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The University of Chicago

15 ARTICLES PUBLISHED IN JoVE

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Biology

Intraperitoneal Injection into Adult Zebrafish
Mary D. Kinkel 1, Stefani C. Eames 2, Louis H. Philipson 2,3, Victoria E. Prince 1
1Department of Organismal Biology and Anatomy, The University of Chicago, 2Committee on Molecular Metabolism and Nutrition, The University of Chicago, 3Department of Medicine, The University of Chicago

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 μl NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

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Biology

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA
Yujing Li 1, Chun-Xiao Song 2, Chuan He 2, Peng Jin 1
1Department of Human Genetics, Emory University School of Medicine, 2Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago

Described is a two-step labeling process using β-glucosyltransferase (β-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

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JoVE Journal

Fast Imaging Technique to Study Drop Impact Dynamics of Non-Newtonian Fluids
Qin Xu 1,2, Ivo Peters 2, Sam Wilken 1,2, Eric Brown 3, Heinrich Jaeger 1,2
1Department of Physics, The University of Chicago, 2James Franck Institute, The University of Chicago, 3Department of Mechanical Engineering and Materials Science, Yale University

Drop impact of non-Newtonian fluids is a complex process since different physical parameters influence the dynamics over a very short time (less than one tenth of a millisecond). A fast imaging technique is introduced in order to characterize the impact behaviors of different non-Newtonian fluids.

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Immunology and Infection

Scalable High Throughput Selection From Phage-displayed Synthetic Antibody Libraries
Shane Miersch 1,2, Zhijian Li 1,2, Rachel Hanna 1,2, Megan E. McLaughlin 1,2, Michael Hornsby 1,3, Tet Matsuguchi 1,3, Marcin Paduch 1,4, Annika Sääf 1,4, Jim Wells 1,3, Shohei Koide 1,4, Anthony Kossiakoff 1,4, Sachdev S. Sidhu 1,2
1The Recombinant Antibody Network, 2The Banting and Best Department of Medical Research, University of Toronto, 3Antibiome Center, University of California, San Francisco at Mission Bay, 4Department of Biochemistry and Molecular Biology, The University of Chicago

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

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JoVE Core

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose
Venkatesh Krishnan 1, Robert Clark 1, Marina Chekmareva 2, Amy Johnson 1, Sophia George 3, Patricia Shaw 4, Victoria Seewaldt 4,5, Carrie Rinker-Schaeffer 1
1Section of Urology, Department of Surgery, The University of Chicago, 2Department of Pathology, Robert Wood Johnson Medical School, 3Campbell Family Institute for Breast Cancer Research, Princess Margaret Cancer Centre, University Health Network, 4Department of Laboratory Medicine and Pathobiology, Campbell Family Institute for Breast Cancer Research, Princess Margaret Cancer Centre, University of Toronto, University Health Network, 5Departments of Medicine, Pharmacology, and Cancer Biology, Duke University Medical Center

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

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Biology

Filtration Isolation of Nucleic Acids: A Simple and Rapid DNA Extraction Method
Sally M. McFall 1, Mário F. Neto 1, Jennifer L. Reed 1, Robin L. Wagner 2
1Center for Innovation in Global Health Technologies (CIGHT), Department of Biomedical Engineering, Northwestern University, 2Pritzker School of Medicine, The University of Chicago

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

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Cancer Research

Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones
Go Oshima *1,2, Melinda E. Stack *1, Sean C. Wightman 1, Darren Bryan 1, Elizabeth Poli 1, Lai Xue 1, Kinga B. Skowron 1, Abhineet Uppal 1, Sean P. Pitroda 2, Xiaona Huang 2, Mitchell C. Posner 1, Samuel Hellman 2, Ralph R. Weichselbaum 2, Nikolai N. Khodarev 2
1Department of Surgery, The University of Chicago, 2Department of Radiation and Cellular Oncology and Ludwig Center for Metastasis Research, The University of Chicago

The ability of metastatic clones to colonize distant sites depends on their proliferation capacity and/or their ability to survive in the host microenvironment without significant proliferation. Here, we present an animal model that allows quantitative visualization of both types of liver colonization by metastatic clones.

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Medicine

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases
Pedro Brugarolas 1, Mohammed Bhuiyan 2, Anna Kucharski 2, Richard Freifelder 2
1Department of Neurology, The University of Chicago, 2Department of Radiology, The University of Chicago

We demonstrate the semi-automated radiochemical synthesis of [18F]3F4AP and quality control procedures.

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Immunology and Infection

Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish
Margaret M. Pruitt 1,2, Wilfredo Marin 1, Michael R. Waarts 1, Jill L. O. de Jong 1
1Department of Pediatrics, Section of Hematology-Oncology, The University of Chicago, 2Department of Biology and Biotechnology, Worcester Polytechnic Institute

Here, we describe a technique to isolate the side population cells from a zebrafish model of myc-induced T-cell acute lymphoblastic leukemia (T-ALL). This side population assay is highly sensitive and is described for zebrafish T-ALL, but it may be applicable to other malignant and non-malignant zebrafish cell types.

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Immunology and Infection

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies
Paul E. Leon 1,2, Teddy John Wohlbold 1,2, Wenqian He 1,2, Mark J. Bailey 1,2, Carole J. Henry 3, Patrick C. Wilson 3, Florian Krammer 1, Gene S. Tan 1
1Department of Microbiology, Icahn School of Medicine at Mount Sinai, 2Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, 3The Department of Medicine, Section of Rheumatology, The Knapp Center for Lupus and Immunology Research, The University of Chicago

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

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Biochemistry

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy
Jillian Rosenberg 1, Jun Huang 1,2
1Committee on Cancer Biology, The University of Chicago, 2Pritzker School of Molecular Engineering, The University of Chicago

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.

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JoVE Core

Assembly and Characterization of Polyelectrolyte Complex Micelles
Alexander E. Marras 1, Jeffrey R. Vieregg 1, Matthew V. Tirrell 1
1Pritzker School of Molecular Engineering, The University of Chicago

We provide protocols and representative data for designing, assembling, and characterizing polyelectrolyte complex micelles, core-shell nanoparticles formed by polyelectrolytes and hydrophilic charged-uncharged block copolymers.

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Bioengineering

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling
Menahem Y. Rotenberg *1, Erik N. Schaumann *2, Aleksander Prominski 1,2, Bozhi Tian 1,2,3
1The James Franck Institute, The University of Chicago, 2Department of Chemistry, The University of Chicago, 3The Institute for Biophysical Dynamics, The University of Chicago

This protocol describes the use of silicon nanowires for intracellular optical bio-modulation of cell in a simple and easy to perform method. The technique is highly adaptable to diverse cell types and can be used for in vitro as well as in vivo applications.

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Biology

Characterizing Epithelial Wound Healing In Vivo Using the Cnidarian Model Organism Clytia hemisphaerica
Elizabeth E. L. Lee 1, Emily Watto 1, Jocelyn Malamy 1
1Department of Molecular Genetics and Cell Biology, The University of Chicago

This paper describes a method to create wounds in the epithelium of a live Clytia hemisphaerica medusa and image wound healing at a high resolution in vivo. Additionally, a technique to introduce dyes and drugs to perturb signaling processes in the epithelial cells and extracellular matrix during wound healing is presented.

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Bioengineering

Using Vertically Aligned Carbon Nanofiber Arrays on Rigid or Flexible Substrates for Delivery of Biomolecules and Dyes to Plants
Jessica M. Morgan 1, Joanna Jelenska 2, Dale K. Hensley 3, Pengju Li 4, Bernadeta R. Srijanto 3, Scott T. Retterer 3,5, Robert F. Standaert 6, Jennifer L. Morrell-Falvey 5, Jean T. Greenberg 2
1Biophysical Sciences, The University of Chicago, 2Molecular Genetics and Cell Biology, The University of Chicago, 3Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, 4Pritzker School of Molecular Engineering, The University of Chicago, 5Biosciences Division, Oak Ridge National Laboratory, 6Department of Chemistry, East Tennessee State University

Here we describe methods for microfabricating vertically aligned carbon nanofibers (VACNFs), transferring VACNFs to flexible substrates, and applying VACNFs on both rigid and flexible substrates to plants for biomolecule and dye delivery.

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