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Thomas Jefferson University

17 ARTICLES PUBLISHED IN JoVE

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Biology

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs
Hui Zhang 1,2, Niko G. Gubernator 3,4, Minerva Yue 1, Roland G. W. Staal 1, Eugene V. Mosharov 1, Daniela Pereira 1, Vojtech Balsanek 3, Paul A. Vadola 3, Bipasha Mukherjee 5, Robert H. Edwards 5, David Sulzer 1,2,6, Dalibor Sames 3
1Departments of Neurology, Columbia University, 2Departments of Psychiatry and Pharmacology, Columbia University, 3Department of Chemistry, Columbia University, 4eMolecules, Inc., 5Departments of Neurology and Physiology, University of California School of Medicine, San Francisco, 6Division of Molecular Therapeutics, New York Psychiatric Institute

A new means to measure neurotransmission optically using fluorescent dopamine analogs.

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Biology

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples
Chen Lu 1, Joshua L. Wonsidler 1, Jianwei Li 2, Yanming Du 1, Timothy Block 3, Brian Haab 4, Songming Chen 1
1Institute for Hepatitis and Virus Research, 2Department of Microbiology and Immunology, Thomas Jefferson University, 3Drexel University College of Medicine, 4Van Andel Research Institute

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

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Medicine

Non-invasive Assessment of Microvascular and Endothelial Function
Cynthia Cheng 1, Constantine Daskalakis 2, Bonita Falkner 3
1Department of Family and Community Medicine, Thomas Jefferson University , 2Department of Pharmacology and Experimental Therapeutics, Biostatistics Division, Thomas Jefferson University, 3Department of Internal Medicine, Thomas Jefferson University

Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function.

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Developmental Biology

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment
Janice L. Walker 1, Brigid M. Bleaken 1, Iris M. Wolff 1, A. Sue Menko 1
1Pathology, Anatomy and Cell Biology, Thomas Jefferson University

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

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Medicine

A Reproducible Computerized Method for Quantitation of Capillary Density using Nailfold Capillaroscopy
Cynthia Cheng *1, Chadd W. Lee *2, Constantine Daskalakis 3
1Department of Family and Community Medicine, Thomas Jefferson University, 2Sidney Kimmel Medical College, Thomas Jefferson University, 3Department of Pharmacology and Experimental Therapeutics, Thomas Jefferson University

Capillaroscopy is a non-invasive, efficient, relatively inexpensive and easy-to-learn methodology for directly visualizing capillaries in the microcirculation. However, only one publication to date describes the reliability of a complex software program available for quantitating capillaroscopy data. Here, we present a simple, reliable protocol for quantitating capillaries using a standardized algorithm.

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Education

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I
Diego Marin *1,2, Alexandra L. Nguyen *3, Richard T. Scott, Jr. 1,2, Karen Schindler 3
1IVI-RMA New Jersey, 2Jefferson College of Biomedical Sciences, Thomas Jefferson University, 3Department of Genetics, Rutgers, The State University of New Jersey

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

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Cancer Research

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry
Xiaoyu An *1,2, Xuesong Ouyang *1, Hui Zhang 1, Tingting Li 1, Yu-yang Huang 1, Zhiyuan Li 1, Demin Zhou 2, Qi-Xang Li 1,2
1Crown Bioscience Inc., 2State Key Laboratory of Natural and Biomimetic Drugs, Peking University

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200–600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

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Immunology and Infection

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue
Maria Steblyanko 1, Nadia Anikeeva 1, Marcus Buggert 2,3, Michael R. Betts 2, Yuri Sykulev 4
1Department of Microbiology and Immunology, Thomas Jefferson University, 2Department of Microbiology and Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, 3Department of Medicine Huddinge, Karolinska Institutet, Karolinska University Hospital Huddinge, 4Departments of Microbiology and Immunology and Medical Oncology, Sidney Kimmel Cancer Center, Thomas Jefferson University

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

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JoVE Journal

Sampling Soils in a Heterogeneous Research Plot
Jianwei Li 1
1Department of Agriculture and Environmental Science, Tennessee State University

The traditional soil-sampling procedure determines the number of soil samples arbitrarily. Here, we provide a simple yet efficient clustered soil-sampling design to demonstrate soil spatial heterogeneity and quantitatively determine the number of soil samples required and the associated sampling accuracy.

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Cancer Research

Generation of a Liver Orthotopic Human Uveal Melanoma Xenograft Platform in Immunodeficient Mice
Ken Kageyama 1,3, Shinji Ozaki 2,3, Takami Sato 3
1Department of Diagnostic and Interventional Radiology, Osaka City University Graduate School of Medicine, 2Department of Surgery, National Hospital Organization, Kure Medical Cancer Center, 3Department of Medical Oncology, Sidney Kimmel Cancer Center, Thomas Jefferson University

Orthotopic human liver metastatic uveal melanoma xenograft mouse models were created using surgical orthotopic implantation techniques with patient-derived tumor chunk and needle injection techniques with cultured human uveal melanoma cell lines.

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Genetics

Combining Laser Capture Microdissection and Microfluidic qPCR to Analyze Transcriptional Profiles of Single Cells: A Systems Biology Approach to Opioid Dependence
Sean J. O'Sullivan 1,2, Beverly A.S. Reyes 3, Rajanikanth Vadigepalli 1, Elisabeth J. Van Bockstaele 3, James S. Schwaber 1
1Daniel Baugh Institute for Functional Genomics and Computational Biology, Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, 2Sidney Kimmel Medical College, Thomas Jefferson University, 3Department of Pharmacology & Physiology, Drexel University College of Medicine

This protocol explains how to collect single neurons, microglia, and astrocytes from the central nucleus of the amygdala with high accuracy and anatomic specificity using laser capture microdissection. Additionally, we explain our use of microfluidic RT-qPCR to measure a subset of the transcriptome of these cells.

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Bioengineering

Contrast-Enhanced Subharmonic Aided Pressure Estimation (SHAPE) Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension
Flemming Forsberg 1, Ipshita Gupta 1,2, Priscilla Machado 1, Colette M. Shaw 1, Jonathan M. Fenkel 3, Kirk Wallace 4, John R. Eisenbrey 1
1Department of Radiology, Thomas Jefferson University, 2School of Biomedical Engineering, Sciences and Health Systems, Drexel University, 3Department of Medicine, Division of Gastroenterology and Hepatology, Thomas Jefferson University, 4GE Global Research

A protocol for noninvasively estimating ambient pressures utilizing subharmonic ultrasound imaging of infused contrast microbubbles (following appropriate calibration) is described with examples from human patients with chronic liver disease.

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Cancer Research

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth
Lorela Ciraku 1, Rebecca A. Moeller 1, Emily M. Esquea 1, Wiktoria A. Gocal 1, Edward J. Hartsough 2,4, Nicole L. Simone 3,4, Joshua G. Jackson 2, Mauricio J. Reginato 1,4
1Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, 2Department of Pharmacology and Physiology, Drexel University College of Medicine, 3Department of Radiation Oncology, Thomas Jefferson University, 4Sidney Kimmel Cancer Center, Thomas Jefferson University

We introduce a protocol for measuring real-time drug and radiation response of breast cancer brain metastatic cells in an organotypic brain slice model. The methods provide a quantitative assay to investigate the therapeutic effects of various treatments on brain metastases from breast cancer in an ex vivo manner within the brain microenvironment interface.

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Neuroscience

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis
Karthik Krishnamurthy *1, Davide Trotti 1, Piera Pasinelli 1, Brigid Jensen *1
1Jefferson Weinberg ALS Center, Thomas Jefferson University

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

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Neuroscience

Measurement of Oxygen Consumption Rate in Acute Striatal Slices from Adult Mice
Lianteng Zhi 1, Jingyu Zhao 1, David Jaffe 1, Yuanxin Chen 1, Ninghan Wang 1,2, Qi Qin 1, Erin L. Seifert 3, Chenjian Li 4, Hui Zhang 1
1Department of Neuroscience, Thomas Jefferson University, 2School of Biomedical Engineering, Drexel University, 3Department of Pathology, MitoCare Center, Thomas Jefferson University, 4School of Life Sciences, Peking University

Oxygen consumption rate (OCR) is a common proxy for mitochondrial function and can be used to study different disease models. We developed a new method using a Seahorse XF analyzer to directly measure the OCR in acute striatal slices from adult mice that is more physiologically relevant than other methods.

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Neuroscience

Investigating Drivers of Antireward in Addiction Behavior with Anatomically Specific Single-Cell Gene Expression Methods
Sean J. O'Sullivan *1,2,3, Ankita Srivastava *1, Rajanikanth Vadigepalli 1, James S. Schwaber 1
1Daniel Baugh Institute for Functional Genomics and Computational Biology, Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, 2Sidney Kimmel Medical College, Thomas Jefferson University, 3Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine

The combination of laser capture microdissection and microfluidic RT-qPCR provides anatomic and biotechnical specificity in measuring the transcriptome in single neurons and glia. Applying creative methods with a system's biology approach to psychiatric disease may lead to breakthroughs in understanding and treatment such as the neuroinflammation antireward hypothesis in addiction.

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Environment

Simulating Temperature in a Soil Incubation Experiment
Jianwei Li 1, Precious Areeveso 1, Xuehan Wang 1, Siyang Jian 1,2, Lahiru Gamage 1
1Department of Agricultural and Environmental Sciences, Tennessee State University, 2Department of Plant Biology and Microbiology, University of Oklahoma, Norman

Laboratory soil warming experiments usually employ two or more constant temperatures in multiple chambers. By presenting a sophisticated environmental chamber, we provide an accurate temperature control method to imitate the magnitude and amplitude of in situ soil temperature and improve the experimental design of soil incubation studies.

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