University of Wisconsin-Madison 29 articles published in JoVE Bioengineering High-Throughput Optogenetics Experiments in Yeast Using the Automated Platform Lustro Zachary P. Harmer1, Megan N. McClean1,2 1Department of Biomedical Engineering, University of Wisconsin-Madison, 2University of Wisconsin Carbone Cancer Center, University of Wisconsin School of Medicine and Public Health This protocol outlines the steps for utilizing the automated platform Lustro to perform high-throughput characterization of optogenetic systems in yeast. Genetics A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration Michael Landowski1,2, Samuel Grindel1, Ying Hao3, Sakae Ikeda1,2, Catherine Bowes Rickman3,4, Akihiro Ikeda1,2 1Department of Medical Genetics, University of Wisconsin-Madison, 2McPherson Eye Research Institute, University of Wisconsin-Madison, 3Department of Ophthalmology, Duke University, 4Department of Cell Biology, Duke University Mouse models can be useful tools for investigating the biology of the retinal pigmented epithelium (RPE). It has been established that mice can develop an array of RPE pathologies. Here, we describe a phenotyping protocol to elucidate and quantify RPE pathologies in mice using light, transmission electron, and confocal microscopy. Genetics The Serial Anesthesia Array for the High-Throughput Investigation of Volatile Agents Using Drosophila melanogaster Zachariah P. G. Olufs1, Dena Johnson-Schlitz1, David A. Wassarman2, Misha Perouansky1 1Department of Anesthesiology, University of Wisconsin-Madison, 2Department of Medical Genetics, University of Wisconsin-Madison The fruit fly (Drosophila melanogaster) is widely used for biological and toxicological research. To expand the utility of flies, we developed an instrument, the serial anesthesia array, that simultaneously exposes multiple fly samples to volatile general anesthetics (VGAs), making it possible to investigate the collateral effects (toxic and protective) of VGAs. Behavior Four Temporary Waterslide Designs Adapted to Different Slope Conditions to Encourage Child Socialization in Playgrounds Zhudi Hua1, Ting Tao1, Risa Akita1, Tomofusa Akita1, Yoshiaki Hayakawa1, Masanori Hariyama2, Hayato Sakurai3, Ricki Colman4, Mamiko Koshiba1,2,3 1Graduate School of Sciences and Technology for Innovation, Yamaguchi University, 2Graduate School of Information Sciences, Tohoku University, 3Pediatrics, Saitama Medical University Hospital, 4Department of Cell and Regenerative Biology, University of Wisconsin-Madison Early life social learning is enhanced by interactions with effectively designed environments. Four events were held at different city parks using inexpensive, temporary waterslides to stimulate social learning. This study describes the prototypes used and the evaluation of the children's interactions. Biology Maintaining Laboratory Cultures of Gryllus bimaculatus, a Versatile Orthopteran Model for Insect Agriculture and Invertebrate Physiology Martin K. Ventura1, Valerie J. Stull2, Susan M. Paskewitz1 1Department of Entomology, University of Wisconsin-Madison, 2Global Health Institute, University of Wisconsin-Madison This paper outlines basic methods to standardize important factors such as density, feed availability, hydration source, and environmental controls for the long-term rearing of laboratory cultures of the edible cricket, Gryllus bimaculatus. Chemistry Multi-Faceted Mass Spectrometric Investigation of Neuropeptides in Callinectes sapidus Ashley Phetsanthad*1, Nhu Q. Vu*1, Lingjun Li1,2 1Department of Chemistry, University of Wisconsin-Madison, 2School of Pharmacy, University of Wisconsin-Madison Mass spectrometric characterization of neuropeptides provides sequence, quantitation, and localization information. This optimized workflow is not only useful for neuropeptide studies, but also other endogenous peptides. The protocols provided here describe sample preparation, MS acquisition, MS analysis, and database generation of neuropeptides using LC-ESI-MS, MALDI-MS spotting, and MALDI-MS imaging. Cancer Research A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment Brian M. Burkel1, David R. Inman1, María Virumbrales-Muñoz1,2, Erica J. Hoffmann1, Suzanne M. Ponik1,3 1Department of Cell and Regenerative Biology, University of Wisconsin-Madison, 2Department of Pathology, University of Wisconsin-Madison, 3Carbone Cancer Center, University of Wisconsin-Madison The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images. Biochemistry A Spin-Tip Enrichment Strategy for Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues Dylan Nicholas Tabang1, Danqing Wang1, Lingjun Li1,2 1Department of Chemistry, University of Wisconsin-Madison, 2School of Pharmacy, University of Wisconsin-Madison Post-translational modifications (PTMs) change protein structures and functions. Methods for the simultaneous enrichment of multiple PTM types can maximize coverage in analyses. We present a protocol using dual-functional Ti(IV)-immobilized metal affinity chromatography followed by mass spectrometry for the simultaneous enrichment and analysis of protein N-glycosylation and phosphorylation in pancreatic tissues. Genetics Determining the Role of Maternally-Expressed Genes in Early Development with Maternal Crispants Cara E. Moravec1, Gabriella C. Voit1, Francisco Pelegri1 1Laboratory of Genetics, University of Wisconsin-Madison Early development is dependent on maternally-inherited products, and the role of many of these products is currently unknown. Herein, we described a protocol that uses CRISPR-Cas9 to identify maternal-effect phenotypes in a single generation. Bioengineering Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools Yuming Liu1, Kevin W. Eliceiri1,2,3 1Laboratory for Optical and Computational Instrumentation, Center for Quantitative Cell Imaging, University of Wisconsin-Madison, 2Departments of Medical Physics and Biomedical Engineering, University of Wisconsin-Madison, 3Morgridge Institute for Research, Madison, Wisconsin, 4test, test Here, we present a protocol to use a curvelet transform-based, open-source MATLAB software tool for quantifying fibrillar collagen organization in the extracellular matrix of both normal and diseased tissues. This tool can be applied to images with collagen fibers or other types of line-like structures. Immunology and Infection High Throughput Co-culture Assays for the Investigation of Microbial Interactions Mia I. Temkin1, Caitlin M. Carlson1, Aaron L. Stubbendieck2, Cameron R. Currie1, Reed M. Stubbendieck1 1Department of Bacteriology, University of Wisconsin-Madison, 2Independent Scholar The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens. Medicine Ultrasonography of the Adult Male Urinary Tract for Urinary Functional Testing Teresa T. Liu*1,2, Allison C. Rodgers*3, Tristan M. Nicholson4, Jill A. Macoska5,6, Paul C. Marker6,7, Chad M. Vezina6,8, Dale E. Bjorling6,8, Alejandro Roldan-Alzate2,9,10, Diego Hernando10,11, Granville L. Lloyd12, Timothy A. Hacker3, William A. Ricke1,6 1Department of Urology, University of Wisconsin-Madison, 2K12 Kure, University of Wisconsin-Madison, 3Cardiovascular Research Center, Department of Medicine, University of Wisconsin-Madison, 4Department of Urology, University of Washington, 5University of Massachusetts Boston, 6U54 George M. O'Brien Center, University of Wisconsin-Madison, 7College of Pharmacy, University of Wisconsin-Madison, 8School of Veterinary Medicine, University of Wisconsin-Madison, 9Department of Mechanical Engineering, University of Wisconsin-Madison, 10Department of Radiology, University of Wisconsin-Madison, 11Department of Medical Physics, University of Wisconsin-Madison, 12Rocky Mountain Regional VA Medical Center, University of Colorado We describe the use of high frequency ultrasound with contrast imaging as a method to measure bladder volume, bladder wall thickness, urine velocity, void volume, void duration, and urethral diameter. This strategy can be used to assess voiding dysfunction and treatment efficacy in various mouse models of lower urinary tract dysfunction (LUTD). Genetics In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster Olivia Gevedon1, Harris Bolus1, Shu Hui Lye1, Keaton Schmitz1, Jesualdo Fuentes-González1, Kathryn Hatchell2, Lyndsey Bley2, Jason Pienaar1, Carin Loewen2, Stanislava Chtarbanova1 1Department of Biological Sciences, University of Alabama, 2Department of Genetics, University of Wisconsin-Madison We present a protocol using a forward genetic approach to screen for mutants exhibiting neurodegeneration in Drosophila melanogaster. It incorporates a climbing assay, histology analysis, gene mapping and DNA sequencing to ultimately identify novel genes related to the process of neuroprotection. Bioengineering A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator Benjamin Kerzner1, Hannah L. Martin2, Michael Weiser1, Gianluca Fontana1, Nicholas Russell1, William L. Murphy1,2, Erik A. Lund3, Christopher J. Doro1 1Department of Orthopedics and Rehabilitation, University of Wisconsin-Madison, 2Department of Biomedical Engineering, University of Wisconsin-Madison, 3Summit Orthopedics In vivo mammalian models of critical-sized bone defects are essential for researchers studying healing mechanisms and orthopedic therapies. Here, we introduce a protocol for the creation of reproducible, segmental, femoral defects in rats stabilized using external fixation. Developmental Biology Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish Elaine L. Welch*1, Celeste C. Eno*1, Sreelaja Nair2, Robin E. Lindeman3, Francisco Pelegri1 1Laboratory of Genetics, University of Wisconsin-Madison, 2Department of Biological Sciences, Tata Institute of Fundamental Research, 3Department of Genetics, Cell Biology, and Development, University of Minnesota An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here. Bioengineering Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications Cameron J. Stewart1, Megan N. McClean1 1Department of Biomedical Engineering, University of Wisconsin-Madison We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days. Biology High-resolution Optical Mapping of the Mouse Sino-atrial Node Di Lang1, Alexey V. Glukhov1 1Department of Medicine, University of Wisconsin-Madison School of Medicine and Public Health Here, we present a protocol for optical mapping of electrical activity from the mouse right atrium and especially the sino-atrial node, at a high spatial and temporal resolution. Engineering Iridium Oxide-reduced Graphene Oxide Nanohybrid Thin Film Modified Screen-printed Electrodes as Disposable Electrochemical Paper Microfluidic pH Sensors Jiang Yang1,2, Tae-Joon Kwak3, Xiaodong Zhang4, Robert McClain5, Woo-Jin Chang*3,6, Sundaram Gunasekaran*1 1Department of Biological Systems Engineering, University of Wisconsin-Madison, 2Environment, Energy and Natural Resources Center, Department of Environmental Science and Engineering, Fudan University, 3Department of Mechanical Engineering, University of Wisconsin-Milwaukee, 4Department of Physics, School of Science, Tianjin University, 5Department of Chemistry, University of Wisconsin-Madison, 6School of Freshwater Sciences, University of Wisconsin-Milwaukee The study demonstrates the growth of iridium oxide-reduced graphene oxide (IrO2-RGO) nanohybrid thin films on irregular and rough screen-printed carbon substrate through a green electrochemical synthesis, and their implementation as a pH sensor with a patterned paper-fluidic platform. Neuroscience Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia Vishal Chanana1, Abdulfettah Tumturk2, Douglas Kintner1, Eshwar Udho1, Peter Ferrazzano1,3, Pelin Cengiz1,3 1Waisman Center, University of Wisconsin, 2Department of Neurological Surgery, University of Wisconsin, 3Department of Pediatrics, University of Wisconsin Astrocytes are one of the most important key players in the central nervous system (CNS). Here, we are reporting a practical method of sexed hippocampal astrocyte culture protocol in order to study the mechanisms underlying the astrocyte function in male and female neonate pups after in-vitro ischemia. Biochemistry Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes Chen Ji1, Xuelin Lou1 1Department of Neuroscience, University of Wisconsin-Madison PI(4,5)P2 regulates various cellular functions, but its nanoscale organization in the cell plasma membrane is poorly understood. By labeling PI(4,5)P2 with a dual-color fluorescent probe fused to the Pleckstrin Homology domain, we describe a novel approach to study the PI(4,5)P2 spatial distribution in the plasma membrane at the nanometer scale. Genetics Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts Keren B. Turton1, Stephane Esnault2, Larissa P. Delain2, Deane F. Mosher1,2 1Department of Biomolecular Chemistry, University of Wisconsin-Madison, 2Department of Medicine, University of Wisconsin-Madison Tandem splicing events occur at sites less than 12 nucleotides apart. Quantifying ratios of such splice variants is feasible using an absolute quantitative PCR approach. This manuscript describes how splice variants of the gene STAT3, in which two splicing events results in Serine-701 inclusion/exclusion and α/β C-termini, can be quantified. Bioengineering Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo Brian Burkel1,2, Brett A. Morris1, Suzanne M. Ponik1, Kristin M. Riching1, Kevin W. Eliceiri2,3,4, Patricia J. Keely1,2,5 1Department of Cell and Regenerative Biology, University of Wisconsin-Madison, 2Laboratory for Optical and Computational Instrumentation, University of Wisconsin-Madison, 3Department of Biomedical Engineering, University of Wisconsin-Madison, 4Morgridge Institute for Research, University of Wisconsin-Madison, 5Paul P. Carbone Comprehensive Cancer center, University of Wisconsin-Madison Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels. Immunology and Infection Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection Elizabeth A. Harvie1,2, Anna Huttenlocher2,3 1Microbiology Doctoral Training Program, University of Wisconsin-Madison, 2Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, 3Department of Pediatrics, University of Wisconsin-Madison Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle. Behavior Using the Threat Probability Task to Assess Anxiety and Fear During Uncertain and Certain Threat Daniel E. Bradford*1, Katherine P. Magruder*1, Rachel A. Korhumel1, John J. Curtin1 1Department of Psychology, University of Wisconsin-Madison Potentiation of the startle reflex is measured via electromyography of the orbicularis oculi muscle during low (uncertain) and high (certain) probability electric shock threat in the Threat Probability Task. This provides an objective measure of distinct negative emotional states (fear/anxiety) for research on psychopathology, substance use/abuse, and broad affective science. Environment Measurement of Greenhouse Gas Flux from Agricultural Soils Using Static Chambers Sarah M. Collier1, Matthew D. Ruark2, Lawrence G. Oates3,4, William E. Jokela5, Curtis J. Dell6 1Office of Sustainability, University of Wisconsin-Madison, 2Department of Soil Science, University of Wisconsin-Madison, 3Department of Agronomy, University of Wisconsin-Madison, 4Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, 5USDA-ARS Dairy Forage Research Center, 6USDA-ARS Pasture Systems Watershed Management Research Unit This article showcases the static chamber-based method for measurement of greenhouse gas flux from soil systems. With relatively modest infrastructure investments, measurements may be obtained from multiple treatments/locations and over timeframes ranging from hours to years. Biology A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination Joshua C. Neuman1, Nathan A. Truchan2, Jamie W. Joseph3, Michelle E. Kimple2 1Department of Nutrional Sciences, University of Wisconsin-Madison, 2Department of Medicine, Division of Endocrinology, Diabetes, and Metabolism, University of Wisconsin-Madison, 3School of Pharmacy, University of Waterloo Assaying in vitro β-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining β-cell function. Biology MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules Erin Gemperline1, Lingjun Li1,2 1Department of Chemistry, University of Wisconsin- Madison, 2School of Pharmacy, University of Wisconsin- Madison Mass spectrometric imaging (MSI) is a powerful tool that can be used to discover and identify various chemical species in intact tissues, preserving the compounds in their native environments, which can provide new insights into biological processes. Herein a MSI method developed for the analysis of small molecules is described. Biology An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila Daniel T. Babcock1, Barry Ganetzky1 1Department of Genetics, University of Wisconsin-Madison Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening. Medicine Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia Tristan M. Nicholson1,2, Kristen S. Uchtmann1, Conrad D. Valdez1, Ashleigh B. Theberge1,3, Tihomir Miralem1, William A. Ricke1 1Department of Urology, University of Wisconsin-Madison, 2Medical Scientist (MD/PhD) Training Program, University of Rochester School of Medicine & Dentistry, 3Molecular and Environmental Toxicology Center, University of Wisconsin-Madison We describe the manufacture of compressed hormone pellets, as well as subcutaneous surgical implantation into mice. This strategy can be combined with the growth of cell and tissue xenografts under the renal capsule of athymic nude mice to evaluate hormonal carcinogenesis and regulation of benign prostate growth.