University of Florida View Institution's Website 88 articles published in JoVE Medicine Suspension Culture Production and Purification of Adeno-Associated Virus by Iodixanol Density Gradient Centrifugation for In Vivo Applications Kathryn K. Harris1, Oleksandr Kondratov1, Sergei Zolotukhin1 1Department of Pediatrics, University of Florida Adeno-associated virus is produced in suspension cell culture and purified by double iodixanol density gradient centrifugation. Steps are included to increase total virus yield, decrease the risk of virus precipitation, and further concentrate the final virus product. Expected final titers reach 1012 viral particles/mL and are suitable for pre-clinical in vivo use. Biology CorrelationCalculator and Filigree: Tools for Data-Driven Network Analysis of Metabolomics Data Gayatri Iyer1, Marci Brandenburg1,2, Christopher Patsalis1, George Michailidis3, Alla Karnovsky1 1Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, 2Taubman Health Sciences Library, University of Michigan, Ann Arbor, 3Department of Statistics, University of Florida We present CorrelationCalculator and Filigree, two tools for data-driven network construction and analysis of metabolomics data. CorrelationCalculator supports building a single interaction network of metabolites based on expression data, while Filigree allows building a differential network, followed by network clustering and enrichment analysis. Biology Detached Maize Sheaths for Live-Cell Imaging of Infection by Fungal Foliar Maize Pathogens Renata Belisário1, Maria F. Torres1,2, Ester A. S. Buiate1,3, Katia V. Xavier1,4, Etta M. Nuckles1, Lisa J. Vaillancourt1 1Department of Plant Pathology, University of Kentucky, 2Department of Biological Sciences, University of Cincinnati, 3Bayer Crop Science, 4Everglades Research and Education Center, University of Florida This manuscript details an optimized inoculation protocol that uses detached maize leaf sheaths for reproducible cytological, physiological, and molecular studies of maize interactions with fungal plant pathogens. The leaf sheaths facilitate real-time observation of cellular interactions between the living plant and fungus in unfixed tissues. Biology Sieving Fruit Pulp to Detect Immature Tephritid Fruit Flies in the Field Amy L. Roda1, Gary Steck2, Thomas Fezza3, Todd Shelly3, Rita Duncan4, Nicholas Manoukis5, Lori Carvalho5, Abbie Fox6, Paul Kendra7, Daniel Carrillo4 1Animal Plant Health Inspection Service (APHIS), Plant Protection and Quarantine (PPQ), Science and Technology (S&T) Miami, United States Department of Agriculture (USDA), 2Division of Plant Industry, Florida Department of Agriculture and Consumer Services, 3APHIS, PPQ, S&T, USDA, 4IFAS, Tropical Research and Education Center, University of Florida, 5Agricultural Research Service (ARS), Pacific Basin Agricultural Research Center, USDA, 6APHIS, PPQ, Field Operations, USDA, 7ARS, Subtropical Horticulture Research Station, USDA Increasing the detection of immature tephritid fruit flies in the field can trigger timely efforts to eliminate populations of these destructive pests. Detecting late instar larvae is faster and more accurate when mushing host fruit in a bag and passing the pulp through a series of sieves than hand cutting and visual inspection. Biology Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a Min Li*1, Hongkun Qin*1, Yunfei Long1, Meiqin Cheng1, Ling Li1, Aijun Huang2, Nian Wang3, Shuo Duan1 1China-USA Citrus Huanglongbing Joint Laboratory, National Navel Orange Engineering Research Center, Gannan Normal University, 2Department of Biological Sciences, Gannan Normal University, 3Citrus Research and Education Center, Department of Microbiology and Cell Science, University of Florida - Institute of Food and Agricultural Sciences In this work, a rapid, sensitive, and portable detection method for Candidatus Liberibacter asiaticus based on recombinase polymerase amplification combined with CRISPR-Cas12a was developed. Biochemistry Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection Jonathan T. Specker1, Alexander B. Smith2, Orlaith Keenan2, Joseph P. Zackular2,3, Boone M. Prentice1 1Department of Chemistry, University of Florida, 2 A novel sample preparation method is demonstrated for the analysis of agar-based, bacterial macrocolonies via matrix-assisted laser desorption/ionization imaging mass spectrometry. Biology Deploying Community Scientists to Conduct Nondestructive Genetic Sampling of Rare Butterfly Populations Jaret C. Daniels1,2, Caroline G. Storer2, Geena M. Hill3, Amanda Markee2, Christian Couch2, Kristin A. Rossetti2 1Department of Entomology and Nematology, University of Florida, 2Florida Museum of Natural History, University of Florida, 3Florida Natural Areas Inventory Here, we present a straightforward protocol for noninvasive genetic sampling of butterfly populations based on the field collection of residual egg debris. It can be used to confirm species identity and quantify genetic variation. This protocol can be easily adapted to broader groups for community science involvement. Editorial Novel Methodological Perspectives In The Study Of Mosquito Biology Yuemei Dong1, Eric P. Caragata2 1W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Malaria Research Institute, Bloomberg School of Public Health, Johns Hopkins University, 2Department of Entomology & Nematology, Florida Medical Entomology Laboratory, Institute of Food and Agricultural Sciences, University of Florida Behavior Assessment of Nerve Injury-Induced Mechanical Hypersensitivity in Rats Using an Orofacial Operant Pain Assay Basak Donertas-Ayaz1, Ariana C. Brice-Tutt2, Wendi L. Malphurs2, Darrice Montgomery2, Richard H. Mills3, John K. Neubert2, Robert M. Caudle1 1Department of Oral and Maxillofacial Surgery, University of Florida, Gainesville, 2Department of Orthodontics, University of Florida, Gainesville, 3Stoelting Co. Wood Dale This protocol describes the assessment of mechanical hypersensitivity in a rat model of neuropathic orofacial pain using an operant-based orofacial pain assessment device. Biology Combining 3D Magnetic Force Actuator and Multi-Functional Fluorescence Imaging to Study Nucleus Mechanobiology Miao Huang1,5, Heyang Wang1,5, Alfredo A. Delgado1, Tyler A. Reid1, Julian Long2, Shu Wang3,5, Hayley Sussman4, Juan Guan5,6,7, Hitomi Yamaguchi1, Xin Tang1,5,8,9 1Department of Mechanical and Aerospace Engineering, Herbert Wertheim College of Engineering, University of Florida, 2Department of Materials Science and Engineering, University of Florida, 3Department of Biostatistics, University of Florida, 4Department of Biomedical Engineering, College of Engineering (COE), University of Delaware (UD), 5UF Health Cancer Center, University of Florida, 6Department of Physics, College of Liberal Arts and Sciences, University of Florida, 7Department of Anatomy and Cell Biology, College of Medicine, University of Florida, 8J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, 9Department of Physiology and Functional Genomics, University of Florida This study presents a new protocol to directly apply mechanical force on the cell nucleus through magnetic microbeads delivered into the cytoplasm and to conduct simultaneous live-cell fluorescent imaging. Medicine A Murine Model of Hemodialysis Access-Related Hand Dysfunction Kyoungrae Kim1, Erik M. Anderson2,4, Brian J. Fazzone2,4, Kerri A. O’Malley2,4, Scott A. Berceli2,4, Terence E. Ryan1,3, Salvatore T. Scali2,4 1Department of Applied Physiology and Kinesiology, University of Florida, 2Division of Vascular Surgery and Endovascular Therapy, University of Florida, 3Center for Exercise Science, University of Florida, 4Malcom Randall Veteran Affairs Medical Center This protocol details the surgical steps of murine common iliac arteriovenous fistula creation. We developed this model to study hemodialysis access-related limb pathophysiology. Biochemistry Ex Vivo Hepatic Perfusion Through the Portal Vein in Mouse Anthony G. Giacalone1, Matthew E. Merritt1, Mukundan Ragavan1 1Department of Biochemistry and Molecular Biology, University of Florida The protocol describes a straightforward method of resectioning an intact mouse liver for metabolic studies through portal vein perfusion. Bioengineering All-optical Mechanobiology Interrogation of Yes-associated Protein in Human Cancer and Normal Cells using a Multi-functional System Qin Luo*1, Miao Huang*2, Chenyu Liang2, Justin Zhang3, Gaoming Lin1, Sydney Yu1, Mai Tanaka4,5, Sharon Lepler4,5, Juan Guan5,6,7, Dietmar Siemann4,5, Xin Tang2,5 1Department of Electrical and Computer Engineering, Herbert Wertheim College of Engineering, University of Florida, 2Department of Mechanical and Aerospace Engineering, Herbert Wertheim College of Engineering, University of Florida, 3Department of Electrical and Computer Engineering, University of California, 4Department of Radiation Oncology, College of Medicine, University of Florida, 5UF Health Cancer Center, University of Florida, 6Department of Physics, College of Liberal Arts and Sciences, University of Florida, 7Department of Anatomy and Cell Biology, College of Medicine, University of Florida This paper presents a detailed stepwise protocol on how to utilize an integrated multi-functional and user-programmable system that enables automatic multi-channel imaging and mechanobiological analysis to elucidate the mechano-sensitivity of Yes-associated protein (YAP). Biology A Contrast of Three Inoculation Techniques used to Determine the Race of Unknown Fusarium oxysporum f.sp. niveum Isolates James C. Fulton*1, Matthew A. Cullen*1, Kristin Beckham1, Tatiana Sanchez2, Zhuxuan Xu1, Preston Stern1, Gary Vallad3, Geoffrey Meru4,5, Cecilia McGregor6, Nicholas S. Dufault1 1Department of Plant Pathology, University of Florida, 2University of Florida Institute of Food and Agricultural Sciences, 3Gulf Coast Research and Education Center, University of Florida, 4Horticultural Sciences Department, University of Florida, 5Tropical Research and Education Center, University of Florida, 6Department of Horticulture, University of Georgia Managing Fusarium wilt of watermelon requires knowledge of the pathogen races present. Here, we describe the root-dip, infested kernel seeding, and modified tray-dip inoculation methods to demonstrate their efficacy in race-typing of the pathogenic fungus Fusarium oxysporum f. sp niveum (Fon). Biology Automating Aggregate Quantification in Caenorhabditis elegans Alfonso S. Vaziriyan-Sani1, Robert D. Handy1, Alyssa C. Walker1, Carol Navya Pagolu1,2, Samantha M. Enslow1, Daniel M. Czyż1 1Department of Microbiology and Cell Science, University of Florida, 2Department of Computer and Information Science and Engineering, University of Florida The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis. Immunology and Infection Adapting Gastrointestinal Organoids for Pathogen Infection and Single Cell Sequencing under Biosafety Level 3 (BSL-3) Conditions Megan L. Stanifer1,2, Steeve Boulant2,3,4 1Department of Infectious Diseases, Molecular Virology, Heidelberg University Hospital, 2Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, 3Department of Infectious Diseases, Virology, Heidelberg University Hospital, 4Research Group “Cellular Polarity and Viral Infection”, German Cancer Research Center (DKFZ) This protocol describes how to infect human intestinal organoids from either their apical or basolateral side to characterize host/pathogen interactions at the single-cell level using single-cell RNA sequencing (scRNAseq) technology. Immunology and Infection Retrograde Parotid Gland Infusion through Stensen's Duct in a Non-Human Primate for Vectored Gene Delivery Guy El Helou1, Joseph F. Goodman2, Maria Blevins3, David L. Caudell4, Todd A. Ponzio3, John W. Sanders3,5 1Department of Medicine, Division of Infectious Diseases and Global Medicine, University of Florida, 2Department of Otolaryngology, George Washington University School of Medicine, 3Department of Medicine, Section on Infectious Diseases, Wake Forest Baptist Medical Center, 4Department of Pathology, Wake Forest School of Medicine, 5Department of Medicine, Section of Infectious Diseases, Hefner Veterans Affairs Medical Center Salivary glands have been proposed as a tissue target site for gene therapy, especially in the area of vaccination by gene transfer. We demonstrate gene delivery in a non-human primate model utilizing retrograde parotid infusion. Neuroscience A Plate-Based Assay for the Measurement of Endogenous Monoamine Release in Acute Brain Slices Jose A. Pino*1, Nora Awadallah*2, Alessandra M. Norris3, Gonzalo E. Torres2 1Department of Medicine, School of Medicine, Universidad de Atacama, 2Department of Molecular, Cellular, and Biomedical Sciences, City University of New York School of Medicine at City College, 3Department of Pharmacology and Therapeutics, University of Florida College of Medicine This method introduces a simple technique for the detection of endogenous monoamine release using acute brain slices. The setup uses a 48-well plate containing a tissue holder for monoamine release. Released monoamine is analyzed by HPLC coupled with electrochemical detection. Additionally, this technique provides a screening method for drug discovery. Medicine A Preclinical Model of Exertional Heat Stroke in Mice Michelle A. King1, Jamal M. Alzahrani1, Thomas L. Clanton1, Orlando Laitano1,2 1Department of Applied Physiology and Kinesiology, College of Health and Human Performance, University of Florida, 2Department of Nutrition and Integrative Physiology, College of Health and Human Sciences, Florida State University The protocol describes the development of a standardized, repeatable, preclinical model of exertional heat stroke (EHS) in mice free from adverse external stimuli such as electric shock. The model provides a platform for mechanistic, preventative, and therapeutic studies. Biology A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing Tse-Yu Chen1, Adam E. Vorsino2, Kyle J. Kosinski1, Ana L. Romero-Weaver1, Eva A. Buckner1, Joanna C. Chiu3, Yoosook Lee1 1Florida Medical Entomology Laboratory, Department of Entomology and Nematology, Institute of Food and Agricultural Sciences, University of Florida, 2U. S. Fish and Wildlife Service, Pacific Islands Fish and Wildlife Office, 3Department of Entomology and Nematology, University of California Davis Described here is a DNA extraction protocol using magnetic beads to produce high quality DNA extractions from mosquitoes. These extractions are suitable for a downstream next-generation sequencing approach. Immunology and Infection Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices Mollie K. Huber1, Denise M. Drotar2,3,4, Helmut Hiller1, Maria L. Beery1, Paul Joseph1, Irina Kusmartseva1, Stephan Speier2,3,4, Mark A. Atkinson1, Clayton E. Mathews1, Edward A. Phelps5 1Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, 2Paul Langerhans Institute Dresden (PLID) of the Helmholtz Zentrum München at the University Clinic Carl Gustav Carus of Technische Universität Dresden, 3Institute of Physiology, Faculty of Medicine, Technische Universität Dresden, 4German Center for Diabetes Research (DZD), 5J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida This study presents the application of live pancreatic tissue slices to the study of islet physiology and islet-immune cell interactions. Biology Preparing Irradiated and Marked Male Aedes aegypti Mosquitoes for Release in an Operational Sterile Insect Technique Program Bianca J. Moreno1, Robert L. Aldridge1, Seth C. Britch1, Barbara E. Bayer1, Jedidiah Kline1, Daniel A. Hahn2, Chao Chen2, Kenneth J. Linthicum1 1US Department of Agriculture, Agricultural Research Service Center for Medical, Agricultural, & Veterinary Entomology, 2Department of Entomology and Nematology, University of Florida The sterile insect technique (SIT) is used to control specific, medically important mosquito populations that may be resistant to chemical controls. Here, we describe a method of mass rearing and preparation of sterile male mosquitoes for release in an operational SIT program targeting the Aedes aegypti mosquito. Biology Field-Based Thermal Physiology Assay: Cold Shock Recovery under Ambient Conditions Emily S. Khazan1 1School of Natural Resources and Environment, University of Florida Here, a low-cost, accessible protocol is described to evaluate cold shock recovery of butterflies under ambient environmental conditions. Neuroscience Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish Elias T. Lunsford1, James C. Liao1 1Department of Biology, University of Florida, The Whitney Laboratory for Marine Bioscience We describe a protocol to monitor changes in the afferent neuron activity during motor commands in a model vertebrate hair cell system. Genetics Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator Kayli Sieber1, Maya Saar1, Comzit Opachaloemphan2, Matthew Gallitto3, Huan Yang2, Hua Yan1,4 1Department of Biology, University of Florida, 2Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, 3Department of Radiation Oncology, Columbia University Medical Center, 4Center for Smell and Taste, University of Florida Many characteristics of insect eusociality rely on within-colony communication and division of labor. Genetic manipulation of key regulatory genes in ant embryos via microinjection and CRISPR-mediated mutagenesis provides insights into the nature of altruistic behavior in eusocial insects. Neuroscience Low-intensity Blast Wave Model for Preclinical Assessment of Closed-head Mild Traumatic Brain Injury in Rodents Aric F. Logsdon*1,2, Brandon P. Lucke-Wold*3, Ryan C. Turner*4, Sean M. Collins5, Evan L. Reeder5, Jason D. Huber4, Charles L. Rosen6, Matthew J. Robson*5, Florian Plattner*7 1Geriatrics Research Education and Clinical Center, Veterans Affairs, 2Division of Gerontology and Geriatric Medicine, University of Washington, 3Department of Neurosurgery, University of Florida, 4Department of Neurosurgery, West Virginia University, 5Division of Pharmaceutical Sciences, University of Cincinnati, 6Central Illinois Neuro Health Sciences, Bloomington, IL, 7Neuro-research, Dallas, TX We present here a protocol of a blast wave model for rodents to investigate neurobiological and pathophysiological effects of mild to moderate traumatic brain injury. We established a gas-driven, bench-top setup equipped with pressure sensors allowing for reliable and reproducible generation of blast-induced mild to moderate traumatic brain injury. Environment Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification Owen Hudson1, Sumyya Waliullah1, Justin Hand2, Romina Gazis-Seregina3, Fulya Baysal-Gurel4, Md Emran Ali1 1Department of Plant Pathology, Coastal Plain Experiment Station, University of Georgia, 2University of Georgia Cooperative Extension, 3Department of Plant Pathology, Tropical Research & Education Center, University of Florida, 4Department of Agricultural and Environmental Sciences, Otis L. Floyd Nursery Research Center, Tennessee State University We developed a method to detect Phytophthora capsici zoospores in water sources using a filter paper DNA extraction method coupled with a loop-mediated isothermal amplification (LAMP) assay that can be analyzed in the field or in the lab. Biochemistry TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis Silveli Suzuki-Hatano1, Ang-Chen Tsai1, Audrey Daugherty1, Christina A. Pacak1 1Department of Pediatrics, University of Florida We present an optimized tandem mass tag (TMT) labeling protocol that includes detailed information for each of the following steps: protein extraction, quantification, precipitation, digestion, labeling, submission to a proteomics facility, and data analyses. Immunology and Infection Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry Jasmine L. Madrigal1, Melissa K. Jones1 1Microbiology and Cell Science Department, University of Florida The goal of this protocol is to quantify binding of the eukaryotic pathogen human norovirus to bacteria. After performing an initial virus-bacterium attachment assay, flow cytometry is used to detect virally-bound bacteria within the population. Biology At-Risk Butterfly Captive Propagation Programs to Enhance Life History Knowledge and Effective Ex Situ Conservation Techniques Jaret C. Daniels1,2, Geena M. Hill2,3, Kristin A. Rossetti2, Stephanie J. Sanchez2, Jacob A. Hornfeldt1 1Department of Entomology and Nematology, University of Florida, 2Florida Museum of Natural History, University of Florida, 3School of Natural Resources and Environment, University of Florida Here, we present protocols for 1) the laboratory captive propagation of the federally endangered Miami blue butterfly (Cyclargus thomasi bethunebakeri), and 2) assessing basic life history information such as immature development time and number of larval stadia. Both methods can be adapted for use with other ex situ conservation programs. Medicine Updated Technique for Reliable, Easy, and Tolerated Transcranial Electrical Stimulation Including Transcranial Direct Current Stimulation Helen Borges1, Alexandra Dufau1,2, Bhaskar Paneri1, Adam J. Woods3, Helena Knotkova4,5, Marom Bikson1 1Department of Biomedical Engineering, The City College of New York, CUNY, 2Department of Clinical and Health Psychology, Center for Cognitive Aging and Memory, 3McKnight Brain Institute, University of Florida, 4MJHS Institute for Innovation in Palliative Care, 5Department of Family and Social Medicine, Albert Einstein College of Medicine When administering transcranial direct current stimulation (tDCS), reproducible electrode preparation and placement are vital for a tolerated and effective session. The purpose of this article is to demonstrate updated modern setup procedures for the administration of tDCS and related transcranial electrical stimulation techniques, such as transcranial alternating current stimulation (tACS). Biochemistry Rapid Collection of Floral Fragrance Volatiles using a Headspace Volatile Collection Technique for GC-MS Thermal Desorption Sampling Haleigh A. Ray1, Charles J. Stuhl2, Jennifer L. Gillett-Kaufman1 1Entomology and Nematology Department, University of Florida, 2Center for Medical, Agricultural and Veterinary Entomology, Agricultural Research Service U.S. Department of Agriculture Here, we present a protocol for collecting the floral fragrance volatiles from blooming flowers, using a non-destructive sampling procedure. Medicine Porcine Model of Infrarenal Abdominal Aortic Aneurysm Alexander H. Shannon1, J. Michael Cullen1, Jolian J. Dahl1, Erik J. Scott1, Zachary Tyerman1, Michael D. Spinosa1, William G. Montgomery1, W. Forrest Johnston2, Guanyi Lu3, Morgan Salmon*1, Gorav Ailawadi*1,4, Gilbert R. Upchurch Jr.*3 1Department of Surgery, University of Virginia School of Medicine, 2Department of Surgery, Ochsner Medical Center, 3Department of Surgery, University of Florida, 4The Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine This novel model creates robust infrarenal abdominal aortic aneurysms in swine using a combination of balloon angioplasty, elastase/collagenase perfusion, topical elastase application, and oral compound β-aminopropionitrile administration, which interferes with collagen cross-linking. Medicine Murine Surgical Model of Topical Elastase Induced Descending Thoracic Aortic Aneurysm Zachary Tyerman1, Jolian Dahl1, Alexander Shannon1, W. Forrest Johnston2, Nicholas H. Pope1, Guanyi Lu3, Gilbert R. Upchurch Jr.3, Gorav Ailawadi1,4, Morgan Salmon1 1Department of Surgery, University of Virginia School of Medicine, 2Department of Surgery, Ochsner Medical Center, 3Department of Surgery, University of Florida, 4The Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine We describe a surgical protocol to consistently induce robust descending thoracic aortic aneurysms in mice. The procedure involves left thoracotomy, thoracic aorta exposure, and placement of a sponge soaked in porcine pancreatic elastase on the aortic wall. Environment Sampling for Estimating Frankliniella Species Flower Thrips and Orius Species Predators in Field Experiments Joseph Funderburk1, Xavier Martini1, Josh Freeman1, Iris Strzyzewski1, Edward Traczyk1, Thomas Skarlinsky2, Scott Adkins3 1North Florida Research and Education Center, University of Florida, 2Animal and Plant Health Inspection Services, Plant Protection Quarantine, Miami Plant Inspection Station, United States Department of Agriculture, 3Agricultural Research Service, US Horticultural Research Laboratory, United States Department of Agriculture Presented here is a protocol to determine the number of thrips and minute pirate bug predators in crops over multiple dates in field experiments. Also illustrated is how to determine the efficacy of management tactics against thrips and evaluate the benefits of predation by minute pirate bugs. Environment Manipulation of Color Patterns in Jumping Spiders for Use in Behavioral Experiments Malika Ihle1, Lisa Anne Taylor1,2 1Department of Entomology and Nematology, University of Florida, 2Florida Museum of Natural History, University of Florida The goal of this protocol is to manipulate the color patterns of jumping spiders and other very small arthropods with paint in order to study questions related to sexual selection, sexual cannibalism, predation, aposematism, or any other field of animal coloration. Genetics HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries Huacheng Luo1, Amin Sobh2, Christopher D. Vulpe2, Edmond Brewer1, Sinisa Dovat1, Yi Qiu3, Suming Huang1 1Department of Pediatrics, Pennsylvania State University College of Medicine, 2Department of Physiological Sciences, University of Florida, 3Department of Anatomy and Cell Biology, University of Florida A CRISPR/sgRNA library has been applied to interrogating protein-coding genes.However, the feasibility of a sgRNA library to uncover the function of a CTCF boundary in gene regulation remains unexplored. Here, we describe a HOX loci specific sgRNA library to elucidate the function of CTCF boundaries in HOX loci. Bioengineering Construction and Use of an Electrical Stimulation Chamber for Enhancing Osteogenic Differentiation in Mesenchymal Stem/Stromal Cells In Vitro Liudmila Leppik1, Mit B. Bhavsar1, Karla M.C. Oliveira1, Maria Eischen-Loges1, Sahba Mobini1,2, John H. Barker1 1Frankfurt Initiative for Regenerative Medicine, Johann Wolfgang Goethe-University, 2J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida Here we present a protocol for the construction of a cell culture chamber designed to expose cells to various types of electrical stimulation, and its use in treating mesenchymal stem cells to enhance osteogenic differentiation. Immunology and Infection A Controlled Mouse Model for Neonatal Polymicrobial Sepsis Byron Brook*1, Nelly Amenyogbe*1, Rym Ben-Othman2, Bing Cai2, Danny Harbeson1, Freddy Francis1, Aaron C. Liu1, Natallia Varankovich2, James Wynn3,4, Tobias R. Kollmann1,2 1Department of Experimental Medicine, University of British Columbia, 2Department of Pediatrics, Division of Infectious Diseases, University of British Columbia, 3Department of Pediatrics, College of Medicine, University of Florida, 4Department of Pathology, Immunology, and Laboratory Medicine, University of Florida This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice. Biology Reverse Dissection and DiceCT Reveal Otherwise Hidden Data in the Evolution of the Primate Face Anne M. Burrows1,2, Kailey M Omstead1, Ashley R Deutsch3,5, Justin T. Gladman4, Adam Hartstone-Rose3 1Department of Physical Therapy, Duquesne University, 2Department of Anthropology, University of Pittsburgh, 3College of Sciences, North Carolina State University, 4Shared Materials Instrumentation Facility, Duke University, 5Department of Anthropology, University of Florida Facial expressions are a mode of visual communication produced by mimetic muscles. Here, we present protocols for the novel techniques of reverse dissection and DiceCT to fully visualize and assess mimetic muscles. These combined techniques can examine both morphological and physiological aspects of mimetic musculature to determine functional aspects. Neuroscience Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster Daniel R. Swale1, Aaron D. Gross2, Quentin R. R. Coquerel3, Jeffrey R. Bloomquist3 1Department of Entomology, Louisiana State University AgCenter, 2Department of Entomology, Virginia Tech, 3Department of Entomology and Nematology, Emerging Pathogens Institute, University of Florida This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways. Biochemistry A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton Qiao Zhang1, Andrew C. Tamashunas1, Tanmay P. Lele1 1Department of Chemical Engineering, University of Florida In this protocol, we describe a micropipette method to directly apply a controlled force to the nucleus in a living cell. This assay allows interrogation of nuclear mechanical properties in the living, adherent cell. Biology Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation Sheldon Lawrence II1,2,3, Quiying Pang2,3,4, Wenwen Kong2,3, Sixue Chen1,2,3,5 1Plant Molecular and Cellular Biology Program, University of Florida, 2Department of Biology, University of Florida, 3Genetics Institute (UFGI), University of Florida, 4Alkali Soil Natural Environmental Science Center, Northeast Forestry University, 5Interdisciplinary Center for Biotechnology Research (ICBR), University of Florida This protocol describes a method of preparing enriched stomatal guard cells that is useful for physiological and other biological studies. Biochemistry Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1 Ozlem Yaren1, Barry W. Alto2, Kevin M. Bradley3, Patricia Moussatche3, Lyudmyla Glushakova3, Steven A. Benner1,3 1Foundation for Applied Molecular Evolution (FfAME), 2Florida Medical Entomology Laboratory, University of Florida, 3Firebird Biomolecular Sciences LLC Current multiplexed diagnostics to detect Zika, chikungunya, and dengue viruses require complex sample preparation and expensive instrumentation, and are difficult to use in low resource environments. We show a diagnostic that uses isothermal amplification with target-specific strand displaceable probes to detect and differentiate these viruses with high sensitivity and specificity. Bioengineering High Resolution 3D Imaging of the Human Pancreas Neuro-insular Network Elizabeth Butterworth1, Wesley Dickerson2, Vindhya Vijay3, Kristina Weitzel1, Julia Cooper1, Eric W. Atkinson4, Jason E. Coleman5, Kevin J. Otto4, Martha Campbell-Thompson1 1Department of Pathology, Immunology and Experimental Medicine, University of Florida, 2Heller School for Social Policy and Management, Brandeis University, 3Department of Medicine, College of Medicine, University of Florida, 4Department of Biomedical Engineering, College of Engineering, University of Florida, 5Department of Pediatrics, College of Medicine, University of Florida Here, we present a protocol to image human pancreas sections in three dimensions (3D) using optimized passive clearing methods. This manuscript demonstrates these procedures for passive optical clearing followed by multiple immunofluorescence staining to identify key elements of the autonomic and sensory neural networks innervating human islets. Environment Capturing Flow-weighted Water and Suspended Particulates from Agricultural Canals During Drainage Events Jehangir H. Bhadha1, Anne Sexton1, Timothy A. Lang2, Samira H. Daroub1 1Soil and Water Sciences Department, University of Florida, 2Everglades Research and Education Center, University of Florida Nutrients present in particulate form can contribute significantly to the overall loads in agricultural drainage waters. This study describes a novel method to capture flow-weighted water and suspended particulates from farm canal drainage over the entire duration of the drainage event. Bioengineering Metabolic Support of Excised, Living Brain Tissues During Magnetic Resonance Microscopy Acquisition Jeremy J. Flint1,2, Kannan Menon2,3, Brian Hansen4, John Forder2,3,5, Stephen J. Blackband1,2,6 1Department of Neuroscience, University of Florida, 2McKnight Brain Institute, University of Florida, 3Department of Biomedical Engineering, University of Florida, 4Center for Functionally Integrative Neuroscience, Aarhus University, 5Department of Radiology, University of Florida, 6National High Magnetic Field Laboratory, Florida State University The current protocol describes a method by which users can maintain viability of acute hippocampal and cortical slice preparations during the collection of magnetic resonance microscopy data. Environment Controlled-release of Chlorine Dioxide in a Perforated Packaging System to Extend the Storage Life and Improve the Safety of Grape Tomatoes Xiuxiu Sun1,2, Elizabeth Baldwin1, Anne Plotto1, Jan Narciso1, Christopher Ference1, Mark Ritenour2, Ken Harrison3, Joseph Gangemi3, Jinhe Bai1 1USDA, ARS, USHRL, 2Indian River Research and Education Center, University of Florida, 3Worrell Water Technologies, LLC Here, we describe a protocol for the application of a novel, slow-release ClO2 product that reduces spoilage and extends the shelf life of fresh fruit. The slow-release ClO2 product was added to standard commercial grape tomato packaging and tested against Escherichia coli and Alternaria alternata. Environment A Method for Evaluating Insecticide Efficacy against Bed Bug, Cimex lectularius, Eggs and First Instars Brittany E. Campbell1, Dini M. Miller2 1Department of Entomology and Nematology, University of Florida, 2Department of Entomology, Virginia Tech Insecticide evaluations are often targeted against adult insects, rather than immature stages. Here, we present a protocol for evaluating insecticides against bed bug eggs with a comparison to the first nymphal bed bug stage. These protocols could be adjusted for other insects to evaluate insecticide efficacy in non-adult life stages. Cancer Research Endobronchial Ultrasound-guided Intratumoral Injection of Cisplatin for the Treatment of Isolated Mediastinal Recurrence of Lung Cancer Hiren J. Mehta1, Michael A. Jantz1 1Division of Pulmonary/Critical care/Sleep Medicine, University of Florida College of Medicine The management of isolated recurrent lung cancer in a previously-irradiated field is challenging. Here, we describe an endobronchial ultrasound (EBUS)-guided cisplatin injection for the management of patients with localized lung cancer recurrence in a previously-radiated field. Environment Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays Alvine C. Mehinto1, B. Sumith Jayasinghe2, Darcy R. Vandervort1, Nancy D. Denslow2, Keith A. Maruya1 1Southern California Coastal Water Research Project Authority, 2Department of Physiological Sciences, University of Florida A protocol to screen for endocrine activity in organic extracts of water samples, including treated wastewater effluent and surface (receiving) water, was adapted using commercially available division-arrested ("freeze and thaw") in vitro transactivation bioassays. Medicine A Repetitive Concussive Head Injury Model in Mice Zhihui Yang1,2, Fan Lin1,2, Amanda S. Weissman1,2, Emily Jaalouk1,2, Qing-shan Xue3, Kevin K.W. Wang1,2 1Program for Neurotrauma, Neuroproteomics & Biomarkers Research, University of Florida, 2Department of Psychiatry, University of Florida, 3Department of Neuroscience, University of Florida Concussion presents the most common type of traumatic brain injury. Therefore, a repetitive concussive animal model, which replicates the important features of an injury in patients, may provide a means to study concussion in a rigorous, controlled, and efficient manner. Environment A Push-pull Protocol to Reduce Colonization of Bird Nest Boxes by Honey Bees Caroline A. Efstathion1, William H. Kern, Jr.1 1Department of Entomology and Nematology, University of Florida Preventing colonization of bird nest boxes by invasive Africanized honey bees is important for conservation efforts of nest-site limited birds. We provide an integrated pest management approach to "push" bees away from nest boxes with a repellant insecticide, permethrin, and "pull" them toward pheromone baited swarm traps. Medicine Murine Model of Intestinal Ischemia-reperfusion Injury Ekaterina O. Gubernatorova1,2, Ernesto Perez-Chanona3, Ekaterina P. Koroleva1, Christian Jobin3, Alexei V. Tumanov1,2 1Trudeau Institute, 2Engelhardt Institute of Molecular Biology, 3Departments of Medicine and Infectious Diseases and Pathology, University of Florida Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration. Developmental Biology Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures Heather H. Ross1, Milap S. Sandhu1, Sharareh Sharififar1, David D. Fuller1 1Department of Physical Therapy, University of Florida This article describes the methodology for administering short periods of intermittent hypoxia to postnatal day 1-8 mouse or rat pups. This approach effectively elicits a robust tissue level “priming effect” on cultured neural progenitor cells that are harvested within 30 min of hypoxia exposure. Medicine Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model Dibash K. Das1,2, Michelle K. Naidoo1, Adeodat Ilboudo1, Pascal DuBois1, Victoria Durojaiye1, Chen Liu3, Olorunseun O. Ogunwobi1,2 1Department of Biological Sciences, Hunter College of The City University of New York, 2Departments of Biology and Biochemistry, The Graduate Center of The City University of New York, 3Department of Pathology, Immunology, and Laboratory Medicine, University of Florida Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described. Medicine Minimally Invasive Thumb-sized Pterional Craniotomy for Surgical Clip Ligation of Unruptured Anterior Circulation Aneurysms Eric M Deshaies1,2, Mark R Villwock1, Amit Singla3, Gentian Toshkezi4, David J Padalino1 1Neurovascular and Stroke Center, Crouse Neuroscience Institute, 2Department of Biology, Syracuse University, 3Department of Neurosurgery, University of Florida, 4Department of Neurosurgery, SUNY Upstate Medical University Minimally invasive thumb-sized pterional craniotomy for aneurysm clipping has afforded our patients with a shorter hospital stay at a lower cost compared to the national average. Neuroscience An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents Joy A. Franco1, Heidi E. Kloefkorn2, Shawn Hochman3, Katherine A. Wilkinson4 1Department of Mechanical Engineering, San José State University, 2J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, 3Department of Physiology, Emory University School of Medicine, 4Department of Biological Sciences, San José State University Muscle sensory neurons are involved in proprioceptor signaling and also report on metabolic state and injury related events. We describe an adult mouse in vitro muscle-nerve preparation for studies on stretch-activated muscle afferents. Neuroscience Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction Ji-Yoen Kim*1, Stacy D. Grunke*1, Yona Levites2, Todd E. Golde2, Joanna L. Jankowsky1,3 1Department of Neuroscience, Baylor College of Medicine, 2Department of Neuroscience, Center for Translational Research in Neurodegenerative Disease, University of Florida, 3Department of Neurology and Department of Neurosurgery, Baylor College of Medicine Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes. Environment Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water Bledar Bisha1, Jaclyn A. Adkins2, Jana C. Jokerst3, Jeffrey C. Chandler1, Alma Pérez-Méndez4, Shannon M. Coleman4, Adrian O. Sbodio5, Trevor V. Suslow5, Michelle D. Danyluk6, Charles S. Henry2, Lawrence D. Goodridge7 1Department of Animal Science, University of Wyoming, 2Department of Chemistry, Colorado State University, 3Department of Environmental and Radiological Health Sciences, Colorado State University, 4Department of Animal Sciences, Colorado State University, 5Department of Plant Sciences, University of California, Davis, 6Department of Food Science and Human Nutrition, Citrus Research and Education Center, University of Florida, 7Department of Food Science and Agricultural Chemistry, McGill University A protocol involving integrated concentration, enrichment, and end-point colorimetric detection of foodborne pathogens in large volumes of agricultural water is presented here. Water is filtered through Modified Moore Swabs (MMS), enriched with selective or non-selective media, and detection is performed using paper-based analytical devices (µPAD) imbedded with bacterial-indicative colorimetric substrates. Biology Transmitting Plant Viruses Using Whiteflies Jane E. Polston1, H. Capobianco1 1Deptartment of Plant Pathology, University of Florida Improving our understanding of and our ability to manage many of the insect-transmitted plant viruses requires the use of the vector. Insect transmission of plant viruses is a tritrophic interaction, and as such requires the manipulation of insects, virus, and plant. Vectors must be reared in large numbers and manipulated in such a way as to insure high rates of transmission to test plants. The basics of rearing and manipulating the whitefly, Bemisa tabaci (Hemiptera: Aleyrodidae), a vector of plant viruses belonging to several emerging plant virus genera, representing a large number of economically significant viruses is presented. Bioengineering Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves Sharan Ramaswamy1, Manuel Salinas1, Rob Carrol2, Karla Landaburo1, Xavier Ryans1, Cynthia Crespo1, Ailyn Rivero1, Faris Al-Mousily3,4, Curt DeGroff3, Mark Bleiweis3, Hitomi Yamaguchi2 1Tissue Engineered Mechanics, Imaging and Materials Laboratory, Department of Biomedical Engineering, Florida International University, 2Department of Mechanical and Aerospace Engineering, University of Florida, 3College of Medicine, University of Florida, 4King Faisal Specialty Hospital and Research Center, Jeddah, Saudi Arabia There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions. Immunology and Infection Isolation and Th17 Differentiation of Naïve CD4 T Lymphocytes Simone K. Bedoya*1, Tenisha D. Wilson*1, Erin L. Collins1, Kenneth Lau1, Joseph Larkin III1 1Department of Microbiology and Cell Science, The University of Florida With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether naïve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response. Behavior Assessing Forelimb Function after Unilateral Cervical SCI using Novel Tasks: Limb Step-alternation, Postural Instability and Pasta Handling Zin Z. Khaing*1, Sydney A. Geissler*1, Timothy Schallert2, Christine E. Schmidt1,3 1Department of Biomedical Engineering, The University of Texas at Austin, 2Department of Psychology, The University of Texas at Austin, 3The J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida Three new behavioral tests (forelimb step-alternation, postural instability test, pasta handling test) for evaluating forelimb function after cervical spinal cord injury in rodents are described. Behavior Use of the Operant Orofacial Pain Assessment Device (OPAD) to Measure Changes in Nociceptive Behavior Ethan M. Anderson1,2, Richard Mills3, Todd A. Nolan4, Alan C. Jenkins4, Golam Mustafa1, Chris Lloyd3, Robert M. Caudle1,2, John K. Neubert4 1Department of Oral and Maxillofacial Surgery, University of Florida College of Dentistry, 2Department of Neuroscience, McKnight Brain Institute, University of Florida College of Medicine, 3Stoelting Co., 4Department of Orthodontics, University of Florida We present a user-friendly, high-throughput operant system for the evaluation of pain behaviors in awake, conscious rodents. The Orofacial Pain Assessment Device (OPAD) can assess pain through a reward/conflict paradigm thus providing a more humane way of testing. This protocol will yield more clinically relevant and translational data from rodents. Biology Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature Andrew J. McElrone1,2, Brendan Choat3, Dilworth Y. Parkinson4, Alastair A. MacDowell4, Craig R. Brodersen5 1U.S. Department of Agriculture, 2Department of Viticulture and Enology, University of California - Davis, 3Hawkesbury Institute for the Environment, University of Western Sydney, 4Advanced Light Source, Lawrence Berkeley National Lab, 5Citrus Research & Education Center, University of Florida High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species. Biology Purification of Transcripts and Metabolites from Drosophila Heads Kurt Jensen1, Jonatan Sanchez-Garcia1, Caroline Williams2, Swati Khare1, Krishanu Mathur1, Rita M. Graze3, Daniel A. Hahn2, Lauren M. McIntyre3, Diego E. Rincon-Limas1,4, Pedro Fernandez-Funez1,4 1Department of Neurology, McKnight Brain Institute, University of Florida, 2Department of Entomology and Nematology, University of Florida, 3Genetics Institute, Department of Molecular Genetics and Microbiology, University of Florida, 4McKnight Brain Institute, Department of Neuroscience, Genetics Institute, Center for Translational Research on Neurodegenerative Diseases, and Center for Movement Disorders and Neurorestoration, University of Florida We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects. Medicine Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion Robert W. Regenhardt1, Saeed Ansari2, Hassan Azari3, Kenneth J. Caldwell2, Adam P. Mecca1 1Department of Physiology and Functional Genomics, University of Florida, 2Department of Neurosurgery, McKnight Brain Institute, University of Florida, 3Department of Anatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo. Medicine Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat Saeed Ansari1, Hassan Azari2, Kenneth J. Caldwell1, Robert W. Regenhardt3, Vishnumurthy S. Hedna4, Micheal F. Waters4, Brian L. Hoh1, Adam P. Mecca3 1Department of Neurosurgery, University of Florida, 2Department of Anatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran, 3Department of Physiology and Functional Genomics, University of Florida, 4Department of Neurology, University of Florida Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed. Neuroscience Vertical T-maze Choice Assay for Arthropod Response to Odorants Lukasz Stelinski1, Siddharth Tiwari1 1Entomology and Nematology Department, University of Florida A vertical, T-maze olfactometer is described for assaying the behavioral response of arthropods. The olfactometer allows the experimenter to measure choices performed by test subjects when subjected to two potential odor fields. Both attraction to and repulsion from odorants can be measured with this device. Neuroscience Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry Kaustuv Saha1, Jarod Swant1, Habibeh Khoshbouei1,2 1Department of Neuroscience, University of Florida, 2Department of Psychiatry, University of Florida The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine. Medicine Staining Protocols for Human Pancreatic Islets Martha L. Campbell-Thompson1, Tiffany Heiple1, Emily Montgomery1, Li Zhang1, Lynda Schneider1 1Department of Pathology, Immunology, and Laboratory Medicine, University of Florida This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide. Medicine Collection Protocol for Human Pancreas Martha L. Campbell-Thompson1, Emily L. Montgomery1, Robin M. Foss1, Kerwin M. Kolheffer1, Gerald Phipps1, Lynda Schneider1, Mark A. Atkinson1 1Department of Pathology, Immunology, and Laboratory Medicine, University of Florida This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density. Biology GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil Chao Liang1,2, Harry W. Read2, Teri C. Balser2,3 1DOE-Great Lakes Bioenergy Research Center, University of Wisconsin, Madison, 2Department of Soil Science, University of Wisconsin, Madison, 3Department of Soil and Water Science, University of Florida We describe a method protocol for the GC-based analysis of the aldonitrile acetate derivatives of glucosamine and muramic acid extracted from soil. For elucidation of the chemical mechanism, we also present a strategy to confirm the structure of the derivative and the ion fragments formed upon electron ionization. Medicine Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE) Loic P. Deleyrolle1, Mark R. Rohaus1, Jeff M. Fortin1, Brent A. Reynolds1, Hassan Azari1,2 1Department of Neurosurgery, The University of Florida, 2Department of Anatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division. Neuroscience The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry Hassan Azari1,2, Sharareh Sharififar1, Jeff M. Fortin1, Brent A. Reynolds1 1Department of Neurosurgery, The University of Florida, 2Laboratory for Stem Cell Research, Department of Anatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology. Biology Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry Jennifer Parker1, Ning Zhu2, Mengmeng Zhu2, Sixue Chen1,2,3,4 1Plant Molecular and Cellular Biology Program, University of Florida, 2Department of Biology, University of Florida, 3Interdisciplinary Center for Biotechnology Research, University of Florida, 4Genetics Institute, University of Florida Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves. Biology Pouring and Running a Protein Gel by reusing Commercial Cassettes Alexander C. Hwang1, Paris H. Grey1, Katrina Cuddy1, David G. Oppenheimer1,2,3 1Department of Biology, University of Florida, 2UF Genetics Institute, University of Florida, 3Plant Molecular & Cellular Biology Program, University of Florida Our protocol demonstrates how to pour multiple protein gels at a time by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4°C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment. Neuroscience Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay Hassan Azari1,2, Sebastien Millette1, Saeed Ansari1, Maryam Rahman1, Loic P. Deleyrolle1, Brent A. Reynolds1 1Department of Neurosurgery, University of Florida, 2Department of Anatomical Sciences, Shiraz University of Medical Sciences This video protocol demonstrates the isolation and expansion of stem like cells from surgically resected human glioblastoma mutliforme (GBM) tumor tissue using the neurosphere assay culture method. Immunology and Infection Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes Jing Chen1, Scott Grieshaber1, Clayton E. Mathews1 1Departments of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail. Biology High-throughput Screening and Biosensing with Fluorescent C. elegans Strains Chi K. Leung1, Andrew Deonarine1, Kevin Strange2, Keith P. Choe1 1Department of Biology, University of Florida, 2Mount Desert Island Biological Laboratory A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants. Medicine Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice Saeed Ansari*1, Hassan Azari*1,2, Douglas J. McConnell1, Aqeela Afzal1, J Mocco1 1Department of Neurosurgery, University of Florida, 2Department of Anatomical Sciences, Shiraz University of Medical Sciences The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results. Medicine High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors Chen Ling1, Yuan Lu1, Binbin Cheng1, Katherine E. McGoogan1, Samantha W.Y. Gee1, Wenqin Ma1, Baozheng Li1, George V. Aslanidi1, Arun Srivastava1 1Department of Pediatrics, Division of Cellular and Molecular Therapy, University of Florida In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells. Neuroscience Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells Hassan Azari1,2, Sharon A. Louis3, Sharareh Sharififar1, Vinata Vedam-Mai1, Brent A. Reynolds1 1Department of Neurosurgery, University of Florida, 2Department of Anatomical Sciences, Shiraz University of Medical Sciences, 3STEMCELL Technologies, Inc. This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay. Neuroscience Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay Hassan Azari1,2, Sharareh Sharififar2, Maryam Rahman2, Saeed Ansari2, Brent A. Reynolds2 1Department of Anatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran, 2Department of Neurosurgery, The University of Florida This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain. Neuroscience Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay Hassan Azari1,2, Maryam Rahman2, Sharareh Sharififar2, Brent A. Reynolds2 1 Department of Anatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran, 2Department of Neurosurgery, University of Florida This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. Biology Cargo Loading onto Kinesin Powered Molecular Shuttles Yolaine Jeune-Smith1, Ashutosh Agarwal2, Henry Hess2 1Department of Materials Science and Engineering, University of Florida, 2Department of Biomedical Engineering, Columbia University Molecular shuttles consisting of functionalized microtubules gliding on surface-adhered kinesin motor proteins can serve as a nanoscale transport system. Here, the assembly of a typical shuttle system is described. Neuroscience The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice Kelly A. Tennant1, Aaron L. Asay2, Rachel P. Allred3, Angela R. Ozburn4, Jeffrey A. Kleim5, Theresa A. Jones1,2 1Institute for Neuroscience, University of Texas at Austin, 2Department of Psychology, University of Texas at Austin, 3Department of Neurology, University of Florida, 4Department of Psychiatry, University of Texas Southwestern Medical Center, 5Department of Neuroscience, McKnight Brain Institute, University of Florida The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.
