University of Pennsylvania View Institution's Website 90 articles published in JoVE Behavior Tracking Sugar-Elicited Local Searching Behavior in Drosophila Manal Shakeel1,2, Shivam Kaushik3, Teiichi Tanimura4, Axel Brockmann1, Pinky Kain3,5 1National Centre for Biological Sciences, 2The University of Trans-Disciplinary Health Sciences and Technology, 3Regional Centre for Biotechnology, 4Neural Circuit Group, Division of Biological Science, Graduate School of Science, Nagoya University, 5Perelman School of Medicine, University of Pennsylvania This protocol describes a behavioral assay for recording sugar-elicited search behavior using Drosophila melanogaster. The assay can be utilized to study feeding and foraging-related behaviors, as well as the underlying neuronal mechanisms. Bioengineering Finite Element Analysis Model for Assessing Expansion Patterns from Surgically Assisted Rapid Palatal Expansion Jia-Hong Lin*1, Guan-Lin Wu*2, Chun-Kai Chiu2, Steven Wang3, Chun-Hsi Chung1, Chenshuang Li1 1Department of Orthodontics, School of Dental Medicine, University of Pennsylvania, 2Department of Biomedical Engineering, College of Engineering, National Cheng Kung University, 3Department of Oral and Maxillofacial Surgery/Pharmacology, School of Dental Medicine, University of Pennsylvania A set of novel finite element models of surgically assisted rapid palatal expansion (SARPE) that could perform a clinically required amount of expander activation with various angles of buccal osteotomy was created for further analysis of the expansion patterns of the hemimaxillae in all three dimensions. Immunology and Infection Infection of Primary Nasal Epithelial Cells Grown at an Air-Liquid Interface to Characterize Human Coronavirus-Host Interactions Clayton J. Otter1,2, Alejandra Fausto1,2, Li Hui Tan3,4, Susan R. Weiss1,2, Noam A. Cohen3,4 1Department of Microbiology, University of Pennsylvania, 2Penn Center for Research on Coronaviruses and Other Emerging Pathogens, Perelman School of Medicine, University of Pennsylvania, 3Otorhinolaryngology-Head and Neck Surgery, University of Pennsylvania, 4Corporal Michael J. Crescenz VA Medical Center The nasal epithelium is the primary barrier site encountered by all respiratory pathogens. Here, we outline methods to use primary nasal epithelial cells grown as air-liquid interface (ALI) cultures to characterize human coronavirus-host interactions in a physiologically relevant system. Bioengineering An Intra-Tissue Radiometry Microprobe for Measuring Radiance In Situ in Living Tissue Amanda L. Holt1,3, Yakir Luc Gagnon2,4, Alison M. Sweeney1,3 1Department of Physics, Yale University, 2Lund Vision Group, Department of Biology, Lund University, 3Department of Physics and Astronomy, University of Pennsylvania, 4Department of Biology, Duke University In this paper, a method for measuring radiance in situ in living tissue is described. This work includes details of the construction of micro-scale probes for different measurements of radiance and irradiance, provides guidance for mounting tissue for the characterization of radiance, and outlines computational methods for analyzing the resulting data. Bioengineering Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing Rakan El-Mayta1, Marshall S. Padilla1, Margaret M. Billingsley1, Xuexiang Han1, Michael J. Mitchell1 1Department of Bioengineering, University of Pennsylvania Here, a protocol for formulating lipid nanoparticles (LNPs) that encapsulate mRNA encoding firefly luciferase is presented. These LNPs were tested for their potency in vitro in HepG2 cells and in vivo in C57BL/6 mice. Developmental Biology Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes Chantal K. Diallo1, Andrew J. Modzelewski1 1Department of Biomedical Sciences, School of Veterinary Medicine, The University of Pennsylvania Here, we describe a simple technique intended for the efficient generation of genetically modified mice called CRISPR RNP Electroporation of Zygotes (CRISPR-EZ). This method delivers editing reagents by electroporation into embryos at an efficiency approaching 100%. This protocol is effective for point mutations, small genomic insertions, and deletions in mammalian embryos. Biology Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices Emily A. Gardea1, Destiny DeNicola1, Samuel Freitas1, Will Peterson1, Hope Dang1, Karissa Shuck1, Christopher Fang-Yen2, George L. Sutphin1 1Department of Molecular & Cellular Biology, University of Arizona, 2Department of Bioengineering, School of Engineering and Applied Sciences, University of Pennsylvania Presented here is an optimized protocol for culturing isolated individual nematodes on solid media in microfabricated multi-well devices. This approach allows individual animals to be monitored throughout their lives for a variety of phenotypes related to aging and health, including activity, body size and shape, movement geometry, and survival. Biochemistry Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection Jonathan T. Specker1, Alexander B. Smith2, Orlaith Keenan2, Joseph P. Zackular2,3, Boone M. Prentice1 1Department of Chemistry, University of Florida, 2 A novel sample preparation method is demonstrated for the analysis of agar-based, bacterial macrocolonies via matrix-assisted laser desorption/ionization imaging mass spectrometry. Bioengineering Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications Victoria G. Muir1, Margaret E. Prendergast1, Jason A. Burdick1,2,3 1Department 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 Ishiyama1,2,3,4, Casey Kissel5, Xin Guo1, Alexis Howard6, Harumi Saeki7, Tomoaki Ito8, Polina Sysa-Shah9, Hajime Orita10, Kazuhiro Sakamoto4, Kathleen Gabrielson1,2 1Department 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 Zhang1 1Department 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. Levine1,2, Carl H. June1,2 1Center 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. Schwab1,2, Alex Miller2, Ann-Marie Raphail3, Ari Levine2, Christopher Haslam2, H. Branch Coslett1,2, Roy H. Hamilton1,2,4 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. Biochemistry Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays Ananya Tripathi1, Charles Bond1,2, James R. Sellers1, Neil Billington1, Yasuharu Takagi1 1Laboratory 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. Alferiev1,2, Robert J. Levy1,2, 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 Anllo1 1Cell 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 1Envision 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. Goldman1 1Pennsylvania 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 Driscoll1,2,3, Kathleen Maleski4,5, Andrew G. Richardson2,6, Brendan Murphy1,2,3, Babak Anasori4,5, Timothy H. Lucas2,6, Yury Gogotsi4,5, Flavia Vitale1,2,3,7,8 1Department 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 Sor2,3, Ahron J. Flowers2,3, Cynthia Clendenin2,3, Katelyn T. Byrne1,4 1Department 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 Gadue1,2,3, Deborah L. French1,2,3 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. Wangensteen1,2 1Department 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 Luca1 1Department 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. Carlson1 1Insitute 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 You1 1Department 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 Fritsch1,2, Jean-Francois Pierre Gout3,4, Marc Vermulst1 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 Chen1 1Thayer 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 Song1,2 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-Vélez1,2,4, John C. O'Donnell1,2, Yeri J. Song1,5, Nicole S. Hernandez1,5, Laura A. Struzyna1,2,4, D. Kacy Cullen1,2,5 1Center 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. Tischfield1,2, Stewart A. Anderson1 1Neuroscience 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. Khurana1 1Department 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-Vélez1,2,3, Michael R. Grovola2,3, Justin C. Burrell2,3, Kritika S. Katiyar2,3,4, Dmitriy Petrov2,3, James P. Harris2,3, D. Kacy Cullen2,3 1Department 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. Anguera1 1Department 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. Raman1 1Davis 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-Wu1 1Department 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 Wachter5 1Department 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 Bae1,2, Shu-lin Liu1,2, Fitzroy J. Byfield3, Paul A. Janmey3, Richard K. Assoian1,2 1Program 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 Alley2,3, Alison Loren2,3, Jennifer J. D. Morrissette1 1Department 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. Way1,2, Maura Southwell1, Scott P. McRobert1 1Department 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. Garcia1 1Epigenetics 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. Parry1,2, Qingqing Wang2,3, Kevin P. Gillespie2,3, Noelle N. Saillant4, Carrie Sims4, Clementina Mesaros1,2, Nathaniel W. Snyder5, Ian A. Blair1,2 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. Chemistry Cooling Rate Dependent Ellipsometry Measurements to Determine the Dynamics of Thin Glassy Films Ethan C. Glor1, Zahra Fakhraai1 1Department 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. Sales1 1Civil, 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. Patel1 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. 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 Tsourkas1 1Department 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-Schill1 1Department 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. Kheir1 1Department 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 Tchou4,5,6, Jose R. Conejo-Garcia1 1Tumor 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. Volkov1,2, Anatoly V. Zaytsev3, Ekaterina L. Grishchuk3 1Center 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. Root1,2, David S. Green1,3, Kang I. Ko1,4, Jing W. Wang1 1Neurobiology 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. McCarren1,2,4, Jason T. Moore1,3,4, Max B. Kelz1,2,3,4 1Department 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 Bryant1,2, Mirella L Meyer-Ficca3, Vanessa M Dang1, Shelley L Berger1,4,5, Ralph G Meyer3 1Department 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 Garcia1,2, Catherine Norise1,2, Olufunsho Faseyitan1,2, Margaret A. Naeser3,4,5, Roy H. Hamilton1,2 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. 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. Oslin2 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. 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. Blair1 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. Biology Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq) Sandra Deliard1, Jianhua Zhao1, Qianghua Xia1, Struan F.A. Grant1,2 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. Neuroscience Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles Ruifeng Yang1, Xiaowei Xu1 1Department 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. Barton3 1Department 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. Wallace1 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. Immunology and Infection Quantitative Measurement of the Immune Response and Sleep in Drosophila Tzu-Hsing Kuo1, Arun Handa1, Julie A. Williams1 1Center 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. Smith1,2, Brian N. Johnson1, Jaclynn A. Elkind1, Jill M. See1, Guoxiang Xiong1, Akiva S. Cohen1,3 1Division 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 Levitan1 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. 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. Kao1 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. Biology Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion Jessica W. Wen1, Abby L. Olsen2, Maryna Perepelyuk2, Rebecca G. Wells2 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. 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. Nair1 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. 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 Alavi2 1Division 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. Achneck1 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. Bioengineering Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis Stacy L. Gelhaus1,2, A. Clementina Mesaros1,2, Ian A. Blair1,2 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). Biology Purification of Hsp104, a Protein Disaggregase Elizabeth A. Sweeny1, Morgan E. DeSantis1, James Shorter1 1Department 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. Achneck3 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. Neuroscience Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice Lauren H. Sansing1,2,3, Scott E. Kasner2, Louise McCullough1, Puneet Agarwal2, Frank A. Welsh4, Katalin Kariko4 1Department 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. Gitler2 1Neuroscience 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 Schindler1 1Department 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 Skordalakes1 1Gene 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 Devor1,4, Eng H. Lo3, Sergei A. Vinogradov2, David A. Boas1 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. Neuroscience Transcriptome Analysis of Single Cells Jacqueline Morris*1, Jennifer M. Singh*1, James H. Eberwine1,2 1Department 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. Falk1,2, Meera Rao*1, Julian Ostrovsky*1, Evgueni Daikhin1, Ilana Nissim1, Marc Yudkoff1,2 1Department 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. Kushner1 1Division 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 Scholler1 1Penn 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 (MOV1KAT) 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 Feldman3,4,5, Joseph G. Brand*4,6 1Department 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 Cherry1 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. Biology Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels Alexandra Cretu1, Paola Castagnino1, Richard Assoian1 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. Biology Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences Erika Kague1, Christopher Weber1, Shannon Fisher1 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. Biology Dissection of Organs from the Adult Zebrafish Tripti Gupta1, Mary C. Mullins1 1Department 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. Vinogradov1 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. Biology Optical Recording of Electrical Activity in Guinea-pig Enteric Networks using Voltage-sensitive Dyes Ana L. Obaid1, B. M. Salzberg1,2 1Department 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 Burdick2 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. Biology Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture Jamie L. Ifkovits1, Harini G. Sundararaghavan1, Jason A. Burdick1 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. Biology Neutrophil Isolation Protocol Hana Oh1, Brian Siano1, Scott Diamond1 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. 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. Chen1 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.