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Auburn University

27 ARTICLES PUBLISHED IN JoVE

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Neuroscience

Isolation and Culture of Hippocampal Neurons from Prenatal Mice
Michael L. Seibenhener 1, Marie W. Wooten 1
1Department of Biological Sciences, Auburn University

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

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JoVE Methods Collections: Current Research Methods in Rabies Diagnosis, Prevention, Treatment, and Control
Charles E. Rupprecht 1, Wei-Cheng Hsu 2, Feng Ye 3, Megan Golding 4, Luca Zaeck 5, Laurent Dacheux 6, Noel Tordo 7, Jodie A. Jarvis 8, Erin Patrick 9, Are Berentsen 10
1LYSSA LLC, 2Animal Health Research Institute, Council of Agriculture, Executive Yuan, 3Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Institute of Military Veterinary Medicine, Academy of Military Medical Sciences, 4Wildlife Zoonoses & Vector-Borne Diseases Research Group, Animal and Plant Health Agency, 5Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, 6Unit Lyssavirus Epidemiology and Neuropathology, National Reference Center for Rabies and WHO Collaborating Center for Reference and Research on Rabies, Institut Pasteur, 7Unit Antiviral Strategies, OIE Reference Laboratories for Rift Valley Fever Virus & Crimean Congo Hemorrhagic Fever Virus, Institut Pasteur, Institut Pasteur de Guinea, Conakry, 8Rabies Laboratory, Wadsworth Center, New York State Department of Health, 9Animal and Plant Health Inspection Service, Wildlife Services, Knoxville, TN, US Department of Agriculture, 10Animal and Plant Health Inspection Service, Wildlife Services, National Wildlife Research Center, US Department of Agriculture

JoVE Methods Collections: Current Research Methods in Rabies Diagnosis, Prevention, Treatment, and Control

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Bioengineering

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria
Rajesh Guntupalli 1, Iryna Sorokulova 1, Eric Olsen 2, Ludmila Globa 1, Oleg Pustovyy 1, Vitaly Vodyanoy 1
1Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University , 2Clinical Research Laboratory, 81st Medical Group, Keesler Air Force Base

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

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Immunology and Infection

Assays for the Identification of Novel Antivirals against Bluetongue Virus
Linlin Gu 1, Stewart W. Schneller 2, Qianjun Li 1
1Division of Infectious Diseases, Department of Medicine, University of Alabama at Birmingham, 2Molette Laboratory for Drug Discovery, Department of Chemistry and Biochemistry, Auburn University

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

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Behavior

Use of the Open Field Maze to Measure Locomotor and Anxiety-like Behavior in Mice
Michael L. Seibenhener 1, Michael C. Wooten 1
1Department of Biological Sciences, Auburn University

A protocol is provided to use an Open Field Maze to access general locomotor activity, anxiety and emotionality in a laboratory mouse model.

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Neuroscience

Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals
Feng Liu 1, Nannan Liu 1
1Department of Entomology and Plant Pathology, Auburn University

Bed bugs rely on olfactory receptor neurons housed in their antennal olfactory sensilla to detect semiochemicals in the environment. Utilizing single sensillum recording, we demonstrate a method to evaluate bed bug response to semiochemicals and explore the coding process involved.

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Environment

Sediment Core Sectioning and Extraction of Pore Waters under Anoxic Conditions
Alison R. Keimowitz 1, Yan Zheng 2, Ming-Kuo Lee 3, Michael Natter 3, Jeffrey Keevan 3
1Department of Chemistry, Vassar College, 2Division of Geochemistry, Lamont-Doherty Earth Observatory, 3Department of Geosciences, Auburn University

A protocol for sectioning sediment cores and extracting pore waters under anoxic conditions in order to permit analysis of redox sensitive species in both solids and fluids is presented.

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JoVE Core

Printing Fabrication of Bulk Heterojunction Solar Cells and In Situ Morphology Characterization
Feng Liu 1, Sunzida Ferdous 2, Xianjian Wan 1, Chenhui Zhu 3, Eric Schaible 3, Alexander Hexemer 3, Cheng Wang 3, Thomas P. Russell 1,2
1Materials Sciences Division, Lawrence Berkeley National Laboratory, 2Department of Polymer Science and Engineering, University of Massachusetts, Amherst, 3Advanced Light Source, Lawrence Berkeley National Laboratory

Here, we present a protocol to fabricate organic thin film solar cells using a mini-slot die coater and related in-line structure characterizations using synchrotron scattering techniques.

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Immunology and Infection

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain
Iryna Sorokulova 1, Ludmila Globa 1, Oleg Pustovyy 1, Vitaly Vodyanoy 1
1Department of Anatomy, Physiology and Pharmacology, Auburn University

We described a protocol for prevention of heat stress effects in rats by oral pre-treatment with beneficial bacteria. This protocol can be modified and used for various routes of administration and for analysis of different compounds.

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Environment

Using Pharmacological Manipulation and High-precision Radio Telemetry to Study the Spatial Cognition in Free-ranging Animals
Timothy C. Roth *1, Aaron R. Krochmal *2, William B. Gerwig, IV 1, Sage Rush 3, Nathaniel T. Simmons 2, Jeffery D. Sullivan 4, Katrina Wachter 5
1Department of Psychology, Franklin and Marshall College, 2Department of Biology, Washington College, 3University of Pennsylvania, 4School of Forestry and Wildlife Sciences, Auburn University, 5Morsani College of Medicine, University of South Florida

This paper describes a novel protocol that combines the pharmacological manipulation of memory and radio telemetry to document and quantify the role of cognition in navigation.

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Neuroscience

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models
Holly C. Hunsberger 1, Sharay E. Setti 2, Ryan T. Heslin 2, Jorge E. Quintero 3, Greg A. Gerhardt 3, Miranda N. Reed 2
1Department of Psychology, West Virginia University, 2Department of Drug Discovery & Development, Auburn University, 3Department of Neuroscience, University of Kentucky Medical Center

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

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Genetics

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing
Ahmed Elaswad *1,2, Karim Khalil *1,3, David Cline 1, Patrick Page-McCaw 4, Wenbiao Chen 4, Maximilian Michel 5, Roger Cone 5, Rex Dunham 1
1School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, 2Department of Animal Wealth Development, Faculty of Veterinary Medicine, Suez Canal University, 3Anatomy and Embryology Department, Faculty of Veterinary Medicine, Cairo University, 4Department of Molecular Physiology and Biophysics, Vanderbilt University, 5Life Science Institute, University of Michigan

A simple and efficient microinjection protocol for gene editing in channel catfish embryos using the CRISPR/Cas9 system is presented. In this protocol, guide RNAs and Cas9 protein were microinjected into the yolk of one-cell embryos. This protocol has been validated by knocking out two channel catfish immune-related genes.

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Developmental Biology

Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis
Junhua Lv 1,2, Feng Liu 1,2
1State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, 2University of Chinese Academy of Sciences

This protocol describes using cultured Aorta-Gonad-Mesonephros for expression analyses, colony-forming units in the culture and spleen, and long-term reconstitution to determine the effect of regulatory factors and signaling pathways on hematopoietic stem cell development. This has been demonstrated as an effective system for studying hematopoietic stem cell biology and function.

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Immunology and Infection

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy
Peter B. Rogers 1, Elizabeth Hiltbold Schwartz 1
1Department of Biological Sciences, Auburn University

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

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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.

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Biochemistry

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica
Xuechun Feng 1, Nannan Liu 1
1Department of Entomology and Plant Pathology, Auburn University

Here, we present a protocol to produce house fly carboxylesterase proteins in vitro with a baculovirus mediated insect cell expression system and later functionally characterize their roles in metabolizing permethrin, thereby, conferring pyrethroid resistance by conducting cell-based MTT assay and in vitro metabolic studies.

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Environment

Cultivation of Green Microalgae in Bubble Column Photobioreactors and an Assay for Neutral Lipids
Qichen Wang 1, Haixin Peng 1, Brendan T. Higgins 1
1Department of Biosystems Engineering, Auburn University

Here, we present a protocol to construct lab-scale bubble column photobioreactors and use them to culture microalgae. It also provides a method for the determination of the culture growth rate and neutral lipid content.

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Medicine

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice
Fuliang Luo 1, Xin Wang 1, Xiaokang Luo 1, Bo Li 1, Desheng Zhu 2, Hansong Sun 3, Yue Tang 1
1Beijing Key Laboratory of Pre-clinical Research and Evaluation for Cardiovascular Implant Materials, State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Centre for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, 2Laboratory Animal Center, Peking University, 3Center of Cardiac Surgery for Adults, State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Centre for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College

Here, we present a protocol to perform an invasive hemodynamic assessment of the right ventricle and pulmonary artery in mice using an open-chest surgery approach.

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Behavior

Training Dogs for Awake, Unrestrained Functional Magnetic Resonance Imaging
Lily R. Strassberg *1, L. Paul Waggoner *2, Gopikrishna Deshpande *1,3,4,5, Jeffrey S. Katz *1,3,4,5
1Department of Psychology, Auburn University, 2Canine Performance Sciences Program, College of Veterinary Medicine, Auburn University, 3AU MRI Research Center, Department of Electrical & Computer Engineering, Auburn University, 4Alabama Advanced Imaging Consortium, 5Center for Neuroscience, Auburn University

Magnetic resonance imaging (MRI) on unrestrained awake dogs is a new method with several advantages over imaging with physical or chemical restraint. This protocol introduces a cost-effective training method that minimizes training in the MRI environment, which can be expensive, and maximizes the subject pool available for canine functional MRI.

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Medicine

Technical Modification of the Terminal Ureter During Total Transperitoneal Laparoscopic Nephroureterectomy for Upper Urinary Tract Urothelial Carcinoma
Chunlong Fu *1,2, Yaqun Zhang *1, Xin Wang 1, Shengcai Zhu 1, Huimin Hou 1, Shengjie Liu 1, Cheng Pang 1, Xinda Song 1, Jia Chen 1, Jianye Wang 1, Ming Liu 1,2
1(Urology) Beijing Hospital, National Center of Gerontology, The Joint Unit of the Chinese Academy of Medical Sciences, 2Graduate School of Peking Union Medical College, Beijing Hospital

Here, we present a protocol to increase the surgical field of view and reduce the difficulty of total transperitoneal laparoscopic nephroureterectomy surgery by precutting the umbilical ligament before treating the terminal ureter.

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Developmental Biology

Microwaving and Fluorophore-Tyramide for Multiplex Immunostaining on Mouse Adrenals − Using Unconjugated Primary Antibodies from the Same Host Species
Qiongxia Lyu 1,2, Huifei Sophia Zheng 2, Karly Laprocina 2, Chen-Che Jeff Huang 2
1College of Animal Science & Technology, Henan University of Science and Technology, 2Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University

Several different methods have been established for multiplex immunostaining using primary antibodies from the same host species. Here, we describe the use of microwave-mediated antibody stripping and of fluorophore-tyramide to block antibody cross-reactivity during multiplex immunostaining on formalin-fixed paraffin-embedded mouse adrenal sections.

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Genetics

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus
Michelle Bui 1, Ming Li 1, Robyn R Raban 1, Nannan Liu 2, Omar S Akbari 1,3
1Section of Cell and Developmental Biology, University of California, San Diego, 2Department of Entomology and Plant Patholog, Auburn University, 3Tata Institute for Genetics and Society, University of California, San Diego

This protocol describes microinjection procedures for Culex quinquefasciatus embryos that are optimized to work with CRISPR/Cas9 gene editing tools. This technique can efficiently generate site-specific, heritable, germline mutations that can be used for building genetic technologies in this understudied disease vector.

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Neuroscience

Heterologous Expression and Functional Analysis of Aedes aegypti Odorant Receptors to Human Odors in Xenopus Oocytes
Xin Wang *1, Zhou Chen *1,2, Yifan Wang 1, Feng Liu 1,3, Shanshan Jiang 1, Nannan Liu 1
1Department of Entomology and Plant Pathology, Auburn University, 2Departments of Biochemistry & Biophysics, and Cellular & Molecular Pharmacology, University of California, San Francisco, 3Department of Biological Sciences, Vanderbilt University

A protocol is presented that functionally characterizes mosquito ORs in response to human odors using a Xenopus oocyte expression system coupled with a two-electrode voltage clamp, providing a powerful new technique for exploring the responses of mosquitoes ORs to exposure to human odors.

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Biology

Development of a Mobile Mitochondrial Physiology Laboratory for Measuring Mitochondrial Energetics in the Field
Hailey A. Parry 1, Kang Nian Yap 2, Geoffrey E. Hill 2, Wendy R. Hood 2, L. Bruce Gladden 1, Michael Eddy 3, Andreas N. Kavazis 1
1School of Kinesiology, Auburn University, 2Department of Biological Sciences, Auburn University, 3Department of Electrical and Computer Engineering, Auburn University

We designed and constructed a mobile laboratory to measure respiration rates in isolated mitochondria of wild animals captured at field locations. Here, we describe the design and outfitting of a mobile mitochondrial laboratory and the associated laboratory protocols.

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Biology

Isolate Cell-Type-Specific RNAs from Snap-Frozen Heterogeneous Tissue Samples without Cell Sorting
Huifei Sophia Zheng 1, Chen-Che Jeff Huang 1
1Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University

This protocol aims to isolate cell-type-specific translating ribosomal mRNAs using the NuTRAP mouse model.

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Neuroscience

Confocal Microscopy to Measure Three Modes of Fusion Pore Dynamics in Adrenal Chromaffin Cells
Sue Han *1, Xin Wang *1, Nicholas Cordero 1, Ling-Gang Wu 1
1National Institute of Neurological Disorders and Stroke

This protocol describes a confocal imaging technique to detect three fusion modes in bovine adrenal chromaffin cells. These fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving fused vesicle shrinkage.

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Methods Collection In Lyssavirus Detection, Diagnosis and Research
Charles E. Rupprecht 1
1College of Forestry, Wildlife and Environment, Auburn University

Methods Collection In Lyssavirus Detection, Diagnosis and Research

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