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23 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques
Hong He 1, Amy N. Courtney 1, Eric Wieder 2, K. Jagannadha Sastry 1
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.

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Neuroscience

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents
Lynne A. Fieber 1, Stephen L. Carlson 1, Andrew T. Kempsell 1, Justin B. Greer 1, Michael C. Schmale 1
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.

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Biology

Imaging Through the Pupal Case of Drosophila melanogaster
Mark B Keroles 1, Sonya K Dusseault 1, Chuchu Liu 1, Masood R Mohammed 1, Christy M Vadakkan 1, Jessica H Amiel 1, Samantha N Abel 1, Elena R Bensoussan 1, Benjamin L Russell 1, James Baker 1
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.

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Cancer Research

Capture and Release of Viable Circulating Tumor Cells from Blood
Siddarth Rawal 1, Zheng Ao 2, Ashutosh Agarwal 1,3
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.

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Neuroscience

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery
Ana R. Leda 1, Levy Dygert 1, Luc Bertrand 1, Michal Toborek 1,2
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.

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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 Dygert 2, Michal Toborek 1,3
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.

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Immunology and Infection

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining
Lucas Gonzalez-Nieto 1, Aline Domingues 1, Michael Ricciardi 1, Martin J. Gutman 1, Helen S. Maxwell 1, Nuria Pedreño-Lopez 1, Varian Bailey 1, Diogo M. Magnani 1, Mauricio A. Martins 1
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.

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Behavior

A Computational Method to Quantify Fly Circadian Activity
Andrey Lazopulo 1, Sheyum Syed 1
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.

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Biology

Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets
Madina Makhmutova 1, Tao Liang 2, Herbert Gaisano 2, Alejandro Caicedo 1, Joana Almaça 1
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.

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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 Scheetz 2, Kevin M. Collins 1,2
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.  

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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
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.

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JoVE Core

Expression of Cementitious Pore Solution and the Analysis of Its Chemical Composition and Resistivity Using X-ray Fluorescence
Marisol Tsui Chang 1, Luca Montanari 2, Prannoy Suraneni 3, W. Jason Weiss 1
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.

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Behavior

Assessing the Influence of Personality on Sensitivity to Magnetic Fields in Zebrafish
Alessandro Cresci 1, Rosario De Rosa 2,3, Claudio Agnisola 4
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.

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Environment

Investigation of Plant Interactions Across Common Mycorrhizal Networks Using Rotated Cores
Joanna Weremijewicz 1,2, David P. Janos 2
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.

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Biology

Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations
Grace A. Snyder 1, William E. Browne 2, 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.

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Bioengineering

Detergent-Free Decellularization of the Human Pancreas for Soluble Extracellular Matrix (ECM) Production
Riccardo Tamburrini 1,2,3, Deborah Chaimov 1,3, Amish Asthana 1,3, Carlo Gazia 3,4, Kevin Enck 3, Sean M. Muir 5, Justine Mariam Aziz 6, Sandrine Lablanche 7, Emily Tubbs 7, Alice A. Tomei 8,9, Mark Van Dyke 10, Shay Soker 3, Emmanuel C. Opara 3, Giuseppe Orlando 1,3
1Department of Surgery, Wake Forest Baptist Medical Center, 2Department of General Surgery, PhD Program in Experimental Medicine, University of Pavia, 3Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, 4Department of Surgery, Tor Vergata University of Rome, 5Wake Forest University College of Arts and Science, 6Wake Forest University School of Medicine, 7Laboratory of Fundamental and Applied Bioenergetics (LBFA), and Environmental and System Biology (BEeSy), Grenoble Alps University, 8Department of Biomedical Engineering, University of Miami, 9Diabetes Research Institute, University of Miami Miller School of Medicine, 10Department of Biomedical Engineering and Mechanics, School of Biomedical Engineering and Sciences, Virginia Polytechnic Institute and State University

The protocol described in this manuscript explains the steps for the fabrication of a soluble extracellular matrix (ECM) from the human pancreas. The solubilized ECM powder obtained through this protocol may be used for the recapitulation of pancreatic islets’ microenvironment in vitro and, potentially, in vivo settings.

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Environment

A Field Primer for Monitoring Benthic Ecosystems Using Structure-From-Motion Photogrammetry
Ty N. F. Roach 1, Shreya Yadav 1, Carlo Caruso 1, Jenna Dilworth 1,2, Catherine M. Foley 1, Joshua R. Hancock 1, Joel Huckeba 1, Ariana S. Huffmyer 1, Kira Hughes 1, Valerie A. Kahkejian 1, Elizabeth M.P. Madin 1, Shayle B. Matsuda 1, Michael McWilliam 1, Spencer Miller 1,3, Erika P. Santoro 4, Mariana Rocha de Souza 1, Damaris Torres-Pullizaa 1, Crawford Drury 1, Joshua S. Madin 1
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.

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Medicine

High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model
Antoine J. Ribieras 1, Yulexi Y. Ortiz 1,2, Sophie Shrestha 3, C. Theodore Huerta 1, Hongwei Shao 1,2, Maria E. Boulina 4, Roberto I. Vazquez-Padron 1,2, Zhao-Jun Liu 1,2, Omaida C. Velazquez 1,2
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.

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Biochemistry

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro
Nikolai Fattakhov 1, Silvia Torices 1, Sarah Becker 1, Timea Teglas 1, Oandy Naranjo 1, Michal Toborek 1,2
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.

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Neuroscience

TACI: An ImageJ Plugin for 3D Calcium Imaging Analysis
Alisa A. Omelchenko 1,2, Hua Bai 1, Sibtain Hussain 1, Jordan J. Tyrrell 1,3, Mason Klein 4, Lina Ni 1
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.

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Biology

Collecting Marine Gnathiid Isopod Fish Parasites with Light Traps
Mary O. Shodipo 1, Ranelagh Yzabelle Lauguico 1, Klaus M. Stiefel 1,2, Paul C. Sikkel 3,4
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.

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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. Walker 1, Heather A. Nelson 2, Matthew Ishahak 3, Gregory Poffenberger 2, Radhika Aramandla 2, Conrad Reihsmann 2, Ashutosh Agarwal 3, Alvin C. Powers 1,2,4, Marcela Brissova 2
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.

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Biology

In Vivo Imaging of Liver Spheroids Engrafted in the Anterior Chamber of the Mouse Eye
Francesca Lazzeri-Barcelo 1, Pierpaolo Ciardo 1, Barbara Leibiger 1, Ingo B. Leibiger 1, 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.

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