling3, Susan V. Brooks1,4 1Department of Biomedical Engineering, University of Michigan, 2Department of Surgery, Section of Plastic Surgery, University of Michigan, 3Departments of Pediatrics and Neurology, University of Michigan, 4Department of Molecular and Integrative Physiology, University of Michigan Force measurements can be used to demonstrate changes in muscle function due to development, injury, disease, treatment or chemical toxicity. In this video, we demonstrate a method to measure force during a maximal contraction of zebrafish larval trunk muscle. Bioengineering Fabrication of Carbon Nanotube High-Frequency Nanoelectronic Biosensor for Sensing in High Ionic Strength Solutions Girish S. Kulkarni1, Zhaohui Zhong1 1Department of Electrical Engineering and Computer Science, University of Michigan - Ann Arbor We describe the device fabrication and measurement protocol for carbon nanotube based high frequency biosensors. The high frequency sensing technique mitigates the fundamental ionic (Debye) screening effect and allows nanotube biosensor to be operated in high ionic strength solutions where conventional electronic biosensors fail. Our technology provides a unique platform for point-of-care (POC) electronic biosensors operating in physiologically relevant conditions. Medicine Technique and Considerations in the Use of 4x1 Ring High-definition Transcranial Direct Current Stimulation (HD-tDCS) Mauricio F. Villamar1,2, Magdalena Sarah Volz1,3, Marom Bikson4, Abhishek Datta1,4, Alexandre F. DaSilva*5, Felipe Fregni*1 1Laboratory of Neuromodulation, Department of Physical Medicine & Rehabilitation, Spaulding Rehabilitation Hospital and Massachusetts General Hospital, Harvard Medical School, 2School of Medicine, Pontifical Catholic University of Ecuador, 3Charité University Medicine Berlin, 4The City College of The City University of New York, 5Headache & Orofacial Pain Effort (H.O.P.E.), Biologic & Materials Sciences, School of Dentistry, University of Michigan High-definition transcranial direct current stimulation (HD-tDCS), with its 4x1-ring montage, is a noninvasive brain stimulation technique that combines both the neuromodulatory effects of conventional tDCS with increased focality. This article provides a systematic demonstration of the use of 4x1 HD-tDCS, and the considerations needed for safe and effective stimulation. Engineering Design, Fabrication, and Experimental Characterization of Plasmonic Photoconductive Terahertz Emitters Christopher Berry1, Mohammad Reza Hashemi1, Mehmet Unlu1, Mona Jarrahi1 1Electrical Engineering and Computer Science Department, University of Michigan We describe methods for the design, fabrication, and experimental characterization of plasmonic photoconductive emitters, which offer two orders of magnitude higher terahertz power levels compared to conventional photoconductive emitters. Engineering High-speed Particle Image Velocimetry Near Surfaces Louise Lu1, Volker Sick1 1Department of Mechanical Engineering, University of Michigan A procedure for studying transient flows near boundaries using high-resolution, high-speed particle image velocimetry (PIV) is described here. PIV is a non-intrusive measurement technique applicable to any optically accessible flow by optimizing several parameter constraints such as the image and recording properties, the laser sheet properties, and analysis algorithms. Biology Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain Kerry Flegel*1, Dan Sun*1, Olga Grushko*1, Yiqin Ma1, Laura Buttitta1 1Molecular, Cellular and Developmental Biology, University of Michigan A protocol for cell cycle analysis of live Drosophila tissues using the Attune Acoustic Focusing Cytometer is described. This protocol simultaneously provides information about relative cell size, cell number, DNA content and cell type via lineage tracing or tissue specific expression of fluorescent proteins in vivo. Medicine In vitro Organoid Culture of Primary Mouse Colon Tumors Xiang Xue1, Yatrik M. Shah1,2 1Department of Molecular & Integrative Physiology, University of Michigan, 2Department of Internal Medicine, Division of Gastroenterology, University of Michigan A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not. Medicine Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies Jacqueline R. Dauch1, Chelsea N. Lindblad1, John M. Hayes1, Stephen I. Lentz2, Hsinlin T. Cheng1 1Department of Neurology, University of Michigan, 2Department of Internal Medicine, University of Michigan In order to study the changes of nociceptive intraepidermal nerve fibers (IENFs) in painful neuropathies (PN), we developed protocols that could directly examine three-dimensional morphological changes observed in nociceptive IENFs. Three-dimensional analysis of IENFs has the potential to evaluate the morphological changes of IENF in PN. Bioengineering Cell Co-culture Patterning Using Aqueous Two-phase Systems John P. Frampton1, Joshua B. White1, Abin T. Abraham1, Shuichi Takayama1,2 1Department of Biomedical Engineering, University of Michigan, 2Department of Macromolecular Science and Engineering, University of Michigan Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions. Biology Measurement of Lifespan in Drosophila melanogaster Nancy J. Linford1, Ceyda Bilgir1, Jennifer Ro2, Scott D. Pletcher1 1Department of Molecular and Integrative Physiology, University of Michigan, 2Cellular and Molecular Biology Program, University of Michigan Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them. Medicine High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease Bryan Fiema*1, Andrew C. Harris*1, Aurelie Gomez1, Praechompoo Pongtornpipat1, Kelly Lamiman1, Mark T. Vander Lugt1, Sophie Paczesny1 1Pediatric Blood and Marrow Transplant Program, University of Michigan High throughput validation of multiple candidate biomarkers can be performed by sequential ELISA in order to minimize freeze/thaw cycles and use of precious plasma samples. Here, we demonstrate how to sequentially perform ELISAs for six different validated plasma biomarkers1-3 of graft-versus-host disease (GVHD)4 on the same plasma sample. Immunology and Infection Plaque Assay for Murine Norovirus Mariam B. Gonzalez-Hernandez1, Juliana Bragazzi Cunha1, Christiane E. Wobus1 1Department of Microbiology and Immunology, University of Michigan, Ann Arbor Here we describe a method to quantify infectious particles of murine norovirus (MNV), which is the only norovirus that efficiently replicates in cell culture. The plaque assay takes advantage of MNV’s tropism for murine macrophages and can be adapted for use with biological or environmental samples containing MNV. Neuroscience Whole Animal Perfusion Fixation for Rodents Gregory J. Gage1, Daryl R. Kipke1, William Shain2 1Biomedical Engineering, University of Michigan, 2Department of Neurological Surgery, University of Washington School of Medicine Here we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain. Engineering Fabrication, Densification, and Replica Molding of 3D Carbon Nanotube Microstructures Davor Copic1, Sei Jin Park1, Sameh Tawfick1, Michael De Volder2, A. John Hart1 1Mechanosynthesis Group, Department of Mechanical Engineering, University of Michigan, 2IMEC, Belgium We present methods for fabrication of patterned microstructures of vertically aligned carbon nanotubes (CNTs), and their use as master molds for production of polymer microstructures with organized nanoscale surface texture. The CNT forests are densified by condensation of solvent onto the substrate, which significantly increases their packing density and enables self-directed formation of 3D shapes. Medicine Corneal Donor Tissue Preparation for Endothelial Keratoplasty Maria A. Woodward1, Michael Titus2, Kyle Mavin2, Roni M. Shtein1 1Department of Ophthalmology, University of Michigan, 2MidWest Eye Banks Endothelial corneal transplantation is a surgical technique for treatment of posterior corneal diseases. Mechanical microkeratome dissection to prepare tissue results in thinner, more symmetric grafts with less endothelial cell loss and improved outcomes. Dissections can be performed at the eye bank prior to corneal transplantation surgery. Biology Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster Martin J. Tinkerhess1, Sara Ginzberg1, Nicole Piazza1, Robert J. Wessells1 1Department of Geriatric Medicine, University of Michigan Medical School We describe the first endurance training protocol for an important genetic model species, Drosophila melanogaster, and outline several assays to chart improvements in mobility following training. Neuroscience Voltage Biasing, Cyclic Voltammetry, & Electrical Impedance Spectroscopy for Neural Interfaces Seth J. Wilks1, Tom J. Richner2, Sarah K. Brodnick2, Daryl R. Kipke3, Justin C. Williams2, Kevin J. Otto1,4 1Weldon School of Biomedical Engineering, Purdue University, 2Biomedical Engineering, University of Wisconsin-Madison, 3Biomedical Engineering, University of Michigan, 4Department of Biological Sciences, Purdue University The electrode-tissue interface of neural recording electrodes can be characterized with electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Application of voltage biasing changes the electrochemical properties of the electrode-tissue interface and can improve recording capability. Voltage biasing, EIS, CV, and neural recordings are complementary. Neuroscience Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals Gregory J. Gage1, Colin R. Stoetzner1, Thomas Richner2, Sarah K. Brodnick2, Justin C. Williams2, Daryl R. Kipke1,3 1Biomedical Engineering, University of Michigan, 2Biomedical Engineering, University of Wisconsin-Madison, 3NeuroNexus Technologies We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model. Biology A Protocol for Computer-Based Protein Structure and Function Prediction Ambrish Roy1,2, Dong Xu1, Jonathan Poisson1, Yang Zhang1,2 1Center for Computational Medicine and Bioinformatics, University of Michigan, 2Center for Bioinformatics and Department of Molecular Bioscience, University of Kansas Guidelines for computer based structural and functional characterization of protein using the I-TASSER pipeline is described. Starting from query protein sequence, 3D models are generated using multiple threading alignments and iterative structural assembly simulations. Functional inferences are thereafter drawn based on matches to proteins with known structure and functions. Bioengineering Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers Krishnan Raghunathan1, Joshua N. Milstein2, Jens -Christian Meiners2 1LSA Biophysics, University of Michigan, 2LSA Biophysics, Department of Physics, University of Michigan We illustrate the use of a constant force axial optical tweezers to explore the mechanical properties of short DNA molecules. By stretching DNA axially, we minimize steric hindrances and artifacts arising in conventional lateral manipulation, allowing us to study DNA molecules as short as ~100 nm. Medicine High-Resolution Endocardial and Epicardial Optical Mapping in a Sheep Model of Stretch-Induced Atrial Fibrillation David Filgueiras-Rama1, Raphael Pedro Martins1, Steven R. Ennis1, Sergey Mironov1, Jiang Jiang1, Masatoshi Yamazaki1, Jérôme Kalifa1, Josè Jalife1, Omer Berenfeld1 1Center for Arrhythmia Research. Internal Medicine, University of Michigan This report provides a detailed description of the methodology and results of simultaneous endocardial and epicardial optical mapping of electrical excitation in the intact left atrium of a Langendorff-perfused sheep heart during stretch-induced atrial fibrillation. Medicine Electrolytic Inferior Vena Cava Model (EIM) of Venous Thrombosis Jose A. Diaz1, Shirley K. Wrobleski1, Angela E. Hawley1, Benedict R. Lucchesi2, Thomas W. Wakefield1, Daniel D. Myers, Jr.1 1Conrad Jobst Vascular Research Laboratories, Section of Vascular Surgery, University of Michigan, 2Department of Pharmacology, University of Michigan The electrolytic induction of endothelial activation to the internal surface of the Inferior Vena Cava results in venous type thrombus formation due to endothelial activation and partial blood stasis, two components of Virchow's triad. Medicine Mouse Complete Stasis Model of Inferior Vena Cava Thrombosis Shirley K. Wrobleski1, Diana M. Farris1, José A. Diaz1, Daniel D. Myers Jr.1, Thomas W. Wakefield1 1Conrad Jobst Vascular Research Laboratories, Section of Vascular Surgery, University of Michigan The mouse complete stasis model of inferior vena cava thrombosis yields quantifiable amounts of vein wall tissue and thrombus. It has proven useful for evaluating interactions between the vein wall and the occlusive thrombus and in assessing the progression from acute to chronic inflammation. Biology Solid Plate-based Dietary Restriction in Caenorhabditis elegans Tsui-Ting Ching1, Ao-Lin Hsu1,2 1Department of Internal Medicine, Division of Geriatric Medicine, University of Michigan, 2Department of Molecular and Integrative Physiology, University of Michigan Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria. Neuroscience Electrode Positioning and Montage in Transcranial Direct Current Stimulation Alexandre F. DaSilva1, Magdalena Sarah Volz2,3, Marom Bikson4, Felipe Fregni2 1Headache & Orofacial Pain Effort (H.O.P.E.), Biologic & Material Sciences, School of Dentistry, University of Michigan, 2Laboratory of Neuromodulation, Department of Physical Medicine & Rehabilitation, Spaulding Rehabilitation Hospital and Massachusetts General Hospital, Harvard Medical School, 3Charité, University Medicine Berlin, 4Department of Biomedical Engineering, The City College of New York Transcranial direct current stimulation (tDCS) is an established technique to modulate cortical excitability1,2. It has been used as an investigative tool in neuroscience due to its effects on cortical plasticity, easy operation, and safe profile. One area that tDCS has been showing encouraging results is pain alleviation 3-5. Biology Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software Koen J. C. Verbrugghe1, Raymond C. Chan1 1Human Genetics, University of Michigan The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software. Neuroscience The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds Michelle K. Leach1, Zhang-Qi Feng2, Caitlyn C. Gertz3, Samuel J. Tuck3, Tara M. Regan3, Youssef Naim3, Andrea M. Vincent3, Joseph M. Corey1,3,4 1Department of Biomedical Engineering, University of Michigan, 2State Key Laboratory of Bioelectronics, Southeast University, 3Department of Neurology, University of Michigan, 4Geriatric Research, Education and Clinical Center, Veterans Affairs Ann Arbor Health System Aligned electrospun fibers direct the growth of neurons in vitro and are a potential component of nerve regeneration scaffolds. We describe a procedure for preparing electrospun fiber substrates and the serum-free culture of primary rat E15 sensory (DRG) and motor neurons. Visualization of neurons by immunocytochemistry is also included. Bioengineering Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters Michelle K. Leach1, Zhang-Qi Feng1,2, Samuel J. Tuck3, Joseph M. Corey1,3,4 1Department of Biomedical Engineering, University of Michigan, 2State Key Laboratory of Bioelectronics, Southeast University, 3Department of Neurology, University of Michigan, 4Geriatrics Research, Education and Clinical Center, Veterans Affairs Ann Arbor Healthcare Center Electrospinning techniques can create a variety of nanofibrous scaffolds for tissue engineering or other applications. We describe here a procedure to optimize the parameters of the electrospinning solution and apparatus to obtain fibers with the desired morphology and alignment. Common problems and troubleshooting techniques are also presented. Biology Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development Katie E. Holmes1, Matthew J. Wyatt2, Yu-chi Shen2, Deborah A. Thompson2,3, Kate F. Barald2,4 1Department of Veterinary Science, University of Wisconsin, Madison, 2Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, 3Present address: Department of Pulmonary Medicine, University of Michigan, Ann Arbor, MI, 4Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear. Immunology and Infection The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis Kelly Schwartz1, Rachel Stephenson1, Margarita Hernandez1, Nicolays Jambang1, Blaise R. Boles1 1Molecular, Cellular, and Developmental Biology, University of Michigan Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail. Neuroscience Visualization of Mitochondrial DNA Replication in Individual Cells by EdU Signal Amplification Kristine M. Haines1, Eva L. Feldman2, Stephen I. Lentz3 1Michigan Research Community, Undergraduate Research Opportunity Program, University of Michigan, 2Department of Neurology, University of Michigan, 3Department of Internal Medicine, Division of Metabolism, Endocrinology, and Diabetes, University of Michigan We developed a sensitive technique to label newly synthesized mitochondrial DNA (mtDNA) in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of EdU together with a tyramide signal amplification (TSA) protocol to visualize mtDNA replication within subcellular compartments of neurons. Neuroscience Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats Esther Y. Maier1,2, Sean T. Ma3, Allison Ahrens2,4, Timothy J. Schallert2,4,5, Christine L. Duvauchelle1,2,4 1College of Pharmacy, Division of Pharmacology and Toxicology, University of Texas at Austin, 2The Waggoner Center of Addiction and Alcohol Research, University of Texas at Austin, 3Department of Psychology, University of Michigan, 4Institute for Neuroscience, University of Texas at Austin, 5Department of Psychology, University of Texas at Austin Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience. Biology Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis Andrew D. Klocko1, Kaleena M. Crafton1, Brian W. Walsh1, Justin S. Lenhart1, Lyle A. Simmons1 1Department of Molecular, Cellular, and Developmental Biology, University of Michigan-Ann Arbor A detailed protocol is described for imaging the real time formation of DNA repair complexes in Bacillus subtilis cells. Biology Exploring Cognitive Functions in Babies, Children & Adults with Near Infrared Spectroscopy Mark H. Shalinsky1, Iouila Kovelman1, Melody S. Berens2, Laura-Ann Petitto2 1Department of Psychology, University of Michigan, Ann Arbor, 2Department of Psychology, University of Toronto Scarborough Here we describe a data collection and data analysis method for functional Near Infrared Spectroscopy (fNIRS), a novel non-invasive brain imaging system used in cognitive neuroscience, particularly in studying child brain development. This method provides a universal standard of data acquisition and analysis vital to data interpretation and scientific discovery.