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28 ARTICLES PUBLISHED IN JoVE

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Biology

Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces
Ravi A. Desai 1, Michael T. Yang 1, Nathan J. Sniadecki 2, Wesley R. Legant 1, Christopher S. Chen 1
1Department of Bioengineering, University of Pennsylvania , 2University of Washington

In this video, we demonstrate how to fabricate and utilize microfabricated post array detectors (mPADs) to assess modulations of cellular contractility.

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Biology

Neutrophil Isolation Protocol
Hana Oh 1, Brian Siano 1, Scott Diamond 1
1Institute for Medicine and Engineering, University of Pennsylvania

Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.

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Biology

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture
Jamie L. Ifkovits 1, Harini G. Sundararaghavan 1, Jason A. Burdick 1
1Department of Bioengineering, University of Pennsylvania

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

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Biology

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering
Sudhir Khetan 1, Jason Burdick 2
1Department of Bioengineering, University of Pennsylvania , 2Department of Bioengineering, University of Pennsylvania-School of Medicine

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

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Neuroscience

Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence
Sava Sakadžić 1, Emmanuel Roussakis 2, Mohammad A. Yaseen 1, Emiri T. Mandeville 3, Vivek J. Srinivasan 1, Ken Arai 3, Svetlana Ruvinskaya 1, Weicheng Wu 1, Anna Devor 1,4, Eng H. Lo 3, Sergei A. Vinogradov 2, David A. Boas 1
1Optics Division, Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital and Harvard Medical School, 2Department of Biochemistry and Biophysics, University of Pennsylvania, 3Neuroprotection Research Laboratory, Departments of Radiology and Neurology, Massachusetts General Hospital and Harvard Medical School, 4Departments of Neurosciences and Radiology, University of California

We present an experimental procedure for measuring the partial pressure of oxygen (pO2) in cerebral vasculature based on oxygen-dependent quenching of phosphorescence. Animal preparation and imaging procedures were outlined for both large field of view CCD-based imaging of pO2 in rats and 2-photon excitation based imaging of pO2 in mice.

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Biology

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences
Erika Kague 1, Christopher Weber 1, Shannon Fisher 1
1Department of Cell and Developmental Biology, University of Pennsylvania

We demonstrate our approach to finding potential enhancer elements from developmentally regulated genes and evaluating their function through mosaic zebrafish transgenesis.

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Biology

Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems
Louise E. Sinks 1, Emmanuel Roussakis 1, Tatiana V. Esipova 1, Sergei A. Vinogradov 1
1Department of Biochemistry and Biophysics, University of Pennsylvania

We present principles of oxygen measurements by phosphorescence quenching and review design of porphyrin-based dendritic nanosensors for oxygen imaging in biological systems.

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Biology

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels
Alexandra Cretu 1, Paola Castagnino 1, Richard Assoian 1
1Department of Pharmacology, University of Pennsylvania

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

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Immunology and Infection

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells
Theresa S. Moser 1, Leah R. Sabin 1, Sara Cherry 1
1Department of Microbiology, Penn Genome Frontiers Institute, University of Pennsylvania

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

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Bioengineering

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model
Alexandra E. Jantzen 1, Whitney O. Lane 2, Shawn M. Gage 3, Justin M. Haseltine 1, Lauren J. Galinat 1, Ryan M. Jamiolkowski 4, Fu-Hsiung Lin 3, George A. Truskey 1, Hardean E. Achneck 3
1Department of Biomedical Engineering, Duke University , 2School of Medicine, Duke University , 3Department of Surgery, Duke University Medical Center, 4School of Medicine, University of Pennsylvania

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

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Bioengineering

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress
Whitney O. Lane 1, Alexandra E. Jantzen 2, Tim A. Carlon 2, Ryan M. Jamiolkowski 3, Justin E. Grenet 1, Melissa M. Ley 1, Justin M. Haseltine 2, Lauren J. Galinat 2, Fu-Hsiung Lin 1, Jason D. Allen 4, George A. Truskey 2, Hardean E. Achneck 1
1Department of Surgery, Duke University Medical Center, 2Department of Biomedical Engineering, Duke University , 3School of Medicine, University of Pennsylvania , 4Department of Medicine, Division of Cardiology, Duke University Medical Center

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

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Bioengineering

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis
Stacy L. Gelhaus 1,2, A. Clementina Mesaros 1,2, Ian A. Blair 1,2
1Centers for Cancer Pharmacology and Excellence in Environmental Toxicology, University of Pennsylvania , 2Department of Pharmacology, University of Pennsylvania

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

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Biology

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion
Jessica W. Wen 1, Abby L. Olsen 2, Maryna Perepelyuk 2, Rebecca G. Wells 2
1Division of Gastroenterology, Hepatology & Nutrition, Department of Pediatrics, The Children's Hospital of Philadelphia, 2Department of Medicine, University of Pennsylvania

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

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Bioengineering

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness
Myung-Jin Oh 1, Frank Kuhr 1, Fitzroy Byfield 2, Irena Levitan 1
1Section of Respiratory, Critical Care and Sleep Medicine, Department of Medicine, University of Illinois, 2Institute for Medicine and Engineering, University of Pennsylvania

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

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Immunology and Infection

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation
Karen L. Joyce 1, Will Morgan 2, Robert Greenberg 2, Meera G. Nair 1
1Institute of Immunology, Department of Microbiology, Perelman School of Medicine, University of Pennsylvania , 2Pathobiology, School of Veterinary Medicine, University of Pennsylvania

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

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Medicine

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme
Brian C. Baumann 1, Jay F. Dorsey 1, Joseph L. Benci 1, Daniel Y. Joh 1, Gary D. Kao 1
1Department of Radiation Oncology, University of Pennsylvania

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

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Biology

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes
Joseph D. Tario Jr. 1, Kristen Humphrey 1, Andrew D. Bantly 2, Katharine A. Muirhead 3, Jonni S. Moore 4, Paul K. Wallace 1
1Department of Flow and Image Cytometry, Roswell Park Cancer Institute, 2Flow Cytometry & Cell Sorting Resource Laboratory, University of Pennsylvania , 3SciGro, Inc., 4Department of Pathology and Laboratory Medicine, University of Pennsylvania

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

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Biology

Isolating Primary Melanocyte-like Cells from the Mouse Heart
Hayoung Hwang 1, Fang Liu 1, Mark D. Levin 1, Vickas V. Patel 1
1Penn Cardiovascular Institute, Perelman School of Medicine, University of Pennsylvania

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. 

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Medicine

Minimal Erythema Dose (MED) Testing
Carolyn J. Heckman 1, Rachel Chandler 2, Jacqueline D. Kloss 3, Amy Benson 2, Deborah Rooney 2, Teja Munshi 1, Susan D. Darlow 1, Clifford Perlis 4, Sharon L. Manne 5, David W. Oslin 2
1Cancer Prevention and Control Program, Fox Chase Cancer Center , 2Department of Psychiatry, University of Pennsylvania , 3Department of Psychology, Drexel University , 4Department of Medicine, Fox Chase Cancer Center , 5Cancer Prevention and Control Program, The Cancer Institute of New Jersey

This article describes how to conduct minimal erythema dose (MED) testing in order to determine the lowest dose of ultraviolet radiation that will cause erythema (burning) when administered to an individual.

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Medicine

Utilizing Repetitive Transcranial Magnetic Stimulation to Improve Language Function in Stroke Patients with Chronic Non-fluent Aphasia
Gabriella Garcia 1,2, Catherine Norise 1,2, Olufunsho Faseyitan 1,2, Margaret A. Naeser 3,4,5, Roy H. Hamilton 1,2
1Department of Neurology, Perelman School of Medicine, University of Pennsylvania , 2Center for Cognitive Neuroscience, University of Pennsylvania , 3Veterans Affairs Boston Healthcare System, 4Harold Goodglass Aphasia Research Center, Boston University School of Medicine, 5Department of Neurology, Boston University School of Medicine

We explore the use of repetitive transcranial magnetic stimulation (rTMS) to improve language abilities in patients with chronic stroke and non-fluent aphasia. After identifying a site in the right frontal gyrus for each patient that responds optimally to stimulation, we target this site during ten days of rTMS treatment.

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Biology

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)
Sandra Deliard 1, Jianhua Zhao 1, Qianghua Xia 1, Struan F.A. Grant 1,2
1Division of Human Genetics, Children's Hospital of Philadelphia Research Institute, 2Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

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Chemistry

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)
Nathaniel W. Snyder 1, Maya Khezam 1, Clementina A. Mesaros 1, Andrew Worth 1, Ian A. Blair 1
1Centers for Cancer Pharmacology and Excellence in Environmental Toxicology, Department of Pharmacology, University of Pennsylvania

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

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Bioengineering

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds
Tonya J. Whitehead 1, Harini G. Sundararaghavan 1
1Biomedical Engineering, Wayne State University

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

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Biology

Isolation of Neonatal Extrahepatic Cholangiocytes
Sara Karjoo 1, Rebecca G. Wells 2
1Division of Gastroenterology, Hepatology, and Nutrition, The Children's Hospital of Philadelphia, 2Department of Medicine, The Perelman School of Medicine at the University of Pennsylvania

A technique to isolate cholangiocytes from the extrahepatic bile ducts of neonatal mice is described. The ducts are meticulously dissected, and then cells are isolated by outgrowth in thick collagen gels. This method provides a useful tool for studying extrahepatic bile duct development and pathology.

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Engineering

Preparation and Evaluation of Hybrid Composites of Chemical Fuel and Multi-walled Carbon Nanotubes in the Study of Thermopower Waves
Hayoung Hwang *1, Taehan Yeo *1, Yonghwan Cho 1, Dongjoon Shin 1, Wonjoon Choi 1
1School of Mechanical Engineering, Korea University

A protocol for conducting thermopower wave experiments is presented. The synthesis of hybrid composites of a chemical fuel and micro/nanostructured material, manufacturing of a thermopower wave generator, and methods for measuring the corresponding physical phenomena are described.

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Education

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism
Andrew J. Worth *1,2, Dylan M. Marchione *2,3, Robert C. Parry 1,2, Qingqing Wang 2,3, Kevin P. Gillespie 2,3, Noelle N. Saillant 4, Carrie Sims 4, Clementina Mesaros 1,2, Nathaniel W. Snyder 5, Ian A. Blair 1,2
1Center for Cancer Pharmacology, University of Pennsylvania, 2Center for Excellence in Environmental Toxicology, University of Pennsylvania, 3Penn SRP and Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, 4Division of Traumatology, Department of Surgery, Critical Care and Acute Care Surgery, University of Pennsylvania, 5A.J. Drexel Autism Institute, Drexel University

Here we show isolated human platelets can be used as an accessible ex vivo model to study metabolic adaptations in response to the complex I inhibitor rotenone. This approach employs isotopic tracing and relative quantification by liquid chromatography-mass spectrometry and can be applied to a variety of study designs.

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Medicine

Optimized Griess Reaction for UV-Vis and Naked-eye Determination of Anti-malarial Primaquine
Yalan Wu *1, Shengjun Wu *1, Xin-an Huang 1, Qingping Zeng 1, Tao Deng 1, Fang Liu 1
1Institute of Tropical Medicine, Guangzhou University of Chinese Medicine

This protocol describes a novel colorimetric method for antimalarial primaquine (PMQ) detection in synthetic urines and human serums.

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Medicine

Virtual Reality Tools for Assessing Unilateral Spatial Neglect: A Novel Opportunity for Data Collection
Peter J. Schwab 1,2, Alex Miller 2, Ann-Marie Raphail 3, Ari Levine 2, Christopher Haslam 2, H. Branch Coslett 1,2, Roy H. Hamilton 1,2,4
1Department of Neurology, University of Pennsylvania, 2Laboratory for Cognition and Neural Stimulation, University of Pennsylvania, 3Department of Psychology, Drexel University, 4Department of Physical Medicine and Rehabilitation, University of Pennsylvania

The goal was to design, build, and pilot a novel virtual reality task to detect and characterize unilateral spatial neglect, a syndrome affecting 23-46% of acute stroke survivors, expanding the role of virtual reality in the study and management of neurologic disease.

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