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University of Nebraska-Lincoln

31 ARTICLES PUBLISHED IN JoVE

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Biology

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ
Qingsheng Li 1, Pamela J. Skinner 2, Lijie Duan 1, Ashley T. Haase 1
1Department of Microbiology, Medical School, University of Minnesota, 2Department of Veterinary and Biomedical Sciences, University of Minnesota

A technique combining in situ tetramer staining and in situ hybridization (ISTH) enables visualization, mapping and analysis of the spatial proximity of virus-specific CD8+ T cells to their virus-infected targets, and determination of the quantitative relationships between these immune effectors and targets to infection outcomes.

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Medicine

A Protocol for Comprehensive Assessment of Bulbar Dysfunction in Amyotrophic Lateral Sclerosis (ALS)
Yana Yunusova 1,2, Jordan R. Green 3, Jun Wang 3, Gary Pattee 4, Lorne Zinman 2,5
1Department of Speech-Language Pathology, University of Toronto, 2ALS/ MN Clinic, Sunnybrook Health Science Centre, 3Department of Special Education and Communication Disorders, University of Nebraska-Lincoln, 4Department of Neurology, Munroe-Meyer Institute, University of Nebraska Medical Center, 5Department of Neurology, University of Toronto

Objective assessments of the physiological mechanisms that support speech are needed to monitor disease onset and progression in persons with ALS and to quantify treatment effects in clinical trials. In this video, we present a comprehensive, instrumentation-based protocol for quantifying speech motor performance in clinical populations.

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Bioengineering

Impulsive Pressurization of Neuronal Cells for Traumatic Brain Injury Study
Matthew Nienaber *1, Jeong Soon Lee *1, Ruqiang Feng 1, Jung Yul Lim 1
1Department of Engineering Mechanics, University of Nebraska-Lincoln

A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate the molecular/cellular mechanisms of blast-induced traumatic brain injury.

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Bioengineering

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth
Evan T. Curtis 1, Simeng Zhang 1, Vahid Khalilzad-Sharghi 1, Thomas Boulet 2, Shadi F. Othman 1
1Department of Biological Systems Engineering, University of Nebraska-Lincoln, 2Department of Engineering Mechanics, University of Nebraska-Lincoln

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (μMRE).

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JoVE Core

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies
Denise K. Zinniel 1, Robert J. Fenton 1, Steven Halouska 2, Robert Powers 2, Raul G. Barletta 1
1School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, 2Department of Chemistry, University of Nebraska-Lincoln

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

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Bioengineering

Monitoring the Wall Mechanics During Stent Deployment in a Vessel
Brian D. Steinert 1, Shijia Zhao 1, Linxia Gu 1
1Department of Mechanical and Materials Engineering, University of Nebraska-Lincoln

Stent-induced arterial strain distributions are characterized using an optical surface strain measurement system. This visualization technique is used to gain insights into the impact of stent implantation on the host vessel.

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Medicine

Noninvasive Assessment of Cardiac Abnormalities in Experimental Autoimmune Myocarditis by Magnetic Resonance Microscopy Imaging in the Mouse
Chandirasegaran Massilamany *1, Vahid Khalilzad-Sharghi *2, Arunakumar Gangaplara 1, David Steffen 1, Shadi F. Othman 2, Jay Reddy 1
1School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, 2Department of Biological Systems Engineering, University of Nebraska-Lincoln

This study demonstrates the successful establishment of magnetic resonance microscopy imaging as a non-invasive tool to assess the cardiac abnormalities in mice affected with autoimmune myocarditis. The data indicate that the technique can be used to monitor the disease-progression in live animals.

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Immunology and Infection

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers
Chandirasegaran Massilamany 1, Arunakumar Gangaplara 1, Ting Jia 1, Christian Elowsky 2, Qingsheng Li 3, You Zhou 2, Jay Reddy 1
1School of Veterinary Medicine and Biomedical Sciences, University of Nebraska, Lincoln, 2Center for Biotechnology, University of Nebraska, Lincoln, 3Nebraska Center for Virology and School of Biological Sciences, University of Nebraska, Lincoln

The protocol to detect self-reactive CD4 T cells in brain and heart by direct staining with major histocompatibility complex class II dextramers has been described in this report. For comprehensive analysis, a reliable method to enumerate the frequencies of antigen-specific CD4+ T cells in situ is also devised.

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Biology

Toxicological Assays for Testing Effects of an Epigenetic Drug on Development, Fecundity and Survivorship of Malaria Mosquitoes
Atashi Sharma 1, Troy D. Anderson 1, Igor V. Sharakhov 1
1Department of Entomology, Virginia Tech

A protocol is developed to examine the effects of an epigenetic drug DZNep on the development, fecundity and survivorship of mosquitoes. Here we describe procedures for the aqueous exposure of DZNep to immature mosquitoes and a blood-based exposure of DZNep to adult mosquitoes in addition to measuring SAH hydrolase inhibition.

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Chemistry

Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray
Lisa S. Ramirez 1,2, Jun Wang 1,2
1Department of Chemistry, University at Albany, State University of New York, 2Multiplex Biotechnology Laboratory, Cancer Research Center

This protocol outlines the fabrication of a large-scale, multiplexed two-dimensional DNA or antibody array, with potential applications in cell signaling studies and biomarker detection.

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Biology

Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function
Scott T. O'Neal 1, Troy D. Anderson 1
1Department of Entomology, Virginia Tech

The abdominal dorsal vessel of the honey bee and other insects serves as the functional equivalent of the mammalian heart and plays an important role in nutrient transport, waste removal, immune function, and more. Here we describe a protocol for the visualization and pharmacological manipulation of bee heart rate.

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Environment

Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees
Nicholas R. Larson 1, Troy D. Anderson 2
1Entomology, Virginia Tech, 2Entomology, University of Nebraska-Lincoln

The loss of honey bee colonies presents a challenge to crop pollination services. Current pollinator protection practices warrant an alternative approach to minimize the contact of honey bees to harmful pesticides using repellent chemistries. Here, we provide detailed methods for a visual tracking protocol to screen deterrents for bees.

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Medicine

Construction and Evaluation of a Murine Calvarial Osteolysis Model by Exposure to CoCrMo Particles in Aseptic Loosening
Hui Jiang *1, Yicun Wang *1, Zhantao Deng *1,2,3, Jiewen Jin 2,3, Jia Meng 1, Shuo Chen 1, Jun Wang 1, Yang Qiu 1, Ting Guo 1, Jianning Zhao 1
1Department of Orthopedics, Jinling Hospital, School of Medicine, Nanjing University, 2Center for Translational Medicine, Nanjing University Medical School, 3Jiangsu Key Laboratory for Molecular Medicine, Nanjing University Medical School

This manuscript describes a murine calvarial osteolysis model by exposure to CoCrMo particles, which constitutes an ideal animal model for assessing the interactions between wear particles and various cells in aseptic loosening.

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Developmental Biology

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation
Qinggang Dai *1, Yujiao Han *2, Furong Xie *1, Xuhui Ma 3, Zhan Xu 2, Xiao Liu 1, Weiguo Zou 2, Jun Wang 1
1Department of Pediatric Dentistry, Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai Key Laboratory of Stomatology & Shanghai Research Institute of Stomatology, National Clinical Research Center of Stomatology, 2State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, 3Department of Oral and Maxillofacial-Head and Neck Oncology, Ninth People's Hospital, Shanghai Jiaotong University School of Medicine

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 in osteoclast formation.

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Chemistry

Fabrication of Spherical and Worm-shaped Micellar Nanocrystals by Combining Electrospray, Self-assembly, and Solvent-based Structure Control
Xinyi Ding 1,2,3, Yuxiang Sun 1,2,3, Yanming Chen 1,2,3, Wanchuan Ding 1,2,3, Steven Emory 1,2,3,4, Tianhao Li 1,2,3, Zixing Xu 1,2,3, Ning Han 1,2,3, Jun Wang 1,2,3, Gang Ruan 1,2,3
1Department of Biomedical Engineering, College of Engineering and Applied Sciences, Nanjing University, 2Institute of Materials Engineering, College of Engineering and Applied Sciences, Nanjing University, 3Collaborative Innovation Center of Chemistry for Life Sciences, Nanjing University, 4Department of Chemistry, Western Washington University

The present work describes a method to fabricate micellar nanocrystals, an emerging major class of nanobiomaterials. This method combines top-down electrospray, bottom-up self-assembly, and solvent-based structure control. The fabrication method is largely continuous, can produce high quality products, and possesses an inexpensive means of structure control.

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Environment

Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments
Morgan R. McPherson 1, Peng Wang 1, Ellen L. Marsh 1, Robert B. Mitchell 2, Daniel P. Schachtman 1
1Department of Agronomy and Horticulture and Center for Plant Science Innovation, University of Nebraska-Lincoln, 2USDA-ARS, Wheat, Sorghum and Forage Research Unit, University of Nebraska-Lincoln East Campus

Excavation of plant roots from the field as well as processing of samples into endosphere, rhizosphere, and soil are described in detail, including DNA extraction and data analysis methods. This paper is designed to enable other laboratories to use these techniques for the study of soil, endosphere, and rhizosphere microbiomes.

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Genetics

Culturing and Manipulation of O9-1 Neural Crest Cells
Bao H. Nguyen 1, Mamoru Ishii 2, Robert E. Maxson 2, Jun Wang 1
1Molecular Physiology and Biophysics, Baylor College of Medicine, 2Department of Biochemistry and Molecular Biology, University of Southern California

O9-1 is a multipotent mouse neural crest cell line. Here we describe detailed step-by-step protocols for culturing O9-1 cells, differentiating O9-1 cells into specific cell types, and genetically manipulating O9-1 cells by using siRNA-mediated knockdown or CRISPR-Cas9 genome editing.

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Chemistry

Inkjet Printing All Inorganic Halide Perovskite Inks for Photovoltaic Applications
Dylan Richmond 1, Mason McCormick 2, Thilini K. Ekanayaka 3, Jacob D. Teeter 2, Benjamin L. Swanson 1, Nicole Benker 3, Guanhua Hao 3, Sharmin Sikich 4, Axel Enders 5, Alexander Sinitskii 2, Carolina C. Ilie 1, Peter A. Dowben 3, Andrew J. Yost 3
1Department of Physics, State University of New York-Oswego, 2Department of Chemistry, University of Nebraska-Lincoln, 3Department of Physics, University of Nebraska-Lincoln, 4Department of Chemistry, Doane University, 5Physikalisches Institut, Universität Bayreuth

A protocol for synthesizing inorganic-lead-halide hybrid perovskite quantum dot inks for inkjet printing and the protocol for preparing and printing the quantum dot inks in an inkjet printer with post characterization techniques are presented.

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Engineering

A Silicon-tipped Fiber-optic Sensing Platform with High Resolution and Fast Response
Guigen Liu *1, Qiwen Sheng *1, Weilin Hou 2, Matthew L. Reinke 3, Ming Han 1,4
1Department of Electrical and Computer Engineering, Michigan State University, 2Naval Research Laboratory, Stennis Space Center, 3Oak Ridge National Laboratory, 4Department of Electrical and Computer Engineering, University of Nebraska-Lincoln

This work reports an innovative silicon-tipped fiber-optic sensing platform (Si-FOSP) for high-resolution and fast-response measurement of a variety of physical parameters, such as temperature, flow, and radiation. Applications of this Si-FOSP span from oceanographic research, mechanical industry, to fusion energy research.

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Genetics

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes
Kelsey R. Timme 1, Jennifer R. Wood 1
1Department of Animal Science, University of Nebraska-Lincoln

To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.

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Behavior

Application of 3D Printing in the Construction of Burr Hole Ring for Deep Brain Stimulation Implants
Jiazhi Chen *1,2, Xinyu Chen *3, Siyuan Lv 2, Yuzhen Zhang 1, Hao Long 1, Kaijun Yang 1, Songtao Qi 1, Wangming Zhang 2, Jun Wang 1
1Department of Neurosurgery, Nanfang Hospital, Southern Medical University, 2Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, 3Faculty of Brain Sciences, University of College London

Here, we present a protocol to demonstrate 3D printing in the construction of deep brain stimulation implants.

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Bioengineering

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression
Donghee Lee 1, Alek Erickson 2, Andrew T. Dudley 1, Sangjin Ryu 3,4
1Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, 2Department of Physiology and Pharmacology, Karolinska Institutet, 3Department of Mechanical and Materials Engineering, University of Nebraska-Lincoln, 4Nebraska Center for Materials and Nanoscience, University of Nebraska-Lincoln

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

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Biochemistry

A Double Humanized BLT-mice Model Featuring a Stable Human-Like Gut Microbiome and Human Immune System
Lance Daharsh 1,2, Jianshui Zhang 1,2, Amanda Ramer-Tait 3, Qingsheng Li 1,2
1Nebraska Center for Virology, 2School of Biological Sciences, University of Nebraska-Lincoln, 3Department of Food Science and Technology, University of Nebraska-Lincoln

We describe a novel method for generating double humanized BLT-mice that feature a functional human immune system and a stable engrafted human-like gut microbiome. This protocol can be followed without the need for germ-free mice or gnotobiotic facilities.

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Biology

Bovine Ovarian Cortex Tissue Culture
Courtney M. Sutton *1, Shelby A. Springman *1, Mohamed A. Abedal-Majed 2, Andrea S. Cupp 1
1Department of Animal Science, University of Nebraska-Lincoln, 2Department of Animal Production, School of Agriculture, University of Jordan

In vitro culture of bovine ovarian cortex and the effect of nutritional Stair-step diet on ovarian microenvironment is presented. Ovarian cortex pieces were cultured for seven days and steroids, cytokines, and follicle stages were evaluated. The Stair-Step diet treatment had increased steroidogenesis resulting in follicle progression in culture.

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Bioengineering

Functional Transcranial Doppler Ultrasound for Monitoring Cerebral Blood Flow
Benjamin D. Hage 1, Edward J. Truemper 2, Gregory R. Bashford 3
1Department of Biological Systems Engineering, University of Nebraska-Lincoln, 2Department of Biological Systems Engineering, University of Nebraska-Lincoln, 3Department of Biological Systems Engineering, University of Nebraska-Lincoln

Functional transcranial Doppler ultrasound complements other functional imaging modalities, with its high temporal resolution measurement of stimulus-induced changes in cerebral blood flow within the basal cerebral arteries. This Methods paper gives step-by-step instructions for using functional transcranial Doppler ultrasound to perform a functional imaging experiment. 

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Developmental Biology

An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells
Tram P. Le *1, Xiaolei Zhao *1, Shannon Erhardt 1,2, Jianhua Gu 3, Huie Wang 3, Tina O. Findley 1, Jun Wang 1,2
1Department of Pediatrics, McGovern Medical School, The University of Texas Health Science Center at Houston, 2The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, The University of Texas Health Science Center at Houston, 3SEM AFM Core in the Houston Methodist Hospital Research Institute

Detailed step-by-step protocols are described here for studying mechanical signals in vitro using multipotent O9-1 neural crest cells and polyacrylamide hydrogels of varying stiffness.

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Genetics

Heuristic Mining of Hierarchical Genotypes and Accessory Genome Loci in Bacterial Populations
Natasha Pavlovikj *1, Joao Carlos Gomes-Neto *2,3, Andrew K. Benson 2,3
1Department of Computer Science and Engineering, University of Nebraska-Lincoln, 2Department of Food Science and Technology, University of Nebraska-Lincoln, 3Nebraska Food for Health Center, University of Nebraska-Lincoln

This analytical computational platform provides practical guidance for microbiologists, ecologists, and epidemiologists interested in bacterial population genomics. Specifically, the work presented here demonstrated how to perform: i) phylogeny-guided mapping of hierarchical genotypes; ii) frequency-based analysis of genotypes; iii) kinship and clonality analyses; iv) identification of lineage differentiating accessory loci.

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Medicine

Microscopic Replantation of Penile Glans Amputation Due to Circumcision
Da-Chun Jin *1, Bo Zhou *1, Jun Li 2, Cong-Cong Bao 1, Yong Luo 1, Yong Zhang 1, Peng Wang 1, Gang Bi 1, Yan-Feng Li 1
1Department of Urology, Daping Hospital, Army Medical University, 2Department of Urology, Chongqing 13th People's Hospital

The present protocol describes the emergency management of microscopic replantation of penile glans amputation due to circumcision.

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JoVE Core

Determination of the Mechanical Properties of Flexible Connectors for Use in Insulated Concrete Wall Panels
Fray F. Pozo-Lora 1, Marc Maguire 2
1Civil & Environmental Engineering Department, Pontificia Universidad Catolica Madre y Maestra, 2Durham School of Architectural Engineering and Construction, University of Nebraska-Lincoln

We propose a testing protocol that can be combined with widely available analytical methods to assess the mechanical properties of shear connectors for use in the design of insulated concrete wall panels to predict full-scale insulated panel behavior.

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Bioengineering

Harvesting, Embedding, and Culturing Dorsal Root Ganglia in Multi-compartment Devices to Study Peripheral Neuronal Features
Benjamin M. Gane 1, Sydney M. Caparaso 1, Fei San Lee 1, Adan L. Redwine 1, Rebecca A. Wachs 1
1Department of Biological Systems Engineering, University of Nebraska-Lincoln

This protocol provides a technique to harvest and culture explanted dorsal root ganglion (DRG) from adult Sprague Dawley rats in a multi-compartment (MC) device.

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Bioengineering

Porous Substrate-Based Electroporation with Transepithelial Electrical Impedance Monitoring
Sawyer R. Lorenzen 1,2, Justin R. Brooks 1, Tyler C. Heiman 1, Ruiguo Yang 1,2,3
1Department of Mechanical and Materials Engineering, University of Nebraska-Lincoln, 2Department of Biomedical Engineering, Michigan State University, 3Institute for Quantitative Health Science and Engineering (IQ), Michigan State University

Porous substrate electroporation (PSEP) pairs consistent, high throughput delivery with high cell viability. Introduction of transepithelial electrical impedance (TEEI) measurements provides insight into the intermediate processes of PSEP and allows for label-free delivery. This article discusses a method for performing PSEP delivery experiments and TEEI measurement analysis simultaneously.

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