University of Pennsylvania

82 articles published in JoVE

• Bioengineering

  
  Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications Victoria G. Muir¹, Margaret E. Prendergast¹, Jason A. Burdick^{1,2,3 1}Department of Bioengineering, School of Engineering and Applied Sciences, University of Pennsylvania, ²BioFrontiers Institute, University of Colorado Boulder, ³Department of Chemical and Biological Engineering, College of Engineering and Applied Science, University of Colorado Boulder This work describes straightforward, adaptable, and low-cost methods to fabricate microgels with extrusion fragmentation, process the microgels into injectable granular hydrogels, and apply the granular hydrogels as extrusion printing inks for biomedical applications.

• Cancer Research

  
  A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate Shun Ishiyama^1,2,3,4, Casey Kissel⁵, Xin Guo¹, Alexis Howard⁶, Harumi Saeki⁷, Tomoaki Ito⁸, Polina Sysa-Shah⁹, Hajime Orita¹⁰, Kazuhiro Sakamoto⁴, Kathleen Gabrielson^{1,2 1}Department of Molecular and Comparative Pathobiology, The Johns Hopkins University School of Medicine, ²Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, ³Department of Surgery, Johns Hopkins University School of Medicine, ⁴Department of Coloproctological Surgery, Juntendo University Faculty of Medicine, ⁵Program for Comparative Medicine, Gene Therapy Program, Perelman School of Medicine, University of Pennsylvania, ⁶Tuskegee College of Veterinary Medicine, ⁷Department of Human Pathology, Juntendo University Faculty of Medicine, ⁸Department of Surgery, Juntendo University Shizuoka Hospital, Juntendo University School of Medicine, ⁹Department of Urology, The Johns Hopkins School of Medicine, ¹⁰Department of Gastroenterology and Minimally Invasive Surgery, Juntendo University Faculty of Medicine Here, a syngeneic orthotopic implantation followed by an amputation procedure of the osteosarcoma with spontaneous pulmonary metastasis that can be used for preclinical investigation of metastasis biology and development of novel therapeutics is described.

• Biology

  
  Chemical Dimerization-Induced Protein Condensates on Telomeres Rongwei Zhao¹, David M. Chenoweth², Huaiying Zhang^{1 1}Department of Biological Sciences, Mellon College of Science, Carnegie Mellon University, ²Department of Chemistry, School of Arts and Sciences, University of Pennsylvania This protocol illustrates a chemically induced protein dimerization system to create condensates on chromatin.  The formation of promyelocytic leukemia (PML) nuclear body on telomeres with chemical dimerizers is demonstrated. Droplet growth, dissolution, localization and composition are monitored with live cell imaging, immunofluorescence (IF) and fluorescence in situ hybridization (FISH).

• Immunology and Infection

  
  Production of Human CRISPR-Engineered CAR-T Cells Sangya Agarwal*^1,2, Nils Wellhausen*^1,2, Bruce L. Levine^1,2, Carl H. June^{1,2 1}Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, ²Parker Institute for Cancer Immunotherapy, University of Pennsylvania Here, we present a protocol for gene editing in primary human T cells using CRISPR Cas Technology to modify CAR-T cells.

• Medicine

  
  Virtual Reality Tools for Assessing Unilateral Spatial Neglect: A Novel Opportunity for Data Collection Peter J. Schwab^1,2, Alex Miller², Ann-Marie Raphail³, Ari Levine², Christopher Haslam², H. Branch Coslett^1,2, Roy H. Hamilton^{1,2,4 1}Department 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.

• Biochemistry

  
  Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays Ananya Tripathi¹, Charles Bond^1,2, James R. Sellers¹, Neil Billington¹, Yasuharu Takagi^{1 1}Laboratory of Molecular Physiology, Cell and Developmental Biology Center, National Heart, Lung and Blood Institute, National Institutes of Health, ²Department of Physiology, Perelman School of Medicine, University of Pennsylvania Presented here is a procedure to express and purify myosin 5a followed by a discussion of its characterization, using both ensemble and single molecule in vitro fluorescence microscopy-based assays, and how these methods can be modified for the characterization of nonmuscle myosin 2b.

• Bioengineering

  
  Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization Vaishali V. Inamdar¹, Emmett G. Fitzpatrick¹, Ivan S. Alferiev^1,2, Robert J. Levy^1,2, Stanley J. Stachelek*^1,2, Ilia Fishbein*^{1,2 1} Presented here is a protocol for appending peptide CD47 (pepCD47) to metal stents using polybisphosphonate chemistry. Functionalization of metal stents using pepCD47 prevents the attachment and activation of inflammatory cells thus improving their biocompatibility.

• Developmental Biology

  
  Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad Kara A. Nelson¹, Bailey N. Warder¹, Stephen DiNardo¹, Lauren Anllo^{1 1}Cell and Developmental Biology Department and the Penn Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania Here, we provide a dissection protocol required to live-image the late embryonic Drosophila male gonad. This protocol will permit observation of dynamic cellular processes under normal conditions or after transgenic or pharmacological manipulation.

• Behavior

  
  Assessing Binocular Central Visual Field and Binocular Eye Movements in a Dichoptic Viewing Condition Rajkumar N. Raveendran*¹, Arun K. Krishnan*^{1,2 1}Envision Research Institute, ²University of Pennsylvania Presented here is a protocol for assessing binocular eye movements and gaze-controlled central visual field screening in participants with central vision loss.

• Bioengineering

  
  Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy Kevin Y. Chen¹, Ryan M. Jamiolkowski¹, Alyssa M. Tate¹, Shane A. Fiorenza², Shawn H. Pfeil², Yale E. Goldman^{1 1}Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, ²Department of Physics, West Chester University Described here is a nanosphere lithography method for parallel fabrication of zero mode waveguides, which are arrays of nanoapertures in a metal-clad glass microscopy coverslip for single molecule imaging at nano- to micromolar concentrations of fluorophores. The method takes advantage of colloidal crystal self-assembly to create a waveguide template.

• Bioengineering

  
  Fabrication of Ti₃C₂ MXene Microelectrode Arrays for In Vivo Neural Recording Nicolette Driscoll^1,2,3, Kathleen Maleski^4,5, Andrew G. Richardson^2,6, Brendan Murphy^1,2,3, Babak Anasori^4,5, Timothy H. Lucas^2,6, Yury Gogotsi^4,5, Flavia Vitale^{1,2,3,7,8 1}Department of Bioengineering, University of Pennsylvania, ²Center for Neuroengineering and Therapeutics, University of Pennsylvania, ³Corporal Michael J. Crescenz Veterans Affairs Medical Center, ⁴Department of Materials Science and Engineering, Drexel University, ⁵A.J. Drexel Nanomaterials Institute, Drexel University, ⁶Department of Neurosurgery, University of Pennsylvania, ⁷Department of Neurology, University of Pennsylvania, ⁸Department of Physical Medicine and Rehabilitation We describe here a method for fabricating Ti3C2 MXene microelectrode arrays and utilizing them for in vivo neural recording.

• Cancer Research

  
  Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma Ceire A. Hay¹, Rina Sor^2,3, Ahron J. Flowers^2,3, Cynthia Clendenin^2,3, Katelyn T. Byrne^{1,4 1}Department of Medicine, Perelman School of Medicine, University of Pennsylvania, ²Pancreatic Cancer Mouse Hospital, Perelman School of Medicine, University of Pennsylvania, ³Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, ⁴Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

• Genetics

  
  Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells Fabian L. Cardenas-Diaz*¹, Jean Ann Maguire*¹, Paul Gadue^1,2,3, Deborah L. French^{1,2,3 1} This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

• Biology

  
  Cell Type-specific Gene Expression Profiling in the Mouse Liver Amber W. Wang¹, Adam M. Zahm¹, Kirk J. Wangensteen^{1,2 1}Department of Genetics, University of Pennsylvania, ²Department of Medicine, University of Pennsylvania Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

• Neuroscience

  
  Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry Hélène Léger¹, Evelyn Santana², William A Beltran², Francis C Luca^{1 1}Department of Biomedical Sciences, University of Pennsylvania School of Veterinary Medicine, ²Division Experimental Retinal Therapies, Department of Clinical Sciences and Advanced Medicine, University of Pennsylvania School of Veterinary Medicine This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

• Behavior

  
  Experimental Paradigm for Measuring the Effects of Self-distancing in Young Children Amanda Grenell¹, Rachel E. White², Emily O. Prager¹, Catherine Schaefer¹, Ethan Kross³, Angela L. Duckworth⁴, Stephanie M. Carlson^{1 1}Insitute of Child Development, University of Minnesota-Twin Cities, ²Department of Psychology, Hamilton College, ³Department of Psychology, University of Michigan, ⁴Department of Psychology, University of Pennsylvania An experimental paradigm was created to measure the effects of self-distancing in young children (4-6-year-olds). Self-distancing is a process through which individuals adopt a less egocentric perspective. This paradigm has been used to examine the effects of self-distancing on young children's self-regulation.

• Immunology and Infection

  
  Merkel Cell Polyomavirus Infection and Detection Wei Liu*¹, Nathan A. Krump*¹, Christopher B. Buck², Jianxin You^{1 1}Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, ²Laboratory of Cellular Oncology, National Cancer Institute Here, we present a protocol to infect primary human dermal fibroblast with MCPyV. The protocol includes isolation of dermal fibroblasts, preparation of MCPyV virions, virus infection, immunofluorescence staining, and fluorescence in situ hybridization. This protocol can be extended for characterizing MCPyV-host interactions and discovering other cell types infectable by MCPyV.

• Genetics

  
  Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms Clark Fritsch^1,2, Jean-Francois Pierre Gout^3,4, Marc Vermulst^{1 1} This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

• Bioengineering

  
  Probing the Roles of Physical Forces in Early Chick Embryonic Morphogenesis Yan Li*¹, Hannah Grover*¹, Eric Dai², Kevin Yang¹, Zi Chen^{1 1}Thayer School of Engineering, Dartmouth College, ²Department of Bioengineering, University of Pennsylvania Here, we present a protocol introducing a set of new ex-ovo experiments and physical modeling approaches for studying the mechanics of morphogenesis during early chick embryonic brain torsion.

• Medicine

  
  A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System Dan Li*¹, Feng Li*¹, Pavithran Guttipatti¹, Yuanquan Song^{1,2 1} Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

• Bioengineering

  
  Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration Kritika S. Katiyar*^1,2,3, Carla C. Winter*^1,2,4, Wisberty J. Gordián-Vélez^1,2,4, John C. O'Donnell^1,2, Yeri J. Song^1,5, Nicole S. Hernandez^1,5, Laura A. Struzyna^1,2,4, D. Kacy Cullen^{1,2,5 1}Center for Brain Injury & Repair, Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, ²Center for Neurotrauma, Neurodegeneration & Restoration, Michael J. Crescenz Veterans Affairs Medical Center, ³School of Biomedical Engineering, Drexel University, ⁴Department of Bioengineering, School of Engineering and Applied Sciences, University of Pennsylvania, ⁵Neuroscience Graduate Group, Perelman School of Medicine, University of Pennsylvania We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered "living scaffolds", exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

• Neuroscience

  
  Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors David J. Tischfield^1,2, Stewart A. Anderson^{1 1}Neuroscience Graduate Group, Perelman School of Medicine, University of Pennsylvania, ²Department of Psychiatry, Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

• Medicine

  
  In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue Emanuele Loro*¹, Stephen H. Wang*², Richard J. Schwab², Tejvir S. Khurana^{1 1}Department of Physiology and Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, ²Center for Sleep & Circadian Neurobiology, University of Pennsylvania We describe a surgical procedure in an anesthetized rat model for determining the muscle tone and viscoelastic properties of the tongue. The procedure involves specific stimulation of the hypoglossal nerves and application of passive Lissajous force/deformation curves to the muscle.

• Neuroscience

  
  Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling Laura A. Struzyna*^1,2,3, Dayo O. Adewole*^1,2,3, Wisberty J. Gordián-Vélez^1,2,3, Michael R. Grovola^2,3, Justin C. Burrell^2,3, Kritika S. Katiyar^2,3,4, Dmitriy Petrov^2,3, James P. Harris^2,3, D. Kacy Cullen^{2,3 1}Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvania, ²Center for Brain Injury & Repair, Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, ³Center for Neurotrauma, Neurodegeneration & Restoration, Michael J. Crescenz Veterans Affairs Medical Center, ⁴School of Biomedical Engineering, Drexel University This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

• Developmental Biology

  
  In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells Jianle Wang¹, Montserrat C. Anguera^{1 1}Department of Biomedical Sciences, School of Veterinary Medicine, University of Pennsylvania Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

• Medicine

  
  Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle Vidhya Kumar¹, Henry Chang¹, David A. Reiter², David P. Bradley³, Martha Belury⁴, Shana E. McCormack⁵, Subha V. Raman^{1 1}Davis Heart and Lung Research Institute, The Ohio State University, ²Laboratory of Clinical Investigation, National Institute on Aging, ³Division of Endocrinology, Diabetes and Metabolism, The Ohio State University, ⁴Department of Human Sciences, Human Nutrition, The Ohio State University, ⁵Division of Endocrinology and Diabetes, Department of Pediatrics, University of Pennsylvania This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (³¹PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

• Neuroscience

  
  High-density Electroencephalographic Acquisition in a Rodent Model Using Low-cost and Open-source Resources Andrzej Z. Wasilczuk¹, Alexander Proekt¹, Max B. Kelz¹, Andrew R. McKinstry-Wu^{1 1}Department of Anesthesiology and Critical Care, Perelman School of Medicine, University of Pennsylvania Instructions for the low-cost construction and surgical implantation of a chronic transcranial high-density electroencephalographic montage into mice are provided. Signal recording, extraction, and processing techniques are also described.

• Environment

  
  Using Pharmacological Manipulation and High-precision Radio Telemetry to Study the Spatial Cognition in Free-ranging Animals Timothy C. Roth*¹, Aaron R. Krochmal*², William B. Gerwig, IV¹, Sage Rush³, Nathaniel T. Simmons², Jeffery D. Sullivan⁴, Katrina Wachter^{5 1}Department of Psychology, Franklin and Marshall College, ²Department of Biology, Washington College, ³University of Pennsylvania, ⁴School of Forestry and Wildlife Sciences, Auburn University, ⁵Morsani College of Medicine, University of South Florida This paper describes a novel protocol that combines the pharmacological manipulation of memory and radio telemetry to document and quantify the role of cognition in navigation.

• Biology

  
  Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy Yong Ho Bae^1,2, Shu-lin Liu^1,2, Fitzroy J. Byfield³, Paul A. Janmey³, Richard K. Assoian^{1,2 1}Program in Translational Biomechanics, Institute of Translational Medicine and Therapeutics, University of Pennsylvania, ²Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, ³Department of Physiology, University of Pennsylvania We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

• Cancer Research

  
  Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors Alan J. Fox¹, Matthew C. Hiemenz¹, David B. Lieberman¹, Shrey Sukhadia¹, Barnett Li¹, Joseph Grubb¹, Patrick Candrea¹, Karthik Ganapathy¹, Jianhua Zhao¹, David Roth¹, Evan Alley^2,3, Alison Loren^2,3, Jennifer J. D. Morrissette^{1 1}Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, ²Division of Hematology/Oncology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, ³Abramson Cancer Center This manuscript describes clinical protocols for two next-generation sequencing panels. One panel interrogates hematologic malignancies while the other panel targets genes commonly mutated in solid tumors. Molecular classification of driver mutations in human malignancies offers valuable prognostic and predictive information.

• Medicine

  
  Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes Gregory P. Way^1,2, Maura Southwell¹, Scott P. McRobert^{1 1}Department of Biology, Saint Joseph’s University, ²Genomics and Computational Biology, University of Pennsylvania This manuscript describes the setup, implementation, and analysis of boldness, aggression, and shoaling in zebrafish and testing for the presence of a behavioral syndrome. A standardized approach for behavioral quantification will allow for easier comparison across studies. Modifications to this protocol are possible as each assay can be run individually.

• Biology

  
  Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis Simone Sidoli¹, Natarajan V. Bhanu¹, Kelly R. Karch¹, Xiaoshi Wang¹, Benjamin A. Garcia^{1 1}Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

• Environment

  
  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. Saillant⁴, Carrie Sims⁴, Clementina Mesaros^1,2, Nathaniel W. Snyder⁵, Ian A. Blair^{1,2 1}Center 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.

• Chemistry

  
  Cooling Rate Dependent Ellipsometry Measurements to Determine the Dynamics of Thin Glassy Films Ethan C. Glor¹, Zahra Fakhraai^{1 1}Department of Chemistry, University of Pennsylvania Here, we present a protocol for cooling rate dependent ellipsometry experiments, which can determine the glass transition temperature (Tg), average dynamics, fragility and the expansion coefficient of the super-cooled liquid and glass for a variety of glassy materials.

• Bioengineering

  
  A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities Jacob R. Price¹, Wen K. Shieh², Christopher M. Sales^{1 1}Civil, Architectural, and Environmental Engineering, Drexel University, ²Chemical and Biomolecular Engineering, University of Pennsylvania A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

• Biology

  
  Isolating Primary Melanocyte-like Cells from the Mouse Heart Hayoung Hwang¹, Fang Liu¹, Mark D. Levin¹, Vickas V. Patel^{1 1}Penn 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. 

• Biology

  
  Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons Yang Song*¹, Xuemei Zhang*¹, Lingyan Huang², Mark A. Behlke², Andrew Tsourkas^{1 1}Department of Bioengineering, University of Pennsylvania, ²Integrated DNA Technologies, Inc. Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

• Neuroscience

  
  Using Informational Connectivity to Measure the Synchronous Emergence of fMRI Multi-voxel Information Across Time Marc N. Coutanche¹, Sharon L. Thompson-Schill^{1 1}Department of Psychology, University of Pennsylvania Informational connectivity measures the correspondence between time courses of multi-voxel information across different brain regions. Multi-voxel pattern discriminability time series are extracted from regions and compared, revealing networks that are not identified in a typical functional connectivity approach.

• Bioengineering

  
  Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration Lindsay M. Thomson¹, Brian D. Polizzotti¹, Frances X. McGowan², John N. Kheir^{1 1}Department of Cardiology, Boston Children's Hospital, Harvard Medical School, ²Department of Anesthesiology and Critical Care, Children's Hospital of Philadelphia, University of Pennsylvania We describe methods for the manufacture of large volumes of lipid-based oxygen microbubbles (LOMs) designed for intravenous oxygen delivery using high-shear homogenization and serial concentration.

• Medicine

  
  Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer Melanie R. Rutkowski*¹, Michael J. Allegrezza*¹, Nikolaos Svoronos¹, Amelia J. Tesone¹, Tom L. Stephen¹, Alfredo Perales-Puchalt¹, Jenny Nguyen¹, Paul J. Zhang², Steven N. Fiering³, Julia Tchou^4,5,6, Jose R. Conejo-Garcia^{1 1}Tumor Microenvironment and Metastasis Program, Wistar Institute, ²Department of Pathology and Lab Medicine, Perelman School of Medicine, University of Pennsylvania, ³Department of Microbiology and Immunology and Department of Genetics, Geisel School of Medicine at Dartmouth, ⁴Division of Endocrine and Oncologic Surgery, Department of Surgery, Perelman School of Medicine, University of Pennsylvania, ⁵Rena Rowan Breast Center, Abramson Cancer Center, University of Pennsylvania, ⁶Center for Advanced Medicine, University of Pennsylvania Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

• Biology

  
  Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends Vladimir A. Volkov^1,2, Anatoly V. Zaytsev³, Ekaterina L. Grishchuk^{3 1}Center for Theoretical Problems of Physicochemical Pharmacology, Russian Academy of Sciences, ²Federal Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russia, ³Physiology Department, Perelman School of Medicine, University of Pennsylvania Microtubules are inherently unstable polymers, and their switching between growth and shortening is stochastic and difficult to control. Here we describe protocols using segmented microtubules with photoablatable stabilizing caps. Depolymerization of segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting analysis of motions with the disassembling microtubule ends.

• Neuroscience

  
  A Single-fly Assay for Foraging Behavior in Drosophila Orel A. Zaninovich¹, Susy M. Kim¹, Cory R. Root^1,2, David S. Green^1,3, Kang I. Ko^1,4, Jing W. Wang^{1 1}Neurobiology Section, Division of Biological Sciences, University of California-San Diego, ²Department of Neuroscience, Columbia University, ³Dart NeuroScience, ⁴School of Dental Medicine, University of Pennsylvania In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

• Medicine

  
  Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention Hilary S. McCarren^1,2,4, Jason T. Moore^1,3,4, Max B. Kelz^{1,2,3,4 1}Department of Anesthesiology and Critical Care, Perelman School of Medicine, University of Pennsylvania, ²Department of Pharmacology, Perelman School of Medicine, University of Pennsylvania, ³Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, ⁴Center for Sleep and Circadian Neurobiology, Perelman School of Medicine, University of Pennsylvania Loss of the righting reflex has long served as a standard behavioral surrogate for unconsciousness, also called hypnosis, in laboratory animals. Alterations in volatile anesthetic sensitivity caused by pharmacological interventions can be detected with a carefully controlled high-throughput assessment system, which may be adapted for delivery of any inhaled therapeutic.

• Biology

  
  Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation Jessica M Bryant^1,2, Mirella L Meyer-Ficca³, Vanessa M Dang¹, Shelley L Berger^1,4,5, Ralph G Meyer^{3 1}Department of Cell and Developmental Biology, University of Pennsylvania, ²Biomedical Graduate Studies, University of Pennsylvania, ³Department of Animal Biology and Center for Animal Transgenesis and Germ Cell Research, University of Pennsylvania, ⁴Department of Genetics, University of Pennsylvania, ⁵Department of Biology, University of Pennsylvania The STA-PUT method allows for the separation of different populations of spermatogenic cells based on size and density.

• 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 1}Department 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.

• Medicine

  
  Minimal Erythema Dose (MED) Testing Carolyn J. Heckman¹, Rachel Chandler², Jacqueline D. Kloss³, Amy Benson², Deborah Rooney², Teja Munshi¹, Susan D. Darlow¹, Clifford Perlis⁴, Sharon L. Manne⁵, David W. Oslin^{2 1}Cancer 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.

• Chemistry

  
  Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS) Nathaniel W. Snyder¹, Maya Khezam¹, Clementina A. Mesaros¹, Andrew Worth¹, Ian A. Blair^{1 1}Centers 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.

• Biology

  
  Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq) Sandra Deliard¹, Jianhua Zhao¹, Qianghua Xia¹, Struan F.A. Grant^{1,2 1}Division 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.

• Neuroscience

  
  Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles Ruifeng Yang¹, Xiaowei Xu^{1 1}Department of Pathology and Laboratory Medicine, School of Medicine, University of Pennsylvania This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

• Biology

  
  Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies Catherine Moorwood¹, Min Liu², Zuozhen Tian², Elisabeth R. Barton^{3 1}Department of Anatomy and Cell Biology, School of Dental Medicine, University of Pennsylvania, ²Department of Physiology, Perelman School of Medicine, University of Pennsylvania, ³Department of Anatomy and Cell Biology, School of Dental Medicine, School of Dental Medicine, University of Pennsylvania Muscle function measurements contribute to the evaluation of potential therapeutics for muscle pathology, as well as to the determination of mechanisms underlying physiology of this tissue. We will demonstrate the preparation of the extensor digitorum longus and diaphragm muscles for functional testing. Protocols for isometric and eccentric contractions will be shown, as well as differences in results between dystrophic muscles, representing a pathological state, and wildtype muscles.

• Biology

  
  Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes Joseph D. Tario Jr.¹, Kristen Humphrey¹, Andrew D. Bantly², Katharine A. Muirhead³, Jonni S. Moore⁴, Paul K. Wallace^{1 1}Department 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.

• Immunology and Infection

  
  Quantitative Measurement of the Immune Response and Sleep in Drosophila Tzu-Hsing Kuo¹, Arun Handa¹, Julie A. Williams^{1 1}Center for Sleep and Circadian Neurobiology, University of Pennsylvania Perelman School of Medicine To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NFκB.

• Neuroscience

  
  Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury Colin J. Smith^1,2, Brian N. Johnson¹, Jaclynn A. Elkind¹, Jill M. See¹, Guoxiang Xiong¹, Akiva S. Cohen^{1,3 1}Division of Neurology, Children's Hospital of Philadelphia, ²Neuroscience Graduate Group, Perelman School of Medicine at the University of Pennsylvania, ³Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

• Bioengineering

  
  Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness Myung-Jin Oh¹, Frank Kuhr¹, Fitzroy Byfield², Irena Levitan^{1 1}Section 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.

• Medicine

  
  Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme Brian C. Baumann¹, Jay F. Dorsey¹, Joseph L. Benci¹, Daniel Y. Joh¹, Gary D. Kao^{1 1}Department 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.

• Biology

  
  Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion Jessica W. Wen¹, Abby L. Olsen², Maryna Perepelyuk², Rebecca G. Wells^{2 1}Division 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.

• Immunology and Infection

  
  Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation Karen L. Joyce¹, Will Morgan², Robert Greenberg², Meera G. Nair^{1 1}Institute 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.

• Medicine

  
  Quantification of Atherosclerotic Plaque Activity and Vascular Inflammation using [18-F] Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography (FDG-PET/CT) Nehal N. Mehta¹, Drew A. Torigian², Joel M. Gelfand³, Babak Saboury², Abass Alavi^{2 1}Division of Cardiovascular Medicine, University of Pennsylvania, Perelman School of Medicine, ²Department of Radiology, University of Pennsylvania, Perelman School of Medicine, ³Department of Dermatology, University of Pennsylvania, Perelman School of Medicine There is great need to identify atherosclerosis non-invasively, and here we demonstrate how FDG-PET/CT can be used to detect and quantify atherosclerotic plaque activity and vascular inflammation.

• Bioengineering

  
  Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress Whitney O. Lane¹, Alexandra E. Jantzen², Tim A. Carlon², Ryan M. Jamiolkowski³, Justin E. Grenet¹, Melissa M. Ley¹, Justin M. Haseltine², Lauren J. Galinat², Fu-Hsiung Lin¹, Jason D. Allen⁴, George A. Truskey², Hardean E. Achneck^{1 1}Department 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.

• 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 1}Centers 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).

• Biology

  
  Purification of Hsp104, a Protein Disaggregase Elizabeth A. Sweeny¹, Morgan E. DeSantis¹, James Shorter^{1 1}Department of Biochemistry and Biophysics, University of Pennsylvania Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

• Bioengineering

  
  Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model Alexandra E. Jantzen¹, Whitney O. Lane², Shawn M. Gage³, Justin M. Haseltine¹, Lauren J. Galinat¹, Ryan M. Jamiolkowski⁴, Fu-Hsiung Lin³, George A. Truskey¹, Hardean E. Achneck^{3 1}Department 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.

• Neuroscience

  
  Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice Lauren H. Sansing^1,2,3, Scott E. Kasner², Louise McCullough¹, Puneet Agarwal², Frank A. Welsh⁴, Katalin Kariko^{4 1}Department of Neurology, University of Connecticut Health Center, ²Department of Neurology, School of Medicine, University of Pennsylvania, ³Department of Neurosurgery, Hartford Hospital, ⁴Department of Neurosurgery, School of Medicine, University of Pennsylvania The autologous blood injection model of intracerebral hemorrhage in mice described in this protocol uses the double injection technique to minimize risk of blood reflux up the needle track, no anticoagulants in the pumping system, and eliminates all dead space and expandable tubing in the system.

• Biology

  
  High-throughput Yeast Plasmid Overexpression Screen Michael S. Fleming¹, Aaron D. Gitler^{2 1}Neuroscience Graduate Group, University of Pennsylvania School of Medicine, ²Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

• Biology

  
  Mouse Oocyte Microinjection, Maturation and Ploidy Assessment Paula Stein¹, Karen Schindler^{1 1}Department of Biology, University of Pennsylvania Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

• Biology

  
  In vitro Reconstitution of the Active T. castaneum Telomerase Anthony P. Schuller¹, Michael J. Harkisheimer¹, Emmanuel Skordalakes^{1 1}Gene Expression and Regulation, The Wistar Institute, University of Pennsylvania Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

• Neuroscience

  
  Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence Sava Sakadžić¹, Emmanuel Roussakis², Mohammad A. Yaseen¹, Emiri T. Mandeville³, Vivek J. Srinivasan¹, Ken Arai³, Svetlana Ruvinskaya¹, Weicheng Wu¹, Anna Devor^1,4, Eng H. Lo³, Sergei A. Vinogradov², David A. Boas^{1 1}Optics 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.

• Neuroscience

  
  Transcriptome Analysis of Single Cells Jacqueline Morris*¹, Jennifer M. Singh*¹, James H. Eberwine^{1,2 1}Department of Pharmacology, University of Pennsylvania, ²The Penn Genome Frontiers Institute, University of Pennsylvania In this article we describe a simple method for the harvesting of single cells from rat primary neuronal cultures and subsequent transcriptome analysis using aRNA amplification. This approach is generalizable to any cell type.

• Biology

  
  Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans Marni J. Falk^1,2, Meera Rao*¹, Julian Ostrovsky*¹, Evgueni Daikhin¹, Ilana Nissim¹, Marc Yudkoff^{1,2 1}Department of Pediatrics, The Children's Hospital of Philadelphia, ²Department of Pediatrics, University of Pennsylvania Stable isotopic profiling by gas chromatography mass spectrometric analysis of intermediary metabolic flux is described in the nematode, Caenorhabditis elegans. Methods are detailed for assessing isotopic enrichment in carbon dioxide, organic acids, and amino acids following isotope exposure either during development on agar plates or during adulthood in liquid culture.

• Biology

  
  Immunofluorescent Detection of Two Thymidine Analogues (CldU and IdU) in Primary Tissue Alex H. Tuttle*¹, Matthew M. Rankin*¹, Monica Teta¹, Daniel J. Sartori¹, Geneva M. Stein¹, Gina J. Kim¹, Cristina Virgilio¹, Anne Granger¹, Di Zhou¹, Simon H. Long¹, Alisa B. Schiffman¹, Jake A. Kushner^{1 1}Division of Endocrinology and Diabetes, Children’s Hospital of Philadelphia, Institute of Diabetes Obesity and Metabolism, Institute for Regenerative Medicine, Department of Pediatrics, University of Pennsylvania-School of Medicine We have derived a strategy to detect sequential incorporation of thymidine analogues (CldU and IdU) into tissues of adult mice to quantify two successive rounds of cell division. This strategy is useful to detect cell turnover of long-lived tissues, oncogenic transformation, or transit-amplifying cells.

• Immunology and Infection

  
  An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses Selene Nunez-Cruz¹, Denise C. Connolly², Nathalie Scholler^{1 1}Penn Ovarian Cancer Research Center, Center for Research on Reproduction and Womans Health, Department of Obstetrics and Gynecology, University of Pennsylvania-School of Medicine, ²Women's Cancer Program, Fox Chase Cancer Center To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1^KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.

• Medicine

  
  Technique to Collect Fungiform (Taste) Papillae from Human Tongue Andrew I. Spielman*¹, M. Yanina Pepino², Roy Feldman^3,4,5, Joseph G. Brand*^{4,6 1}Department of Basic Science and Craniofacial Biology, College of Dentistry, New York University, ²Department of Internal Medicine and Department of Psychiatry, School of Medicine, Washington University in St. Louis, ³Veterans Affairs Medical Center, ⁴School of Dental Medicine, Department of Biochemistry, University of Pennsylvania-School of Medicine, ⁵Monell Chemical Senses Center, ⁶Monell Chemical Senses Center Knowledge of molecular mechanisms underlying gustatory transduction has recently enjoyed significant advances, largely due to using animal models. However, the wide diversity in taste sensitivity and specificity among mammals warrants studies in human tissue. We describe a biopsy technique to collect living taste cells from the papillae on human tongue.

• Immunology and Infection

  
  RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells Theresa S. Moser¹, Leah R. Sabin¹, Sara Cherry^{1 1}Department 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.

• Biology

  
  Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels Alexandra Cretu¹, Paola Castagnino¹, Richard Assoian^{1 1}Department of Pharmacology, University of Pennsylvania The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

• Biology

  
  Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences Erika Kague¹, Christopher Weber¹, Shannon Fisher^{1 1}Department 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.

• Biology

  
  Dissection of Organs from the Adult Zebrafish Tripti Gupta¹, Mary C. Mullins^{1 1}Department of Cell and Developmental Biology, University of Pennsylvania-School of Medicine This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

• Biology

  
  Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems Louise E. Sinks¹, Emmanuel Roussakis¹, Tatiana V. Esipova¹, Sergei A. Vinogradov^{1 1}Department 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.

• Biology

  
  Optical Recording of Electrical Activity in Guinea-pig Enteric Networks using Voltage-sensitive Dyes Ana L. Obaid¹, B. M. Salzberg^{1,2 1}Department of Neuroscience, University of Pennsylvania-School of Medicine, ²Department of Physiology, University of Pennsylvania-School of Medicine This protocol illustrates how voltage-sensitive dyes enable optical recording of electrical activity from intact neural networks such as the plexuses of the guinea-pig enteric nervous system, with an adjustable resolution that ranges from single-cells to multi-ganglionic circuitry.

• Biology

  
  Cellular Encapsulation in 3D Hydrogels for Tissue Engineering Sudhir Khetan¹, Jason Burdick^{2 1}Department 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.

• Biology

  
  Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture Jamie L. Ifkovits¹, Harini G. Sundararaghavan¹, Jason A. Burdick^{1 1}Department 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.

• Biology

  
  Neutrophil Isolation Protocol Hana Oh¹, Brian Siano¹, Scott Diamond^{1 1}Institute 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.

• Biology

  
  Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces Ravi A. Desai¹, Michael T. Yang¹, Nathan J. Sniadecki², Wesley R. Legant¹, Christopher S. Chen^{1 1}Department 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.