University of Tennessee

24 articles published in JoVE

• Developmental Biology

  
  Isolation of Preadipocytes from Broiler Chick Embryos Minjeong Kim¹, Usuk Jung¹, Elizabeth Shepherd¹, Robert Mihelic¹, Brynn H. Voy^{1 1}Department of Animal Science, University of Tennessee The present protocol describes a simple method for isolating preadipocytes from adipose tissue in broiler embryos. This method enables isolation with high yield, primary culture, and adipogenic differentiation of preadipocytes. Oil Red O staining and lipid/DNA stain measured the adipogenic ability of isolated cells induced with differentiation media.

• Engineering

  
  Forming Micro-and Nano-Plastics from Agricultural Plastic Films for Employment in Fundamental Research Studies Anton F. Astner¹, Douglas G. Hayes¹, Hugh M. O'Neill², Barbara R. Evans³, Sai Venkatesh Pingali², Volker S. Urban², Timothy M. Young^{4 1}Department of Biosystems Engineering and Soil Science, University of Tennessee, ²Neutron Scattering, Oak Ridge National Laboratory, ³Chemical Sciences Divisions, Oak Ridge National Laboratory, ⁴Center for Renewable Carbon, The University of Tennessee We show the formation and dimensional characterization of micro- and nanoplastics (MPs and NPs, respectively) using a stepwise process of mechanical milling, grinding, and imaging analysis.

• Bioengineering

  
  Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems Jaime Lorenzo N. Dinglasan^1,3, David T. Reeves^2,3, Robert L. Hettich³, Mitchel J. Doktycz^{3 1}Graduate School of Genome Science & Technology, University of Tennessee Knoxville, ²Bredesen Center for Interdisciplinary Research, University of Tennessee Knoxville, ³Biosciences Division, Oak Ridge National Laboratory The protocols describe high-performance liquid chromatography methods coupled to refractive index or mass spectrometric detection for studying metabolic reactions in complex lysate-based cell-free systems.

• Bioengineering

  
  Temperature-Controlled Assembly and Characterization of a Droplet Interface Bilayer Jessie D. Ringley¹, Stephen Andrew Sarles^{1 1}Department of Mechanical, Aerospace and Biomedical Engineering, University of Tennessee This protocol details the use of a feedback temperature-controlled heating system to promote lipid monolayer assembly and droplet interface bilayer formation for lipids with elevated melting temperatures, and capacitance measurements to characterize temperature-driven changes in the membrane.

• Environment

  
  Evaluating the Impact of Hydraulic Fracturing on Streams using Microbial Molecular Signatures Jeremy R. Chen See^1,2, Olivia Wright¹, Lavinia V. Unverdorben^1,2, Nathan Heibeck¹, Stephen M. Techtmann³, Terry C. Hazen^4,5, Regina Lamendella^{1,2 1}Department of Biology, Juniata College, ²Wright Labs, LLC, ³Department of Biological Sciences, Michigan Technological University, ⁴Biosciences Division, Oak Ridge National Laboratory, ⁵Department of Civil and Environmental Engineering, University of Tennessee Here, we present a protocol to investigate the impacts of hydraulic fracturing on nearby streams by analyzing their water and sediment microbial communities.

• Neuroscience

  
  Computer-based Multitaper Spectrogram Program for Electroencephalographic Data Christopher B. O'Brien¹, Helen A. Baghdoyan^1,2,3, Ralph Lydic^{1,2,3 1}Department of Psychology, University of Tennessee, ²Department of Anesthesiology, University of Tennessee, ³Oak Ridge National Laboratory This protocol provides an open source, compiled MATLAB program that generates multitaper spectrograms for electroencephalographic data.

• Environment

  
  Modeling the Size Spectrum for Macroinvertebrates and Fishes in Stream Ecosystems Daniel J. McGarvey¹, Taylor E. Woods^1,2, Andrew J. Kirk^{3 1}Center for Environmental Studies, Virginia Commonwealth University, ²Department of Ecology and Evolutionary Biology, University of Tennessee, ³Department of Environmental Quality This is a protocol to model the size spectrum (scaling relationship between individual mass and population density) for combined fish and invertebrate data from wadable streams and rivers. Methods include: field techniques to collect quantitative fish and invertebrate samples; lab methods to standardize the field data; and statistical data analysis.

• Bioengineering

  
  Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes Joseph S. Najem^1,2, Graham J. Taylor^2,3, Nick Armendarez⁴, Ryan J. Weiss⁵, Md Sakib Hasan⁵, Garrett S. Rose⁵, Catherine D. Schuman⁶, Alex Belianinov⁷, Stephen A. Sarles², C. Patrick Collier^{2,3,7 1}Joint Institute for Biological Sciences, Oak Ridge National Laboratory, ²Department of Mechanical, Aerospace and Biomedical Engineering, University of Tennessee, ³Bredesen Center for Interdisciplinary Research, University of Tennessee, ⁴Department of Biosystems and Agriculture Engineering, University of Kentucky, ⁵Department of Electrical Engineering and Computer Science, University of Tennessee, ⁶Computer Science and Mathematics Division, Oak Ridge National Laboratory, ⁷Center for Nanophase Materials Sciences, Oak Ridge National Laboratory Soft, low-power, biomolecular memristors leverage similar composition, structure, and switching mechanisms of bio-synapses. Presented here is a protocol to assemble and characterize biomolecular memristors obtained from insulating lipid bilayers formed between water droplets in oil. The incorporation of voltage-activated alamethicin peptides results in memristive ionic conductance across the membrane.

• Chemistry

  
  Monitoring the Effects of Illumination on the Structure of Conjugated Polymer Gels Using Neutron Scattering Brian Morgan¹, Samantha J. Rinehart¹, Mark D. Dadmun^{1,2 1}Department of Chemistry, University of Tennessee, Knoxville, ²Chemical Sciences Division, Oak Ridge National Laboratory A protocol for the analysis of gels formed from the optoelectronic conjugated polymer poly(3-hexylthiophene-2,5-diyl) (P3HT) using small and ultra-small angle neutron scattering in both the presence and absence of illumination is presented.

• Bioengineering

  
  Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform Jayde A. Aufrecht^1,2, Jennifer M. Ryan³, Sahar Hasim⁴, David P. Allison^2,3, Andreas Nebenführ³, Mitchel J. Doktycz^1,2, Scott T. Retterer^{1,2 1}Bredesen Center for Interdisciplinary Research and Graduate Education, University of Tennessee, ²Bioscience Division and Center for Nanophase Materials Sciences, Oak Ridge national Laboratory, ³Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, ⁴Department of Microbiology, University of Tennessee This article demonstrates how to culture Arabidopsis thaliana seedlings in a two-layer microfluidic platform that confines the main root and root hairs to a single optical plane. This platform can be used for real-time optical imaging of fine root morphology as well as for high-resolution imaging by other means.

• Bioengineering

  
  Assembly and Tracking of Microbial Community Development within a Microwell Array Platform Andrea C. Timm¹, Michelle C. Halsted², Jared L. Wilmoth¹, Scott T. Retterer^{1,3 1}Biosciences Division, Oak Ridge National Laboratory, ²Bredesen Center for Interdisciplinary Research and Graduate Education, University of Tennessee, ³Center for Nanophase Materials Sciences, Oak Ridge National Laboratory The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

• Biology

  
  Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast Bin Wei¹, Brian S. Hercyk¹, Julius Habiyaremye¹, Maitreyi Das^{1 1}Department of Biochemistry & Cellular and Molecular Biology, University of Tennessee The fission yeast, Schizosaccharomyces pombe is an excellent model system to study cytokinesis, the final stage in cell division. Here we describe a microscopy approach to analyze different cytokinetic events in live fission yeast cells.

• Genetics

  
  A Robotic Platform for High-throughput Protoplast Isolation and Transformation Elizabeth M. Dlugosz¹, Scott C. Lenaghan^2,3, C. Neal Stewart, Jr.^{1 1}Department of Plant Sciences, University of Tennessee, Knoxville, ²Center for Renewable Carbon, University of Tennessee, Knoxville, ³Department of Mechanical, Aerospace, and Biomedical Engineering, University of Tennessee, Knoxville A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

• Chemistry

  
  Sulfate Separation by Selective Crystallization with a Bis-iminoguanidinium Ligand Charles A. Seipp^1,2, Neil J. Williams^1,3, Radu Custelcean^{1 1}Chemical Sciences Division, Oak Ridge National Laboratory, ²Department of Chemistry, The University of Texas at Austin, ³Department of Chemistry, The University of Tennessee A protocol for in situ aqueous synthesis of a bis(iminoguanidinium) ligand and its utilization in selective separation of sulfate is presented.

• Engineering

  
  Production of Synthetic Nuclear Melt Glass Joshua J. Molgaard¹, John D. Auxier II^2,3, Andrew V. Giminaro^2,3, Colton J. Oldham², Jonathan Gill², Howard L. Hall^{2,3,4 1}Department of Physics and Nuclear Engineering, United States Military Academy, ²Department of Nuclear Engineering, University of Tennessee, ³Radiochemistry Center of Excellence (RCoE), University of Tennessee, ⁴Institute for Nuclear Security, University of Tennessee A protocol for the production of synthetic nuclear melt glass, similar to trinitite, is presented.

• Bioengineering

  
  Sealable Femtoliter Chamber Arrays for Cell-free Biology Sarah Elizabeth Norred^1,2, Patrick M. Caveney^1,2, Scott T. Retterer^1,2, Jonathan B. Boreyko^1,2, Jason D. Fowlkes^2,3, Charles Patrick Collier², Michael L. Simpson^{1,2,3 1}Bredesen Center, University of Tennessee, Knoxville, ²Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, ³Department of Materials Science and Engineering, University of Tennessee, Knoxville A microfabricated device with sealable femtoliter-volume reaction chambers is described. This report includes a protocol for sealing cell-free protein synthesis reactants inside these chambers for the purpose of understanding the role of crowding and confinement in gene expression.

• Biology

  
  Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy Krzysztof Bobik¹, John R. Dunlap², Tessa M. Burch-Smith^{1 1}Department of Biochemical, Cellular and Molecular Biology, University of Tennessee, Knoxville, ²Advanced Microscopy and Imaging Facility, University of Tennessee, Knoxville Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.

• Bioengineering

  
  Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas Jaana Mannik*¹, Alex Meyers*², Paul Dalhaimer^{1,2 1}Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, ²Department of Chemical and Biomolecular Engineering, University of Tennessee Two techniques for isolating cellular lipid droplets from 1) yeast cells and 2) human placentas are presented. The centerpiece of both procedures is density gradient centrifugation, where the resulting floating layer containing the droplets can be readily visualized by eye, extracted, and quantified by Western Blot analysis for purity.

• Biology

  
  Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity Tingting Xu¹, Dan M. Close², James D. Webb³, Steven A. Ripp^2,3, Gary S. Sayler^{1,2,3 1}The Joint Institute for Biological Sciences, Oak Ridge National Laboratory, ²490 BioTech, Inc., ³The Center for Environmental Biotechnology, The University of Tennessee, Knoxville Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

• Engineering

  
  Fabrication of Spatially Confined Complex Oxides Hangwen Guo^1,2, Thomas Z. Ward^{1 1}Materials Science and Technology Division, Oak Ridge National Laboratory, ²Department of Physics & Astronomy, University of Tennessee, Knoxville We describe the use of pulsed laser deposition (PLD), photolithography and wire-bonding techniques to create micrometer scale complex oxides devices. The PLD is utilized to grow epitaxial thin films. Photolithography and wire-bonding techniques are introduced to create practical devices for measurement purposes.

• Neuroscience

  
  Profiling Voltage-gated Potassium Channel mRNA Expression in Nigral Neurons using Single-cell RT-PCR Techniques Shengyuan Ding¹, Fu- Ming Zhou^{1 1}Department of Pharmacology, University of Tennessee College of Medicine Neurons are first characterized electrophysiologically. Then the cytoplasm from the recorded neuron is aspirated and subjected to reverse transcription-PCR analysis to detect the expression of mRNAs for neurotransmitter synthesis enzymes, ion channels, and receptors.

• Biology

  
  Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity Michael R. Duff, Jr.¹, Jordan Grubbs¹, Elizabeth E. Howell^{1 1}Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee A general protocol for the use of isothermal titration calorimetry to monitor the binding thermodynamics for biological systems with moderate binding affinities is presented.

• Bioengineering

  
  Bacterial Immobilization for Imaging by Atomic Force Microscopy David P. Allison^1,2, Claretta J. Sullivan³, Ninell Pollas Mortensen^1,2, Scott T. Retterer^1,4, Mitchel Doktycz^{1,4 1}Biological and Nanoscale Systems Group, Biosciences Division, Oak Ridge National Laboratory, ²Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, ³Department of Surgery, Eastern Virginia Medical School, ⁴Center for Nanophase Materials Sciences Division, Oak Ridge National Laboratory Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

• Immunology and Infection

  
  Estimating Virus Production Rates in Aquatic Systems Audrey R. Matteson¹, Charles R. Budinoff¹, Claire E. Campbell¹, Alison Buchan¹, Steven W. Wilhelm^{1 1}Department of Microbiology, University of Tennessee The turnover rate of viruses in marine and freshwater systems can be estimated by a reduction and reoccurrence technique. The data allow researchers to infer rates of virus-mediated microbial mortality in aquatic systems.