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The University of North Carolina at Chapel Hill

10 ARTICLES PUBLISHED IN JoVE

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Biology

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface
Loretta Müller 1, Luisa E. Brighton 1, Johnny L. Carson 1,2, William A. Fischer II 1,3, Ilona Jaspers 1,2,4
1Center for Environmental Medicine, Asthma, and Lung Biology, The University of North Carolina at Chapel Hill, 2Department of Pediatrics, The University of North Carolina at Chapel Hill, 3Pulmonary Diseases and Critical Care, The University of North Carolina at Chapel Hill, 4Curriculum in Toxicology, The University of North Carolina at Chapel Hill

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

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Neuroscience

Deep Brain Stimulation with Simultaneous fMRI in Rodents
John Robert Younce 1,2,5, Daniel L Albaugh 1,2,4, Yen-Yu Ian Shih 1,2,3,4
1Department of Neurology, University of North Carolina, 2Biomedical Research Imaging Center, University of North Carolina, 3Department of Biomedical Engineering, University of North Carolina, 4Curriculum in Neurobiology, University of North Carolina, 5School of Medicine, University of North Carolina

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

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Biology

Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy
Johnny L. Carson 1
1Department of Pediatrics, Center for Environmental Medicine, Asthma, and Lung Biology, The University of North Carolina at Chapel Hill

Basic techniques and refinements of freeze-fracture processing of biological specimens and nanomaterials for examination by transmission electron microscopy are described. This technique is a preferred method for revealing ultrastructural features and specializations of biological membranes and for obtaining ultrastructural level dimensional and spatial data in materials sciences and nanotechnology products.

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Neuroscience

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis
Cortney C. Winkle 1, Christopher C. Hanlin 2, Stephanie L. Gupton 1,2
1Curriculum in Neurobiology, The University of North Carolina at Chapel Hill, 2Department of Cell Biology and Physiology, The University of North Carolina at Chapel Hill

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

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Biology

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins
Kathryn A. Ramsey 1,2, Zachary L. Rushton 1, Camille Ehre 1,3
1Marsico Lung Institute/CF Center, University of North Carolina at Chapel Hill, 2Telethon Kids Institute, University of Western Australia, 3Department of Pediatrics, University of North Carolina at Chapel Hill

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

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Behavior

Behavioral Assessment of Hearing in 2 to 4 Year-old Children: A Two-interval, Observer-based Procedure Using Conditioned Play-based Responses
Angela Yarnell Bonino 1,2, Lori J. Leibold 3
1Department of Allied Health Sciences, The University of North Carolina at Chapel Hill, 2Department of Speech, Language, and Hearing Sciences, University of Colorado Boulder, 3Center for Hearing Research, Boys Town National Research Hospital

This paper describes a procedure for measuring hearing sensitivity in 2 to 4 year-old children. Children are trained to perform play-based responses when they hear a target signal. Thresholds are then estimated in a two-interval, two-alternative forced-choice paradigm, based on observations of the child's behaviors.

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Developmental Biology

A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells
Kimberly A. Peters 1, Elizabeth Detmar 1, Liz Sepulveda 2, Corrina Del Valle 2, Ruth Valsquier 2, Anna Ritz 2, Stephen L. Rogers 1,3,4, Derek A. Applewhite 2
1Department of Biology, The University of North Carolina at Chapel Hill, 2Department of Biology, Reed College, 3Carolina Center for Genome Sciences, The University of North Carolina at Chapel Hill, 4Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill

Here we describe a contractility assay using Drosophila S2R+ cells. The application of an exogenous ligand, folded gastrulation (Fog), leads to the activation of the Fog signaling pathway and cellular contractility. This assay can be used to investigate the regulation of cellular contractility proteins in the Fog signaling pathway.

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Biology

Automated Detection and Analysis of Exocytosis
Fabio Urbina 1, Stephanie L. Gupton 1
1UNC Neuroscience Center, Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill

We developed automated computer vision software to detect exocytic events marked by pH-sensitive fluorescent probes. Here, we demonstrate the use of a graphical user interface and RStudio to detect fusion events, analyze and display spatiotemporal parameters of fusion, and classify events into distinct fusion modes.

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Medicine

Rapid Viscoelastic Characterization of Airway Mucus Using a Benchtop Rheometer
Jason A. Wykoff 1, Kendall M. Shaffer 1, Kenza C. Araba 1, Matthew R. Markovetz 1, Jérémy Patarin 2, Matthieu Robert de Saint Vincent 2, Scott H. Donaldson 1,3, Camille Ehre 1,4
1Marsico Lung Institute / CF Center, The University of North Carolina at Chapel Hill, 2Rheonova, 3Department of Pulmonary and Critical Care Medicine, The University of North Carolina at Chapel Hill, 4Department of Pediatric, Pediatric Pulmonology Division, The University of North Carolina at Chapel Hill

The viscoelastic properties of mucus play a critical role in mucociliary clearance. However, traditional mucus rheological techniques require complex and time-consuming approaches. This study provides a detailed protocol for the use of a benchtop rheometer that can rapidly and reliably perform viscoelastic measurements.

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Neuroscience

Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy
Felix A. Kyere *1,2, Ian Curtin *1,2, Oleh Krupa 1,2, Carolyn M. McCormick 1,2, Mustafa Dere 3, Sarah Khan 3,7, Minjeong Kim 7, Tzu-Wen Winnie Wang 4,5,6, Qiuhong He 4,5,6, Guorong Wu 3, Yen-Yu Ian Shih 4,5,6, Jason L. Stein 1,2
1UNC Neuroscience Center, University of North Carolina, Chapel Hill, 2Department of Genetics, University of North Carolina, Chapel Hill, 3Department of Psychiatry, University of North Carolina, Chapel Hill, 4Center for Animal Magnetic Resonance Imaging, The University of North Carolina at Chapel Hill, 5Biomedical Research Imaging Center, The University of North Carolina at Chapel Hill, 6Department of Neurology, The University of North Carolina at Chapel Hill, 7Department of Computer Science, The University of North Carolina at Greensboro

This protocol describes methods for conducting magnetic resonance imaging, clearing, and immunolabeling of intact mouse brains using iDISCO+, followed by a detailed description of imaging using light-sheet microscopy, and downstream analyses using NuMorph.

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