University of Nebraska, Lincoln View Institution's Website 22 articles published in JoVE Bioengineering Functional Transcranial Doppler Ultrasound for Monitoring Cerebral Blood Flow Benjamin D. Hage1, Edward J. Truemper2, Gregory R. Bashford3 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. Biology Bovine Ovarian Cortex Tissue Culture Courtney M. Sutton*1, Shelby A. Springman*1, Mohamed A. Abedal-Majed2, Andrea S. Cupp1 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. Chemistry Chromatographic Fingerprinting by Template Matching for Data Collected by Comprehensive Two-Dimensional Gas Chromatography Federico Stilo1, Chiara Cordero1, Carlo Bicchi1, Daniela Peroni2, Qingping Tao3, Stephen E. Reichenbach3,4 1Dipartimento di Scienza e Tecnologia del Farmaco, Università degli Studi di Torino, 2SRA Instruments, 3GC Image LLC, 4Computer Science and Engineering Department, University of Nebraska This protocol presents an approach to fingerprint and explore multi-dimensional data collected by comprehensive two-dimensional gas chromatography coupled to mass spectrometry. Dedicated pattern recognition algorithms (template matching) are applied to explore the chemical information encrypted in the extra-virgin olive oil volatile fraction (i.e., volatilome). Bioengineering A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression Donghee Lee1, Alek Erickson2, Andrew T. Dudley1, Sangjin Ryu3,4 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. Biochemistry A Double Humanized BLT-mice Model Featuring a Stable Human-Like Gut Microbiome and Human Immune System Lance Daharsh1,2, Jianshui Zhang1,2, Amanda Ramer-Tait3, Qingsheng Li1,2 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. Developmental Biology Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans Edward M. Germany1, Nataliya Zahayko1, Oleh Khalimonchuk1,2,3,4 1Department of Biochemistry, University of Nebraska, 2Nebraska Redox Biology Center, University of Nebraska, 3Nebraska Center for Integrated Biomolecular Communication, University of Nebraska, 4Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center Here, we present a protocol for simple isolation of specific groups of live neuronal cells expressing green fluorescent protein from transgenic Caenorhabditis elegans lines. This method enables a variety of ex vivo studies focused on specific neurons and has the capacity to isolate cells for further short-term culturing. Genetics Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes Kelsey R. Timme1, Jennifer R. Wood1 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. Chemistry Inkjet Printing All Inorganic Halide Perovskite Inks for Photovoltaic Applications Dylan Richmond1, Mason McCormick2, Thilini K. Ekanayaka3, Jacob D. Teeter2, Benjamin L. Swanson1, Nicole Benker3, Guanhua Hao3, Sharmin Sikich4, Axel Enders5, Alexander Sinitskii2, Carolina C. Ilie1, Peter A. Dowben3, Andrew J. Yost3 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. Engineering A Silicon-tipped Fiber-optic Sensing Platform with High Resolution and Fast Response Guigen Liu*1, Qiwen Sheng*1, Weilin Hou2, Matthew L. Reinke3, Ming Han1,4 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. Environment Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments Morgan R. McPherson1, Peng Wang1, Ellen L. Marsh1, Robert B. Mitchell2, Daniel P. Schachtman1 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. Biochemistry Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion Fatima Cabral1, Colton M. Miller1, Katrina M. Kudrna1, Blake E. Hass1, Jocelyn G. Daubendiek1, Brianna M. Kellar1, Edward N. Harris1 1Department of Biochemistry, University of Nebraska The goal of this protocol is to obtain high viability and high yield of hepatocytes and sinusoidal endothelial cells from liver. This is accomplished by perfusing the liver with a type IV collagenase solution via the portal vein, followed by differential centrifugation to obtain hepatocytes and sinusoidal endothelial cells. Environment Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees Nicholas R. Larson1, Troy D. Anderson2 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. Immunology and Infection In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers Chandirasegaran Massilamany1, Arunakumar Gangaplara1, Ting Jia1, Christian Elowsky2, Qingsheng Li3, You Zhou2, Jay Reddy1 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. 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 Gangaplara1, David Steffen1, Shadi F. Othman2, Jay Reddy1 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. Biology Introducing an Angle Adjustable Cutting Box for Analyzing Slice Shear Force in Meat Tom Whitesell1, Carmen Avilés2, Jennifer L. Aalhus1, Chris R. Calkins3, Ivy L. Larsen1, Manuel Juárez1 1Lacombe Research Centre, Agriculture and Agri-Food Canada, 2Grupo de investigación MERAGEM, Universidad de Córdoba, 3Department of Animal Science, University of Nebraska Slice shear force is a reference method for beef texture analysis. Using an angle adjustable cutting box could increase its accuracy for research purposes. The results from different locations within the longissimus muscle show a high correlation with Warner-Bratzler shear force methodology and high potential adaptability for different muscles. Immunology and Infection Protein Misfolding Cyclic Amplification of Prions Samuel E. Saunders1, Jason C. Bartz2, Ronald A. Shikiya2 1Department of Civil Engineering, University of Nebraska at Lincoln, 2Department of Medical Microbiology and Immunology, Creighton University Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay. Immunology and Infection Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies Denise K. Zinniel1, Robert J. Fenton1, Steven Halouska2, Robert Powers2, Raul G. Barletta1 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. Bioengineering Monitoring the Wall Mechanics During Stent Deployment in a Vessel Brian D. Steinert1, Shijia Zhao1, Linxia Gu1 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. Bioengineering Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth Evan T. Curtis1, Simeng Zhang1, Vahid Khalilzad-Sharghi1, Thomas Boulet2, Shadi F. Othman1 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). Biology In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion Sandhya Gopalakrishnan1, Edward N. Harris1 1Department of Biochemistry, University of Nebraska, Lincoln The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation. Bioengineering Impulsive Pressurization of Neuronal Cells for Traumatic Brain Injury Study Matthew Nienaber*1, Jeong Soon Lee*1, Ruqiang Feng1, Jung Yul Lim1 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. Medicine A Protocol for Comprehensive Assessment of Bulbar Dysfunction in Amyotrophic Lateral Sclerosis (ALS) Yana Yunusova1,2, Jordan R. Green3, Jun Wang3, Gary Pattee4, Lorne Zinman2,5 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.
