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University of Texas Southwestern Medical Center at Dallas

13 ARTICLES PUBLISHED IN JoVE

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Biology

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue
Meirigeng Qi 1, Barbara Barbaro 1, Shusen Wang 1, Yong Wang 1, Mike Hansen 1, Jose Oberholzer 1
1Department of Surgery, University of Illinois, Chicago

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

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Biology

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets
Meirigeng Qi 1, Barbara Barbaro 1, Shusen Wang 1, Yong Wang 1, Mike Hansen 1, Jose Oberholzer 1
1Department of Surgery, University of Illinois, Chicago

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

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Biology

A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device
Adeola F. Adewola 1, Yong Wang 1, Tricia Harvat 1, David T. Eddington 2, Dongyoung Lee 1, Jose Oberholzer 1,2
1Department of Surgery, University of Illinois, Chicago, 2Department of Bioengineering, University of Illinois, Chicago

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence imaging of calcium influx and mitochondrial potential changes.

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Biology

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging
W. Ryan Williamson 1, P. Robin Hiesinger 1
1Department of Physiology and Green Center for Systems Biology, University of Texas Southwestern Medical Center

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

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Biology

Recombineering Homologous Recombination Constructs in Drosophila
Arnaldo Carreira-Rosario 1, Shane Scoggin 1, Nevine A. Shalaby 1, Nathan David Williams 1, P. Robin Hiesinger 2,3, Michael Buszczak 1
1Department of Molecular Biology, University of Texas Southwestern Medical Center at Dallas, 2Department of Physiology, University of Texas Southwestern Medical Center at Dallas, 3Green Center for Systems Biology, University of Texas Southwestern Medical Center at Dallas

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

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Biology

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies
Sungsoo Lee *1, Hui Zheng *1, Liang Shi 1, Qiu-Xing Jiang 1
1Department of Cell Biology, University of Texas Southwestern Medical Center at Dallas

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

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Bioengineering

Quantitative and Temporal Control of Oxygen Microenvironment at the Single Islet Level
Joe Fu-Jiou Lo 1, Yong Wang 2,3, Zidong Li 1, Zhengtuo Zhao 1, Di Hu 1, David T. Eddington 3, Jose Oberholzer 2,3
1Department of Mechanical Engineering, University of Michigan-Dearborn, 2Department of Surgery/Transplant, University of Illinois at Chicago, 3Department of Bioengineering, University of Illinois at Chicago

Microfluidic oxygen control confers more than just convenience and speed over hypoxic chambers for biological experiments. Especially when implemented via diffusion through a membrane, microfluidic oxygen can provide simultaneous liquid and gas phase modulations at the microscale-level. This technique enables dynamic multi-parametric experiments critical for studying islet pathophysiology.

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Biology

Fluorescence Imaging with One-nanometer Accuracy (FIONA)
Yong Wang *1,2, En Cai *1,2, Janet Sheung 1,2, Sang Hak Lee 1,2, Kai Wen Teng 2,3, Paul R. Selvin 1,2,3
1Department of Physics, University of Illinois at Urbana-Champaign, 2Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, 3Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

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Medicine

A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish
Xiao Ma *1,2,3, Yonghe Ding *2,3, Yong Wang 2,3,4, Xiaolei Xu 1,2,3
1Clinical and Translational Sciences Track, Mayo Clinic Graduate School of Biomedical Sciences, 2Department of Biochemistry and Molecular Biology, Mayo Clinic, 3Division of Cardiovascular Diseases, Mayo Clinic, 4Institute of Life Science, Beijing University of Chinese Medicine

A method to generate a doxorubicin-induced cardiomyopathy model in adult zebrafish (Danio rerio) is described here. Two alternative ways of intraperitoneal injection are presented and conditions to reduce variations among different experimental groups are discussed.

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Developmental Biology

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies
Wei Zheng 1,2, Zhiguo Chen 1,2
1Cell Therapy Center, Beijing Institute of Geriatrics, Xuanwu Hospital, Capital Medical University, and Key Laboratory of Neurodegeneration, Ministry of Education, 2Center of Neural Injury and Repair, Beijing Institute for Brain Disorders

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson's disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

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Developmental Biology

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development
Yong Wang *1, Ding Zhang *2, Fang Zhou 2, Meijun Zhou 2, Qiujie Li 1, Jingyu Chen 3, Jun Yang 1,2
1Department of Physiology and Department of Cardiology of the Second Affiliated Hospital, Zhejiang University School of Medicine, 2Department of Cell Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, 3Wuxi Lung Transplant Center, Wuxi People's Hospital affiliated to Nanjing Medical University

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

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JoVE Core

High-Temperature and High-Pressure In situ Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy
Nicholas R. Jaegers 1, Wenda Hu 2, Yong Wang 1, Jian Zhi Hu 1
1Pacific Northwest National Laboratory, 2Washington State University

The molecular structures and dynamics of solids, liquids, gases, and mixtures are of critical interest to diverse scientific fields. High-temperature, high-pressure in situ MAS NMR enables detection of the chemical environment of constituents in mixed phase systems under tightly controlled chemical environments.

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Neuroscience

Whole Central and Peripheral Nervous System Mice Dissection
Krystina Rhéaume *1, Zhiguo Chen *2, Yong Wang *2, Camille Plante 1, Dhanushka Hewa Bostanthirige 1, Maude Lévesque 1, Sameh Geha 3,4, Lu Q. Le 2,5,6,7, Jean-Philippe Brosseau 1,4
1Department of Biochemistry and Functional Genomics, Université de Sherbrooke, 2Department of Dermatology, University of Texas Southwestern Medical Center at Dallas, 3Department of Pathology, Université de Sherbrooke, 4Centre de Recherche du Centre Hospitalier de Universitaire de Sherbrooke, Université de Sherbrooke, 5Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center at Dallas, 6UTSW Comprehensive Neurofibromatosis Clinic, University of Texas Southwestern Medical Center at Dallas, 7Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center at Dallas

Here, we present a protocol to complete the dissection of central and peripheral nervous systems of mice. The dissected tissues are further analyzed in downstream applications, such as taking gross pictures or histology.

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