S'identifier

University of Chicago

30 ARTICLES PUBLISHED IN JoVE

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Biology

Understanding Cerebellar Pattern Formation
Kathleen J. Millen 1
1Department of Neurology, University of Chicago

Understanding Cerebellar Pattern Formation

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Biology

In Ovo Electroporations of HH Stage 10 Chicken Embryos
Marissa C. Blank 1, Victor Chizhikov 2, Kathleen J. Millen 2
1Department of Molecular Genetics and Cell Biology, University of Chicago, 2Department of Human Genetics, University of Chicago

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

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Biology

In situ Quantification of Pancreatic Beta-cell Mass in Mice
Abraham Kim 1, German Kilimnik 1, Manami Hara 1
1Department of Medicine, University of Chicago

The following protocol outlines the process of pancreatic dissection for virtual slice imaging, and the subsequent quantification of all GFP-tagged beta-cells in the entire pancreas.

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Biology

Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays
John Zaborske 1, Tao Pan 1
1Department of Biochemistry and Molecular Biology, University of Chicago

We describe a method for microarray analysis to determine relative aminoacylation levels of all tRNAs from S. cerivisiae.

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Bioengineering

Preparation of Complaint Matrices for Quantifying Cellular Contraction
Yvonne Aratyn-Schaus 1, Patrick W. Oakes 1, Jonathan Stricker 2, Stephen P. Winter 3, Margaret L. Gardel 1,2
1Institute for Biophysical Dynamics, University of Chicago, 2Physics Department - James Franck Institute, University of Chicago, 3Interdisciplinary Scientist Training Program, University of Chicago

In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.

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Neuroscience

Combining Computer Game-Based Behavioural Experiments With High-Density EEG and Infrared Gaze Tracking
Keith J. Yoder 1,2, Matthew K. Belmonte 1,3
1Department of Human Development, Cornell University, 2Social Sciences Division, University of Chicago, 3National Brain Research Centre, Manesar, India

Procedures for recording high-density EEG and gaze data during computer game-based cognitive tasks are described. Using a video game to present cognitive tasks enhances ecological validity without sacrificing experimental control.

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Medicine

Generation of Comprehensive Thoracic Oncology Database - Tool for Translational Research
Mosmi Surati 1, Matthew Robinson 2, Suvobroto Nandi 2, Leonardo Faoro 2, Carley Demchuk 2, Rajani Kanteti 2, Benjamin Ferguson 1, Tara Gangadhar 2, Thomas Hensing 3, Rifat Hasina 2, Aliya Husain 4, Mark Ferguson 5, Theodore Karrison 6, Ravi Salgia 2
1Pritzker School of Medicine, University of Chicago, 2Department of Medicine, University of Chicago, 3Department of Medicine, Northshore University Health Systems, 4Department of Pathology, University of Chicago, 5Department of Surgery, University of Chicago, 6Department of Biostatistics, University of Chicago

A thoracic oncology database was developed to serve as a comprehensive repository for clinical and laboratory data for the purposes of translational research. The database will serve translational cancer researchers within the Thoracic Oncology Research Program. This database is adaptable to other cancer models, as well as other human diseases.

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Biology

Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture
Abraham Kim 1, German Kilimnik 1, Charles Guo 1, Joshua Sung 1, Junghyo Jo 2, Vipul Periwal 2, Piotr Witkowski 3, Philip Dilorio 4, Manami Hara 1
1Department of Medicine, University of Chicago, 2Laboratory of Biological Modeling, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 3Department of Surgery, University of Chicago, 4Diabetes Division, University of Massachusetts

Novel computer-assisted methods of large-scale procurement and analysis of immunohistochemically stained pancreatic specimens are described: (1) Virtual Slice capture of the entire section; (2) Mass analysis of large-scale data; (3) Reconstruction of 2D Virtual Slices; (4) 3D islet mapping; and (5) Mathematical analysis.

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Neuroscience

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence
Oksana Polesskaya 1, Anita Sun 2, Gheorghe Salahura 2, Jharon N. Silva 1, Stephen Dewhurst 1, Karl Kasischke 3
1Department of Microbiology and Immunology, University of Rochester Medical Center, 2Center for Neural Development and Disease, University of Rochester Medical Center, 3Deptartment of Neurology, Center for Neural Development and Disease, University of Rochester Medical Center

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

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Medicine

Murine Fetal Echocardiography
Gene H. Kim 1
1Department of Medicine, Section of Cardiology, Institute for Cardiovascular Research, University of Chicago

Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development. High-frequency ultrasound imaging has improved 2-D resolution and can provide excellent information on early cardiac development and is an ideal method to detect the impact on cardiac structure and function prior to death.

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Engineering

Concurrent Quantitative Conductivity and Mechanical Properties Measurements of Organic Photovoltaic Materials using AFM
Maxim P. Nikiforov 1, Seth B. Darling 1,2
1Center for Nanoscale Materials, Argonne National Laboratory, 2Institute for Molecular Engineering, University of Chicago

Organic photovoltaic (OPV) materials are inherently inhomogeneous at the nanometer scale. Nanoscale inhomogeneity of OPV materials affects performance of photovoltaic devices. In this paper, we describe a protocol for quantitative measurements of electrical and mechanical properties of OPV materials with sub-100 nm resolution.

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Medicine

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells
Yi Cai 1, Steven Kregel 2, Donald J. Vander Griend 1,2
1Department of Surgery, Section of Urology, University of Chicago, 2Committee on Cancer Biology, University of Chicago

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

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Engineering

Synthesis and Microdiffraction at Extreme Pressures and Temperatures
Barbara Lavina 1, Przemyslaw Dera 2, Yue Meng 3
1High Pressure Science and Engineering Center, Department of Physics and Astronomy, University of Nevada, Las Vegas, 2GeoSoilEnviroCARS, University of Chicago, 3High Pressure Collaborative Access Team, Carnegie Institution of Washington

The laser heated diamond anvil cell combined with synchrotron micro-diffraction techniques allows researchers to explore the nature and properties of new phases of matter at extreme pressure and temperature (PT) conditions. Heterogeneous samples can be characterized in situ under high pressure by 2D mapping and combined powder, single-crystal and multigrain diffraction approaches.

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Chemistry

Seeded Synthesis of CdSe/CdS Rod and Tetrapod Nanocrystals
Karthish Manthiram *1,4, Brandon J. Beberwyck *2,4, Dmitri V. Talapin 5,6, A. Paul Alivisatos 2,3,4
1Department of Chemical Engineering, UC Berkeley, 2Department of Materials Science and Engineering, UC Berkeley, 3Department of Chemistry, UC Berkeley, 4Materials Sciences Division, Lawrence Berkeley National Laboratory, 5Department of Chemistry, University of Chicago, 6Center for Nanoscale Materials, Argonne National Laboratory

A protocol for the seeded synthesis of rod-shaped and tetrapod-shaped multicomponent nanostructures consisting of CdS and CdSe is presented.

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Neuroscience

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits
Charles C. Lee 1, Ying-Wan Lam 2, Kazuo Imaizumi 1, S. Murray Sherman 2
1Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, 2Department of Neurobiology, University of Chicago

We describe a method for characterizing the functional topography and synaptic properties of forebrain circuits using an optogenetic approach to photostimulate neuronal populations in vitro.

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Biology

Surface Spreading and Immunostaining of Yeast Chromosomes
Jennifer Grubb 1, M. Scott Brown 1,2, Douglas K. Bishop 1,2
1Department of Radiation and Cellular Oncology, University of Chicago, 2Department of Molecular Genetics and Cell Biology, University of Chicago

A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.

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Medicine

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity
Pamela N. Peters 1, Elizabeth M. Schryver 1, Ernst Lengyel 1, Hilary Kenny 1
1Department of Obstetrics and Gynecology, Section of Gynecologic Oncology, University of Chicago

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

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Neuroscience

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation
Christopher Nishimura 1, Oksana Polesskaya 2, Stephen Dewhurst 1, Jharon N. Silva 1
1Department of Microbiology and Immunology, University of Rochester Medical Center, 2Department of Human Genetics, University of Chicago

This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

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Chemistry

High Pressure Single Crystal Diffraction at PX^2
Dongzhou Zhang 1, Przemyslaw K. Dera 1, Peter J. Eng 2, Joanne E. Stubbs 2, Jin S. Zhang 1, Vitali B. Prakapenka 2, Mark L. Rivers 2
1Hawai’i Institute of Geophysics and Planetology, University of Hawai’i at Manoa, 2Center for Advanced Radiation Sources, University of Chicago

In this report, we describe detailed procedures for carrying out single crystal X-ray diffraction experiments with a diamond anvil cell at the GSECARS 13-BM-C beamline at the Advanced Photon Source. ATREX and RSV programs are used to analyze the data.

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Neuroscience

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior
Xiao Sun 1, Ellie S. Heckscher 1,2
1Committee on Development, Regeneration, and Stem Cell Biology, University of Chicago, 2Department of Molecular Genetics and Cell Biology, University of Chicago

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

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Bioengineering

Conducting Multiple Imaging Modes with One Fluorescence Microscope
Seongjin Park 1, Jiacheng Zhang 2, Matthew A. Reyer 2, Joanna Zareba 1,3, Andrew A. Troy 4, Jingyi Fei 1,2
1Department of Biochemistry and Molecular Biology, University of Chicago, 2The Institute for Biophysical Dynamics, University of Chicago, 3Faculty of Chemistry, Wrocław University of Science and Technology, 4Nikon Instruments Inc.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

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Biology

4D Microscopy of Yeast
Natalie Johnson 1, Benjamin S. Glick 1
1Department of Molecular Genetics and Cell Biology, University of Chicago

This protocol describes the analysis of fluorescently labeled intracellular compartments in budding yeast using multi-color 4D (time-lapse 3D) confocal microscopy. The imaging parameters are chosen to capture adequate signals while limiting photodamage. Custom ImageJ plugins allow labeled structures to be tracked and quantitatively analyzed.

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Neuroscience

Real-time Video Projection in an MRI for Characterization of Neural Correlates Associated with Mirror Therapy for Phantom Limb Pain
Faddi G. Saleh Velez 1,2, Camila B. Pinto 1,3, Emma S. Bailin 4, Marionna Münger 1, Andrew Ellison 5, Beatriz T. Costa 1, David Crandell 6, Nadia Bolognini 7,8, Lotfi B. Merabet 4, Felipe Fregni 1
1Laboratory of Neuromodulation & Center for Clinical Research Learning, Department of Physical Medicine and Rehabilitation, Harvard Medical School, Spaulding Rehabilitation Hospital, 2University of Chicago Medical Center, Department of Neurology, University of Chicago, 3Department of Neuroscience and Behavior, Psychology Institute, University of Sao Paulo, 4The Laboratory for Visual Neuroplasticity, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, 5Center for Biomedical Imaging, Department of Anatomy and Neurobiology, Boston University School of Medicine, 6Spaulding Rehabilitation Hospital, Harvard Medical School, 7Department of Psychology & Milan Center for Neuroscience, University of Milano-Bicocca, 8Neuropsychological Laboratory, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Istituto Auxologico Italiano

We present a novel combined behavioral and neuroimaging protocol employing real-time video projection for the purpose of characterizing the neural correlates associated with mirror therapy within the magnetic resonance imaging scanner environment in leg amputee subjects with phantom limb pain.

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Engineering

Procedure for the Transfer of Polymer Films Onto Porous Substrates with Minimized Defects
Lorenzo Guio *1, Claire Liu *1, Dean Boures *1, Patrick T. Getty *1, Ruben Waldman 1,2, Xiaoying Liu 1, Seth B. Darling 1,2,3,4
1Institute for Molecular Engineering, University of Chicago, 2Chemical Sciences and Engineering Division, Argonne National Laboratory, 3Institute for Molecular Engineering, Argonne National Laboratory, 4Advanced Materials for Energy-Water Systems (AMEWS) Energy Frontier Research Center, Argonne National Laboratory

We present a procedure for highly controlled and wrinkle-free transfer of block copolymer thin films onto porous support substrates using a 3D-printed drain chamber. The drain chamber design is of general relevance to all procedures involving transfer of macromolecular films onto porous substrates, which is normally done by hand in an irreproducible fashion.

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Biology

Quantification of Proliferative and Dead Cells in Enteroids
Hua-Shan Li *1, Shao-Fang Xu *1, Jian-Ying Sheng *1, Zhi-Hui Jiang 1, Jing Wang 1, Ning Ding 1, Tao Wang 1, Matthew A. Odenwald 2, Jerrold R. Turner 2,3, Wei-Qi He 1, Hong Xu 1, Juan-Min Zha 1
1Jiangsu Key Laboratory of Neuropsychiatric Diseases and Cambridge-Suda (CAM-SU) Genomic Resource Center, Medical College of Soochow University, Department of Oncology, The First Affiliated Hospital of Soochow University, 2Department of Pathology, University of Chicago, 3Department of Pathology, Brigham and Women's Hospital–Harvard Medical School

The presented protocol uses flow cytometry to quantify the number of proliferating and dead cells in cultured mouse enteroids. This method is helpful to evaluate the effects of drug treatment on organoid proliferation and survival.

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Chemistry

An Externally-Heated Diamond Anvil Cell for Synthesis and Single-Crystal Elasticity Determination of Ice-VII at High Pressure-Temperature Conditions
Xiaojing Lai 1,2, Feng Zhu 2, Jin S. Zhang 3, Dongzhou Zhang 2,4, Sergey Tkachev 4, Vitali B. Prakapenka 4, Bin Chen 2
1Gemmological Institute, China University of Geosciences (Wuhan), 2Hawai'i Institute of Geophysics and Planetology, University of Hawai'i at Mānoa, 3Department of Earth and Planetary Sciences, University of New Mexico, 4Center for Advanced Radiation Sources, University of Chicago

This work focuses on the standard protocol for preparing the externally-heated diamond anvil cell (EHDAC) for generating high-pressure and high-temperature (HPHT) conditions. The EHDAC is employed to investigate materials in Earth and planetary interiors under extreme conditions, which can be also used in solid state physics and chemistry studies.

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Bioengineering

An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug
Aarushi Bhargava 1, Samuel A. Hendley 2, Kenneth B. Bader 1,2
1Department of Radiology, University of Chicago, 2Graduate Program in Medical Physics, University of Chicago

Histotripsy-aided lytic delivery or lysotripsy is under development for the treatment of deep vein thrombosis. An in vitro procedure is presented here to assess the efficacy of this combination therapy. Key protocols for the clot model, image guidance, and assessment of treatment efficacy are discussed.

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Engineering

Building an Enhanced Flight Mill for the Study of Tethered Insect Flight
Anastasia Bernat 1
1Department of Ecology and Evolution, University of Chicago

This protocol uses three-dimensional (3D) printers and laser cutters found in makerspaces in order to create a more flexible flight mill design. By using this technology, researchers can reduce costs, enhance design flexibility, and generate reproducible work when constructing their flight mills for tethered insect flight studies.

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Developmental Biology

Eye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual System
Olivia L. Hagen 1, Yehyun Kim 1, Elaine Kushkowski 1,2, Hannah Rouse 3, Kara L. Cerveny 1
1Department of Biology, Reed College, 2Committee on Development, Regeneration, and Stem Cell Biology, University of Chicago, 3Department of Cell and Developmental Biology, University College London

The article explains how to surgically remove eyes from living zebrafish larvae as the first step toward investigating how retinal input influences optic tectum growth and development. In addition, the article provides information about larval anesthetization, fixation, and brain dissection, followed by immunohistochemistry and confocal imaging.

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Cancer Research

Far-Red Fluorescent Senescence-Associated β-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry
Amy Flor 1, Joanna Pagacz 1, DeShawn Thompson 1, Stephen Kron 1
1Department of Molecular Genetics and Cell Biology, University of Chicago

A protocol for fluorescent, flow cytometric quantification of senescent cancer cells induced by chemotherapy drugs in cell culture or in murine tumor models is presented. Optional procedures include co-immunostaining, sample fixation to facilitate large batch or time point analysis, and the enrichment of viable senescent cells by flow cytometric sorting.

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