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35 ARTICLES PUBLISHED IN JoVE

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Biology

RhoC GTPase Activation Assay
Michelle Lucey 1, Heather Unger 1, Kenneth L. van Golen 1
1Department of Biological Sciences, The Center for Translational Cancer Research, University of Delaware

This protocol utilizes a pull down assay to determine the levels of active RhoC GTPase within cells.

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Biology

Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects
Gary Balian 1, Gina Beck 1, Vedavathi Madhu 1, Robert Sikes 2, Quanjun Cui 3, Haixiang Liang 1, Joshua Bush 1
1Orthopaedics Research, University of Virginia, 2Biological Sciences, University of Delaware, 3Orthopaedic Surgery, University of Virginia

A phage display library was used to identify peptide sequences that target bone. The objective was to investigate the effect of these peptides on mesenchymal cell differentiation and to determine their effect on bone regeneration.

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Engineering

Measuring Material Microstructure Under Flow Using 1-2 Plane Flow-Small Angle Neutron Scattering
A. Kate Gurnon 1, P. Douglas Godfrin 1, Norman J. Wagner 1, Aaron P. R. Eberle 1,2, Paul Butler 2, Lionel Porcar 1,3
1Center for Neutron Science, Department of Chemical and Biomolecular Engineering, University of Delaware, 2NIST Center for Neutron Research, National Institute of Standards and Technology, 3Institut Laue-Langevin

A shear cell is developed for small-angle neutron scattering measurements in the velocity-velocity gradient plane of shear and is used to characterize complex fluids. Spatially resolved measurements in the velocity gradient direction are possible for studying shear-banding materials. Applications include investigations of colloidal dispersions, polymer solutions, and self-assembled structures.

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Bioengineering

Construction and Characterization of a Novel Vocal Fold Bioreactor
Aidan B. Zerdoum 1, Zhixiang Tong 2, Brendan Bachman 2, Xinqiao Jia 1,2
1Biomedical Engineering Program, University of Delaware, 2Department of Materials Science and Engineering, Delaware Biotechnology Institute, University of Delaware

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(ε-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

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Medicine

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders
Dennis J. Wu 1, Neha Dixit 1, Erika Suzuki 1, Thanh Nguyen 2, Hyun Seock Shin 1, Jack Davis 2, Emanual Maverakis 3, Iannis E. Adamopoulos 1,2
1Division of Rheumatology, Allergy and Clinical Immunology, University of California, Davis, 2Institute for Pediatric Regenerative Medicine, Shriners Hospitals for Children - Northern California, 3Department of Dermatology, University of California, Davis

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

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Neuroscience

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue
Jaclyn M. Schwarz 1
1Department of Psychological and Brain Sciences, University of Delaware

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

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Bioengineering

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications
Lisa A. Sawicki 1, April M. Kloxin 1,2
1Department of Chemical and Biomolecular Engineering, University of Delaware, 2Department of Materials Science and Engineering, University of Delaware

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

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Chemistry

Synthesis of Hierarchical ZnO/CdSSe Heterostructure Nanotrees
Zhengxin Li 1,2, Jesus Nieto-Pescador 1,2, Alexander J. Carson 1,2, Jolie C. Blake 1,2, Lars Gundlach 1,2
1Department of Chemistry & Biochemistry, University of Delaware, 2Department of Physics & Astronomy, University of Delaware

Here, we prepare and characterize novel tree-like hierarchical ZnO/CdSSe nanostructures, where CdSSe branches are grown on vertically aligned ZnO nanowires. The resulting nanotrees are a potential material for solar energy conversion and other opto-electronic devices.

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Behavior

Wheel Running and Environmental Complexity as a Therapeutic Intervention in an Animal Model of FASD
Zachary H. Gursky 1, Anna Y. Klintsova 1
1Department of Psychological and Brain Sciences, University of Delaware

Cardiovascular exercise and stimulating experiences in a complex environment have positive benefits on multiple measures of neuroplasticity within the rodent brain. This article will discuss the implementation of these interventions as a "superintervention" which combines wheel running and environmental complexity and will address the limitations of these interventions.

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Bioengineering

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks
Keely A. Heintz 1, David Mayerich 2, John H. Slater 1,3
1Department of Biomedical Engineering, University of Delaware, 2Department of Electrical and Computer Engineering, University of Houston, 3Delaware Biotechnology Institute

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

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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.

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Environment

Functionalization and Dispersion of Carbon Nanomaterials Using an Environmentally Friendly Ultrasonicated Ozonolysis Process
Eudora S. Y. Yeo 1, Gary I. Mathys 2, Narelle Brack 3, Erik T. Thostenson 4,5,6, Andrew N. Rider 1
1Aerospace Division, Defence Science and Technology Group, 2Maritime Division, Defence Science and Technology Group, 3Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, 4Department of Mechanical Engineering, University of Delaware, 5Department of Materials Science and Engineering, University of Delaware, 6Center for Composite Materials, University of Delaware

Here, a novel method for the functionalization and stable dispersion of carbon nanomaterials in aqueous environments is described. Ozone is injected directly into an aqueous dispersion of carbon nanomaterial that is continuously recirculated through a high-powered ultrasonic cell.

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Bioengineering

Predicting Gene Silencing Through the Spatiotemporal Control of siRNA Release from Photo-responsive Polymeric Nanocarriers
Chad T. Greco 1, Thomas H. Epps, III 1,2, Millicent O. Sullivan 1
1Department of Chemical and Biomolecular Engineering, University of Delaware, 2Department of Materials Science and Engineering, University of Delaware

We present a novel method that uses photo-responsive block copolymers for more efficient spatiotemporal control of gene silencing with no detectable off-target effects. Additionally, changes in gene expression can be predicted using straightforward siRNA release assays and simple kinetic modeling.

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Genetics

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells
Natalia Rivera-Torres 1,2, Eric B. Kmiec 1,2
1Gene Editing Institute, Helen F. Graham Cancer Center and Research Institute, Christiana Care Health Services, 2Department of Medical Sciences, University of Delaware

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site—in essence, analyzing onsite mutagenesis.

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Neuroscience

Neuroimaging Field Methods Using Functional Near Infrared Spectroscopy (NIRS) Neuroimaging to Study Global Child Development: Rural Sub-Saharan Africa
Kaja K. Jasińska 1,2, Sosthène Guei 3
1Linguistics and Cognitive Science Department, University of Delaware, 2Haskins Laboratories, 3Centre de Recherche et d'Action pour la Paix

Portable neuroimaging approaches (functional Near Infrared Spectroscopy) provide advances to the study of the brain in previously inaccessible regions; here, rural Côte d'Ivoire. Innovation in methods and development of culturally-appropriate neuroimaging protocols permits novel study of the brain's development and children's learning outcomes in environments with significant poverty and adversity.

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Behavior

Using a Real-Time Locating System to Measure Walking Activity Associated with Wandering Behaviors Among Institutionalized Older Adults
Mary E. Bowen 1,2, William Kearns 3, Jeremy R. Crenshaw 4, Steven J. Stanhope 4
1School of Nursing, University of Delaware, 2Corporeal Michael J. Crescenz VA Medical Center, 3Child and Family Studies, University of South Florida, 4Department of Kinesiology and Applied Physiology, University of Delaware

This paper discusses the use of a continuous and objective real-time locating system to measure walking activity associated with wandering behaviors, focusing on older adults with cognitive impairment. Walking activity is measured by walking distance, sustained walking distance, and sustained gait speed. Also assessed are gait quality and balance ability.

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Neuroscience

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain
Brianna Kimmelmann-Shultz 1, Negin Mohmammadmirzaei 1, Jeffrey Caplan 2, Dayan Knox 1
1Department of Psychological and Brain Sciences, University of Delaware, 2Plant and Soil Sciences, Delaware Biotechnology Institute, University of Delaware

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

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Immunology and Infection

Electrophysiological Recordings of Single-cell Ion Currents Under Well-defined Shear Stress
Ibra S. Fancher 1, Irena Levitan 1
1Department of Medicine, Division of Pulmonary, Critical Care, Sleep and Allergy, University of Illinois at Chicago

The goal of this protocol is to describe a modified parallel plate flow chamber for use in investigating real time activation of mechanosensitive ion channels by shear stress.

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Bioengineering

Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform
Omar A. Banda 1, John H. Slater 1,2,3
1Department of Biomedical Engineering, University of Delaware, 2Department of Materials Science & Engineering, University of Delaware, 3Delaware Biotechnology Institute

This protocol provides instructions for implementing multiphoton lithography to fabricate three-dimensional arrays of fluorescent fiducial markers embedded in poly(ethylene glycol)-based hydrogels for use as reference-free, traction force microscopy platforms. Using these instructions, measurement of 3D material strain and calculation of cellular tractions is simplified to promote high-throughput traction force measurements.

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JoVE Core

Measuring Statistical Learning Across Modalities and Domains in School-Aged Children Via an Online Platform and Neuroimaging Techniques
Julie M. Schneider 1, Anqi Hu 1, Jennifer Legault 1, Zhenghan Qi 1
1Department of Linguistics and Cognitive Science, University of Delaware

Presented here is a protocol introducing a set of child-friendly statistical learning tasks geared towards examining children’s learning of temporal statistical patterns across domains and sensory modalities. The developed tasks collect behavioral data using the web-based platform and task-based functional magnetic resonance imaging (fMRI) data for examining neural engagement during statistical learning.

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Bioengineering

Evaluating Regional Pulmonary Deposition using Patient-Specific 3D Printed Lung Models
Emma L. Peterman 1, Emily L. Kolewe 1, Catherine A. Fromen 1
1Department of Chemical and Biomolecular Engineering, University of Delaware

We present a high-throughput, in vitro method for quantifying regional pulmonary deposition at the lobe level using CT scan-derived, 3D printed lung models with tunable air flow profiles.

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Environment

A Method to Preserve Wetland Roots and Rhizospheres for Elemental Imaging
Angelia L. Seyfferth 1, Matt A. Limmer 1, Ryan Tappero 2
1Department of Plant and Soil Sciences, University of Delaware, 2National Synchrotron Radiation Lightsource-II, Brookhaven National Laboratory

We describe a protocol to sample, preserve, and section intact roots and the surrounding rhizosphere soil from wetland environments using rice (Oryza sativa L.) as a model species. Once preserved, the sample can be analyzed using elemental imaging techniques, such as synchrotron X-ray fluorescence (XRF) chemical speciation imaging.

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Bioengineering

Area-based Image Analysis Algorithm for Quantification of Macrophage-fibroblast Cocultures
Tohn Borjigin 1, Anuraag Boddupalli 1, Millicent O. Sullivan 1
1Department of Chemical and Biomolecular Engineering, University of Delaware

We present a method, which utilizes a generalizable area-based image analysis approach to identify cell counts. Analysis of different cell populations exploited the significant cell height and structure differences between distinct cell types within an adaptive algorithm.

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Immunology and Infection

Efficient SARS-CoV-2 Quantitative Reverse Transcriptase PCR Saliva Diagnostic Strategy utilizing Open-Source Pipetting Robots
Rachel E. Ham *1, Austin R. Smothers *1,2, Kylie L. King 1, Justin M. Napolitano 1, Theodore J. Swann 3, Lesslie G. Pekarek 4, Mark A. Blenner *1,5,6, Delphine Dean *1,2
1Center for Innovative Medical Devices and Sensors (REDDI Lab), Clemson University, 2Department of Bioengineering, Clemson University, 3Swann Medicine, 4Student Health Services, Clemson University, 5Department of Chemical & Biomolecular Engineering, Clemson University, 6Department of Chemical & Biomolecular Engineering, University of Delaware

The protocol describes a SARS-CoV-2 diagnostic method that utilizes open-source automation to perform RT-qPCR molecular testing of saliva samples. This scalable approach can be applied to clinical public health surveillance as well as to increase the capacity of smaller university laboratories.

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Biology

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications
Thanh Nguyen 1, Sang Joon Ahn 2, Richard West 3, Ibra S. Fancher 1
1Department of Kinesiology and Applied Physiology, College of Health Sciences, University of Delaware, 2Division of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, University of Illinois at Chicago, 3University of Delaware Flow Cytometry Core, Delaware Biotechnology Institute, University of Delaware

This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction.

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Genetics

Measuring Caenorhabditis elegans Sensitivity to the Acetylcholine Receptor Agonist Levamisole
Allison N. Davis 1, Jessica E. Tanis 1
1Department of Biological Sciences, University of Delaware

The present protocol describes an assay to determine response to levamisole, a pharmacological agonist of one class of Caenorhabditis elegans acetylcholine receptors. In this liquid levamisole swim assay, researchers visually observe and quantitate the time-dependent paralysis of animals cultivated in 24-well plates.

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Engineering

Optimizing Magnetic Force Microscopy Resolution and Sensitivity to Visualize Nanoscale Magnetic Domains
Audrey C. Parker *1, Olivia O. Maryon *1, Mojtaba T. Kaffash 2, M. Benjamin Jungfleisch 2, Paul H. Davis 1,3
1Micron School of Materials Science & Engineering, Boise State University, 2Department of Physics and Astronomy, University of Delaware, 3Center for Advanced Energy Studies

Magnetic force microscopy (MFM) employs a vertically magnetized atomic force microscopy probe to measure sample topography and local magnetic field strength with nanoscale resolution. Optimizing MFM spatial resolution and sensitivity requires balancing decreasing lift height against increasing drive (oscillation) amplitude, and benefits from operating in an inert atmosphere glovebox.

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Neuroscience

Assessing Corticospinal Excitability During Goal-Directed Reaching Behavior
Isaac N. Gomez 1, Serena R. Orsinger 1, Hyosub E. Kim 2, Ian Greenhouse 1
1Department of Human Physiology, University of Oregon, 2Department of Physical Therapy/Psychological & Brain Sciences, University of Delaware

Reaching is a fundamental skill that allows humans to interact with the environment. Several studies have aimed to characterize reaching behavior using a variety of methodologies. This paper offers an open-source application of transcranial magnetic stimulation to assess the state of corticospinal excitability in humans during reaching task performance.

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Developmental Biology

Visualizing Early Infection Sites of Rice Blast Disease (Magnaporthe oryzae) on Barley (Hordeum vulgare) Using a Basic Microscope and a Smartphone
Jessica G. Cooper 1, Nicole M. Donofrio 1, Jeffrey L. Caplan 1, Timothy R. Chaya 1
1Department of Plant and Soil Sciences, University of Delaware

This is a straightforward protocol of a barley leaf sheath assay using minimal reagents and common laboratory equipment (including a basic smartphone). The purpose is to visualize the early infection process of blast disease in labs without access to advanced microscopy equipment.

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Genetics

Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans
Ji Kent Kwah 1, Aimee Jaramillo-Lambert 1
1Department of Biological Sciences, University of Delaware

Here, we present a general method to determine the embryonic viability and total number of embryos produced (brood) using the model organism C. elegans.

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Neuroscience

Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior
Nicole G. Pastorino 1, Saori Tomatsu 1, Amy Lin 1, Jackson Doerr 1, Zachary Waterman 1, Krisztina Sershen 1, Pulak Ray 2, Analiz Rodriguez 3, Deni S. Galileo 1
1Department of Biological Sciences, University of Delaware, 2Helen F. Graham Cancer Center and Research Institute, Christiana Care, 3Department of Neurosurgery, Winthrop P. Rockefeller Cancer Institute, University of Arkansas for Medical Sciences

Chick embryos are used for studying human glioblastoma (GBM) brain tumors in ovo and in ex vivo brain slice co-cultures. GBM cell behavior can be recorded by time-lapse microscopy in ex vivo co-cultures, and both preparations can be analyzed at the experimental endpoint by detailed 3D confocal analysis.

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Medicine

Measurement of Healthy and Injured Triceps Surae Morphology
Hayley Powell Smitheman 1, Kayla D. Seymore 1, Morgan N. Potter 1, Andy K. Smith 1, Susanna Aufwerber 2,3, Karin Grävare Silbernagel 1
1Department of Physical Therapy, University of Delaware, 2Department of Molecular Medicine and Surgery, Karolinska Institute, 3Women’s Health and Allied Health Professionals Theme, Medical Unit of Occupational Therapy and Physiotherapy, Karolinska University Hospital

Ultrasound imaging is becoming more accessible in clinical and research settings, and a consistent protocol will be beneficial for comparison between studies and for clinical interpretations. This protocol for ultrasound evaluation is a valid and reliable method to evaluate Achilles tendon morphology in healthy, tendinopathic, and ruptured tendons.

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Biology

Whole Mount Imaging to Visualize and Quantify Peripheral Lens Structure, Cell Morphology, and Organization
Grace Emin *1, Sadia T. Islam *1, Rylee E. King 1, Velia M. Fowler 1, Catherine Cheng 2, Justin Parreno 1,3
1Department of Biological Sciences, University of Delaware, 2School of Optometry and Vision Science Program, Indiana University, 3Department of Biomedical Engineering, University of Delaware

The present protocols describe novel whole mount imaging for the visualization of peripheral structures in the ocular lens with methods for image quantification. These protocols can be used in studies to better understand the relationship between lens microscale structures and lens development/function.

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Biology

Modeling Cataract Surgery in Mice
Leah M. O'Neill 1, Yan Wang 1, Melinda K. Duncan 1
1Department of Biological Sciences, University of Delaware

This method models cataract surgery in vivo by removing lens fiber cells from adult mice and leaving behind the capsular bag with attached lens epithelial cells (LECs). The injury response is then assessed at various times post-surgery using molecular and morphological criteria.

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Biology

Separation of Avian Preovulatory Follicle Granulosa and Theca Cell Layers for Downstream Applications
Ashley E. Kramer *1, Kathryn M. Ellwood *1, Erin M. Brannick 1, Aditya Dutta 2
1Department of Animal & Food Sciences, University of Delaware, 2Departments of Animal & Food Sciences, Biological Sciences, Medical & Molecular Sciences, and Microbiology Graduate Program, University of Delaware

Here, we describe a protocol for separating yolk, granulosa cells, and theca cells in avian preovulatory follicles. This precision handling enables critical investigations into the role of these layers in reproductive function, aiding the understanding of follicular development, hormonal regulation, and disease research for enhanced agricultural yield and biomedical insights.

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