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94 ARTICLES PUBLISHED IN JoVE

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Biology

Fabrication of Myogenic Engineered Tissue Constructs
Christina A. Pacak 1,2, Douglas B. Cowan 1,2
1Department of Anesthesiology, Children's Hospital Boston and Harvard Medical School, 2Perioperative and Pain Medicine, Children's Hospital Boston and Harvard Medical School

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

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Biology

Implantation of Engineered Tissue in the Rat Heart
Bjoern Sill 1, Ivan V. Alpatov 2, Christina A. Pacak 2, Douglas B. Cowan 2
1Department of Anesthesiology, Perioperative and Pain Medicine, Children's Hospital Boston and Harvard Medical School, 2Department of Anesthesiology, Perioperative and Pain Medicine, Children’s Hospital Boston

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

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Biology

Cargo Loading onto Kinesin Powered Molecular Shuttles
Yolaine Jeune-Smith 1, Ashutosh Agarwal 2, Henry Hess 2
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.

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Neuroscience

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice
Kelly A. Tennant 1, Aaron L. Asay 2, Rachel P. Allred 3, Angela R. Ozburn 4, Jeffrey A. Kleim 5, Theresa A. Jones 1,2
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.

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Neuroscience

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay
Hassan Azari 1,2, Maryam Rahman 2, Sharareh Sharififar 2, 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.

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Neuroscience

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay
Hassan Azari 1,2, Sharareh Sharififar 2, Maryam Rahman 2, Saeed Ansari 2, Brent A. Reynolds 2
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.

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Medicine

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors
Chen Ling 1, Yuan Lu 1, Binbin Cheng 1, Katherine E. McGoogan 1, Samantha W.Y. Gee 1, Wenqin Ma 1, Baozheng Li 1, George V. Aslanidi 1, Arun Srivastava 1
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.

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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. Louis 3, Sharareh Sharififar 1, Vinata Vedam-Mai 1, Brent A. Reynolds 1
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.

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Immunology and Infection

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes
Jing Chen 1, Scott Grieshaber 1, Clayton E. Mathews 1
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.

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Biology

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains
Chi K. Leung 1, Andrew Deonarine 1, Kevin Strange 2, Keith P. Choe 1
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.

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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. McConnell 1, Aqeela Afzal 1, J Mocco 1
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.

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Education

Voltage Biasing, Cyclic Voltammetry, & Electrical Impedance Spectroscopy for Neural Interfaces
Seth J. Wilks 1, Tom J. Richner 2, Sarah K. Brodnick 2, Daryl R. Kipke 3, Justin C. Williams 2, Kevin J. Otto 1,4
1Weldon School of Biomedical Engineering, Purdue University, 2Biomedical Engineering, University of Wisconsin-Madison, 3Biomedical Engineering, University of Michigan , 4Department of Biological Sciences, Purdue University

The electrode-tissue interface of neural recording electrodes can be characterized with electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Application of voltage biasing changes the electrochemical properties of the electrode-tissue interface and can improve recording capability. Voltage biasing, EIS, CV, and neural recordings are complementary.

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Neuroscience

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay
Hassan Azari 1,2, Sebastien Millette 1, Saeed Ansari 1, Maryam Rahman 1, Loic P. Deleyrolle 1, Brent A. Reynolds 1
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.

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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 Sharififar 1, Jeff M. Fortin 1, Brent A. Reynolds 1
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.

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Biology

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry
Jennifer Parker 1, Ning Zhu 2, Mengmeng Zhu 2, Sixue Chen 1,2,3,4
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.

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Medicine

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)
Loic P. Deleyrolle 1, Mark R. Rohaus 1, Jeff M. Fortin 1, Brent A. Reynolds 1, Hassan Azari 1,2
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.

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Medicine

Collection Protocol for Human Pancreas
Martha L. Campbell-Thompson 1, Emily L. Montgomery 1, Robin M. Foss 1, Kerwin M. Kolheffer 1, Gerald Phipps 1, Lynda Schneider 1, Mark A. Atkinson 1
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.

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Medicine

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat
Saeed Ansari 1, Hassan Azari 2, Kenneth J. Caldwell 1, Robert W. Regenhardt 3, Vishnumurthy S. Hedna 4, Micheal F. Waters 4, Brian L. Hoh 1, Adam P. Mecca 3
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.

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Medicine

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion
Robert W. Regenhardt 1, Saeed Ansari 2, Hassan Azari 3, Kenneth J. Caldwell 2, Adam P. Mecca 1
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.

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Neuroscience

Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue
Andrew J. Woolley 1, Himanshi A. Desai 1, Janak Gaire 2, Andrew L. Ready 1, Kevin J. Otto 1,2
1Weldon School of Biomedical Engineering, Purdue University, 2Department of Biological Sciences, Purdue University

Here we present a histological method for capturing, labeling, optically clearing, and imaging the intact brain tissue interface around chronically implanted microdevices in rodent brain tissue. Results from the techniques comprising this method are useful for understanding the impact of various penetrating brain-implants on their surrounding tissue.

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Biology

Purification of Transcripts and Metabolites from Drosophila Heads
Kurt Jensen 1, Jonatan Sanchez-Garcia 1, Caroline Williams 2, Swati Khare 1, Krishanu Mathur 1, Rita M. Graze 3, Daniel A. Hahn 2, Lauren M. McIntyre 3, Diego E. Rincon-Limas 1,4, Pedro Fernandez-Funez 1,4
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.

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Bioengineering

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves
Sharan Ramaswamy 1, Manuel Salinas 1, Rob Carrol 2, Karla Landaburo 1, Xavier Ryans 1, Cynthia Crespo 1, Ailyn Rivero 1, Faris Al-Mousily 3,4, Curt DeGroff 3, Mark Bleiweis 3, Hitomi Yamaguchi 2
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.

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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 Schallert 2, Christine E. Schmidt 1,3
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.

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Bioengineering

An Improved Method for the Preparation of Type I Collagen From Skin
Christina A. Pacak 1,2, Allison A. MacKay 1, Douglas B. Cowan 1,2
1Department of Anesthesiology, Perioperative and Pain Medicine, Boston Children's Hospital, 2Department of Anesthesia, Harvard Medical School

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

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Environment

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water
Bledar Bisha 1, Jaclyn A. Adkins 2, Jana C. Jokerst 3, Jeffrey C. Chandler 1, Alma Pérez-Méndez 4, Shannon M. Coleman 4, Adrian O. Sbodio 5, Trevor V. Suslow 5, Michelle D. Danyluk 6, Charles S. Henry 2, Lawrence D. Goodridge 7
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.

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Medicine

Minimally Invasive Thumb-sized Pterional Craniotomy for Surgical Clip Ligation of Unruptured Anterior Circulation Aneurysms
Eric M Deshaies 1,2, Mark R Villwock 1, Amit Singla 3, Gentian Toshkezi 4, David J Padalino 1
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.

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Biology

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration
Janine M. Preble 1, Christina A. Pacak 2, Hiroshi Kondo 1, Allison A. MacKay 2, Douglas B. Cowan 2, James D. McCully 1
1Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School, 2Department of Anesthesiology, Perioperative and Pain Medicine, Boston Children's Hospital and Department of Anesthesia, Harvard Medical School

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is <30 min compared to 60 - 100 min using alternative methods.

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Neuroscience

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction
Ji-Yoen Kim *1, Stacy D. Grunke *1, Yona Levites 2, Todd E. Golde 2, Joanna L. Jankowsky 1,3
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.

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Neuroscience

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents
Joy A. Franco 1, Heidi E. Kloefkorn 2, Shawn Hochman 3, Katherine A. Wilkinson 4
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.

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JoVE Journal

A Multi-detection Assay for Malaria Transmitting Mosquitoes
Yoosook Lee 1, Allison M. Weakley 1, Catelyn C. Nieman 1, Julia Malvick 2, Gregory C. Lanzaro 1
1Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California - Davis, 2Veterinary Genetics Laboratory, School of Veterinary Medicine, University of California, Davis

Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

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Developmental Biology

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures
Heather H. Ross 1, Milap S. Sandhu 1, Sharareh Sharififar 1, David D. Fuller 1
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.

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Medicine

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model
Dibash K. Das 1,2, Michelle K. Naidoo 1, Adeodat Ilboudo 1, Pascal DuBois 1, Victoria Durojaiye 1, Chen Liu 3, Olorunseun O. Ogunwobi 1,2
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.

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Medicine

Murine Model of Intestinal Ischemia-reperfusion Injury
Ekaterina O. Gubernatorova 1,2, Ernesto Perez-Chanona 3, Ekaterina P. Koroleva 1, Christian Jobin 3, Alexei V. Tumanov 1,2
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.

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Environment

A Push-pull Protocol to Reduce Colonization of Bird Nest Boxes by Honey Bees
Caroline A. Efstathion 1, 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.

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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 Xue 3, Kevin K.W. Wang 1,2
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.

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Environment

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays
Alvine C. Mehinto 1, B. Sumith Jayasinghe 2, Darcy R. Vandervort 1, Nancy D. Denslow 2, Keith A. Maruya 1
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.

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Environment

A Method for Evaluating Insecticide Efficacy against Bed Bug, Cimex lectularius, Eggs and First Instars
Brittany E. Campbell 1, Dini M. Miller 2
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.

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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 Baldwin 1, Anne Plotto 1, Jan Narciso 1, Christopher Ference 1, Mark Ritenour 2, Ken Harrison 3, Joseph Gangemi 3, Jinhe Bai 1
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.

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Environment

Capturing Flow-weighted Water and Suspended Particulates from Agricultural Canals During Drainage Events
Jehangir H. Bhadha 1, Anne Sexton 1, Timothy A. Lang 2, Samira H. Daroub 1
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.

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Bioengineering

Metabolic Support of Excised, Living Brain Tissues During Magnetic Resonance Microscopy Acquisition
Jeremy J. Flint 1,2, Kannan Menon 2,3, Brian Hansen 4, John Forder 2,3,5, Stephen J. Blackband 1,2,6
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.

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Bioengineering

High Resolution 3D Imaging of the Human Pancreas Neuro-insular Network
Elizabeth Butterworth 1, Wesley Dickerson 2, Vindhya Vijay 3, Kristina Weitzel 1, Julia Cooper 1, Eric W. Atkinson 4, Jason E. Coleman 5, Kevin J. Otto 4, Martha Campbell-Thompson 1
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.

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Biochemistry

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1
Ozlem Yaren 1, Barry W. Alto 2, Kevin M. Bradley 3, Patricia Moussatche 3, Lyudmyla Glushakova 3, Steven A. Benner 1,3
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.

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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
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.

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Biochemistry

A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton
Qiao Zhang 1, Andrew C. Tamashunas 1, Tanmay P. Lele 1
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.

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Neuroscience

Minimally Invasive Endoscopic Intracerebral Hemorrhage Evacuation
Jonathan Pan 1, Alexander G. Chartrain 1, Jacopo Scaggiante 1, Olivia S. Allen 1, Danny Hom 1, Joshua B. Bederson 1, J Mocco 1, Christopher P. Kellner 1
1Department of Neurosurgery, Icahn School of Medicine at Mount Sinai

This paper details the surgical protocol for minimally invasive endoscopic intracerebral hemorrhage evacuation using the SCUBA technique.

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Neuroscience

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster
Daniel R. Swale 1, Aaron D. Gross 2, Quentin R. R. Coquerel 3, Jeffrey R. Bloomquist 3
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.

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Biology

Reverse Dissection and DiceCT Reveal Otherwise Hidden Data in the Evolution of the Primate Face
Anne M. Burrows 1,2, Kailey M Omstead 1, Ashley R Deutsch 3,5, Justin T. Gladman 4, Adam Hartstone-Rose 3
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.

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Immunology and Infection

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis
Byron Brook *1, Nelly Amenyogbe *1, Rym Ben-Othman 2, Bing Cai 2, Danny Harbeson 1, Freddy Francis 1, Aaron C. Liu 1, Natallia Varankovich 2, James Wynn 3,4, Tobias R. Kollmann 1,2
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.

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Biochemistry

Rapid Collection of Floral Fragrance Volatiles using a Headspace Volatile Collection Technique for GC-MS Thermal Desorption Sampling
Haleigh A. Ray 1, Charles J. Stuhl 2, Jennifer L. Gillett-Kaufman 1
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.

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Medicine

Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer
Shizuko Tachibana 1, Chao Chen 1, Oliver R. Zhang 1, Sarah V. Schurr 1, Cameron Hill 1, Ruixia Li 1, Ana M. Manso 1, Jianlin Zhang 1, Aleksander Andreyev 2, Anne N. Murphy 2, Robert S. Ross 1,3, Yoshitake Cho 1
1Division of Cardiology, Department of Medicine, University of California San Diego, 2Department of Pharmacology, University of California San Diego, 3Cardiology Section, Department of Medicine, Veterans Administration Healthcare, San Diego

The goal of this protocol is to illustrate how to use mouse neonatal cardiomyocytes as a model system to examine how various factors can alter oxygen consumption in the heart.

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Bioengineering

Construction and Use of an Electrical Stimulation Chamber for Enhancing Osteogenic Differentiation in Mesenchymal Stem/Stromal Cells In Vitro
Liudmila Leppik 1, Mit B. Bhavsar 1, Karla M.C. Oliveira 1, Maria Eischen-Loges 1, Sahba Mobini 1,2, John H. Barker 1
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.

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Medicine

Updated Technique for Reliable, Easy, and Tolerated Transcranial Electrical Stimulation Including Transcranial Direct Current Stimulation
Helen Borges 1, Alexandra Dufau 1,2, Bhaskar Paneri 1, Adam J. Woods 3, Helena Knotkova 4,5, Marom Bikson 1
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).

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Medicine

Assessment and Characterization of Hyaloid Vessels in Mice
Zhongxiao Wang 1, Chi-Hsiu Liu 1, Shuo Huang 1, Jing Chen 1,2
1Department of Ophthalmology, Boston Children's Hospital, Harvard Medical School, 2The Manton Center for Orphan Disease Research, Boston Children's Hospital, Harvard Medical School

This protocol describes both in vivo and ex vivo methods to fully visualize and characterize hyaloid vessels, a model of vascular regression in mouse eyes, using optical coherence tomography and fundus fluorescein angiography for the live imaging and ex vivo isolation and subsequent flat mount of hyaloid for quantitative analysis.

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Genetics

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries
Huacheng Luo 1, Amin Sobh 2, Christopher D. Vulpe 2, Edmond Brewer 1, Sinisa Dovat 1, Yi Qiu 3, Suming Huang 1
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.

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Environment

Manipulation of Color Patterns in Jumping Spiders for Use in Behavioral Experiments
Malika Ihle 1, Lisa Anne Taylor 1,2
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.

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Environment

Sampling for Estimating Frankliniella Species Flower Thrips and Orius Species Predators in Field Experiments
Joseph Funderburk 1, Xavier Martini 1, Josh Freeman 1, Iris Strzyzewski 1, Edward Traczyk 1, Thomas Skarlinsky 2, Scott Adkins 3
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.

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Medicine

Murine Surgical Model of Topical Elastase Induced Descending Thoracic Aortic Aneurysm
Zachary Tyerman 1, Jolian Dahl 1, Alexander Shannon 1, W. Forrest Johnston 2, Nicholas H. Pope 1, Guanyi Lu 3, Gilbert R. Upchurch Jr. 3, Gorav Ailawadi 1,4, Morgan Salmon 1
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.

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Medicine

Porcine Model of Infrarenal Abdominal Aortic Aneurysm
Alexander H. Shannon 1, J. Michael Cullen 1, Jolian J. Dahl 1, Erik J. Scott 1, Zachary Tyerman 1, Michael D. Spinosa 1, William G. Montgomery 1, W. Forrest Johnston 2, Guanyi Lu 3, 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.

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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. Rossetti 2, Stephanie J. Sanchez 2, Jacob A. Hornfeldt 1
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.

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Biochemistry

TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis
Silveli Suzuki-Hatano 1, Ang-Chen Tsai 1, Audrey Daugherty 1, Christina A. Pacak 1
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.

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Immunology and Infection

Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry
Jasmine L. Madrigal 1, Melissa K. Jones 1
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.

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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. Collins 5, Evan L. Reeder 5, Jason D. Huber 4, Charles L. Rosen 6, 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.

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Cancer Research

Creating Matched In vivo/In vitro Patient-Derived Model Pairs of PDX and PDX-Derived Organoids for Cancer Pharmacology Research
Xiaoxi Xu 1, Limei Shang 1, Philip Wang 2, Jun Zhou 2, Xuesong Ouyang 2, Meiling Zheng 1, Binchen Mao 2, Likun Zhang 2, Bonnie Chen 1, Jingjing Wang 2, Jing Chen 3, Wubin Qian 2, Sheng Guo 2, Yujun Huang 2, Qi-Xiang Li 3
1Crown Bioscience Inc., Changping District, Beijing, China, 2Crown Bioscience Inc., Taicang, Jiangsu, China, 3Crown Bioscience Inc., San Diego, CA, USA

A method is described to create organoids using patient-derived xenografts (PDX) for in vitro screening, resulting in matched pairs of in vivo/in vitro models. PDX tumors were harvested/processed into small pieces mechanically or enzymatically, followed by the Clevers’ method to grow tumor organoids that were passaged, cryopreserved and characterized against the original PDX.

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Environment

Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification
Owen Hudson 1, Sumyya Waliullah 1, Justin Hand 2, Romina Gazis-Seregina 3, Fulya Baysal-Gurel 4, Md Emran Ali 1
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.

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Genetics

Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator
Kayli Sieber 1, Maya Saar 1, Comzit Opachaloemphan 2, Matthew Gallitto 3, Huan Yang 2, Hua Yan 1,4
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.

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Immunology and Infection

Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices
Mollie K. Huber 1, Denise M. Drotar 2,3,4, Helmut Hiller 1, Maria L. Beery 1, Paul Joseph 1, Irina Kusmartseva 1, Stephan Speier 2,3,4, Mark A. Atkinson 1, Clayton E. Mathews 1, Edward A. Phelps 5
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.

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Biology

Field-Based Thermal Physiology Assay: Cold Shock Recovery under Ambient Conditions
Emily S. Khazan 1
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.

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Biology

Preparing Irradiated and Marked Male Aedes aegypti Mosquitoes for Release in an Operational Sterile Insect Technique Program
Bianca J. Moreno 1, Robert L. Aldridge 1, Seth C. Britch 1, Barbara E. Bayer 1, Jedidiah Kline 1, Daniel A. Hahn 2, Chao Chen 2, Kenneth J. Linthicum 1
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.

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Cancer Research

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells
Ziyi Hu *1, Tingting Zhou *1, Fangrong Wu 1, Fan Lin 1,2
1Department of Cell Biology, School of Basic Medical Sciences, Nanjing Medical University, 2Institute for Brain Tumors & Key Laboratory of Rare Metabolic Diseases, Nanjing Medical University; Nanjing Medical University Affiliated Cancer Hospital; Key Laboratory of Human Functional Genomics of Jiangsu Province

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in glioblastoma (GBM), the most prevalent and devastating primary brain tumor. The presence of GSCs makes the GBM very refractory to most of individual targeted agents, so high-throughput screening methods are required to identify potential effective combination therapeutics. The protocol describes a simple workflow to enable rapid screening for potential combination therapy with synergistic interaction. The general steps of this workflow consist of establishing luciferase-tagged GSCs, preparing matrigel coated plates, combination drug screening, analyzing, and validating the results.

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Biology

A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing
Tse-Yu Chen 1, Adam E. Vorsino 2, Kyle J. Kosinski 1, Ana L. Romero-Weaver 1, Eva A. Buckner 1, Joanna C. Chiu 3, Yoosook Lee 1
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.

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Immunology and Infection

Retrograde Parotid Gland Infusion through Stensen's Duct in a Non-Human Primate for Vectored Gene Delivery
Guy El Helou 1, Joseph F. Goodman 2, Maria Blevins 3, David L. Caudell 4, Todd A. Ponzio 3, John W. Sanders 3,5
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.

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Medicine

A Preclinical Model of Exertional Heat Stroke in Mice
Michelle A. King 1, Jamal M. Alzahrani 1, Thomas L. Clanton 1, Orlando Laitano 1,2
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.

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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 Liang 2, Justin Zhang 3, Gaoming Lin 1, Sydney Yu 1, Mai Tanaka 4,5, Sharon Lepler 4,5, Juan Guan 5,6,7, Dietmar Siemann 4,5, Xin Tang 2,5
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).

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Biology

Automating Aggregate Quantification in Caenorhabditis elegans
Alfonso S. Vaziriyan-Sani 1, Robert D. Handy 1, Alyssa C. Walker 1, Carol Navya Pagolu 1,2, Samantha M. Enslow 1, Daniel M. Czyż 1
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.

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Neuroscience

Implantation and Control of Wireless, Battery-free Systems for Peripheral Nerve Interfacing
Hongkai Wang 1,2, Dom D’Andrea 1, Yeon Sik Choi 3,4, Yasmine Bouricha 1, Grace Wickerson 3,4, Hak-Young Ahn 3, Hexia Guo 3,4, Yonggang Huang 3,4,5,6, Milap S. Sandhu 7, Sumanas W. Jordan 8, John A. Rogers 3,4,6,9,10,11,12, Colin K. Franz 1,3,13
1Laboratory of Regenerative Rehabilitation, Shirley Ryan AbilityLab, Department of Physical Medicine and Rehabilitation, Northwestern University Feinberg School of Medicine, 2Northwestern University Interdepartmental Neuroscience Program, 3Center for Bio-integrated Electronics, Querrey Simpson Institute for Bioelectronics, Northwestern University, 4Department of Materials Science and Engineering, Northwestern University, 5Department of Civil and Environmental Engineering, Northwestern University, 6Department of Mechanical Engineering, Northwestern University, 7Arms and Hands Lab, Shirley Ryan AbilityLab, Department of Physical Medicine and Rehabilitation, Northwestern University Feinberg School of Medicine, 8Division of Plastic and Reconstructive Surgery, Biologics, Shirley Ryan AbilityLab, Northwestern University, 9Department of Biomedical Engineering, Northwestern University, 10Department of Neurological Surgery, Northwestern University, 11Department of Chemistry, Northwestern University, 12Department of Electrical and Computer Engineering, Northwestern University, 13The Ken and Ruth Davee Department of Neurology, Northwestern University Feinberg School of Medicine

This is a protocol for the surgical implantation and operation of a wirelessly powered interface for peripheral nerves. We demonstrate the utility of this approach with examples from nerve stimulators placed on either the rat sciatic or phrenic nerve.

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Biochemistry

Ex Vivo Hepatic Perfusion Through the Portal Vein in Mouse
Anthony G. Giacalone 1, Matthew E. Merritt 1, Mukundan Ragavan 1
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.

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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 Beckham 1, Tatiana Sanchez 2, Zhuxuan Xu 1, Preston Stern 1, Gary Vallad 3, Geoffrey Meru 4,5, Cecilia McGregor 6, Nicholas S. Dufault 1
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).

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Biology

Deploying Community Scientists to Conduct Nondestructive Genetic Sampling of Rare Butterfly Populations
Jaret C. Daniels 1,2, Caroline G. Storer 2, Geena M. Hill 3, Amanda Markee 2, Christian Couch 2, Kristin A. Rossetti 2
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.

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Measuring Cerebral Vasospasm after Subarachnoid Hemorrhage in Mice
William S. Dodd 1, Brandon Lucke-Wold 1, Koji Hosaka 1, Brian L. Hoh 1
1Department of Neurosurgery, College of Medicine, University of Florida

The present protocol provides a detailed procedure for inducing subarachnoid hemorrhage in mice via autologous blood injection to the anterior circulation and measuring delayed cerebral vasospasm by vascular gel casting.

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Medicine

A Murine Model of Hemodialysis Access-Related Hand Dysfunction
Kyoungrae Kim 1, Erik M. Anderson 2,4, Brian J. Fazzone 2,4, Kerri A. O’Malley 2,4, Scott A. Berceli 2,4, Terence E. Ryan 1,3, Salvatore T. Scali 2,4
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.

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Biology

Embryo Rescue Protocol for Interspecific Hybridization in Squash
Yuqing Fu 1, Swati Shrestha 1, Pamela Moon 1, Geoffrey Meru 1
1UF/IFAS Tropical Research and Education Center

The article describes an embryo rescue protocol for the regeneration of immature embryos derived from the interspecific hybridization of Cucurbita pepo and Cucurbita moschata. The protocol can be easily replicated and will be an important resource for squash breeding programs.

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Medicine

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability
Kiran Bora *1, Zhongxiao Wang *1, Felix Yemanyi 1, Meenakshi Maurya 1, Alexandra K. Blomfield 1, Yohei Tomita 1, Jing Chen 1
1Department of Ophthalmology, Boston Children’s Hospital, Harvard Medical School

This protocol illustrates an in vitro endothelial cell transcytosis assay as a model to evaluate inner blood-retinal barrier permeability by measuring the ability of human retinal microvascular endothelial cells to transport horseradish peroxidase across cells in caveolae-mediated transcellular transport processes.

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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. Delgado 1, Tyler A. Reid 1, Julian Long 2, Shu Wang 3,5, Hayley Sussman 4, Juan Guan 5,6,7, Hitomi Yamaguchi 1, Xin Tang 1,5,8,9
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.

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Biochemistry

Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection
Jonathan T. Specker 1, Alexander B. Smith 2, Orlaith Keenan 2, Joseph P. Zackular 2,3, Boone M. Prentice 1
1Department of Chemistry, University of Florida, 2Division of Protective Immunity, Children’s Hospital of Philadelphia, 3Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania

A novel sample preparation method is demonstrated for the analysis of agar-based, bacterial macrocolonies via matrix-assisted laser desorption/ionization imaging mass spectrometry.

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Novel Methodological Perspectives In The Study Of Mosquito Biology
Yuemei Dong 1, Eric P. Caragata 2
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

Novel Methodological Perspectives In The Study Of Mosquito Biology

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Use of Minimally Invasive Methods to Assess Fuel Utilization and Circadian Rhythms in Older Adults

Use of Minimally Invasive Methods to Assess Fuel Utilization and Circadian Rhythms in Older Adults
Christian McLaren 1,2, Carly Bohlman 1, Armin Ezzati 1, Karyn Esser 1, Christiaan Leeuwenburgh 1, Todd Manini 3, Marco Pahor 1, Stephen Anton 1,2, Stephanie E. Wohlgemuth 1
1Department of Physiology and Aging, University of Florida, 2Department of Clinical and Health Psychology, College of Public Health and Health Professions, University of Florida, 3Department of Health Outcomes and Bioinformatics, University of Florida

A novel and minimally invasive protocol is presented to evaluate the synergistic impact of fuel utilization and circadian rhythms on aging individuals, utilizing peripheral blood mononuclear cells.

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Medicine

Micro-Ultrasound Guided Transperineal Prostate Biopsy: A Clinic-Based Procedure
Rani Ashouri 1, Brianna Nguyen 1, Jeremy Archer 2, Paul Crispen 1, Padraic O’Malley 1, Li-Ming Su 1, Joseph Grajo 1,3, Sara M. Falzarano 4, Yahya Acar 5,6,7, David Lizdas 5,6, Samsun Lampotang 1,5,6, Wayne Brisbane 1,6
1Department of Urology, University of Florida College of Medicine, 2Division of Urology, North Mississippi Medical Center, 3Department of Radiology, University of Florida College of Medicine, 4Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, 5Department of Anesthesiology, University of Florida College of Medicine, 6Center for Safety, Simulation & Advanced Learning Technologies, University of Florida, 7Department of Emergency Medicine, Gulhane School of Medicine

This manuscript outlines the steps for performing a micro-ultrasound-guided transperineal prostate biopsy under local anesthesia.

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Biology

Sieving Fruit Pulp to Detect Immature Tephritid Fruit Flies in the Field
Amy L. Roda 1, Gary Steck 2, Thomas Fezza 3, Todd Shelly 3, Rita Duncan 4, Nicholas Manoukis 5, Lori Carvalho 5, Abbie Fox 6, Paul Kendra 7, Daniel Carrillo 4
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.

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Biology

CorrelationCalculator and Filigree: Tools for Data-Driven Network Analysis of Metabolomics Data
Gayatri Iyer 1, Marci Brandenburg 1,2, Christopher Patsalis 1, George Michailidis 3, Alla Karnovsky 1
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.

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Biology

Detached Maize Sheaths for Live-Cell Imaging of Infection by Fungal Foliar Maize Pathogens
Renata Belisário 1, Maria F. Torres 1,2, Ester A. S. Buiate 1,3, Katia V. Xavier 1,4, Etta M. Nuckles 1, Lisa J. Vaillancourt 1
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.

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JoVE Core

Comparative Study of Basement-Membrane Matrices for Human Stem Cell Maintenance and Intestinal Organoid Generation
Janny Pineiro-Llanes 1, Lais da Silva 1, John Huang 2, Rodrigo Cristofoletti 1
1Center for Pharmacometrics & Systems Pharmacology, Department of Pharmaceutics, University of Florida, 2TheWell Bioscience, Inc

Organoids have become valuable tools for disease modeling. The extracellular matrix (ECM) guides cell fate during organoid generation, and using a system that resembles the native tissue can improve model accuracy. This study compares the generation of induced pluripotent stem cells-derived human intestinal organoids in animal-derived ECM and xeno-free hydrogels.

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Medicine

Suspension Culture Production and Purification of Adeno-Associated Virus by Iodixanol Density Gradient Centrifugation for In Vivo Applications
Kathryn K. Harris 1, Oleksandr Kondratov 1, Sergei Zolotukhin 1
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.

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Medicine

A Preclinical Model of Sepsis-Induced Myopathy with Disuse in Mice
Franccesco P. Boeno 1, Diana C. Muller 1, Ali Aldakkan 1, Zhuoxin Li 1, Gisienne Reis 1, Elisabeth R. Barton 1, Orlando Laitano 1
1Department of Applied Physiology and Kinesiology, College of Health and Human Performance, University of Florida

This murine model combines a septic insult with hindlimb muscle disuse to recapitulate the bedridden feature of the typical septic patient. The model represents a significant departure from previous models to study muscle dysfunction in sepsis and is a reproducible approach to addressing therapeutic strategies to treat this condition.

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Biology

A Rapid Method to Confine and Safely Handle Bees in the Field
Chase B. Kimmel 1, Jaret C. Daniels 1,2
1Florida Museum of Natural History, University of Florida, 2Department of Entomology and Nematology, University of Florida

We demonstrate a tested method for safely handling field-collected bees. This method allows swift manipulation, identification, genetic sampling, and confirmation of plant-insect interactions via pollen collected while sampling. Easily adaptable, this approach offers a cost-effective, non-lethal means to study rare insect groups.

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