University of Michigan, Ann Arbor

120 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. Rao^{1,2,3,4,5,6,7,8}, Bryan L. Betz^{1 1}Department 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 Brandenburg^1,2, Christopher Patsalis¹, George Michailidis³, Alla Karnovsky^{1 1}Department 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’Ancona^1,2, Vaibhav Sidarala¹, Scott A. Soleimanpour^{1,3,4 1}Department 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.

• 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. Carroll^{1,2 1}Hollings 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. Leiser^{1,2 1}Molecular 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. Miller^{1,2 1}Kellogg 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. Herron^{1 1}Frankel 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. Gottula^1,2,3, Suhas Devangam¹, Jessica L. Koehler², Matthew J. Sigakis^{1 1}Department 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 Noly^1,2, Wei Huang¹, Suyash Naik¹, Paul Tang¹, Ienglam Lei^{1 1}University 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 Saini^{1 1}Department 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. Westfall^{1 1}Department 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 Wang^{1 1}Department 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)26Sor^{tm38(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 Zhang^{1 1}Department 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. Cederna^1,2, Stephen W. P. Kemp^{1,2 1}Department 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 Han^1,6, Elias Georgas^7,8, John Erby Wilkinson⁹, Harrison Seidner¹, Leonie Foerschner¹, David A. Tuveson¹, Yi-Xian Qin⁸, Mikala Egeblad^{1 1}Cold 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. Liu^{1,4,5,6 1}Department 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. Chestek^{1,4,5,6 1}Department 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 Li^2,3,4, David B. Lombard^{1,4,5 1}Department 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. Allard^{5 1}Center 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 Koch^3,4, Renae Lyons¹, Asma Nusrat¹, Charles A. Parkos^{1 1}Department 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. Parkos^{1 1}Department 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 Quiros^{1 1}Department 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 Levenberg^{1 1}Faculty 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. Castro^2,3,4, Sofia D. Merajver^{1,2 1}Department 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. Hegedus^{1 1}Department 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. Cederna^1,2, Stephen W. P. Kemp^{1,2 1}Department 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 Vesia^{1 1}School 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. Roberts^{1 1}Kresge 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. Scott^{2,3 1}Department 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. Sharkins^2,4, Allen H. Hunter⁵, Amir Avishai⁶, Evon S. Ereifej^{2,3,4 1}Department 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-Barboza^1,2, Dominic Polsinelli⁵, Phillip J. Webster^1,2, Oliver A. Varban¹, Carey N. Lumeng^2,3,4, Robert W. O'Rourke^{1,6 1}Department 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 Frankel^{1 1}Department 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 Leventhal^{2,3,4 1}Neuroscience 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 Mehta^{1,2,3,4 1}Department 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 Mishina^{2 1}The 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. Ludlow^{1 1}School 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. Soleimanpour^{1,2 1}Department 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. Merchant^1,2, Linda C. Samuelson^1,2, Nataliya Razumilava^{1 1}Departments 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. Carlson^{1 1}Insitute 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. Núñez^1,2, Flor M. Mendez², Maria B. Garcia-Fabiani^1,2, Joaquín Pardo^1,3, Marta Edwards^1,2, Pedro R. Lowenstein^1,2, Maria G. Castro^{1,2 1}Department 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. Smaldino^{1 1}Department 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 Guan^1,2, Shiyuan Wang¹, Minjun Jin², Haotian Xu³, Qiong Yang^{1,4 1}Department 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. Hays^1,2, Julia L. Hays^1,2, Hasan B. Alam^{1 1}Department 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 Wang^1,4, Ramon Ocadiz-Ruiz¹, Sinju Sundaresan¹, Lin Ding¹, Michael Hayes¹, Nirakar Sahoo³, Haoxing Xu^1,2, Juanita L. Merchant^{1,2,5 1}Department 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. Heron^{1 1}Department 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 Zavros^{1 1}Department 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. Beg^{1 1}Department 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 Thurston^1,2, Lisa Burlingame^1,2, Patrick A. Lester^1,2, Jennifer Lofgren^{1,2 1}Unit 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 Zhang^2,3, Van Phuc Nguyen¹, Xueding Wang^2,4, Yannis M. Paulus^{1,2 1}Kellogg 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 Dunham^{1 1}School 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. Spence^1,2, Vincent B. Young^{1,3 1}Department 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. Lentz^{3 1}University 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. Bohnsack^{1 1}Department 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 Zhang^1,5, Jinyao Tang⁴, L. Jay Guo^2,3, Wen-Di Li^{1,5 1}Department 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. Peace^1,2, Sarah K. Abey¹, LeeAnne B. Sherwin¹, John W. Wiley³, Wendy A. Henderson^{1 1}Digestive 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 Saito^1,2, Kumiko Iwabuchi³, Noemi Fusaki^4,5, Fumito Ito^{3,6 1}Department 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 Koldewey^1,2, James C. A. Bardwell^1,2, Ursula Jakob^{1 1}Department 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 Sivaramakrishnan^{1 1}Genetics, 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 Sandkvist^{1 1}Department 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 Ono^{1 1}Department 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 1}Dermatology, 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 Wen^{2 1}Department 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. Morrow^{2 1}Neuroscience 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. Davidson^1,2, James Dowling^{1,2,4 1}Program 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. Baker^1,2, Maria G. Castro^1,2, Pedro R. Lowenstein^{1,2 1}Department 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. AbouAlaiwi^{1 1}Department 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 Romero^1,2,3,4, Sonia S. Hassan^1,5, Nardhy Gomez-Lopez^{1,5,6 1}Perinatology 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. Ochyl^1,2, James J Moon^{1,2,3 1}Department 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 Betel^1,2, Alicia Alonso¹, Christopher E. Mason³, Maria E. Figueroa⁴, Ari M. Melnick^{1 1}Department 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. Lowenstein^1,3, Maria G. Castro^{1,3 1}Department 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 Wang^{1 1}Cardiac 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. Lipschutz^{2 1}Department 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. Rickard^{1 1}Department 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 Dawid^{1,2 1}Microbiology 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. Mian^{1 1}Department 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 Kolterud^{2 1}Cell 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. Balis^{2 1}Laboratory 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. Martikainen^1,3, 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 Zubieta^{3 1}Headache & 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 Lee^2,3, Jing Liu³, Xudong Fan³, Tal Carmon², Gaurav Bahl^{1 1}Mechanical 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. Kleer^{2 1}Department 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. Fletcher^4,5, Allen P. Liu^{1,2 1}Department 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 Ye^3,4, Nikos Chronis^2,5, Catherine A. Collins^{1 1}Department 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 3^rd 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. Wicha^{2 1}Department 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 Chen^{1 1}Center 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 Wang^2,3, Zidong Li¹, Zhengtuo Zhao¹, Di Hu¹, David T. Eddington³, Jose Oberholzer^{2,3 1}Department 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. Dowling^{1 1}Departments 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. Claflin^1,2, James J. Dowling³, Susan V. Brooks^{1,4 1}Department 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 Zhong^{1 1}Department 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. Villamar^1,2, Magdalena Sarah Volz^1,3, Marom Bikson⁴, Abhishek Datta^1,4, Alexandre F. DaSilva*⁵, Felipe Fregni*^{1 1}Laboratory 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 Jarrahi^{1 1}Electrical 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 Sick^{1 1}Department 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 Buttitta^{1 1}Molecular, 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. Shah^{1,2 1}Department 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. Cheng^{1 1}Department 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 Takayama^{1,2 1}Department 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. Pletcher^{1 1}Department 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 Paczesny^{1 1}Pediatric 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 biomarkers^1-3 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. Wobus^{1 1}Department 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 Shain^{2 1}Biomedical 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 Hart^{1 1}Mechanosynthesis 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. Shtein^{1 1}Department 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. Wessells^{1 1}Department 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. Otto^{1,4 1}Weldon 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. Kipke^{1,3 1}Biomedical 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 Roy^1,2, Dong Xu¹, Jonathan Poisson¹, Yang Zhang^{1,2 1}Center 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 Meiners^{2 1}LSA 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 Berenfeld^{1 1}Center 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 1}Conrad 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. Wakefield^{1 1}Conrad 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 Hsu^{1,2 1}Department 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 Volz^2,3, Marom Bikson⁴, Felipe Fregni^{2 1}Headache & 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 excitability^1,2. 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. Chan^{1 1}Human 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. Corey^{1,3,4 1}Department 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 Feng^1,2, Samuel J. Tuck³, Joseph M. Corey^{1,3,4 1}Department 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. Thompson^2,3, Kate F. Barald^{2,4 1}Department 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. Boles^{1 1}Molecular, 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. Lentz^{3 1}Michigan 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. Maier^1,2, Sean T. Ma³, Allison Ahrens^2,4, Timothy J. Schallert^2,4,5, Christine L. Duvauchelle^{1,2,4 1}College 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. Simmons^{1 1}Department 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 Petitto^{2 1}Department 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.