56 articles published in JoVE

• Genetics

  
  Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq) Lauren Woodward¹, Pooja Gangras¹, Guramrit Singh^{1 1}Department of Molecular Genetics, Center for RNA Biology, The Ohio State University Here, we present a protocol to enrich endogenous RNA binding sites or "footprints" of RNA:protein (RNP) complexes from mammalian cells. This approach involves two immunoprecipitations of RNP subunits and is therefore dubbed RNA immunoprecipitation in tandem (RIPiT).

• Cancer Research

  
  Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures Brandon M. Murphy¹, Tirzah J. Weiss¹, Christin E. Burd^{1,2 1}Department of Cancer Biology and Genetics, The Ohio State University, ²Department of Molecular Genetics, The Ohio State University This protocol outlines a rapid method to simultaneously generate melanocyte and fibroblast cultures from the skin of 0-4 day old mice. These primary cultures can be maintained and manipulated in vitro to study a variety of physiologically relevant processes, including skin cell biology, pigmentation, wound healing and melanoma.

• Immunology and Infection

  
  Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice Kyle Caution¹, Kacy Yount¹, Rajendar Deora^1,2, Purnima Dubey^{1 1}Department of Microbial Infection & Immunity, The Ohio State University, ²Department of Microbiology, The Ohio State University Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

• Bioengineering

  
  Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels Abdulaziz S. Fakhouri^1,2,3, Jennifer L. Leight^{1,2 1}Department of Biomedical Engineering, The Ohio State University, ²The Ohio State University Comprehensive Cancer Center, Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, ³Biomedical Technology Department, King Saud University Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

• Cancer Research

  
  Detection of Protease Activity by Fluorescent Peptide Zymography Ameya A. Deshmukh^1,2, Jessica L. Weist¹, Jennifer L. Leight^{1,2 1}Comprehensive Cancer Center, James Cancer Hospital and Solove Research Center, Ohio State University, ²Department of Biomedical Engineering, College of Engineering, Ohio State University Here, we present a detailed protocol for a modified zymographic technique in which fluorescent peptides are used as the degradable substrate in place of native proteins. Electrophoresis of biological samples in fluorescent peptide zymograms enables detection of a wider range of proteases than previous zymographic techniques.

• Immunology and Infection

  
  High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells Jonathan G.T. Lam^1,2,3, Chi Song⁴, Stephanie Seveau^{1,2,3 1}Department of Microbial Infection and Immunity, The Ohio State University, ²Department of Microbiology, The Ohio State University, ³Infectious Diseases Institute, The Ohio State University, ⁴Division of Biostatistics, College of Public Health, The Ohio State University Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

• Behavior

  
  Novel Object Recognition and Object Location Behavioral Testing in Mice on a Budget Jiyeon K. Denninger¹, Bryon M. Smith¹, Elizabeth D. Kirby^{1,2,3 1}Department of Psychology, Ohio State University, ²Department of Neuroscience, Ohio State University, ³Center for Chronic Brain Injury, Ohio State University Here we provide a protocol which includes comprehensive instructions for the economical establishment of murine object location and novel object recognition behavioral testing, including the design, cost, and construction of required equipment as well as execution of behavioral testing, data collection, and analysis.

• Immunology and Infection

  
  The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis Matthew J. Dunn¹, Andrew L. Woodruff¹, Matthew Z. Anderson^{1,2 1}Department of Microbiology, The Ohio State University, ²Department of Microbial Infection and Immunity, The Ohio State University Galleria mellonella serves as an invertebrate model for disseminated candidiasis. Here, we detail the infection protocol and provide supporting data for the model's effectiveness.

• Behavior

  
  Gaze in Action: Head-mounted Eye Tracking of Children's Dynamic Visual Attention During Naturalistic Behavior Lauren K. Slone¹, Drew H. Abney¹, Jeremy I. Borjon¹, Chi-hsin Chen², John M. Franchak³, Daniel Pearcy¹, Catalina Suarez-Rivera¹, Tian Linger Xu¹, Yayun Zhang¹, Linda B. Smith¹, Chen Yu^{1 1}Department of Psychological and Brain Sciences, Indiana University, ²Department of Otolaryngology-Head and Neck Surgery, The Ohio State University, ³Department of Psychology, University of California, Riverside Young children do not passively observe the world, but rather actively explore and engage with their environment. This protocol provides guiding principles and practical recommendations for using head-mounted eye trackers to record infants' and toddlers' dynamic visual environments and visual attention in the context of natural behavior.

• Medicine

  
  Ferric Chloride-induced Canine Carotid Artery Thrombosis: A Large Animal Model of Vascular Injury Allyson L. Huttinger¹, Debra G. Wheeler¹, Surya Gnyawali², David Dornbos III¹, Juliana M. Layzer³, Nicholas Venetos¹, Spencer Talentino¹, Nicholas J. Musgrave¹, Cheyenne Jones¹, Camille Bratton¹, Matthew E. Joseph⁴, Chandan Sen², Bruce A. Sullenger³, Shahid M. Nimjee^{1 1}Department of Neurological Surgery, Ohio State University, ²Department of Surgery, Ohio State University, ³Department of Surgery, Duke University, ⁴Department of Internal Medicine, Ohio State University Here, we present the modifications necessary to a well characterized and commonly used small animal ferric chloride-induced (FeCl3) carotid artery injury model for use in a large animal vascular injury model. The resulting model can be utilized for pre-clinical trial assessment of both prophylactic and thrombolytic pharmacological and mechanical interventions.

• Biochemistry

  
  Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis Kang-Yi Su*^1,2, Steven D. Goodman*³, Hung-Ming Lai¹, Rong-Syuan Yen¹, Wei-Yao Hu¹, Wern-Cherng Cheng², Liang-In Lin^1,2, Ya-Chien Yang^1,2, Woei-Horng Fang^{1,2 1}Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, ²Department of Laboratory Medicine, National Taiwan University Hospital, ³Center for Microbial Pathogenesis, Nationwide Children's Hospital and the Department of Pediatrics, The Ohio State University A non-labeled, non-radio-isotopic method for DNA polymerase proofreading and a DNA repair assay was developed by using high-resolution MALDI-TOF mass spectrometry and a single nucleotide extension strategy. The assay proved to be very specific, simple, rapid, and easy to perform for proofreading and repair patches shorter than 9-nucleotides.

• Medicine

  
  Collecting Hair Samples for Hair Cortisol Analysis in African Americans Kathy D. Wright¹, Jodi L. Ford¹, Joseph Perazzo², Lenette M. Jones³, Sherrilynn Mahari⁴, Brent A. Sullenbarger^5,6,7, Mark L. Laudenslager^{8 1}College of Nursing, The Ohio State University, ²College of Nursing, University of Cincinnati, ³School of Nursing, University of Michigan, ⁴Licensed Cosmetologist Consultant, ⁵Clinical Research Manager, The Ohio State University, ⁶Biomedical Core Research Coordinator, The Ohio State University, ⁷College of Nursing Biomedical Core Research Laboratory, The Ohio State University, ⁸Department of Psychiatry, Behavioral Immunology & Endocrinology Laboratory, University of Colorado Denver Anschutz Medical Campus Hair cortisol concentration analysis provides an alternative to traditional measures of cortisol; however, to collect hair samples from African Americans, scientists need to be culturally informed and competent. The purpose of this protocol is to demonstrate a culturally informed technique to collect hair samples for cortisol analysis from African Americans.

• Neuroscience

  
  A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons Dustin Servello¹, Yuanzheng Gu^2,3, Chen Gu^{1,2 1}Molecular, Cellular, and Developmental Biology Program, The Ohio State University, ²Department of Biological Chemistry and Pharmacology, The Ohio State University, ³Biogen This protocol describes a physiologically relevant, pressurized fluid approach for rapid and reversible induction of varicosities in neurons.

• Medicine

  
  Mechanical Ventilation Boot Camp Curriculum Jennifer Yee^1,2, Alma Benner², Jared Hammond², Bethany Malone², Charles Fuenning³, Richard George^2,4, Rami A. Ahmed^{2,3,4 1}Department of Emergency Medicine, The Ohio State University, ²Summa Health System, Akron City Hospital, ³Western Reserve Hospital, ⁴Northeast Ohio Medical University This curriculum outlines how to execute a simulation-based boot camp to teach providers how to manage mechanically ventilated patients.

• Environment

  
  The Calibration and Use of Capacitance Sensors to Monitor Stem Water Content in Trees Ashley M. Matheny¹, Steven R. Garrity², Gil Bohrer^{3 1}Department of Geological Sciences, Jackson School of Geosciences, University of Texas at Austin, ²METER Group, Inc., USA, ³Department of Civil, Environmental and Geodetic Engineering, Ohio State University The hydraulic capacitance of biomass is a key component of the vegetation water budget, which serves as a buffer against short and long-term drought stresses. Here, we present a protocol for the calibration and use of soil moisture capacitance sensors to monitor water content in the stems of large trees.

• Biology

  
  Standardized Method for High-throughput Sterilization of Arabidopsis Seeds Benson E. Lindsey III*¹, Luz Rivero*¹, Chistopher S. Calhoun¹, Erich Grotewold^1,2, Jelena Brkljacic^{1 1}Arabidopsis Biological Resource Center, Center for Applied Plant Sciences, The Ohio State University, ²Department of Molecular Genetics, Center for Applied Plant Sciences, The Ohio State University The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

• Genetics

  
  Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow Gajendra W. Suryawanshi*^1,2, Song Xu*³, Yiming Xie¹, Tom Chou³, Namshin Kim⁴, Irvin S. Y. Chen^1,5, Sanggu Kim^{6 1}UCLA AIDS Institute, University of California at Los Angeles (UCLA), ²Department of Microbiology, Immunology, & Molecular Genetics, University of California at Los Angeles (UCLA), ³Departments of Biomathematics and Mathematics, University of California at Los Angeles (UCLA), ⁴Personalized Genomic Medicine Research Center, Division of Strategic Research Groups, Korea Research Institute of Bioscience and Biotechnology, ⁵Department of Medicine, University of California at Los Angeles (UCLA), ⁶Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University (OSU) This manuscript describes the experimental procedure and software analysis for a bidirectional integration site assay that can simultaneously analyze upstream and downstream vector-host junction DNA. Bidirectional PCR products can be used for any downstream sequencing platform. The resulting data are useful for a high-throughput, quantitative comparison of integrated DNA targets.

• Chemistry

  
  NMR Spectroscopy as a Robust Tool for the Rapid Evaluation of the Lipid Profile of Fish Oil Supplements Kathryn Williamson¹, Emmanuel Hatzakis^{1,2 1}Department of Food Science and Technology, The Ohio State University, ²Foods for Health Discovery Theme, The Ohio State University Here, high-resolution ¹H and ¹³C Nuclear Magnetic Resonance (NMR) spectroscopy was used as a rapid and reliable tool for quantitative and qualitative analysis of encapsulated fish oil supplements.

• Medicine

  
  Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle Vidhya Kumar¹, Henry Chang¹, David A. Reiter², David P. Bradley³, Martha Belury⁴, Shana E. McCormack⁵, Subha V. Raman^{1 1}Davis Heart and Lung Research Institute, The Ohio State University, ²Laboratory of Clinical Investigation, National Institute on Aging, ³Division of Endocrinology, Diabetes and Metabolism, The Ohio State University, ⁴Department of Human Sciences, Human Nutrition, The Ohio State University, ⁵Division of Endocrinology and Diabetes, Department of Pediatrics, University of Pennsylvania This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (³¹PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

• Biochemistry

  
  Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution Gatikrushna Singh*¹, Sarah M. Fritz*², Arnaz Ranji², Deepali Singh³, Kathleen Boris-Lawrie^{1,2 1}Department of Veterinary & Biomedical Sciences, University of Minnesota, ²Department of Veterinary Biosciences, Ohio State University, ³School of Biotechnology, Gautam Buddha University This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

• Medicine

  
  Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System Kelly Regan¹, Soheil Moosavinasab², Philip Payne¹, Simon Lin^{2 1}Department of Biomedical Informatics, The Ohio State University, ² Here we describe a protocol using the web-based drug repurposing hypothesis generation tool: "RE:fine Drugs." This protocol can be modified to a user's preferences at the level of the query type (gene, drug or disease) and/or the range of available advanced options.

• Environment

  
  Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions Brian A. Pace¹, Helen M. Alexander², D. Jason Emry³, Kristin L. Mercer^{1 1}Department of Horticulture and Crop Science, Ohio State University, ²Department of Ecology and Evolutionary Biology, University of Kansas, ³Department of Biology, Washburn University Here we present a protocol for assessing seed survivorship, germination and dormancy under field conditions using buried, labeled seed strips and tetrazolium chloride (TZ) viability testing.

• Behavior

  
  Electrophysiological Motor Unit Number Estimation (MUNE) Measuring Compound Muscle Action Potential (CMAP) in Mouse Hindlimb Muscles W. David Arnold^1,2,3, Kajri A. Sheth¹, Christopher G. Wier⁴, John T. Kissel^1,3, Arthur H. Burghes^1,3,4, Stephen J. Kolb^{1,3,4 1}Department of Neurology, The Ohio State University Wexner Medical Center, ²Department of Physical Medicine and Rehabilitation, The Ohio State University, ³Department of Neuroscience, The Ohio State University Wexner Medical Center, ⁴Department of Biochemistry and Pharmacology, The Ohio State University Wexner Medical Center We present refined protocols that allow in vivo monitoring of motor unit function in the mouse. Techniques to measure compound muscle action potential (CMAP) and motor unit number estimation (MUNE) in the mouse hind limb muscles innervated by the sciatic nerve are described.

• Engineering

  
  Electron Channeling Contrast Imaging for Rapid III-V Heteroepitaxial Characterization Julia I. Deitz¹, Santino D. Carnevale², Steven A. Ringel³, David W. McComb³, Tyler J. Grassman^{1,2 1}Department of Materials Science and Engineering, The Ohio State University, ²Department of Electrical and Computer Engineering, The Ohio State University, ³Institute of Materials Research, The Ohio State University The use of electron channeling contrast imaging in a scanning electron microscope to characterize defects in III-V/Si heteroexpitaxial thin films is described. This method yields similar results to plan-view transmission electron microscopy, but in significantly less time due to lack of required sample preparation.

• Chemistry

  
  Chemical Vapor Deposition of an Organic Magnet, Vanadium Tetracyanoethylene Megan Harberts¹, Yu Lu², Howard Yu¹, Arthur J. Epstein^1,2, Ezekiel Johnston-Halperin^{1 1}Department of Physics, The Ohio State University, ²Department of Chemistry, The Ohio State University We present the synthesis of the organic-based ferrimagnet vanadium tetracyanoethylene (V[TCNE]x, x~2) via low temperature chemical vapor deposition (CVD). This optimized recipe yields an increase in Curie temperature from 400 K to over 600 K and a dramatic improvement in magnetic resonance properties.

• Medicine

  
  Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect Bo Zhang^1,2, Jonathan P. Davis¹, Mark T. Ziolo^{1 1}Department of Physiology & Cell Biology, Davis Heart and Lung Research Institute, The Ohio State University, ²Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

• Behavior

  
  A Protocol for Housing Mice in an Enriched Environment Andrew M. Slater¹, Lei Cao^{1 1}Department of Molecular Virology, Immunology, and Medical Genetics, and Comprehensive Cancer Center, The Ohio State University Environmental enrichment for mice requires a complex and challenging setup, as well as comprehensive husbandry and handling techniques to assure robust metabolic and anti-cancer effects in the mice. This protocol provides detailed procedures to reproduce the above mentioned effects in mice.

• Medicine

  
  Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots Lu Xu¹, Qiwen Shen¹, Zhongqi Mao², L. James Lee³, Ouliana Ziouzenkova^{1 1}Department of Human Sciences, The Ohio State University, ²Department of Minimally Invasive Surgery, The First Affiliated Hospital of Soochow University, ³NSF Nanoscale Science and Engineering Center for Affordable Nanoengineering of Polymeric Biomedical Devices, The Ohio State University Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

• Medicine

  
  Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program Kevin Nelson^1,2, Christopher Bobba^1,2, Emre Eren³, Tyler Spata⁴, Malak Tadres², Don Hayes, Jr.^5,6, Sylvester M. Black^3,7, Samir Ghadiali*^1,2,3, Bryan A. Whitson*^{2,3,4 1}Department of Biomedical Engineering, Ohio State University Wexner Medical Center, ²Davis Heart & Lung Research Institute, Ohio State University Wexner Medical Center, ³The Collaboration for Organ Perfusion, Protection, Engineering and Regeneration (COPPER) Laboratory, Ohio State University Wexner Medical Center, ⁴Division of Cardiac Surgery, Department of Surgery, Ohio State University Wexner Medical Center, ⁵Departments of Pediatrics and Internal Medicine, Ohio State University, ⁶ Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

• Biology

  
  Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes Shubhra Majumder¹, Harold A. Fisk^{1 1}Department of Molecular Genetics, The Ohio State University Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein’s fluorescence intensity to that of an appropriate internal standard.

• Medicine

  
  Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury Stephana Carelli*¹, Toniella Giallongo*¹, Claudio Gerace¹, Anthea De Angelis¹, Michele D. Basso², Anna Maria Di Giulio¹, Alfredo Gorio^{1 1}Laboratory of Pharmacology, Department of Health Sciences, University of Milan, ²Department of Neuroscience, College of Medicine, The Ohio State University Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

• Biology

  
  An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus Monica F. Poelchau¹, Xin Huang¹, Allison Goff¹, Julie Reynolds², Peter Armbruster^{1 1}Department of Biology, Georgetown University, ²Insect Physiology Lab, EEOB, The Ohio State University RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.

• Biology

  
  Intravenous Injections in Neonatal Mice Sara E. Gombash Lampe¹, Brian K. Kaspar², Kevin D. Foust^{1 1}Department of Neuroscience, Ohio State University, ²Center for Gene Therapy, Nationwide Children's Hospital Research Institute, Ohio State University Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.

• Bioengineering

  
  Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules Michelle R. Emond¹, James D. Jontes^{1 1}Department of Neuroscience, Ohio State University Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

• Medicine

  
  Collection and Extraction of Saliva DNA for Next Generation Sequencing Michael R. Goode¹, Soo Yeon Cheong¹, Ning Li¹, William C. Ray^1,2, Christopher W. Bartlett^{1,3 1} DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

• Biology

  
  Isolation of Murine Valve Endothelial Cells Lindsey J. Miller^1,2, Joy Lincoln^{2,3 1}Molecular, Cellular and Developmental Biology Graduate Program, The Ohio State University, ² The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

• Biology

  
  RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue Ana Clara P. Azevedo-Pouly¹, Ola A. Elgamal¹, Thomas D. Schmittgen^{1 1}Pharmaceutics & Pharmaceutical Chemistry, The Ohio State University We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

• Biology

  
  Tissue Triage and Freezing for Models of Skeletal Muscle Disease Hui Meng¹, Paul M.L. Janssen², Robert W. Grange³, Lin Yang⁴, Alan H. Beggs⁵, Lindsay C. Swanson⁵, Stacy A. Cossette^1,6, Alison Frase⁷, Martin K. Childers⁸, Henk Granzier⁹, Emanuela Gussoni⁵, Michael W. Lawlor^{1 1}Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, ²Department of Physiology and Cell Biology, The Ohio State University, ³Department of Human Nutrition, Foods and Exercise, Virginia Tech, ⁴Division of Biomedical Informatics, Department of Biostatistics, Department of Computer Science, University of Kentucky, ⁵ The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

• Neuroscience

  
  Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay Somaira Nowsheen¹, Fen Xia², Eddy S. Yang^{1,3 1}Department of Radiation Oncology, University of Alabama-Birmingham, ²Department of Radiation Oncology, The Ohio State University Medical School, ³Department of Cell Biology, and Pharmacology and Toxicology, Comprehensive Cancer Center, University of Alabama at Birmingham School of Medicine, University of Alabama-Birmingham The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

• Biology

  
  Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment Zachery Oestreicher¹, Steven K. Lower^1,2, Wei Lin³, Brian H. Lower^{2 1}School of Earth Sciences, The Ohio State University, ²School of Environment & Natural Resources, The Ohio State University, ³Institute of Geology and Geophysics, Chinese Academy of Sciences We demonstrate a method to collect magnetotactic bacteria (MTB) that can be applied to natural waters. MTB can be isolated and enriched from sediment samples using a relatively simple setup that takes advantage of the bacteria's natural magnetism. Isolated MTB can then be examined in detail using both light and electron microscopy.

• Biology

  
  Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles Ki Ho Park¹, Leticia Brotto², Oanh Lehoang¹, Marco Brotto², Jianjie Ma¹, Xiaoli Zhao^{1,3 1}Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, ²Muscle Biology Research Group, University of Missouri-Kansas City, ³Pharmacology division, College of Pharmacy, DHLRI, Ohio State University We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca²⁺ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

• Immunology and Infection

  
  Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model Bryan A. Anthony¹, Gregg A. Hadley^{1 1}Department of Surgery, The Ohio State University Medical Center Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

• Biology

  
  Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps Bhavani Gopalakrishnan¹, Kevin M. Nash¹, Murugesan Velayutham¹, Frederick A. Villamena^{1,2 1}The Davis Heart and Lung Research Institute, The Ohio State University, ²Department of Pharmacology, College of Medicine, The Ohio State University Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

• Medicine

  
  Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats Megan Ryan Detloff^1,2, Lesley C. Fisher¹, Rochelle J. Deibert¹, D. Michele Basso^{1 1}Center for Brain and Spinal Cord Repair, School of Allied Medical Professions, The Ohio State University, ²Spinal Cord Research Center, Drexel University College of Medicine We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

• Neuroscience

  
  Enabling High Grayscale Resolution Displays and Accurate Response Time Measurements on Conventional Computers Xiangrui Li¹, Zhong-Lin Lu^{2,3,4,5 1}Center for Cognitive and Behavioral Brain Imaging, The Ohio State University, ²Department of Psychology, University of Southern California, ³Biomedical Engineering, University of Southern California, ⁴Neuroscience Graduate Program, University of Southern California, ⁵Department of Psychology, The Ohio State University Conventional computer hardware can not generate visual stimuli with sufficiently high grayscale resolution and measure response times with sufficient accuracy. We describe how to use the VideoSwitcher to produce high-resolution monochromatic displays, and the RTbox to measure response times with high accuracy on conventional computer hardware.

• Neuroscience

  
  Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons Hee Joo Choi¹, Christophe P. Ribelayga², Stuart C. Mangel^{1 1}Department of Neuroscience, Ohio State University College of Medicine, ²Department of Ophthalmology and Visual Science, University of Texas Medical School An easy and convenient method to determine the extent of gap junction tracer coupling between retinal neurons is described. This technique enables one to investigate the function of the electrical synapses between neurons in the intact retina under different illumination conditions and at different times of the day and night.

• Medicine

  
  Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens Kaushal Joshi¹, Habibe Demir¹, Ryosuke Yamada¹, Takeshi Miyazaki¹, Abhik Ray-Chaudhury², Ichiro Nakano^{1 1}Department of Neurological Surgery, The Ohio State University Medical Center, ²Department of Pathology, The Ohio State University Medical Center Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed “tumor explant method”. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.

• Immunology and Infection

  
  Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging Jenny M. Tam¹, Carlos E. Castro², Robert J. W. Heath³, Michael K. Mansour¹, Michael L. Cardenas¹, Ramnik J. Xavier³, Matthew J. Lang⁴, Jatin M. Vyas^{1 1}Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Harvard Medical School, ²Department of Mechanical and Aerospace Engineering, The Ohio State University, ³Center for Computational and Integrative Biology, Massachusetts General Hospital, Harvard Medical School, ⁴Dept. of Chemical and Biomolecular Engineering, Vanderbilt University A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

• Medicine

  
  A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation Erik J. Zmuda¹, Catherine A. Powell^1,2, Tsonwin Hai^{1,2,3 1}Molecular and Cellular Biochemistry, Center for Molecular Neurobiology, The Ohio State University, ²Integrated Biomedical Science Graduate Program, The Ohio State University, ³Comprehensive Cancer Center, The Ohio State University Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

• Biology

  
  Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1 Jeffrey Parvin¹, Natsuko Chiba², Derek Ransburgh^{1 1}Department of Biomedical Informatics, The Ohio State University, ²Departments of Molecular Immunology and Clinical Oncology, Tohoku University We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

• Medicine

  
  Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function Ronald X. Xu¹, Kun Huang², Ruogu Qin¹, Jiwei Huang¹, Jeff S. Xu¹, Liya Ding², Urmila S. Gnyawali³, Gayle M. Gordillo³, Surya C. Gnyawali^3,4, Chandan K. Sen^{3 1}Department of Biomedical Engineering, The Ohio State University, ²Department of Biomedical Informatics, The Ohio State University, ³Comprehensive Wound Center, The Ohio State University, ⁴Department of Surgery, The Ohio State University A dual-mode imaging system was developed for non-contact assessment of cutaneous tissue oxygenation and vascular function.

• Medicine

  
  High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart Surya C. Gnyawali^1,2, Sashwati Roy^1,2, Jason Driggs^1,2, Savita Khanna^1,2, Thomas Ryan^2,3, Chandan K. Sen^{2 1}Department of Surgery, The Ohio State University, ²Heart and Lung Research Institute, The Ohio State University, ³Department of Cardiovascular Medicine, The Ohio State University High frequency Doppler ultrasound is a novel technology for assessing regional myocardial function. This work presents first evidence demonstrating applicability of this versatile imaging platform for the repeated measure of myocardial strain, dp/dt, and mitral regurgitation in the ischemia-reperfused (IR) murine heart.

• Biology

  
  Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues Carlos M. Hernandez-Garcia¹, Joseph M. Chiera^1,2, John J. Finer^{1 1}Department of Horticulture and Crop Science, The Ohio State University, ²Department of Plant Pathology, North Carolina State University We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

• Biology

  
  Analysis of Gene Expression in Emerald Ash Borer (Agrilus planipennis) Using Quantitative Real Time-PCR Binny Bhandary¹, Swapna Priya Rajarapu¹, Loren Rivera-Vega¹, Omprakash Mittapalli^{1 1}Department of Entomology, The Ohio State University Quantitative real-time PCR (qRT-PCR) is an effective tool to diagnose mRNA levels in different insect tissues and developmental stages. In this report we show the use of qRT-PCR to ascertain mRNA levels in different larval tissues and developmental stages of the invasive insect species, emerald ash borer.

• Biology

  
  Tomato Analyzer: A Useful Software Application to Collect Accurate and Detailed Morphological and Colorimetric Data from Two-dimensional Objects Gustavo R. Rodríguez¹, Jennifer B. Moyseenko¹, Matthew D. Robbins¹, Nancy Huarachi Morejón¹, David M. Francis¹, Esther van der Knaap^{1 1}Department of Horticulture and Crop Science, The Ohio State University Tomato Analyzer (TA) quantifies attributes of two dimensional shapes and color in a reproducible and accurate manner. A step-by-step procedure for obtaining high quality digitalized images of tomato fruit, morphological and color analyses of these images and several applications using the data generated through this software are described.

• Biology

  
  Murine Renal Transplantation Procedure Jiao-Jing Wang¹, Sara Hockenheimer², Alice A. Bickerstaff¹, Gregg A. Hadley^{1 1}Department of Surgery, The Ohio State University, ²Integrated Biomedical Sciences Graduate Program, The Ohio State University Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.