University of Southern California View Institution's Website 61 articles published in JoVE Developmental Biology Using Avian Skin Explants to Study Tissue Patterning and Organogenesis Tingxin Jiang1, Maeve Secor2, Rusty Lansford3, Randall B. Widelitz1, Cheng Ming Chuong1 1Department of Pathology, Keck School of Medicine of University of Southern California, 2Molecular and Computational Biology, University of Southern California, 3Department of Radiology, Keck School of Medicine of University of Southern California Here we describe protocols for three types of avian embryonic skin explant cultures that can be used to examine tissue interactions, 4D imaging timelapse movie (3D plus time), global or local perturbation of molecular function, and systems biology characterization. Neuroscience Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings Megana R. Iyer1, Christopher Ventura1, Daniel Bronson1, Nathaniel Nowak1, Katherine Regalado1, Radha Kalluri1 1Caruso Department of Otolaryngology Head & Neck Surgery, Zilkha Neurogenetics Institute, Hearing and Communications Neuroscience Training Program, University of Southern California Presented here are methods providing detailed instructions for dissecting, dissociating, culturing, and patch-clamp recording from vestibular ganglion and spiral ganglion neurons of the inner ear. Cancer Research Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging Serena L. Cornelius1,2, Megan M. Colonnetta2,3,4,5, Katherine E. Lake1,2, Clayton A. Smith1,2, Yu-An Zhang1,2, Evanthia T. Roussos-Torres6, Sangeetha M. Reddy1,2, Elizabeth H. Chen2,3,4,5, Isaac S. Chan1,2,3,4 1Department of Internal Medicine, Division of Hematology and Oncology, University of Texas Southwestern Medical Center, 2Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, 3Department of Molecular Biology, University of Texas Southwestern Medical Center, 4Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, 5Department of Cell Biology, University of Texas Southwestern Medical Center, 6Division of Medical Oncology, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California This protocol discusses an approach for generating epithelial organoids from primary normal and tumor mammary tissue through differential centrifugation. Furthermore, instructions are included for three-dimensional culturing as well as immunofluorescent imaging of embedded organoids. Neuroscience Social Isolation Model: A Noninvasive Rodent Model of Stress and Anxiety Saki Watanabe1, Alzahra Al Omran1, Amy S. Shao2, Jing Liang1 1Titus Family Department of Clinical Pharmacy, School of Pharmacy, University of Southern California, 2Homer Stryker M.D. School of Medicine, Western Michigan University Presented here is a social isolation (SI)-induced anxiety mouse model that utilizes wild type C56BL/6J mice to induce stress and anxiety-like behavior with minimal handling and no invasive procedures. This model reflects modern life patterns of social isolation and is ideal for studying anxiety and related disorders. Neuroscience Gentle Isolation of Nuclei from the Brain Tissue of Adult African Turquoise Killifish, a Naturally Short-Lived Model for Aging Research Bryan B. Teefy1, Ari Adler1, Rajyk Bhala1, Bérénice A. Benayoun1,2,3,4,5 1Leonard Davis School of Gerontology, University of Southern California, 2Molecular and Computational Biology Department, USC Dornsife College of Letters, Arts and Sciences, 3Biochemistry and Molecular Medicine Department, USC Keck School of Medicine, 4Epigenetics and Gene Regulation, USC Norris Comprehensive Cancer Center, 5USC Stem Cell Initiative Here we present a protocol to isolate nuclei from the brains of the short-lived vertebrate model Nothobranchius furzeri for downstream applications such as single-nucleus RNA sequencing or single-nucleus assay for transposase-accessible chromatin with sequencing (ATAC-seq). Genetics Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans Toni Castro Torres*1, Darius Moaddeli*1, Maxim Averbukh1, Aeowynn J. Coakley1, Naibedya Dutta1, Gilberto Garcia1, Ryo Higuchi-Sanabria1 1Leonard Davis School of Gerontology, University of Southern California Caenorhabditis elegans serve as an excellent model system with robust and low-cost methods for surveying healthspan, lifespan, and resilience to stress. Bioengineering Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors (GPCRs) Ahmed Elbaradei*1, Lucia Caterina Dalle Ore*1, Noah Malmstadt1 1Mork Family Department of Chemical Engineering and Materials Science, University of Southern California This protocol utilizes agarose swelling as a powerful and generalizable technique for incorporating integral membrane proteins (IMPs) into giant unilamellar lipid vesicles (GUVs), as described here for the reconstitution of the human 1A serotonin receptor protein (5-HT1AR), one of the classes of pharmacologically important G protein-coupled receptors. Neuroscience Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis Kasey Rose1, Sowmya Lokappa1, Jeannie Chen1,2 1Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, 2Department of Cell & Neurobiology, Keck School of Medicine, University of Southern California This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers. Biology Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization Xianrui Cheng1,2, James E. Ferrell, Jr.1,3 1Department of Chemical and Systems Biology, Stanford University School of Medicine, 2Current Address: Department of Biological Sciences, University of Southern California, 3Department of Biochemistry, Stanford University School of Medicine We describe a method for the preparation and live imaging of undiluted cytoplasmic extracts from Xenopus laevis eggs. Cancer Research The Establishment and Utilization of Patient Derived Xenograft Models of Central Nervous System Metastasis Ben Yi Tew1, Bodour Salhia1 1Department of Translational Genomics, Keck School of Medicine, University of Southern California Central nervous system metastasis PDX models represent the phenotypic and molecular characteristics of human metastasis, making them excellent models for preclinical studies. Described here is how to establish PDX models and the inoculation routes that are best used for preclinical studies. Cancer Research Analyzing Tumor Gene Expression Factors with the CorExplorer Web Portal Shirley Pepke1, William M. Nelson2, Greg Ver Steeg3 1Lyrid LLC, 2Independent consultant, 3Information Sciences Institute, University of Southern California We introduce the CorExplorer web portal, an resource for exploration of tumor RNA sequencing factors found by the machine learning algorithm CorEx (Correlation Explanation), and show how factors can be analyzed relative to survival, database annotations, protein-protein interactions, and one another to gain insight into tumor biology and therapeutic interventions. Biology Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy Wilber Escorcia1,2, Kuo-Fang Shen1, Ji-Ping Yuan1, Susan L. Forsburg1 1Program in Molecular and Computational Biology, University of Southern California, 2Leonard Davis School of Gerontology, University of Southern California Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis. Developmental Biology Fast and Efficient Expression of Multiple Proteins in Avian Embryos Using mRNA Electroporation Martin Tran1, Mohit Dave2,3, Rusty Lansford1,2,3 1Department of Biological Sciences, University of Southern California, Los Angeles, 2 We report messenger RNA (mRNA) electroporation as a method that permits fast and efficient expression of multiple proteins in the quail embryo model system. This method can be used to fluorescently label cells and record their in vivo movements by time-lapse microscopy shortly after electroporation. Medicine Murine Drinking Models in the Development of Pharmacotherapies for Alcoholism: Drinking in the Dark and Two-bottle Choice Nhat Huynh*1, Natalie M Arabian*1, Liana Asatryan1, Daryl L Davies1 1Titus Family Department of Clinical Pharmacy and Pharmaceutical Economics & Policy, School of Pharmacy, University of Southern California Alcohol Use Disorder (AUD) is a major national health problem and the development of more effective treatments is required to offset the needs of this patient population. To this end, the following protocol utilizes two simple rodent drinking models to assess the preclinical efficacy of lead anti-alcohol compounds. Genetics Culturing and Manipulation of O9-1 Neural Crest Cells Bao H. Nguyen1, Mamoru Ishii2, Robert E. Maxson2, Jun Wang1 1Molecular Physiology and Biophysics, Baylor College of Medicine, 2Department of Biochemistry and Molecular Biology, University of Southern California O9-1 is a multipotent mouse neural crest cell line. Here we describe detailed step-by-step protocols for culturing O9-1 cells, differentiating O9-1 cells into specific cell types, and genetically manipulating O9-1 cells by using siRNA-mediated knockdown or CRISPR-Cas9 genome editing. Cancer Research Assessment of Resistance to Tyrosine Kinase Inhibitors by an Interrogation of Signal Transduction Pathways by Antibody Arrays Katrin Tiemann1, Carolina Garri1, Jeffrey Wang1, Lauren Clarke1, Kian Kani1 1Lawrence J. Ellison Institute for Transformative Medicine, Center for Applied Molecular Medicine, University of Southern California Here, we present a protocol for antibody arrays to identify alterations in signaling pathways in various cellular models. These changes, caused by drugs/hypoxia/ultra-violet light/radiation, or by overexpression/downregulation/knockouts, are important for various disease models and can indicate whether a therapy will be effective or can identify mechanisms of drugs resistance. Biology Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-β Signaling Hiroshi I. Suzuki1, Masafumi Horie2,3, Hajime Mihira4, Akira Saito2 1David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 2Department of Respiratory Medicine, Graduate School of Medicine, The University of Tokyo, 3Hastings Center for Pulmonary Research, Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, Keck School of Medicine, University of Southern California, 4Department of Molecular Pathology, Graduate School of Medicine, The University of Tokyo A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-β in MS-1 endothelial cells. Cancer Research Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer Larry C. Cheng*1,2, Zhen Li*3, Thomas G. Graeber4, Nicholas A. Graham5, Justin M. Drake1,2,3,6,7 1Graduate Program in Cellular and Molecular Pharmacology, School of Graduate Studies, Rutgers University, The State University of New Jersey, 2Graduate Program in Quantitative Biomedicine, School of Graduate Studies, Rutgers University, The State University of New Jersey, 3Department of Medicine, Division of Medical Oncology, Rutgers Robert Wood Johnson Medical School, 4Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, Jonsson Comprehensive Cancer Center, UCLA Metabolomics Center, and California NanoSystems Institute, David Geffen School of Medicine, University of California, Los Angeles, 5Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, 6Pharmacology, Rutgers Robert Wood Johnson Medical School, 7Cancer Metabolism and Growth Program, Rutgers Cancer Institute of New Jersey This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics. Environment Self-standing Electrochemical Set-up to Enrich Anode-respiring Bacteria On-site Akihiro Okamoto1, Annette Rowe2, Xiao Deng3, Kenneth H. Nealson2 1International Center for Materials Nanoarchitectonics, National Institute for Materials Science, 2Department of Earth Sciences, University of Southern California, 3Department of Applied Chemistry, The University of Tokyo On-site microbial enrichment or in situ cultivation techniques can facilitate the isolation of difficult-to-culture microbial taxa, especially from low-biomass or geochemically extreme environments. Here, we describe an electrochemical set-up without using an external power source to enrich microbial strains that are capable of extracellular electron transport (EET). Medicine Murine Oropharyngeal Aspiration Model of Ventilator-associated and Hospital-acquired Bacterial Pneumonia Travis B. Nielsen1, Jun Yan1, Brian Luna1, Brad Spellberg1,2 1Department of Molecular Microbiology and Immunology, University of Southern California, 2Division of Infectious Diseases, Department of Medicine, LAC+USC Medical Center Infectious pneumonia is among the most common infections in human. An appropriate in vivo model is critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics. With this murine oropharyngeal aspiration pneumonia model, one can examine the pathogenesis and new treatments against these deadly infections. Chemistry Characterizing Electron Transport through Living Biofilms Matthew Yates1, Sarah Strycharz-Glaven1, Joel Golden1, Jared Roy1,2, Stanislav Tsoi3, Jeffrey Erickson1, Mohamed El-Naggar4, Scott Calabrese Barton5, Leonard Tender1 1Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 2George Mason University, 3Chemistry Division, Naval Research Laboratory, 4Departments of Physics, Biological Sciences, and Chemistry, University of Southern California, 5Department of Chemical Engineering and Materials Science, Michigan State University A protocol for measuring electrical conductivity of living microbial biofilms under physiologically relevant conditions is presented. Immunology and Infection The Lambda Select cII Mutation Detection System Ahmad Besaratinia1, Stella Tommasi1 1Department of Preventive Medicine, USC Keck School of Medicine, University of Southern California We describe a detailed protocol for the Lambda Select cII mutation assay in cultured cells of transgenic rodents or the corresponding animals treated with a chemical/physical agent of interest. This approach has been widely used for mutagenicity testing of carcinogens in mammalian cells. Neuroscience Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult Stephanie A. Herrlinger1, Qiang Shao2, Li Ma2, Melinda Brindley3, Jian-Fu Chen2 1Biomedical and Health Sciences Institute, University of Georgia, 2Center for Craniofacial Molecular Biology, University of Southern California, 3Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia Here we describe a method for establishing a model of Zika virus-induced microcephaly in mouse. This protocol includes methods for embryonic, neonatal, and adult-stage intracerebral inoculation of the Zika virus. Biology Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining Wilber Escorcia1, Dana L. Ruter1,2,3, James Nhan1,2, Sean P. Curran1,2 1Leonard Davis School of Gerontology, University of Southern California, 2Molecular & Computational Biology Section, University of Southern California, 3Integrative Program for Biological & Genome Sciences, University of North Carolina at Chapel Hill Nile red staining of fixed Caenorhabditis elegans is a method for quantitative measurement of neutral lipid deposits, while oil red O staining facilitates qualitative assessment of lipid distribution among tissues. Cancer Research Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment Marta H. Kubala1,2, Yves A. DeClerck1,2,3 1Division of Hematology, Oncology and Blood and Marrow Transplantation, Department of Pediatrics, University of Southern California, 2 This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment. Bioengineering Synthesis of Monocyte-targeting Peptide Amphiphile Micelles for Imaging of Atherosclerosis Christopher Poon1, Manjima Sarkar1, Eun Ji Chung1 1Department of Biomedical Engineering, University of Southern California This paper presents a method involving the synthesis and characterization of monocyte-targeting peptide amphiphile micelles and the corresponding assays to test for biocompatibility and ability of the micelle to bind to monocytes. Bioengineering Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy (DHM) Manuel Bedrossian1, Casey Barr2, Chris A. Lindensmith3, Kenneth Nealson2, Jay L. Nadeau1 1Department of Medical Engineering, California Institute of Technology, 2Department of Earth Sciences, University of Southern California, 3Jet Propulsion Laboratory, California Institute of Technology Digital holographic microscopy (DHM) is a volumetric technique that allows imaging samples 50-100X thicker than brightfield microscopy at comparable resolution, with focusing performed post-processing. Here DHM is used for identifying, counting, and tracking microorganisms at very low densities and compared with optical density measurements, plate count, and direct count. Biology Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment Su-Jeong Kim1, Jialin Xiao1, Pinchas Cohen1, Kelvin Yen1 1Leonard Davis School of Gerontology, University of Southern California This protocol describes how to stimulate cells with mitochondrial-derived peptides and assess the signaling cascade and localization of phospho-proteins. Biochemistry A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies Prateek Tripathi1, José L. Pruneda-Paz2, Steve A. Kay1 1Department of Neurology, University of Southern California, 2Section of Cell and Developmental Biology, University of California San Diego The modified yeast one-hybrid assay described here is an extension of the classical yeast one-hybrid (Y1H) assay to study and validate the heteromeric protein complex-DNA interaction in a heterologous system for any functional genomics study. Biology Discrimination and Characterization of Heterocellular Populations Using Quantitative Imaging Techniques Colleen M. Garvey1, Torin A. Gerhart1, Shannon M. Mumenthaler1 1Lawrence J. Ellison Institute for Transformative Medicine, University of Southern California (USC) We have developed a novel protocol for studying heterocellular population dynamics in response to perturbations. This manuscript describes an imaging-based platform that produces quantitative datasets for simultaneous characterization of multiple cellular phenotypes of heterocellular populations in a robust manner. Neuroscience Novel Assay for Cold Nociception in Drosophila Larvae Heather N. Turner1,2,5, Christian Landry3, Michael J. Galko1,2,4 1Department of Genetics, UT MD Anderson Cancer Center, 2Neuroscience Program, Graduate School of Biomedical Sciences at Houston, 3ProDev Engineering, 4Genes and Development Program, Graduate School of Biomedical Sciences at Houston, 5Section of Neurobiology, University of Southern California Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception. Bioengineering Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum Nicholas G. Schultz1, Erik Otárola-Castillo2, Matthew D. Dean1 1Molecular and Computational Biology, University of Southern California (USC), 2Purdue University Many biological structures lack easily definable landmarks, making it difficult to apply modern morphometric methods. Here we illustrate methods to study the mouse baculum (a bone in the penis), including dissection and microCT scanning, followed by computational methods to define semi-landmarks that are used to quantify size and shape variation. Medicine Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease Marie-Victoire Guillot-Sestier1, Tara M. Weitz1, Terrence Town1 1Zilkha Neurogenetic Institute, University of Southern California (USC) We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-β (Aβ) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer's disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo. Biochemistry Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography Andrii Ishchenko1,2, Vadim Cherezov1,2, Wei Liu3 1The Bridge Institute, University of Southern California, 2Department of Chemistry, University of Southern California, 3School of Molecular Sciences, Center for Applied Structural Discovery at the Biodesign Institute, Arizona State University We describe procedures for the preparation and delivery of membrane protein microcrystals in lipidic cubic phase for serial crystallography at X-ray free-electron lasers and synchrotron sources. These protocols can also be applied for incorporation and delivery of soluble protein microcrystals, leading to substantially reduced sample consumption compared to liquid injection. Medicine Utilizing an Orally Dissolving Strip for Pharmacological and Toxicological Studies: A Simple and Humane Alternative to Oral Gavage for Animals Nhat Huynh*1, Natalie Arabian*1, Dustin Lieu1, Liana Asatryan1, Daryl L. Davies1 1Titus Family Department of Clinical Pharmacy and Pharmaceutical Economics & Policy, University of Southern California School of Pharmacy The following protocol describes a novel method for chronic oral drug administration using an orally dissolving strip (ODS) in lieu of the more commonly used oral gavage method. We demonstrate that preclinical, oral drug delivery using the ODS method represents a safe, convenient, and humane alternative to oral gavage. Engineering Fabrication of High Contrast Gratings for the Spectrum Splitting Dispersive Element in a Concentrated Photovoltaic System Yuhan Yao1, He Liu1, Wei Wu1 1Department of Electrical Engineering, University of Sothern California The fabrication of high contrast gratings as the parallel spectrum splitting dispersive element in a concentrated photovoltaic system is demonstrated. Fabrication processes including nanoimprint lithography, TiO2 sputtering and reactive ion etching are described. Reflectance measurement results are used to characterize the optical performance. Behavior Quantifying Learning in Young Infants: Tracking Leg Actions During a Discovery-learning Task Barbara Sargent1, Hendrik Reimann2, Masayoshi Kubo3, Linda Fetters1 1Division of Biokinesiology & Physical Therapy at the Herman Ostrow School of Dentistry, University of Southern California, 2Department of Kinesiology, Temple University, 3Department of Physical Therapy, Niigata University of Health and Welfare A method is described in which 3-4 month old infants learn a task by discovery and their leg movements are captured to quantify the learning process. Medicine Whole Vitreous Humor Dissection for Vitreodynamic Analysis Karthik Murali1, Amir H. Kashani2, Mark S. Humayun1 1Biomedical Engineering, University of Southern California, 2Ophthalmology, University of Southern California The goal of this protocol is to show an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes. Medicine A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair Nikita Tripuraneni*1, Marissa K. Srour*1, John W. Funnell1, Thu Zan Tun Thein1, Francesca V. Mariani1 1Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine, University of Southern California The overall goal of this procedure is to successfully resect a portion of bone from the rib of a mouse. The procedure was developed as a model to study large-scale long bone repair. Bioengineering A Microfluidic Technique to Probe Cell Deformability David J. Hoelzle1,2, Bino A. Varghese1,3, Clara K. Chan1, Amy C. Rowat1 1Department of Integrative Biology and Physiology, University of California, Los Angeles, 2Department of Aerospace and Mechanical Engineering, University of Notre Dame, 3Molecular Imaging Center, University of Southern California We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions. Bioengineering Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface Qichao Ruan1, Janet Moradian-Oldak1 1Center for Craniofacial Molecular Biology, Herman Ostrow School of Dentistry, University of Southern California In this article, we describe a protocol for fabricating an amelogenin-chitosan hydrogel for superficial enamel reconstruction. Organized in situ growth of apatite crystals in the hydrogel formed a dense enamel-restoration interface, which will improve the effectiveness and durability of restorations. Bioengineering Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding Philip Chung1, J. Alex Heller1, Mozziyar Etemadi1, Paige E. Ottoson2, Jonathan A. Liu3, Larry Rand2, Shuvo Roy1 1Department of Bioengineering & Therapeutic Sciences, University of California, San Francisco, 2Department of Obstetrics, Gynecology & Reproductive Sciences, University of California, San Francisco, 3Keck School of Medicine, University of Southern California We have devised a method for low-cost and rapid prototyping of liquid elastomer rubber injection molded devices by using fused deposition modeling 3D printers for mold design and a modified desiccator as a liquid injection system. Immunology and Infection Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production Junjie Zhang1, Lining Zhu1, Pinghui Feng1 1Department of Microbiology and Immunology, Keck School of Medicine, University of Southern California We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (γHV68) that is closely-related to human Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. “Reconstituting” the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production. Medicine The Goeckerman Regimen for the Treatment of Moderate to Severe Psoriasis Rishu Gupta1,2, Maya Debbaneh2,3, Daniel Butler2,4, Monica Huynh2,5, Ethan Levin2, Argentina Leon2, John Koo2, Wilson Liao2 1Keck School of Medicine, University of Southern California, 2Psoriasis and Skin Treatment Center Dermatology, University of California, San Francisco, 3University of California Irvine School of Medicine, 4University of Arizona College of Medicine, 5Chicago College of Osteopathic Medicine Psoriasis is a chronic, immune-mediated inflammatory skin disease. The Goeckerman regimen, formulated for the treatment of psoriasis, consists of exposure to ultraviolet B (UVB) light and application of crude coal tar (CCT). The following protocol is for the administration of Goeckerman therapy for the treatment of moderate-to-severe psoriasis. Immunology and Infection A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells Paul D. Bryson1, Chupei Zhang1, Chi-Lin Lee1, Pin Wang1 1Mork Family Department of Chemical Engineering and Materials Science, Viterbi School of Engineering, University of Southern California, Los Angeles Here, we use retroviral transduction and concatemeric transfection to create a cell line that can express the components of a lentiviral vector (LV) in the absence of tetracycline. This LV encodes GFP and is pseudotyped with a glycoprotein, SVGmu, which is specific for a receptor on dendritic cells. Bioengineering Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors Jae Youn Hwang1, Daniel L. Farkas1, Lali K. Medina-Kauwe2,3 1Department of Biomedical Engineering, University of Southern California, 2Department of Biomedical Sciences, Cedars-Sinai Medical Center, 3Geffen School of Medicine, University of California, Los Angeles This article details the procedures for optical imaging analysis of the tumor-targeted nanoparticle, HerDox. In particular, detailed use of the multimode imaging device for detecting tumor targeting and assessing tumor penetration is described here. Bioengineering Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography Wei Song*1,2, Qing Wei*1, Shuliang Jiao3, Hao F. Zhang1,4 1Department of Biomedical Engineering, Northwestern University, 2Department of Physics, Harbin Institute of Technology, 3Department of Ophthalmology, University of Southern California, 4Department of Ophthalmology, Northwestern University Photoacoustic ophthalmology (PAOM), an optical-absorption-based imaging modality, provides the complementary evaluation of the retina to the currently available ophthalmic imaging technologies. We report the using of PAOM integrated with spectral-domain optical coherence tomography (SD-OCT) for simultaneous multimodal retinal imaging in rats. Bioengineering Tissue Engineering of the Intestine in a Murine Model Erik R. Barthel1, Allison L. Speer1, Daniel E. Levin1, Frédéric G. Sala1, Xiaogang Hou1, Yasuhiro Torashima1, Clarence M. Wigfall1, Tracy C. Grikscheit1 1Children's Hospital Los Angeles, Division of Pediatric Surgery, Saban Research Institute, Keck School of Medicine of the University of Southern California This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach. Biology Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells Jennifer L. Fogel1, Thu Zan Tun Thein1, Francesca V. Mariani1 1Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine, University of Southern California We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker. Medicine Doppler Optical Coherence Tomography of Retinal Circulation Ou Tan1, Yimin Wang1, Ranjith K. Konduru2, Xinbo Zhang1, SriniVas R. Sadda2, David Huang1 1Department of Ophthalmology, Oregon Health and Science University, 2Department of Ophthalmology, University of Southern California Total retinal blood flow is measured by Doppler optical coherence tomography and semi-automated grading software. Medicine Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression Kate Lawrenson1, Barbara Grun2, Simon A. Gayther1 1Department of Preventive Medicine, University of Southern California, 2Institute for Women's Health, University College London We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development. Engineering Fabrication of Silica Ultra High Quality Factor Microresonators Ashley J. Maker1, Andrea M. Armani1,2 1Department of Chemical Engineering and Materials Science, University of Southern California, 2Department of Electrical Engineering-Electrophysics, University of Southern California We describe the use of a carbon dioxide laser reflow technique to fabricate silica resonant cavities, including free-standing microspheres and on-chip microtoroids. The reflow method removes surface imperfections, allowing long photon lifetimes within both devices. The resulting devices have ultra high quality factors, enabling applications ranging from telecommunications to biodetection. Neuroscience Enabling High Grayscale Resolution Displays and Accurate Response Time Measurements on Conventional Computers Xiangrui Li1, Zhong-Lin Lu2,3,4,5 1Center for Cognitive and Behavioral Brain Imaging, The Ohio State University, 2Department of Psychology, University of Southern California, 3Biomedical Engineering, University of Southern California, 4Neuroscience Graduate Program, University of Southern California, 5Department of Psychology, The Ohio State University Conventional computer hardware can not generate visual stimuli with sufficiently high grayscale resolution and measure response times with sufficient accuracy. We describe how to use the VideoSwitcher to produce high-resolution monochromatic displays, and the RTbox to measure response times with high accuracy on conventional computer hardware. Immunology and Infection Measurement of γHV68 Infection in Mice Sara Dolatshahi Pirooz1, Joo-Hyung Lee1, Zhen Zhao1, Duojiao Ni1, Soohwan Oh1, Chengyu Liang1 1Department of Molecular Microbiology and Immunology, University of Southern California, Los Angeles γ-Herpesviruses (γ-HVs) establish life-long persistency in their host. Infection of mice with γ-HV68 provides a genetically tractable in vivo model for the characterization of the lifecycle/pathogenesis of γHVs. This protocol describes the detection and quantitation of γHV68 infection at acute and latent stages following infection by plaque-forming, infectious center, and qPCR assays. Neuroscience Cross-Modal Multivariate Pattern Analysis Kaspar Meyer1, Jonas T. Kaplan1 1Brain and Creativity Institute and Department of Psychology, University of Southern California Classical multivariate pattern analysis predicts sensory stimuli a subject perceives from neural activity in the corresponding cortices (e.g. visual stimuli from activity in visual cortex). Here, we apply pattern analysis cross-modally and show that sound- and touch-implying visual stimuli can be predicted from activity in auditory and somatosensory cortices, respectively. Biology Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model Min Wei1, Federica Madia1, Valter D. Longo1 1Andrus Gerontology Center, Department of Biological Sciences, Department of Molecular and Computational Biology, University of Southern California, Los Angeles Here we describe a set of DNA mutation assays that can be combined with the yeast chronological life span model to study the genes/pathways that regulate or contribute to genomic DNA instability during aging. Biology Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae Lauren Liddell1,2, Glenn Manthey2, Nicholas Pannunzio3, Adam Bailis2 1Irell & Manella Graduate School of Biological Sciences, 2Department of Molecular and Cellular Biology, City of Hope Comprehensive Cancer Center and Beckman Research Institute, 3Department of Biochemistry and Molecular Biology, University of Southern California, Norris Comprehensive Cancer Center The HO-stimulated translocation assay monitors single-strand annealing following the creation of DNA double-strand breaks at multiple loci in diploid Saccharomyces cerevisiae. This mechanism may model genome rearrangements in somatic cells of higher eukaryotes following exposure to high doses of ionizing radiation. Neuroscience Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination Daniel A. Gibson1,2, Le Ma2,3 1Neuroscience Graduate Program, Keck School of Medicine, University of Southern California, 2Zilkha Neurogenetic Institute, University of Southern California, 3Department of Cell and Neurobiology, Neuroscience Graduate Program, Keck School of Medicine, University of Southern California An in vivo method to test gene function in postnatal brain is described. Recombinant AAVs expressing Cre and/or a fluorescent protein are injected into neonatal mouse brain. Mosaic gene inactivation and sparse neuronal labeling are achieved, allowing rapid analysis of gene function in processes critical to neural circuit development. Neuroscience Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation A. Cyrus Arman1, Alapakkam P. Sampath2 1Neurosciences Graduate Program, University of Southern California, 2Zilkha Neurogenetic Institute, Department of Physiology and Biophysics, University of Southern California Keck School of Medicine Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings. Biology Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons Virginia M. Hazen*1,2, Keith Phan*1, Ken Yamauchi*1,2, Samantha J. Butler1,2 1Department of Biological Sciences, University of Southern California, 2Neuroscience Graduate Program, University of Southern California This assay assesses the ability of a signaling molecule, here Bone Morphogenetic Protein 7 (BMP7), to reorient commissural axons. An explant of embryonic dorsal spinal cord is cultured adjacent to an aggregate of COS cells secreting the candidate growth factors. Reoriented commissural axons growing within the explant are visualized by immunohistochemistry. Biology Mouse Adrenal Chromaffin Cell Isolation Aaron Kolski-Andreaco1, Haijiang Cai2,3, D. Spencer Currle4, K. George Chandy1, Robert H. Chow2,3 1Department of Physiology and Biophysics, University of California, Irvine (UCI), 2Department of Physiology and Biophysics, University of Southern California, Keck School of Medicine, 3Zilkha Neurogenetic Institute, University of Southern California, Keck School of Medicine, 4Department of Developmental and Cell Biology, University of California, Irvine (UCI) Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting. This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex. The digestion of the medulla into single chromaffin cells is then demonstrated.
