JoVE Logo

Sign In

University of Maryland

74 ARTICLES PUBLISHED IN JoVE

image

Biology

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)
Jin Liang 1,2, Shawn M. Burgess 1
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.

image

Biology

Recordings of Neural Circuit Activation in Freely Behaving Animals
Jens Herberholz 1
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.

image

Biology

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::β-glucuronidase Reporter Gene Expression
Chloe Mara 1, Boyana Grigorova 1, Zhongchi Liu 1
1Department of Cell Biology and Molecular Genetics, University of Maryland College Park

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::β-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

image

Biology

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun
Courtney A. Hollender 1, Zhongchi Liu 1
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)

image

Biology

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry
Peter Nemes 1, Akos Vertes 1
1Department of Chemistry, George Washington University

Laser ablation electrospray ionization (LAESI) is an atmospheric-pressure ion source for mass spectrometry. In the imaging mode, a mid-infrared laser probes the distributions of molecules across a tissue section or a biofilm. This technique presents a new approach for diverse bioanalytical studies carried out under native experimental conditions.

image

Immunology and Infection

Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut
Toru Kariu 1, Adam S. Coleman 1, John F. Anderson 2, Utpal Pal 1
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.

image

Neuroscience

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement
Dean Dessem 1
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.

image

Medicine

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery
Richard M. Lovering 1,2, Joseph A. Roche 1, Mariah H. Goodall 2, Brett B. Clark 2, Alan McMillan 3
1Department of Physiology, University of Maryland School of Medicine, 2Department of Orthopaedics, University of Maryland School of Medicine, 3Department of Diagnostic Radiology, University of Maryland School of Medicine

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

image

Bioengineering

Bridging the Bio-Electronic Interface with Biofabrication
Tanya Gordonov *1, Benjamin Liba *2, Jessica L. Terrell *1, Yi Cheng 3, Xiaolong Luo 2, Gregory F. Payne 1, William E. Bentley 1
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.

image

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. Webb 1
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.

image

Advanced Biology

ELISPOT Assay: Detection of IFN-γ Secreting Splenocytes
Tonya J. Webb 1
1Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Comprehensive Cancer Center

ELISPOT Assay: Detection of IFN-γ Secreting Splenocytes

image

Advanced Biology

Immunohistochemistry and Immunocytochemistry: Tissue Imaging via Light Microscopy
Michael S. Lee 1, Tonya J. Webb 1
1Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Comprehensive Cancer Center

Immunohistochemistry and Immunocytochemistry: Tissue Imaging via Light Microscopy

image

Advanced Biology

Confocal Fluorescence Microscopy: A Technique to Determine the Localization of Proteins in Mouse Fibroblasts
Dominique R. Bollino 1, Eric A. Legenzov 2, Tonya J. Webb 1
1Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Comprehensive Cancer Center, 2Center for Biomedical Engineering and Technology, University of Maryland School of Medicine

Confocal Fluorescence Microscopy: A Technique to Determine the Localization of Proteins in Mouse Fibroblasts

image

Advanced Biology

Immunoprecipitation-Based Techniques: Purification of Endogenous Proteins Using Agarose Beads
Susannah C. Shissler 1, Tonya J. Webb 1
1Department of Microbiology and Immunology, University of Maryland

Immunoprecipitation-Based Techniques: Purification of Endogenous Proteins Using Agarose Beads

image

JoVE Core

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers
Rasa Ghaffarian 1, Silvia Muro 1,2
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.

image

Engineering

Fabrication of Uniform Nanoscale Cavities via Silicon Direct Wafer Bonding
Stephen R. D. Thomson 1, Justin K. Perron 2,3, Mark O. Kimball 4, Sarabjit Mehta 5, Francis M. Gasparini 1
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.

image

Biology

Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model
Pablo Bermejo-Alvarez 1,2, Ki-Eun Park 1,2, Bhanu P. Telugu 1,2
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.

image

Biology

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment
Tamika K. Samuel 1, Jason W. Sinclair 1, Katherine L. Pinter 1, Iqbal Hamza 1,2
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.

image

Bioengineering

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis
Hadar Ben-Yoav 1, Peter H. Dykstra 1, Tanya Gordonov 2, William E. Bentley 2, Reza Ghodssi 1
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.

image

Bioengineering

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications
Jennifer M. Vojtech 1,2, Juliana Cano-Mejia 1,2, Matthieu F. Dumont 1, Raymond W. Sze 1,3, Rohan Fernandes 1,3,4
1The Sheikh Zayed Institute for Pediatric Surgical Innovation, Children's National Medical Center, 2Fischell Department of Bioengineering, University of Maryland, 3Department of Radiology, George Washington University, 4Department of Pediatrics, George Washington University

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.

image

Stimulus-Independent Analysis of Affective Touch Using fMRI
Molly Lucas 1,2, Laura Anderson 1,3, Danielle Bolling 1, Kevin A Pelphrey 1, Martha D Kaiser 1
1Yale Child Study Center, Yale University, 2School of Continuing Education, Columbia University, 3Clinical / Developmental Psychology, University of Maryland

C-tactile (CT) afferents respond to caress-like touch, which has been found to activate “social brain” regions 1. Using fMRI, we compared neural responses of experiencing and imagining CT-targeted vs. non-CT-targeted touch to explore how the affective component of social touch is imbued.

image

Biology

Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers
Joseph S. Najem 1, Myles D. Dunlap 2, Anthony Yasmann 3, Eric C. Freeman 4, John W. Grant 5, Sergei Sukharev 3, Donald J. Leo 4
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.

image

Bioengineering

High Speed Sub-GHz Spectrometer for Brillouin Scattering Analysis
Kim V. Berghaus 1, Seok H. Yun 2,3, Giuliano Scarcelli 1
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.

image

Neuroscience

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice
Yaneri A. Ayala 1, David Pérez-González 1, Daniel Duque 1,2, Alan R. Palmer 3, Manuel S. Malmierca 1,4
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.

image

Engineering

Experimental Methodology for Estimation of Local Heat Fluxes and Burning Rates in Steady Laminar Boundary Layer Diffusion Flames
Ajay V. Singh 1, Michael J. Gollner 1
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.

image

Biology

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases (TALENs)
Johanna E. Kowalko 1, Li Ma 2, William R. Jeffery 3
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.

image

Bioengineering

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations
Poonam Sharma 1, Julianne D. Twomey 1, Michelle Patkin 1, Adam H. Hsieh 1
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.

image

Biochemistry

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments
Parisa Moghaddam-Taaheri 1, Svetlana P. Ikonomova 2, Zifan Gong 2, Janna Q. Wisniewski 1, Amy J. Karlsson 1,2
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.

image

Genetics

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus
Jie Xiang 1,2, Katie Reding 1, Leslie Pick 1,2
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.

image

Engineering

Dielectric RheoSANS — Simultaneous Interrogation of Impedance, Rheology and Small Angle Neutron Scattering of Complex Fluids
Jeffrey J. Richards 1, Cedric V. L. Gagnon 2, Jeffery R. Krzywon 1, Norman J. Wagner 3, Paul D. Butler 1
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.

image

JoVE Core

Fabrication and Testing of Photonic Thermometers
Nikolai N. Klimov 1,2, Zeeshan Ahmed 2
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.

image

Bioengineering

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System
Anjali Nandal 1,2, Barbara Mallon 3, Bhanu P. Telugu 1,2,4
1Department of Animal and Avian Sciences, University of Maryland, 2Animal Bioscience and Biotechnology Laboratory, ARS, USDA, 3NIH Stem Cell Unit, Bethesda, National Institutes of Health, 4RenOVAte Biosciences Inc

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

image

Neuroscience

Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult
Stephanie A. Herrlinger 1, Qiang Shao 2, Li Ma 2, Melinda Brindley 3, Jian-Fu Chen 2
1Biomedical and Health Sciences Institute, University of Georgia, 2Center for Craniofacial Molecular Biology, University of Southern California, 3Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia

Here we describe a method for establishing a model of Zika virus-induced microcephaly in mouse. This protocol includes methods for embryonic, neonatal, and adult-stage intracerebral inoculation of the Zika virus.

image

Education

Esophageal Heat Transfer for Patient Temperature Control and Targeted Temperature Management
Melissa I. Naiman 1,2, Maria Gray 2, Joseph Haymore 3, Ahmed F. Hegazy 4, Andrej Markota 5, Neeraj Badjatia 6, Erik B. Kulstad 2,7
1Center for Advanced Design, Research, and Exploration, University of Illinois at Chicago, 2Attune Medical, 3University of Maryland School of Nursing, 4University of Western Ontario, 5University Medical Centre Maribor, 6University of Maryland, 7Department of Emergency Medicine, University of Texas, Southwestern Medical Center

This study presents a novel method to provide efficient patient temperature control for cooling or warming patients. A single use, triple lumen device is placed into the esophagus, analogous to a standard orogastric tube, and connects to existing heat exchange units to perform automatic patient temperature management.

image

Chemistry

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog (Xenopus laevis) Embryos
Rosemary M. Onjiko 1, Erika P. Portero 1, Sally A. Moody 2, Peter Nemes 1,3
1Department of Chemistry, George Washington University, 2Department of Anatomy & Regenerative Biology, George Washington University, 3Department of Chemistry & Biochemistry, University of Maryland, College Park

We describe steps that enable fast in situ sampling of a small portion of an individual cell with high precision and minimal invasion using capillary-based micro-sampling, to facilitate chemical characterization of a snapshot of metabolic activity in live embryos using a custom-built single cell capillary electrophoresis and mass spectrometry platform.

image

Neuroscience

Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels
Xiaonan Zhang 1, Quentin Gaudry 1
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.

image

JoVE Journal

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae
Meagan Jezek 1, Alison Jacques 1, Deepika Jaiswal 1, Erin M. Green 1
1Department of Biological Sciences, University of Maryland

Here, we describe a protocol for chromatin immunoprecipitation of modified histones from the budding yeast Saccharomyces cerevisiae. Immunoprecipitated DNA is subsequently used for quantitative PCR to interrogate the abundance and localization of histone post-translational modifications throughout the genome.

image

Bioengineering

Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher
Joshua N. Webb 1, Caroline Dong 1, Andres Bernal 2, Giuliano Scarcelli 1
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.

image

Immunology and Infection

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites
Amrita Sarkar 1, Yousuf A. Khan 1, Maria Fernanda Laranjeira-Silva 1, Norma W. Andrews 1, Bidyottam Mittra 1
1Department of Cell Biology and Molecular Genetics, University of Maryland

All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence.

image

Environment

A Video Surveillance System to Monitor Breeding Colonies of Common Terns (Sterna Hirundo)
Jennifer Lynn Wall *1, Paul R. Marbán *2, David F. Brinker 3, Jeffery D. Sullivan 4, Mia Zimnik 5, Jennifer L. Murrow 6, Peter C. McGowan 7, Carl R. Callahan 7, Diann J. Prosser 8
1Chesapeake Conservation Corps, Chesapeake Bay Trust, 2Department of Marine, Estuarine, and Environmental Science, University of Maryland, 3Natural Heritage Program, Maryland Department of Natural Resources, 4Natural Systems Analyst, 5Department of Biology, Hood College, 6Department of Environmental Science and Technology, University of Maryland, 7U.S. Fish and Wildlife Service Chesapeake Bay Field Office, 8U.S. Geological Survey Patuxent Wildlife Research Center

This paper describes a protocol that uses a remote video monitoring surveillance system to continuously monitor breeding colonies of ground-nesting waterbirds. The system includes five cameras monitoring individual nests and one camera monitoring the colony as a whole, and is powered by car batteries that are recharged via solar panels.

image

Chemistry

Disentangling High Strength Copolymer Aramid Fibers to Enable the Determination of Their Mechanical Properties
Amanda L. Forster 1, Viviana Rodriguez Cardenas 1, Ajay Krishnamurthy 1,2, Zois Tsinas 3, Amy Engelbrecht-Wiggans 1,2, Nolan Gonzalez 1
1Material Measurement Laboratory, National Institute of Standards and Technology, 2Theiss Research, 3University of Maryland

The primary goal of the study is to develop a protocol to prepare consistent specimens for accurate mechanical testing of high strength copolymer aramid fibers, by removing a coating and disentangling the individual fiber strands without introducing significant chemical or physical degradation.

image

Environment

Use of Principal Components for Scaling Up Topographic Models to Map Soil Redistribution and Soil Organic Carbon
Xia Li 1,2, Greg W. McCarty 2
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.

image

Developmental Biology

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing
Lijuan Du 1, Amy Zhou 1, Alex Sohr 1, Sougata Roy 1
1Department of Cell Biology and Molecular Genetics, University of Maryland

Here, we present a method for generating tissue-specific binary transcription systems in Drosophila by replacing the first coding exon of genes with transcription drivers. The CRISPR/Cas9-based method places a transactivator sequence under the endogenous regulation of a replaced gene, and consequently facilitates transctivator expression exclusively in gene-specific spatiotemporal patterns.

image

Medicine

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice
Frederico Gerlinger-Romero 1, Alex B. Addinsall 2, Richard M. Lovering 3, Victoria C. Foletta 4, Chris van der Poel 5, Paul A. Della-Gatta 4, Aaron P. Russell 4
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.

image

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. Goldstein 1,4, Gary R. Whittaker 1, Marco R. Straus 1
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.

image

Immunology and Infection

Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining
Liang-Chun Wang 1, Jacob Wagner 2, Annabelle Capino 2, Elizabeth Nesbit 2, Wenxia Song 2, Daniel C. Stein 2
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.

image

JoVE Core

Cutting Procedures, Tensile Testing, and Ageing of Flexible Unidirectional Composite Laminates
Amy Engelbrecht-Wiggans 1,2, Ajay Krishnamurthy 1,2, Faraz Burni 1,3, William Osborn 1, Amanda L. Forster 1
1Material Measurement Laboratory, National Institute of Standards and Technology, 2Theiss Research, 3Chemical and Biomolecular Engineering Department, University of Maryland

The goal of the study was to develop protocols to prepare consistent specimens for accurate mechanical testing of high-strength aramid or ultra-high-molar-mass polyethylene-based flexible unidirectional composite laminate materials and to describe protocols for performing artificial ageing on these materials.

image

Immunology and Infection

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay
Ashley E. Nazario-Toole 1,2, Louisa P. Wu 2,3
1Department of Biology, University of Maryland, 2Department of Cell Biology and Molecular Genetics, University of Maryland, 3Institute for Bioscience and Biotechnology Research, University of Maryland

This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections.

image

Biology

Camera-based Measurements of Intracellular [Na+] in Murine Atrial Myocytes
Libet Garber 1,2, Humberto C. Joca 1, Andrew K. Coleman 1, Liron Boyman 1, W. Jonathan Lederer 1, Maura Greiser 1
1Center for Biomedical Engineering and Technology and Department of Physiology, University of Maryland School of Medicine, 2Fischell Department of Bioengineering, University of Maryland

The intracellular Na+ concentration ([Na+]i) in cardiac myocytes is altered during cardiac diseases. [Na+]i is an important regulator of intracellular Ca2+. We introduce a novel approach to measure [Na+]i in freshly isolated murine atrial myocytes using an electron multiplying charged coupled device (EMCCD) camera and a rapid, controllable illuminator.

image

Chemistry

Isotopic Effect in Double Proton Transfer Process of Porphycene Investigated by Enhanced QM/MM Method
Zhihui Tu 1,2, Jian Yin 3, Liangxu Xie 4
1State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, 2University of Chinese Academy of Sciences, 3XtalPi Inc. (Shenzhen Jingtai Technology Co., Ltd.), 4Institute of Bioinformatics and Medical Engineering, School of Electrical and Information Engineering, Jiangsu University of Technology

A protocol that uses enhanced QM/MM method to investigate the isotopic effect on the double proton transfer process in porphycene is presented here.

image

Neuroscience

Measuring Neural Mechanisms Underlying Sleep-Dependent Memory Consolidation During Naps in Early Childhood
Tamara Allard *1, Tracy Riggins *1, Arcadia Ewell 1, Benjamin Weinberg 1, Sanna Lokhandwala 2, Rebecca M. C. Spencer 2,3
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.

image

Immunology and Infection

Chronic, Acute, and Reactivated HIV Infection in Humanized Immunodeficient Mouse Models
Federico Perdomo-Celis 1,2, Sandra Medina-Moreno 1, Alonso Heredia 1, Harry Davis 1, Joseph Bryant 1, Juan Carlos Zapata 1
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.

image

Immunology and Infection

In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice
Juliana Ide Aoki 1, Ahyun Hong 1, Ricardo Andrade Zampieri 1, Lucile Maria Floeter-Winter 1, Maria Fernanda Laranjeira-Silva 1
1Department of Physiology, Institute of Bioscience, University of Sao Paulo

Here, we present a compiled protocol to evaluate the cutaneous infection of mice with Leishmania amazonensis. This is a reliable method for studying parasite virulence, allowing a systemic view of the vertebrate host response to the infection.

image

Immunology and Infection

Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy
Liangfei Niu 1, Cong Wang 1, Kaile Zhang 1,2, Miaomiao Kang 1,2, Rui Liang 1, Xiaonan Zhang 1, Bo Yan 1
1Shanghai Public Health Clinical Center, Fudan University, 2School of Life Sciences, Bengbu Medical College

This protocol describes a technique for visualizing macrophage behavior and death in embryonic zebrafish during Mycobacterium marinum infection. Steps for the preparation of bacteria, infection of the embryos, and intravital microscopy are included. This technique may be applied to the observation of cellular behavior and death in similar scenarios involving infection or sterile inflammation.

image

Biochemistry

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans
Jessica A. Espino 1, Lisa M. Jones 1
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.

image

Biochemistry

Characterizing Cellular Proteins with In-cell Fast Photochemical Oxidation of Proteins
Emily E. Chea 1, Aimee Rinas 2, Jessica A. Espino 1, Lisa M. Jones 1
1Department of Pharmaceutical Sciences, University of Maryland Baltimore, 2AIT Bioscience

Here, we characterize protein structure and interaction sites in living cells using a protein footprinting technique termed in-cell fast photochemical oxidation of proteins (IC-FPOP).

image

Biology

Circadian Entrainment of Drosophila Melanogaster
Austin O. Dada 1, Minh Q. Nguyen 1, Shea M. Peterson 1, Vy T. Ngo 1, Dayanne V. Cornelio-Parra 1, Bwaar S. Omer 1, Ada Thapa 2, Sarah R. Rapp 1, Veronica J. Cloud 1, Ryan D. Mohan 1
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.

image

Biology

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9
Megan E. Meuti 1, Robert Harrell 2
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.

image

Developmental Biology

Incremental Temperature Changes for Maximal Breeding and Spawning in Astyanax mexicanus
Li Ma 1, Ruby Dessiatoun 1, Janet Shi 1, William R. Jeffery 1
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.

image

Bioengineering

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks
Swathi Swaminathan 1, Alisa Morss Clyne 2
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.

image

Bioengineering

Cardiac Spheroids as in vitro Bioengineered Heart Tissues to Study Human Heart Pathophysiology
Poonam Sharma 1,2,3,4, Carmine Gentile 2,3,4
1University of Newcastle, 2University of Sydney, 3Kolling Institute of Medical Research, Royal North Shore Hospital, 4University of Technology, Sydney

This protocol aims to fabricate 3D cardiac spheroids (CSs) by co-culturing cells in hanging drops. Collagen-embedded CSs are treated with doxorubicin (DOX, a cardiotoxic agent) at physiological concentrations to model heart failure. In vitro testing using DOX-treated CSs may be used to identify novel therapies for heart failure patients.

image

Biochemistry

Platform Incubator with Movable XY Stage: A New Platform for Implementing In-Cell Fast Photochemical Oxidation of Proteins
Danté Johnson 1, Benjamin Punshon-Smith 2, Jessica A. Espino 1, Anne Gershenson 3, Lisa M. Jones 1
1Department of Pharmaceutical Sciences, University of Maryland Baltimore, 2Technology Research Center, University of Maryland Baltimore County, 3Department of Biochemistry and Molecular Biology, University of Massachusetts

A new static platform is used to characterize protein structure and interaction sites in the native cell environment utilizing a protein footprinting technique called in-cell fast photochemical oxidation of proteins (IC-FPOP).

image

Engineering

Solution Blow Spinning of Polymeric Nano-Composite Fibers for Personal Protective Equipment
Zois Tsinas 1,2, Ran Tao 1,3, Amanda L. Forster 1
1Material Measurement Laboratory, National Institute of Standards and Technology, 2Theiss Research, 3Department of Chemical Engineering, Texas Tech University

The primary goal of this study is to describe a protocol to prepare polymeric fiber mats with consistent morphology via solution blow spinning (SBS). We aim to use SBS to develop novel, tunable, flexible polymeric fiber nanocomposites for various applications, including protective materials, by incorporating nanoparticles in a polymer-elastomer matrix.

image

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 Yin 1, David Bloodgood 1, William Sexton 1, Cheng-I Wei 2, Shunyuan Xiao 1,3
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.

image

Chemistry

Comparison of Two Different Synthesis Methods of Single Crystals of Superconducting Uranium Ditelluride
Sheng Ran 1,2,3, I-Lin Liu 1,2, Shanta R. Saha 1,2, Prathum Saraf 1, Johnpierre Paglione 1,2, Nicholas P. Butch 1,2
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.

image

Chemistry

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo
Aparna B. Baxi 1,2, Leena R. Pade 1, Peter Nemes 1,2
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.

image

Biology

Quantifying the Antifungal Activity of Peptides Against Candida albicans
Wright K. Makambi 1, Svetlana P. Ikonomova 1, Amy J. Karlsson 1
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.

image

Immunology and Infection

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice
Ricardo Gonçalves 1, Gabriela Kaliff Teófilo Murta 1, Izabela Aparecida de Souza 1, David M. Mosser 2
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.

image

JoVE Core

AMEBaS: Automatic Midline Extraction and Background Subtraction of Ratiometric Fluorescence Time-Lapses of Polarized Single Cells
Rafael Badain 1, Daniel S. C. Damineli 1,2, Maria Teresa Portes 3, José Feijó 4, Stefano Buratti 5, Giorgia Tortora 6, Hugo Neves de Oliveira 1, Roberto M. Cesar Jr 1
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.

image

Chemistry

A Synthetic Methodology for Preparing Impregnated and Grafted Amine-Based Silica Composites for Carbon Capture
Charlotte M. Wentz 1,2, Zois Tsinas 1,3, Amanda L. Forster 1
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.

image

Bioengineering

Mechanical Dissociation of Tissues for Single Cell Analysis Using a Motorized Device
Mayowa Amosu 1, Andrew J. Gregory 2, John D. Murtagh 2, Nitay Pavin 2, Carson Taylor Meyers 2, Juan Grano de Oro Fernandez 1, Kaitlyn Moore 1, Katharina Maisel 1
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.

image

Developmental Biology

Monitoring the Mechanical Evolution of Tissue During Neural Tube Closure of Chick Embryo
Chenjun Shi 1, Chenchen Handler 2, Haden Florn 1, Jitao Zhang 1
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.

image

Medicine

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung
Yun Zhu Bai 1, Yuriko Terada 1, Katsutaka Mineura 1, Yuhei Yokoyama 1, Ruben G. Nava 1, Alexander S. Krupnick 2, Andrew E. Gelman 1, Mark J. Miller 3, Daniel Kreisel 1, Wenjun Li 1
1Division of Cardiothoracic Surgery, Washington University in St. Louis, 2Division of Thoracic Surgery, University of Maryland, 3Division of Infectious Diseases, Washington University in St. Louis

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

image

Biology

Wet Beveling of Microinjection Needles Utilizing Constant Air Pressure for Feedback on Needle Opening
Robert A. Harrell II 1
1Insect Transformation Facility, The Institute for Bioscience and Biotechnology Research, University of Maryland

This protocol describes the assembly of a pneumatic system for the delivery of pressurized air to a needle during the process of needle beveling. The protocol further describes the beveling process for creating sharp microinjection needles and how to gauge the relative opening size of the needle.

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved