University of Miami View Institution's Website 27 articles published in JoVE Biology In Vivo Imaging of Liver Spheroids Engrafted in the Anterior Chamber of the Mouse Eye Francesca Lazzeri-Barcelo1, Pierpaolo Ciardo1, Barbara Leibiger1, Ingo B. Leibiger1, Per-Olof Berggren*1,2,3, Noah Moruzzi*1 1The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Karolinska University Hospital, 2Tecnológico de Monterrey, 3Diabetes Research Institute, Miller School of Medicine, University of Miami Here, we describe a platform that allows noninvasive in vivo imaging of liver spheroids engrafted in the anterior chamber of the mouse eye. The workflow spans from generating spheroids from primary liver cells to transplantation into the mouse eye and in vivo imaging at cellular resolution by confocal microscopy. Biology Human Pseudoislet System for Synchronous Assessment of Fluorescent Biosensor Dynamics and Hormone Secretory Profiles Tiffany M. Richardson*1, Yasminye D. Pettway*1, John T. Walker1, Heather A. Nelson2, Matthew Ishahak3, Gregory Poffenberger2, Radhika Aramandla2, Conrad Reihsmann2, Ashutosh Agarwal3, Alvin C. Powers1,2,4, Marcela Brissova2 1Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 2Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University Medical Center, 3Department of Biomedical Engineering, University of Miami, 4VA Tennessee Valley Healthcare System This protocol describes a method for the synchronous acquisition and co-registration of intracellular signaling events and the secretion of insulin and glucagon by primary human pseudoislets using the adenoviral delivery of a cyclic adenosine monophosphate (cAMP) biosensor, a cAMP difference detector in situ (cADDis), and a microperifusion system. Biology Collecting Marine Gnathiid Isopod Fish Parasites with Light Traps Mary O. Shodipo1, Ranelagh Yzabelle Lauguico1, Klaus M. Stiefel1,2, Paul C. Sikkel3,4 1Institute of Environmental and Marine Sciences, Silliman University, 2Neurolinx Research Institute, 3Department of Marine Biology and Ecology, Rosenstiel School of Marine Atmospheric and Earth Sciences, University of Miami, 4Water Research Group, Unit for Environmental Sciences and Management, North-West University We present a method to collect marine gnathiid isopod fish parasites using light traps placed at field sites via breath-hold diving or scuba diving. Neuroscience TACI: An ImageJ Plugin for 3D Calcium Imaging Analysis Alisa A. Omelchenko1,2, Hua Bai1, Sibtain Hussain1, Jordan J. Tyrrell1,3, Mason Klein4, Lina Ni1 1School of Neuroscience, Virginia Polytechnic Institute and State University, 2CMU-Pitt Joint Computational Biology, School of Medicine, University of Pittsburgh, 3Eastern Virginia Medical School, 4Department of Physics, University of Miami TrackMate Analysis of Calcium Imaging (TACI) is an open-source ImageJ plugin for 3D calcium imaging analysis that examines motion on the z-axis and identifies the maximum value of each z-stack to represent a cell's intensity at the corresponding time point. It can separate neurons overlapping in the lateral (x/y) direction but on different z-planes. Biochemistry A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro Nikolai Fattakhov1, Silvia Torices1, Sarah Becker1, Timea Teglas1, Oandy Naranjo1, Michal Toborek1,2 1Department of Biochemistry and Molecular Biology, Miller School of Medicine, University of Miami, 2Institute of Physiotherapy and Health Sciences, The Jerzy Kukuczka Academy of Physical Education Here, we describe the method for establishing a triple cell culture model of the blood-brain barrier based on primary human brain microvascular endothelial cells, astrocytes, and pericytes. This multicellular model is suitable for studies of neurovascular unit dysfunction during ischemic stroke in vitro or for the screening of drug candidates. Medicine High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model Antoine J. Ribieras1, Yulexi Y. Ortiz1,2, Sophie Shrestha3, C. Theodore Huerta1, Hongwei Shao1,2, Maria E. Boulina4, Roberto I. Vazquez-Padron1,2, Zhao-Jun Liu1,2, Omaida C. Velazquez1,2 1Dewitt Daughtry Family Department of Surgery, University of Miami Miller School of Medicine, 2Vascular Biology Institute, University of Miami Miller School of Medicine, 3University of Miami, 4Analytical Imaging Core Facility, University of Miami The present protocol describes a unique, clinically relevant model of peripheral arterial disease that combines femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase inhibitor to induce hindlimb gangrene in FVB mice. Intracardiac DiI perfusion is then used for high-resolution, three-dimensional imaging of the footpad vasculature. Environment A Field Primer for Monitoring Benthic Ecosystems Using Structure-From-Motion Photogrammetry Ty N. F. Roach1, Shreya Yadav1, Carlo Caruso1, Jenna Dilworth1,2, Catherine M. Foley1, Joshua R. Hancock1, Joel Huckeba1, Ariana S. Huffmyer1, Kira Hughes1, Valerie A. Kahkejian1, Elizabeth M.P. Madin1, Shayle B. Matsuda1, Michael McWilliam1, Spencer Miller1,3, Erika P. Santoro4, Mariana Rocha de Souza1, Damaris Torres-Pullizaa1, Crawford Drury1, Joshua S. Madin1 1Hawai'i Institute of Marine Biology, University of Hawai'i Manoa, 2Rosenstiel School of Marine and Atmospheric Science, University of Miami, 3Hawai'i Pacific University, 4Federal University of Rio de Janeiro We provide a detailed protocol for conducting underwater structure-from-motion photogrammetry surveys to generate 3D models and orthomosaics. Biology Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations Grace A. Snyder1, William E. Browne2, Nikki Traylor-Knowles*1, Benyamin Rosental*3 1Department of Marine Biology and Ecology, Rosenstiel School of Marine and Atmospheric Science, University of Miami, 2Department of Biology, University of Miami, 3The Shraga Segal Department of Microbiology, Immunology, and Genetics, Faculty of Health Sciences, and Regenerative Medicine and Stem Cell Research Center, Ben Gurion University of the Negev Corals create biodiverse ecosystems important for both humans and marine organisms. However, we still do not understand the full potential and function of many coral cells. Here, we present a protocol developed for the isolation, labeling, and separation of stony coral cell populations. Environment Investigation of Plant Interactions Across Common Mycorrhizal Networks Using Rotated Cores Joanna Weremijewicz1,2, David P. Janos2 1Department of Biology, North Central College, 2Department of Biology, University of Miami Most plants within communities likely are interconnected by arbuscular mycorrhizal (AM) fungi, but mediation of plant interactions by them has been investigated primarily by growing plants with versus without mycorrhizas. We present a method to manipulate common mycorrhizal networks among mycorrhizal plants to investigate their consequences for plant interactions. Behavior Assessing the Influence of Personality on Sensitivity to Magnetic Fields in Zebrafish Alessandro Cresci1, Rosario De Rosa2,3, Claudio Agnisola4 1Rosenstiel School of Marine and Atmospheric Science, Department of Ocean Sciences, University of Miami, 2Department of Physics, Monte S. Angelo (MSA) Campus, University of Naples Federico II, 3Istituto Nazionale di Fisica Nucleare (INFN), 4Department of Biology, MSA Campus, University of Naples Federico II We describe a behavioral protocol designed to assess how zebrafish’s personalities influence their response to water currents and weak magnetic fields. Fishes with the same personalities are separated based on their explorative behavior. Then, their rheotactic orientation behavior in a swimming tunnel with a low flow rate and under different magnetic conditions is observed. Biology Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy Elisa Oltra1,2, Alejandro Caicedo3 1School of Medicine and Dentistry, Universidad Católica de Valencia San Vicente Mártir, 2Unidad Mixta CIPF-UCV, Centro de Investigación Príncipe Felipe, 3Department of Medicine, University of Miami The goal of the protocol is to show longitudinal intravital real-time tracking of thymocytes by laser scanning microscopy in thymic implants in the anterior chamber of the mouse eye. The transparency of the cornea and vascularization of the graft allows for continuously recording progenitor cell recruitment and mature T-cell egress. Engineering Expression of Cementitious Pore Solution and the Analysis of Its Chemical Composition and Resistivity Using X-ray Fluorescence Marisol Tsui Chang1, Luca Montanari2, Prannoy Suraneni3, W. Jason Weiss1 1Civil and Construction Engineering, Oregon State University, 2SES Group and Associates LLC, Turner-Fairbank Highway Research Center, 3Civil, Architectural and Environmental Engineering, University of Miami This protocol describes the procedure to express fresh pore solution from cementitious systems and the measurement of its ionic composition using X-ray fluorescence. The ionic composition can be used to calculate pore solution electrical resistivity, which can be used, together with concrete electrical resistivity, to determine the formation factor. Neuroscience Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans Bhavya Ravi*1, Layla M. Nassar*1,2, Richard J. Kopchock III*2, Pravat Dhakal*2, Michael Scheetz2, Kevin M. Collins1,2 1Neuroscience Program, University of Miami School of Medicine, 2Department of Biology, University of Miami This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. Behavior A Computational Method to Quantify Fly Circadian Activity Andrey Lazopulo1, Sheyum Syed1 1Department of Physics, University of Miami A method to quantify the main temporal features seen in fly circadian locomotor rhythms is presented. The quantification is achieved by fitting fly activity with a multi-parametric model waveform. The model parameters describe the shape and size of the morning and evening peaks of daily activity. Biology Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets Madina Makhmutova1, Tao Liang2, Herbert Gaisano2, Alejandro Caicedo1, Joana Almaça1 1Department of Medicine, University of Miami, 2Department of Medicine, University of Toronto We describe a protocol for visualization of insulin exocytosis in intact islets using pHluorin, a pH-sensitive green fluorescent protein. Isolated islets are infected with adenovirus encoding pHluorin coupled to the vesicle cargo neuropeptide Y. This allows for the detection of insulin granule fusion events by confocal microscopy. Medicine Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area Luc Bertrand1,2, Levi Dygert2, Michal Toborek1,3 1Department of Biochemistry and Molecular Biology, Miller School of Medicine, University of Miami, 2Miller School of Medicine, University of Miami, 3Jerzy Kukuczka Academy of Physical Education We describe a mouse model of stroke induced by the occlusion of the middle cerebral artery using a silicone coated suture. The protocol can be applied to induce permanent occlusion or a temporary ischemia, followed by reperfusion. Neuroscience Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery Ana R. Leda1, Levy Dygert1, Luc Bertrand1, Michal Toborek1,2 1Department of Biochemistry and Molecular Biology, Miller School of Medicine, University of Miami, 2The Jerzy Kukuczka Academy of Physical Education The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery. Immunology and Infection Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining Lucas Gonzalez-Nieto1, Aline Domingues1, Michael Ricciardi1, Martin J. Gutman1, Helen S. Maxwell1, Nuria Pedreño-Lopez1, Varian Bailey1, Diogo M. Magnani1, Mauricio A. Martins1 1Department of Pathology, University of Miami Here, we present an optimized protocol for enumerating and characterizing rhesus macaque CD8+ T cells against the AIDS virus. This article is useful not only to the field of HIV immunology, but also to other areas of biomedical research where CD8+ T cell responses are known to affect disease outcome. Cancer Research Capture and Release of Viable Circulating Tumor Cells from Blood Siddarth Rawal1, Zheng Ao2, Ashutosh Agarwal1,3 1Department of Pathology, University of Miami, 2SRI International, 3Department of Biomedical Engineering, DJTMF Biomedical Nanotechnology Institute at the University of Miami, University of Miami A protocol to utilize a poly(N-iso-propylacrylamide) (PIPAAm) coated microfilter for effective capture and thermoresponsive release of viable circulating tumor cells (CTC) is presented. This method allows capture of CTC from patients' blood and subsequent release of viable CTC for downstream off-chip culture, analyses and characterization. Biology Imaging Through the Pupal Case of Drosophila melanogaster Mark B Keroles1, Sonya K Dusseault1, Chuchu Liu1, Masood R Mohammed1, Christy M Vadakkan1, Jessica H Amiel1, Samantha N Abel1, Elena R Bensoussan1, Benjamin L Russell1, James Baker1 1Department of Biology, University of Miami This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly. Biology A Method for Culturing Embryonic C. elegans Cells Rachele Sangaletti1, Laura Bianchi1 1Department of Physiology and Biophysics, University of Miami We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells. Neuroscience Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents Lynne A. Fieber1, Stephen L. Carlson1, Andrew T. Kempsell1, Justin B. Greer1, Michael C. Schmale1 1Division of Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Sciences, University of Miami We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique. Medicine Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time Midhat H. Abdulreda1,2, Alejandro Caicedo1,3,4, Per-Olof Berggren1,5 1Diabetes Research Institute, University of Miami Miller School of Medicine, 2Department of Surgery, University of Miami Miller School of Medicine, 3Department of Medicine, University of Miami Miller School of Medicine, 4Department of Physiology & Biophysics, University of Miami Miller School of Medicine, 5The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye. Medicine Near Infrared Optical Projection Tomography for Assessments of β-cell Mass Distribution in Diabetes Research Anna U. Eriksson*1, Christoffer Svensson*1, Andreas Hörnblad1, Abbas Cheddad1, Elena Kostromina1, Maria Eriksson1, Nils Norlin1, Antonello Pileggi2, James Sharpe3, Fredrik Georgsson4, Tomas Alanentalo1, Ulf Ahlgren1 1Umeå Centre for Molecular Medicine, Umeå University, 2Cell Transplant Center, Diabetes Research Institute, University of Miami,, 3EMBL-CRG Systems Biology Program, Centre for Genomic Regulation, Catalan Institute of Research and Advanced Studies, 4Dept. of Computing Science, Umeå University We describe the adaptation of optical projection tomography (OPT)1 to imaging in the near infrared spectrum, and the implementation of a number of computational tools. These protocols enable assessments of pancreatic β-cell mass (BCM) in larger specimens, increase the multichannel capacity of the technique and increase the quality of OPT data. Biology Mutagenesis and Analysis of Genetic Mutations in the GC-rich KISS1 Receptor Sequence Identified in Humans with Reproductive Disorders Luciana Madeira da Silva1, Lauren Vandepas1, Suzy D.C. Bianco1,2 1Division of Endocrinology and Metabolism, Department of Medicine, University of Miami Miller School of Medicine, 2Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine Mutations in the kisspeptin receptor (KISS1R) are associated with reproductive disorders in patients. Here we describe how to introduce mutations of interest in the GC-rich sequence of KISS1R as well as the use of KISS1R constructs to characterize the degradation pathway of the receptor by immunoprecipitation and western blot. Neuroscience Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration Yousuf O. Ali1, Wilfredo Escala1, Kai Ruan1, R. Grace Zhai1 1Department of Molecular and Cellular Pharmacology, University of Miami, Miller School of Medicine Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila. Immunology and Infection Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques Hong He1, Amy N. Courtney1, Eric Wieder2, K. Jagannadha Sastry1 1Department of Immunology, MD Anderson Cancer Center - University of Texas, 2Department of Medicine, University of Miami We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.
