University of Maryland View Institution's Website 53 articles published in JoVE Developmental Biology Monitoring the Mechanical Evolution of Tissue During Neural Tube Closure of Chick Embryo Chenjun Shi1, Chenchen Handler2, Haden Florn1, Jitao Zhang1 1Department of Biomedical Engineering, College of Engineering, Wayne State University, 2Fischell Department of Bioengineering, University of Maryland This protocol was developed to longitudinally monitor the mechanical properties of neural plate tissue during chick embryo neurulation. It is based on the integration of a Brillouin microscope and an on-stage incubation system, enabling live mechanical imaging of neural plate tissue in ex ovo cultured chick embryos. Bioengineering Mechanical Dissociation of Tissues for Single Cell Analysis Using a Motorized Device Mayowa Amosu1, Andrew J. Gregory2, John D. Murtagh2, Nitay Pavin2, Carson Taylor Meyers2, Juan Grano de Oro Fernandez1, Kaitlyn Moore1, Katharina Maisel1 1Fischell Department of Bioengineering, University of Maryland, 2UMD Terrapin Works, University of Maryland A general protocol for the combined enzymatic and semi-automated mechanical dissociation of tissues to generate single-cell suspensions for downstream analyses, such as flow cytometry, is provided. Instructions for the fabrication, assembly, and operation of the low-cost mechanical device developed for this protocol are included. Chemistry A Synthetic Methodology for Preparing Impregnated and Grafted Amine-Based Silica Composites for Carbon Capture Charlotte M. Wentz1,2, Zois Tsinas1,3, Amanda L. Forster1 1Material Measurement Laboratory, National Institute of Standards and Technology (NIST), 2University of Maryland, 3Theiss Research This work aims to facilitate the development of standardized techniques for impregnating or grafting aminated compounds onto silica substrates, which are often broadly described in the literature. Specific amounts of solvent, substrate, amines, and the values of other important experimental parameters will be discussed in detail. Immunology and Infection Isolation and Culture of Bone Marrow-Derived Macrophages from Mice Ricardo Gonçalves1, Gabriela Kaliff Teófilo Murta1, Izabela Aparecida de Souza1, David M. Mosser2 1Department of General Pathology, Institute of Biological Sciences, Federal University of Minas Gerais, 2Department of Cell Biology and Molecular Genetics, University of Maryland The present protocol describes the isolation and culture of bone marrow-derived macrophages from mice. Developmental Biology AMEBaS: Automatic Midline Extraction and Background Subtraction of Ratiometric Fluorescence Time-Lapses of Polarized Single Cells Rafael Badain1, Daniel S. C. Damineli1,2, Maria Teresa Portes3, José Feijó4, Stefano Buratti5, Giorgia Tortora6, Hugo Neves de Oliveira1, Roberto M. Cesar Jr1 1Institute of Mathematics and Statistics, University of São Paulo, 2Center for Mathematics, Computing and Cognition, Federal University of ABC, 3Department of Botany, Institute of Biosciences, University of São Paulo, 4Cell Biology and Molecular Genetics Department, University of Maryland, 5Department of Biosciences, University of Milan, 6Department of Physics, Politecnico di Milano Current methods for analyzing the intracellular dynamics of polarized single cells are often manual and lack standardization. This manuscript introduces a novel image analysis pipeline for automating midline extraction of single polarized cells and quantifying spatiotemporal behavior from time lapses in a user-friendly online interface. Biology Quantifying the Antifungal Activity of Peptides Against Candida albicans Wright K. Makambi1, Svetlana P. Ikonomova1, Amy J. Karlsson1 1Department of Chemical and Biomolecular Engineering, University of Maryland This protocol describes a method for obtaining quantitative data on the antifungal activity of peptides and other compounds, such as small-molecule antifungal agents, against Candida albicans. Its use of optical density rather than counting colony-forming units to quantify growth inhibition saves time and resources. Immunology and Infection Mouse Heterotopic Cervical Cardiac Transplantation Utilizing Vascular Cuffs Wenjun Li1, Hailey M. Shepherd1, Alexander S. Krupnick2, Andrew E. Gelman1,3, Kory J. Lavine4, Daniel Kreisel1,3 1Department of Surgery, Washington University School of Medicine, 2Department of Surgery, The University of Maryland, 3Department of Pathology & Immunology, Washington University School of Medicine, 4Department of Medicine, Washington University School of Medicine Mouse cardiac transplantation models represent valuable research tools for studying transplantation immunology. The present protocol details mouse heterotopic cervical cardiac transplantation that involves the placement of cuffs on the recipient's common carotid artery and the donor's pulmonary artery trunk to allow for laminar blood flow. Chemistry Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo Aparna B. Baxi1,2, Leena R. Pade1, Peter Nemes1,2 1Department of Chemistry & Biochemistry, University of Maryland, 2Department of Anatomy & Cell Biology, The George Washington University Here we describe a mass spectrometry-based proteomic characterization of cell lineages with known tissue fates in the vertebrate Xenopus laevis embryo. Chemistry Comparison of Two Different Synthesis Methods of Single Crystals of Superconducting Uranium Ditelluride Sheng Ran1,2,3, I-Lin Liu1,2, Shanta R. Saha1,2, Prathum Saraf1, Johnpierre Paglione1,2, Nicholas P. Butch1,2 1Maryland Quantum Materials Center, Department of Physics, University of Maryland, 2National Institute of Standards and Technology, 3Department of Physics, Washington University in St. Louis Here, we present a protocol to synthesize two types of UTe2 crystals: those exhibiting robust superconductivity, via chemical vapor transport synthesis, and those lacking superconductivity, via molten metal flux synthesis. Biology An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants Ying Wu*1, Darwin Diaz*1, Jian Yin1, David Bloodgood1, William Sexton1, Cheng-I Wei2, Shunyuan Xiao1,3 1Institute for Bioscience and Biotechnology Research, University of Maryland, 2Department of Nutrition and Food Science, University of Maryland College Park, 3Department of Plant Sciences and Landscape Architecture, University of Maryland College Park We present a protocol for constructing a simple spore-distribution system consisting of an inoculation box with a ~50 µm mesh and a transparent plastic chamber. This can be used to evenly inoculate plants with powdery mildew spores, thereby enabling accurate and reproducible assessment of disease phenotypes of plants under study. Developmental Biology Incremental Temperature Changes for Maximal Breeding and Spawning in Astyanax mexicanus Li Ma1, Ruby Dessiatoun1, Janet Shi1, William R. Jeffery1 1Department of Biology, University of Maryland This article outlines the basic laboratory conditions and protocols for an incremental temperature regime to stimulate maximal spawning in the Mexican tetra Astyanax mexicanus, which is an emerging model for developmental and evolutionary studies. Bioengineering Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks Swathi Swaminathan1, Alisa Morss Clyne2 1Department of Biology, Drexel University, 2Fischell Department of Bioengineering, University of Maryland The goal of this protocol is to directly bioprint breast epithelial cells as multicellular spheroids onto pre-formed endothelial networks to rapidly create 3D breast-endothelial co-culture models which can be used for drug screening studies. Biology Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9 Megan E. Meuti1, Robert Harrell2 1Department of Entomology, The Ohio State University, 2Insect Transformation Facility, Institute for Bioscience and Biotechnology Research, University of Maryland CRISPR/Cas9 is increasingly used to characterize gene function in non-model organisms. This protocol describes how to generate knock-out lines of Culex pipiens, from preparing injection mixes, to obtaining and injecting mosquito embryos, as well as how to rear, cross, and screen injected mosquitoes and their progeny for desired mutations. Biology Circadian Entrainment of Drosophila Melanogaster Austin O. Dada1, Minh Q. Nguyen1, Shea M. Peterson1, Vy T. Ngo1, Dayanne V. Cornelio-Parra1, Bwaar S. Omer1, Ada Thapa2, Sarah R. Rapp1, Veronica J. Cloud1, Ryan D. Mohan1 1School of Biological and Chemical Sciences, University of Missouri - Kansas City, 2School of Public Health, University of Maryland Here, we detail how to synchronize Drosophila to a circadian day. This is the first, and most important step necessary for studying biological rhythms and chronobiology. Biochemistry In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans Jessica A. Espino1, Lisa M. Jones1 1Department of Pharmaceutical Sciences, University of Maryland In vivo fast photochemical oxidation of proteins (IV-FPOP) is a hydroxyl radical protein footprinting technique that allows for mapping of protein structure in their native environment. This protocol describes the assembly and set-up of the IV-FPOP microfluidic flow system. Immunology and Infection Chronic, Acute, and Reactivated HIV Infection in Humanized Immunodeficient Mouse Models Federico Perdomo-Celis1,2, Sandra Medina-Moreno1, Alonso Heredia1, Harry Davis1, Joseph Bryant1, Juan Carlos Zapata1 1Institute of Human Virology, School of Medicine, University of Maryland, 2Grupo Inmunovirología, Facultad de Medicina, Universidad de Antioquia Described here are three experimental approaches for studying the dynamics of HIV infection in humanized mice. The first permits the study of chronic infection events, whereas the two latter allows for the study of acute events after primary infection or viral reactivation. Neuroscience Measuring Neural Mechanisms Underlying Sleep-Dependent Memory Consolidation During Naps in Early Childhood Tamara Allard*1, Tracy Riggins*1, Arcadia Ewell1, Benjamin Weinberg1, Sanna Lokhandwala2, Rebecca M. C. Spencer2,3 1Department of Psychology, University of Maryland, 2Department of Psychological and Brain Sciences, University of Massachusetts, 3Neuroscience and Behavior, University of Massachusetts This protocol describes methods used to examine neural mechanisms underlying sleep-dependent memory consolidation during naps in early childhood. It includes procedures for examining the effect of sleep on behavioral memory performance, as well as the application and recording of both polysomnography and actigraphy. Immunology and Infection Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining Liang-Chun Wang1, Jacob Wagner2, Annabelle Capino2, Elizabeth Nesbit2, Wenxia Song2, Daniel C. Stein2 1Department of Marine Biotechnology and Resources, National Sun Yat-Sen University, 2Department of Cell Biology and Molecular Genetics, University of Maryland A simple ATP-measuring assay and live/dead staining method were used to quantify and visualize Neisseria gonorrhoeae survival after treatment with ceftriaxone. This protocol can be extended to examine the antimicrobial effects of any antibiotic and can be used to define the minimal inhibitory concentration of antibiotics in bacterial biofilms. Medicine Non-invasive Assessment of Dorsiflexor Muscle Function in Mice Frederico Gerlinger-Romero1, Alex B. Addinsall2, Richard M. Lovering3, Victoria C. Foletta4, Chris van der Poel5, Paul A. Della-Gatta4, Aaron P. Russell4 1School of Exercise and Nutrition Sciences, Deakin University, 2Centre for Molecular and Medical Research, School of Medicine, Deakin University, 3Department of Orthopaedics, School of Medicine, University of Maryland, 4Institute for Physical Activity and Nutrition (IPAN), School of Exercise and Nutrition Sciences, Deakin University, 5Department of Physiology, Anatomy and Microbiology, La Trobe University Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse. Biochemistry A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation Javier A. Jaimes*1,2, Jean K. Millet*1,3, Monty E. Goldstein1,4, Gary R. Whittaker1, Marco R. Straus1 1Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, 2Department of Microbiology, College of Agricultural and Life Sciences, Cornell University, 3Virologie et Immunologie Moléculaires, Domaine de Vilvert, INRA, 4Department of Cell Biology and Molecular Genetics, University of Maryland We present a fluorogenic peptide cleavage assay that allows a rapid screening of the proteolytic activity of proteases on peptides representing the cleavage site of viral fusion peptides. This method can also be used on any other amino acid motif within a protein sequence to test for the protease activity. Chemistry Fabrication and Testing of Photonic Thermometers Nikolai N. Klimov1,2, Zeeshan Ahmed2 1Joint Quantum Institute, University of Maryland, 2Physical Measurement Laboratory, National Institute of Standards and Technology We describe the process of fabrication and testing of photonic thermometers. Environment Use of Principal Components for Scaling Up Topographic Models to Map Soil Redistribution and Soil Organic Carbon Xia Li1,2, Greg W. McCarty2 1Department of Geographical Sciences, University of Maryland, 2Hydrology & Remote Sensing Laboratory, Agricultural Research Service, United States Department of Agriculture Landscape processes are critical components of soil formation and play important roles in determining soil properties and spatial structure in landscapes. We propose a new approach using stepwise principal component regression to predict soil redistribution and soil organic carbon across various spatial scales. Bioengineering Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher Joshua N. Webb1, Caroline Dong1, Andres Bernal2, Giuliano Scarcelli1 1Fischell Department of Bioengineering, University of Maryland, 2Bioniko Consulting LLC We present an efficient method of studying lens accommodation by using a manual lens stretcher. The protocol mimics physiological accommodation by pulling the zonules connected around the lens capsule, thereby, stretching the lens. Neuroscience Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels Xiaonan Zhang1, Quentin Gaudry1 1Department of Biology, University of Maryland This article features a method to test the monosynaptic connections between neurons by employing tetrodotoxin and the tetrodotoxin-resistant sodium channel, NaChBac. Engineering Dielectric RheoSANS — Simultaneous Interrogation of Impedance, Rheology and Small Angle Neutron Scattering of Complex Fluids Jeffrey J. Richards1, Cedric V. L. Gagnon2, Jeffery R. Krzywon1, Norman J. Wagner3, Paul D. Butler1 1NIST Center for Neutron Research, National Institute of Standards and Technology, 2Department of Materials Science and Engineering, University of Maryland, 3Center for Neutron Science, Department of Chemical and Biomolecular Engineering, University of Delaware Here, we present a procedure for the measurement of simultaneous impedance, rheology and neutron scattering from soft matter materials under shear flow. Genetics Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus Jie Xiang1,2, Katie Reding1, Leslie Pick1,2 1Entomology Department, University of Maryland, 2Program in Molecular and Cell Biology, University of Maryland Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species. Biochemistry Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments Parisa Moghaddam-Taaheri1, Svetlana P. Ikonomova2, Zifan Gong2, Janna Q. Wisniewski1, Amy J. Karlsson1,2 1Fischell Department of Bioengineering, University of Maryland, 2Department of Chemical and Biomolecular Engineering, University of Maryland We provide a method to simultaneously screen a library of antibody fragments for binding affinity and cytoplasmic solubility by using the Escherichia coli twin-arginine translocation pathway, which has an inherent quality control mechanism for intracellular protein folding, to display the antibody fragments on the inner membrane. Bioengineering Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations Poonam Sharma1, Julianne D. Twomey1, Michelle Patkin1, Adam H. Hsieh1 1Fischell Department of Bioengineering, University of Maryland Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct. Biology Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases (TALENs) Johanna E. Kowalko1, Li Ma2, William R. Jeffery3 1Genetics, Development and Cell Biology, Iowa State University, 2Department of Biological Sciences, University of Cincinnati, 3Department of Biology, University of Maryland Gene-targeting mutagenesis is now possible in a wide range of organisms using genome editing techniques. Here, we demonstrate a protocol for targeted gene mutagenesis using transcription activator like effector nucleases (TALENs) in Astyanax mexicanus, a species of fish that includes surface fish and cavefish. Engineering Experimental Methodology for Estimation of Local Heat Fluxes and Burning Rates in Steady Laminar Boundary Layer Diffusion Flames Ajay V. Singh1, Michael J. Gollner1 1Department of Fire Protection Engineering, University of Maryland We describe the use of micro-thermocouples to estimate local temperature gradients in steady laminar boundary layer diffusion flames. By extension of the Reynolds Analogy, local temperature gradients can be further used to estimate the local mass burning rates and heat fluxes in such flames with high accuracy. Neuroscience Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice Yaneri A. Ayala1, David Pérez-González1, Daniel Duque1,2, Alan R. Palmer3, Manuel S. Malmierca1,4 1Auditory Neuroscience Laboratory, Institute of Neuroscience of Castilla y León, University of Salamanca, 2Neural Systems Laboratory, Institute for Systems Research, University of Maryland, 3Medical Research Council Institute of Hearing Research, 4Department of Cell Biology and Pathology, Faculty of Medicine, University of Salamanca We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest. Bioengineering High Speed Sub-GHz Spectrometer for Brillouin Scattering Analysis Kim V. Berghaus1, Seok H. Yun2,3, Giuliano Scarcelli1 1Fischell Department of Bioengineering, University of Maryland, 2Wellman Center for Photomedicine, Harvard Medical School, Massachusetts General Hospital, 3The Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology Here we present a protocol to build a rapid Brillouin spectrometer. Cascading virtually imaged phase array (VIPA) etalons achieve a measurement speed more than 1,000 times faster than traditional scanning Fabry-Perot spectrometers. This improvement provides the means for Brillouin analysis of tissue and biomaterials at low power levels in vivo. Biology Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers Joseph S. Najem1, Myles D. Dunlap2, Anthony Yasmann3, Eric C. Freeman4, John W. Grant5, Sergei Sukharev3, Donald J. Leo4 1Department of Mechanical Engineering, Virginia Polytechnic Institute and State University, 2School of Biomedical Engineering and Sciences, Virginia Polytechnic Institute and State University, 3Department of Biology, University of Maryland, 4College of Engineering, University of Georgia, 5Department of Engineering Sciences and Mechanics, Virginia Polytechnic Institute and State University Bacterial mechanosensitive channels can be used as mechanoelectrical transducers in biomolecular devices. Droplet interface bilayers (DIBs), cell-inspired building blocks to such devices, represent new platforms to incorporate and stimulate mechanosensitive channels. Here, we demonstrate a new micropipette-based method of forming DIBs, allowing the study of mechanosensitive channels under mechanical stimulation. Bioengineering Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications Jennifer M. Vojtech1,2, Juliana Cano-Mejia1,2, Matthieu F. Dumont1, Raymond W. Sze1,3, Rohan Fernandes1,3,4 1 This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting. Bioengineering A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis Hadar Ben-Yoav1, Peter H. Dykstra1, Tanya Gordonov2, William E. Bentley2, Reza Ghodssi1 1MEMS Sensors and Actuators Laboratory (MSAL), Department of Electrical and Computer Engineering, Institute for Systems Research, University of Maryland, 2Institute for Bioscience and Biotechnology Research, Fischell Department of Bioengineering, University of Maryland We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM. Biology Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment Tamika K. Samuel1, Jason W. Sinclair1, Katherine L. Pinter1, Iqbal Hamza1,2 1Department of Animal and Avian Sciences, University of Maryland, 2Department of Cell Biology and Molecular Genetics, University of Maryland C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications. Biology Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model Pablo Bermejo-Alvarez1,2, Ki-Eun Park1,2, Bhanu P. Telugu1,2 1Animal Bioscience and Biotechnology Laboratory, United States Department of Agriculture, 2Department of Animal and Avian Sciences, University of Maryland Utero-tubal embryo transfer uses the utero-tubal junction as a barrier to prevent the embryo outflow that may occur when performing uterine transfer. Vasectomized males are required to obtain pseudopregnant recipients for embryo transfer. Both techniques are discussed. Engineering Fabrication of Uniform Nanoscale Cavities via Silicon Direct Wafer Bonding Stephen R. D. Thomson1, Justin K. Perron2,3, Mark O. Kimball4, Sarabjit Mehta5, Francis M. Gasparini1 1Department of Physics, The State University of New York at Buffalo, 2Joint Quantum Institute, University of Maryland, 3The National Institute of Standards and Technology, 4Cryogenics and Fluids Branch, NASA Goddard Space Flight Center, 5HRL Laboratories A method for permanently bonding two silicon wafers so as to realize a uniform enclosure is described. This includes wafer preparation, cleaning, RT bonding, and annealing processes. The resulting bonded wafers (cells) have uniformity of enclosure ~1%1,2. The resulting geometry allows for measurements of confined liquids and gasses. Bioengineering Intra-lymph Node Injection of Biodegradable Polymer Particles James I. Andorko*1, Lisa H. Tostanoski*1, Eduardo Solano1, Maryam Mukhamedova1, Christopher M. Jewell1 1Fischell Department of Bioengineering, University of Maryland, College Park Lymph nodes are the immunological tissues that orchestrate immune response and are a critical target for vaccines. Biomaterials have been employed to better target lymph nodes and to control delivery of antigens or adjuvants. This paper describes a technique combining these ideas to inject biocompatible polymer particles into lymph nodes. Bioengineering Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers Rasa Ghaffarian1, Silvia Muro1,2 1Fischell Department of Bioengineering, University of Maryland, 2Institute for Bioscience and Biotechnology Research, University of Maryland Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium. Biology Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds Christina Tam1, Andrew R. Flannery1, Norma Andrews1 1Department of Cell Biology and Molecular Genetics, University of Maryland Live imaging of cells exposed to the lipophilic dye FM1-43 allows precise determination of the kinetics by which pore-forming toxins are removed from the plasma membrane. This is a sensitive assay that can be used to assess requirements for Ca2+, sphingomyelinase and other factors on plasma membrane repair. Immunology and Infection Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells James E. East*1, Wenji Sun*1, Tonya J. Webb1 1Department of Microbiology and Immunology, University of Maryland Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells. Immunology and Infection A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes Ryan D. Heselpoth1, Daniel C. Nelson1 1Institute for Bioscience and Biotechnology Research, University of Maryland A novel directed evolution method specific to the field of thermostability engineering was developed and consequently validated for bacteriolytic enzymes. After only one round of random mutagenesis, an evolved bacteriolytic enzyme, PlyC 29C3, displayed greater than twice the residual activity when compared to the wild-type protein after elevated temperature incubation. Neuroscience Derivation of Glial Restricted Precursors from E13 mice André W. Phillips1,2, Sina Falahati1,2, Roshi DeSilva1,3, Irina Shats2, Joel Marx1, Edwin Arauz1, Douglas A. Kerr4, Jeffrey D. Rothstein2,5, Michael V. Johnston1,2,6, Ali Fatemi1,2,6 1Hugo W. Moser Research Institute at Kennedy Krieger, Johns Hopkins University, 2Department of Neurology, Johns Hopkins School of Medicine, 3University of Maryland, 4Experimental Neurology, Biogen Idec, 5The Brain Science Institute, Johns Hopkins School of Medicine, 6Department of Pediatrics, Johns Hopkins School of Medicine This protocol outlines the derivation of Glial Restricted Precursors from fetal spinal cords and maintained in vitro either for transplantation or for the study of oligodendrocytic lineage. Bioengineering Bridging the Bio-Electronic Interface with Biofabrication Tanya Gordonov*1, Benjamin Liba*2, Jessica L. Terrell*1, Yi Cheng3, Xiaolong Luo2, Gregory F. Payne1, William E. Bentley1 1Fischell Department of Bioengineering, University of Maryland, 2Institute for Bioscience and Biotechnology Research, University of Maryland, 3Department of Materials Science and Engineering, University of Maryland This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications. Neuroscience The Mouse Forced Swim Test Adem Can1, David T. Dao2, Michal Arad1, Chantelle E. Terrillion3, Sean C. Piantadosi1, Todd D. Gould1,3,4 1Department of Psychiatry, University of Maryland School of Medicine, 2Tulane University School of Medicine, 3Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, 4The Program in Neuroscience, University of Maryland The forced swim test is validated as an experimental approach to assess potential antidepressant efficacy in rodents. Experimental animals are placed in a tank of water and escape-related mobility behavior is quantified. The common procedures for the mouse version of this test are described. Neuroscience The Tail Suspension Test Adem Can*1, David T. Dao*1,2, Chantelle E. Terrillion3, Sean C. Piantadosi1, Shambhu Bhat1, Todd D. Gould1,3,4 1Department of Psychiatry, University of Maryland School of Medicine, 2Tulane University School of Medicine, 3The Program in Neuroscience, University of Maryland, 4Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine The tail-suspension test is validated as an experimental procedure to assess antidepressant efficacy of drug treatments in mice. Mice are suspended by their tails for six minutes and escape-related behaviors are assessed. We describe procedures used in conducting the tail suspension test. Biology Chromatographic Purification of Highly Active Yeast Ribosomes Arturas Meskauskas1,2, Jonathan A. Leshin1, Jonathan D. Dinman1 1Department of Cell Biology and Molecular Genetics, University of Maryland, 2Department of Biotechnology and Microbiology, Vilnius University Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system. Neuroscience Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement Dean Dessem1 1Department of Neural and Pain Sciences, University of Maryland A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry. Immunology and Infection Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut Toru Kariu1, Adam S. Coleman1, John F. Anderson2, Utpal Pal1 1Department of Veterinary Medicine, University of Maryland, 2Department of Entomology, Connecticut Agricultural Experiment Station Lyme disease research studies often require generation of ticks infected with the pathogen Borrelia burgdorferi, a process that typically takes several weeks. Here we demonstrate a microinjection-based tick infection procedure that can be accomplished within hours. We also demonstrate an immunofluorescence method for in situ localization of B. burgdorferi within ticks. Biology Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun Courtney A. Hollender1, Zhongchi Liu1 1Dept. Of Cell Biology and Molecular Genetics, University of Maryland This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC) Biology Recordings of Neural Circuit Activation in Freely Behaving Animals Jens Herberholz1 1Department of Psychology, Neuroscience and Cognitive Science Program , University of Maryland Non-invasive measurements of neural activity patterns in freely behaving animals are obtained by combining neurophysiological recordings with high speed videography. Biology Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio) Jin Liang1,2, Shawn M. Burgess1 1Genome Technology Branch, National Human Genome Research Institute, 2Neuroscience and Cognitive Science Program, University of Maryland The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.
