University of Southern California

57 articles published in JoVE

• Neuroscience

  
  Social Isolation Model: A Noninvasive Rodent Model of Stress and Anxiety Saki Watanabe¹, Alzahra Al Omran¹, Amy S. Shao², Jing Liang^{1 1}Titus 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. Benayoun^{1,2,3,4,5 1}Leonard 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-Sanabria^{1 1}Leonard 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 Malmstadt^{1 1}Mork 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 Chen^{1,2 1}Zilkha 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 Cheng^1,2, James E. Ferrell, Jr.^{1,3 1}Department 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

  
  Analyzing Tumor Gene Expression Factors with the CorExplorer Web Portal Shirley Pepke¹, William M. Nelson², Greg Ver Steeg^{3 1}Lyrid 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 Escorcia^1,2, Kuo-Fang Shen¹, Ji-Ping Yuan¹, Susan L. Forsburg^{1 1}Program 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 Dave^2,3, Rusty Lansford^{1,2,3 1}Department 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 Davies^{1 1}Titus 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 Wang^{1 1}Molecular 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 Kani^{1 1}Lawrence 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 Horie^2,3, Hajime Mihira⁴, Akira Saito^{2 1}David 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. Drake^{1,2,3,6,7 1}Graduate 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. Nealson^{2 1}International 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 Spellberg^{1,2 1}Department 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 Roy^1,2, Stanislav Tsoi³, Jeffrey Erickson¹, Mohamed El-Naggar⁴, Scott Calabrese Barton⁵, Leonard Tender^{1 1}Center 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 Tommasi^{1 1}Department 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 Chen^{2 1}Biomedical 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. Ruter^1,2,3, James Nhan^1,2, Sean P. Curran^{1,2 1}Leonard 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. Kubala^1,2, Yves A. DeClerck^{1,2,3 1}Division 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 Chung^{1 1}Department 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. Nadeau^{1 1}Department 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 Yen^{1 1}Leonard 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. Kay^{1 1}Department 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. Mumenthaler^{1 1}Lawrence 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. Turner^1,2,5, Christian Landry³, Michael J. Galko^{1,2,4 1}Department 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. Dean^{1 1}Molecular 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 Town^{1 1}Zilkha 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 Ishchenko^1,2, Vadim Cherezov^1,2, Wei Liu^{3 1}The 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. Davies^{1 1}Titus 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 Wu^{1 1}Department 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 Fetters^{1 1}Division 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. Humayun^{1 1}Biomedical 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. Mariani^{1 1}Eli 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. Hoelzle^1,2, Bino A. Varghese^1,3, Clara K. Chan¹, Amy C. Rowat^{1 1}Department 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-Oldak^{1 1}Center 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 Roy^{1 1}Department 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 Feng^{1 1}Department 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 Gupta^1,2, Maya Debbaneh^2,3, Daniel Butler^2,4, Monica Huynh^2,5, Ethan Levin², Argentina Leon², John Koo², Wilson Liao^{2 1}Keck 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 Wang^{1 1}Mork 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-Kauwe^{2,3 1}Department 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. Zhang^{1,4 1}Department 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. Grikscheit^{1 1}Children'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. Mariani^{1 1}Eli 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 Huang^{1 1}Department 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. Gayther^{1 1}Department 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. Armani^{1,2 1}Department 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 Lu^{2,3,4,5 1}Center 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 Liang^{1 1}Department 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. Kaplan^{1 1}Brain 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. Longo^{1 1}Andrus 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 Liddell^1,2, Glenn Manthey², Nicholas Pannunzio³, Adam Bailis^{2 1}Irell & 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. Gibson^1,2, Le Ma^{2,3 1}Neuroscience 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. Sampath^{2 1}Neurosciences 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. Butler^{1,2 1}Department 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 Cai^2,3, D. Spencer Currle⁴, K. George Chandy¹, Robert H. Chow^{2,3 1}Department 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.