University of Florida

82 articles published in JoVE

• 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 Duan^{1 1}China-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.

• Biology

  
  Deploying Community Scientists to Conduct Nondestructive Genetic Sampling of Rare Butterfly Populations Jaret C. Daniels^1,2, Caroline G. Storer², Geena M. Hill³, Amanda Markee², Christian Couch², Kristin A. Rossetti^{2 1}Department 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. Caragata^{2 1}W. 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. Caudle^{1 1}Department 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 Huang^1,5, Heyang Wang^1,5, Alfredo A. Delgado¹, Tyler A. Reid¹, Julian Long², Shu Wang^3,5, Hayley Sussman⁴, Juan Guan^5,6,7, Hitomi Yamaguchi¹, Xin Tang^{1,5,8,9 1}Department 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.

• Biochemistry

  
  Ex Vivo Hepatic Perfusion Through the Portal Vein in Mouse Anthony G. Giacalone¹, Matthew E. Merritt¹, Mukundan Ragavan^{1 1}Department 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 Tanaka^4,5, Sharon Lepler^4,5, Juan Guan^5,6,7, Dietmar Siemann^4,5, Xin Tang^{2,5 1}Department 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 Meru^4,5, Cecilia McGregor⁶, Nicholas S. Dufault^{1 1}Department 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 Pagolu^1,2, Samantha M. Enslow¹, Daniel M. Czyż^{1 1}Department 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. Stanifer^1,2, Steeve Boulant^{2,3,4 1}Department 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. Sanders^{3,5 1}Department 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. Torres^{2 1}Department 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 Laitano^{1,2 1}Department 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 Lee^{1 1}Florida 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. Drotar^2,3,4, Helmut Hiller¹, Maria L. Beery¹, Paul Joseph¹, Irina Kusmartseva¹, Stephan Speier^2,3,4, Mark A. Atkinson¹, Clayton E. Mathews¹, Edward A. Phelps^{5 1}Department 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. Linthicum^{1 1}US 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. Khazan^{1 1}School 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. Liao^{1 1}Department 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 Yan^{1,4 1}Department 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 1}Geriatrics 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 Ali^{1 1}Department 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. Pacak^{1 1}Department 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. Jones^{1 1}Microbiology 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. Daniels^1,2, Geena M. Hill^2,3, Kristin A. Rossetti², Stephanie J. Sanchez², Jacob A. Hornfeldt^{1 1}Department 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 Dufau^1,2, Bhaskar Paneri¹, Adam J. Woods³, Helena Knotkova^4,5, Marom Bikson^{1 1}Department 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-Kaufman^{1 1}Entomology 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 1}Department 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 Ailawadi^1,4, Morgan Salmon^{1 1}Department 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 Adkins^{3 1}North 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 Taylor^{1,2 1}Department 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 Huang^{1 1}Department 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 Mobini^1,2, John H. Barker^{1 1}Frankfurt 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 Wynn^3,4, Tobias R. Kollmann^{1,2 1}Department 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. Burrows^1,2, Kailey M Omstead¹, Ashley R Deutsch^3,5, Justin T. Gladman⁴, Adam Hartstone-Rose^{3 1}Department 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. Bloomquist^{3 1}Department 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. Lele^{1 1}Department 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 II^1,2,3, Quiying Pang^2,3,4, Wenwen Kong^2,3, Sixue Chen^{1,2,3,5 1}Plant 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. Benner^{1,3 1}Foundation 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-Thompson^{1 1}Department 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. Daroub^{1 1}Soil 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. Flint^1,2, Kannan Menon^2,3, Brian Hansen⁴, John Forder^2,3,5, Stephen J. Blackband^{1,2,6 1}Department 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 Sun^1,2, Elizabeth Baldwin¹, Anne Plotto¹, Jan Narciso¹, Christopher Ference¹, Mark Ritenour², Ken Harrison³, Joseph Gangemi³, Jinhe Bai^{1 1}USDA, 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. Miller^{2 1}Department 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. Jantz^{1 1}Division 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. Maruya^{1 1}Southern 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 Yang^1,2, Fan Lin^1,2, Amanda S. Weissman^1,2, Emily Jaalouk^1,2, Qing-shan Xue³, Kevin K.W. Wang^{1,2 1}Program 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 1}Department 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. Gubernatorova^1,2, Ernesto Perez-Chanona³, Ekaterina P. Koroleva¹, Christian Jobin³, Alexei V. Tumanov^{1,2 1}Trudeau 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. Fuller^{1 1}Department 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. Das^1,2, Michelle K. Naidoo¹, Adeodat Ilboudo¹, Pascal DuBois¹, Victoria Durojaiye¹, Chen Liu³, Olorunseun O. Ogunwobi^{1,2 1}Department 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 Deshaies^1,2, Mark R Villwock¹, Amit Singla³, Gentian Toshkezi⁴, David J Padalino^{1 1}Neurovascular 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. Wilkinson^{4 1}Department 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. Jankowsky^{1,3 1}Department 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. Goodridge^{7 1}Department 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. Capobianco^{1 1}Deptartment 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-Mousily^3,4, Curt DeGroff³, Mark Bleiweis³, Hitomi Yamaguchi^{2 1}Tissue 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 III^{1 1}Department 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. Schmidt^{1,3 1}Department 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. Anderson^1,2, Richard Mills³, Todd A. Nolan⁴, Alan C. Jenkins⁴, Golam Mustafa¹, Chris Lloyd³, Robert M. Caudle^1,2, John K. Neubert^{4 1}Department 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. McElrone^1,2, Brendan Choat³, Dilworth Y. Parkinson⁴, Alastair A. MacDowell⁴, Craig R. Brodersen^{5 1}U.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-Limas^1,4, Pedro Fernandez-Funez^{1,4 1}Department 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. Mecca^{1 1}Department 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. Mecca^{3 1}Department 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 Tiwari^{1 1}Entomology 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 Khoshbouei^{1,2 1}Department 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 Schneider^{1 1}Department 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. Atkinson^{1 1}Department 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 Liang^1,2, Harry W. Read², Teri C. Balser^{2,3 1}DOE-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 Azari^{1,2 1}Department 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 Azari^1,2, Sharareh Sharififar¹, Jeff M. Fortin¹, Brent A. Reynolds^{1 1}Department 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 Chen^{1,2,3,4 1}Plant 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. Oppenheimer^{1,2,3 1}Department 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 Azari^1,2, Sebastien Millette¹, Saeed Ansari¹, Maryam Rahman¹, Loic P. Deleyrolle¹, Brent A. Reynolds^{1 1}Department 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. Mathews^{1 1}Departments 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. Choe^{1 1}Department 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 Mocco^{1 1}Department 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 Srivastava^{1 1}Department 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 Azari^1,2, Sharon A. Louis³, Sharareh Sharififar¹, Vinata Vedam-Mai¹, Brent A. Reynolds^{1 1}Department 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 Azari^1,2, Sharareh Sharififar², Maryam Rahman², Saeed Ansari², Brent A. Reynolds^{2 1}Department 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 Azari^1,2, Maryam Rahman², Sharareh Sharififar², Brent A. Reynolds^{2 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 Hess^{2 1}Department 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. Jones^{1,2 1}Institute 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.