University of Miami

22 articles published in JoVE

• 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 Toborek^{1,2 1}Department 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. Ortiz^1,2, Sophie Shrestha³, C. Theodore Huerta¹, Hongwei Shao^1,2, Maria E. Boulina⁴, Roberto I. Vazquez-Padron^1,2, Zhao-Jun Liu^1,2, Omaida C. Velazquez^{1,2 1}Dewitt 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.

• 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 1}Department 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 Weremijewicz^1,2, David P. Janos^{2 1}Department 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 Rosa^2,3, Claudio Agnisola^{4 1}Rosenstiel 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 Oltra^1,2, Alejandro Caicedo^{3 1}School 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 Weiss^{1 1}Civil 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. Collins^{1,2 1}Neuroscience 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 Syed^{1 1}Department 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 Almaça^{1 1}Department 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 Bertrand^1,2, Levi Dygert², Michal Toborek^{1,3 1}Department 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 Toborek^{1,2 1}Department 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. Martins^{1 1}Department 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 Agarwal^{1,3 1}Department 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 Baker^{1 1}Department 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 Bianchi^{1 1}Department 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. Schmale^{1 1}Division 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. Abdulreda^1,2, Alejandro Caicedo^1,3,4, Per-Olof Berggren^{1,5 1}Diabetes 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 Ahlgren^{1 1}Umeå 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. Bianco^{1,2 1}Division 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 Zhai^{1 1}Department 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 Sastry^{1 1}Department 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.