University of California, San Diego

89 articles published in JoVE

• Immunology and Infection

  
  RNA Fluorescence in situ Hybridization (FISH) to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines Dalaena E. Rivera¹, Vladimir Lažetić², Emily R. Troemel², Robert J. Luallen^{1 1}Department of Biology, San Diego State University, ²School of Biological Sciences, University of California, San Diego Intestinal microbes, including extracellular bacteria and intracellular pathogens like the Orsay virus and microsporidia (fungi), are often associated with wild Caenorhabditis nematodes. This article presents a protocol for detecting and quantifying microbes that colonize and/or infect C. elegans nematodes, and for measuring pathogen load after controlled infections in the lab.

• Medicine

  
  Isolation and Profiling of Human Primary Mesenteric Arterial Endothelial Cells at the Transcriptome Level Naseeb Kaur Malhi*¹, Yingjun Luo*¹, Xiaofang Tang*¹, Kiran Sriram^1,3, Riccardo Calandrelli², Sheng Zhong², Zhen Bouman Chen^{1,3 1}Department of Diabetes Complications and Metabolism, City of Hope, ²Department of Bioengineering, University of California San Diego, ³Irell and Manella Graduate School of Biological Sciences, City of Hope The protocol describes the isolation, culture, and profiling of endothelial cells from human mesenteric artery. Additionally, a method is provided to prepare human artery for spatial transcriptomics. Proteomics, transcriptomics, and functional assays can be performed on isolated cells. This protocol can be repurposed for any medium- or large-size artery.

• Medicine

  
  Self-Administration of Drugs in Mouse Models of Feeding and Obesity Rizaldy C. Zapata¹, Dinghong Zhang¹, Besma Chaudry¹, Olivia Osborn^{1 1}Division of Endocrinology and Metabolism, Department of Medicine, University of California San Diego The overall goal of this procedure is to describe a method for self-administration of drugs that can be used in mouse models of feeding and obesity.

• Immunology and Infection

  
  Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice Valerie P. O'Brien^1,2,6, Matthew S. Joens^3,7, Amanda L. Lewis^1,2,4,8, Nicole M. Gilbert^{2,5 1}Department of Molecular Microbiology, Washington University School of Medicine in Saint Louis, ²Center for Women’s Infectious Disease Research, Washington University School of Medicine in Saint Louis, ³Center for Cellular Imaging, Washington University School of Medicine in Saint Louis, ⁴Department of Obstetrics and Gynecology, Washington University School of Medicine in Saint Louis, ⁵Department of Pediatrics, Washington University School of Medicine in Saint Louis, ⁶Fred Hutchinson Cancer Research Center, ⁷TESCAN USA, Inc., ⁸University of California San Diego A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

• Biology

  
  Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis Elena Dalla Benetta*¹, Duverney Chaverra-Rodriguez*¹, Jason L. Rasgon², Omar S. Akbari^{1 1}Division of Biological Sciences, Section of Cell and Developmental Biology, University of California, San Diego, ²Department of Entomology, The Center for Infectious Disease Dynamics, The Huck Institutes of the Life Sciences, The Pennsylvania State University Here, we describe methods for efficient pupal and adult injections in Nasonia vitripennis as accessible alternatives to embryo microinjection, enabling functional analysis of genes of interest using either RNA-silencing via RNA interference (RNAi) or gene knockout via CRISPR/Cas9 genome editing.

• Engineering

  
  Fabrication and Characterization of Thickness Mode Piezoelectric Devices for Atomization and Acoustofluidics Aditya Vasan¹, William Connacher¹, James Friend^{1 1}Medically Advanced Devices Laboratory, Center for Medical Devices, Department of Mechanical and Aerospace Engineering, Jacobs School of Engineering and Department of Surgery, School of Medicine, University of California San Diego, ²Department of Surgery, School of Medicine, University of California San Diego Fabrication of piezoelectric thickness mode transducers via direct current sputtering of plate electrodes on lithium niobate is described. Additionally, reliable operation is achieved with a transducer holder and fluid supply system and characterization is demonstrated via impedance analysis, laser doppler vibrometry, high-speed imaging, and droplet size distribution using laser scattering.

• Biology

  
  Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing Ziad Sabry¹, Christina Rabeler¹, Danielle Ireland¹, Kevin Bayingana¹, Eva-Maria S. Collins^{1,2 1}Biology Department, Swarthmore College, ²Physics Department, UC San Diego Freshwater planarians exhibit three gaits (gliding, peristalsis, and scrunching) that are distinguishable by quantitative behavioral analysis. We describe a method to induce scrunching using various noxious stimuli, quantification thereof, and distinction from peristalsis and gliding. Using gene knockdown, we demonstrate the specificity of scrunching as a quantitative phenotypic readout.

• Engineering

  
  Fabrication of Surface Acoustic Wave Devices on Lithium Niobate Jiyang Mei¹, Naiqing Zhang¹, James Friend^{1 1}Medically Advanced Devices Laboratory, Center for Medical Devices and Instrumentation, Department of Mechanical and Aerospace Engineering, Jacobs School of Engineering and Department of Surgery, School of Medicine, University of California San Diego Two fabrication techniques, lift-off and wet etching, are described in producing interdigital electrode transducers upon a piezoelectric substrate, lithium niobate, widely used to generate surface acoustic waves now finding broad utility in micro to nanoscale fluidics. The as-produced electrodes are shown to efficiently induce megahertz order Rayleigh surface acoustic waves. 

• Genetics

  
  Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus Michelle Bui¹, Ming Li¹, Robyn R Raban¹, Nannan Liu², Omar S Akbari^{1,3 1}Section of Cell and Developmental Biology, University of California, San Diego, ²Department of Entomology and Plant Patholog, Auburn University, ³Tata Institute for Genetics and Society, University of California, San Diego This protocol describes microinjection procedures for Culex quinquefasciatus embryos that are optimized to work with CRISPR/Cas9 gene editing tools. This technique can efficiently generate site-specific, heritable, germline mutations that can be used for building genetic technologies in this understudied disease vector.

• Neuroscience

  
  Simulator Training for Endovascular Neurosurgery Yasmeen Elsawaf¹, Robert C. Rennert¹, Jeffrey A. Steinberg¹, David R. Santiago-Dieppa¹, Scott E. Olson¹, Alexandar A. Khalessi¹, J. Scott Pannell^{1 1}Department of Neurosurgery, University of California Simulation of complex, high-risk procedures is critical to the education of medical trainees. A protocol for simulator-based endovascular neurosurgery training in a controlled academic environment is described. The protocol includes stepwise guidelines for trainees of varying levels, with a discussion of the advantages and limitations of this model.

• Genetics

  
  A Screening Method for Identification of Heterochromatin-Promoting Drugs Using Drosophila Lin Zhang¹, Kenny Dao¹, Angela Kang¹, Andre C. Loyola¹, Robin Shang¹, Jinghong Li¹, Willis X. Li^{1 1}Department of Medicine, University of California San Diego Drosophila is a widely used experimental model suitable for screening drugs with potential applications for cancer therapy. Here, we describe the use of Drosophila variegated eye color phenotypes as a method for screening small-molecule compounds that promote heterochromatin formation.

• Engineering

  
  Fabrication of Nanoheight Channels Incorporating Surface Acoustic Wave Actuation via Lithium Niobate for Acoustic Nanofluidics Naiqing Zhang¹, James Friend^{1 1}Medically Advanced Devices Laboratory, Center for Medical Devices, Department of Mechanical and Aerospace Engineering, Jacobs School of Engineering, and the Department of Surgery, School of Medicine, University of California San Diego We demonstrate fabrication of nanoheight channels with the integration of surface acoustic wave actuation devices upon lithium niobate for acoustic nanofluidics via liftoff photolithography, nano-depth reactive ion etching, and room-temperature plasma surface-activated multilayer bonding of single-crystal lithium niobate, a process similarly useful for bonding lithium niobate to oxides.

• Cancer Research

  
  Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts Sanghee Lee^1,2, Danielle N. Burner^1,2, Theresa R. Mendoza^1,2, Michelle T. Muldong^1,2, Catalina Arreola^1,2, Christina N. Wu^2,3, Nicholas A. Cacalano⁴, Anna A. Kulidjian^2,5, Christopher J. Kane^1,2, Christina A. M. Jamieson^{1,2 1}Department of Urology, University of California, San Diego, ²Moores Cancer Center, University of California, San Diego, ³Department of Medicine, University of California, San Diego, ⁴Department of Radiation Oncology, University of California, Los Angeles, ⁵Department of Orthopedic Surgery, University of California, San Diego Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

• Cancer Research

  
  Isolation and Characterization of Patient-derived Pancreatic Ductal Adenocarcinoma Organoid Models Hervé Tiriac^1,2, Randall French^1,2, Andrew M. Lowy^{1,2 1}Moores Cancer Center, University of California San Diego, ²Department of Surgery, Division of Surgical Oncology, University of California San Diego Patient-derived organoid cultures of pancreatic ductal adenocarcinoma are a rapidly established 3-dimensional model that represent epithelial tumor cell compartments with high fidelity, enabling translational research into this lethal malignancy. Here, we provide detailed methods to establish and propagate organoids as well as to perform relevant biological assays using these models.

• Developmental Biology

  
  A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development Renat N. Khaliullin^1,2,3, Jeffrey M. Hendel^1,2, Adina Gerson-Gurwitz^1,2, Shaohe Wang^1,4,5, Stacy D. Ochoa^1,6, Zhiling Zhao^1,7, Arshad Desai^1,2, Karen Oegema^1,2, Rebecca A. Green^{1,2 1}Ludwig Institute for Cancer Research, San Diego, ²Department of Cellular and Molecular Medicine, University of California, San Diego, ³Recursion Pharmaceuticals, ⁴Biomedical Sciences Graduate Program, University of California, San Diego, ⁵Cell Biology Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, ⁶Department of Biology, San Diego State University, ⁷Developmental and Stem Cell Biology Graduate Program, University of California, San Francisco This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80–100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

• Neuroscience

  
  Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals Ekaterina Martianova¹, Sage Aronson^2,3, Christophe D. Proulx^{1 1}CERVO Brain Research Center, Department of Psychiatry and Neurosciences, Université Laval, ²Center for Neural Circuits and Behavior, Department of Neuroscience and Section of Neurobiology, Division of Biology, University of California at San Diego, ³Neurophotometrics Ltd. This protocol details how to implement and perform multi-fiber photometry recordings, how to correct for calcium-independent artifacts, and important considerations for dual-color photometry imaging.

• Genetics

  
  Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas Francesca Boscolo Sesillo^1,2,3, Alessandra Sacco^{2 1}Graduate School of Biomedical Sciences, Sanford Burnham Prebys Medical Discovery Institute, ²Development, Aging and Regeneration Program, Sanford Burnham Prebys Medical Discovery Institute, ³Department of Obstetrics, Gynecology, and Reproductive Sciences, Division of Female Pelvic Medicine and Reconstructive Surgery, University California San Diego This protocol describes a reproducible method for isolation of mouse rhabdomyosarcoma primary cells, tumorsphere formation and treatment, and allograft transplantation starting from tumorspheres cultures.

• Neuroscience

  
  Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation Barbara Calabrese^1,2, Regina M. Powers^1,2, Alexandria J. Slepian^1,2, Shelley Halpain^{1,2 1}Division of Biological Sciences, University of California, San Diego, ²Sanford Consortium for Regenerative Medicine This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.

• Behavior

  
  Low-Cost Gait Analysis for Behavioral Phenotyping of Mouse Models of Neuromuscular Disease Virginia Wertman^1,2, Anastasia Gromova^1,2,3, Albert R. La Spada^1,2,4,5, Constanza J. Cortes^{1,2 1}Department of Neurology, Duke University School of Medicine, ²Duke Center for Neurodegeneration & Neurotherapeutics, Duke University School of Medicine, ³Biomedical Sciences Graduate Program, University of California San Diego, ⁴Department of Neurobiology, Duke University School of Medicine, ⁵Department of Cell Biology, Duke University School of Medicine Footprint analysis is a low-cost alternative to digitized gait analysis programs for researchers quantifying movement abnormalities in mice. Because of its speed, simplicity, and longitudinal potential, it is ideal for behavioral phenotyping of mouse models.

• Medicine

  
  Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent Eric T. Geier¹, Rebecca J. Theilmann², Chantal Darquenne¹, G. Kim Prisk¹, Rui Carlos Sá^{1 1}Pulmonary Imaging Laboratory, Department of Medicine, University of California, San Diego, ²Pulmonary Imaging Laboratory, Department of Radiology, University of California, San Diego Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data.

• Neuroscience

  
  An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons Brent Aulston¹, Qing Liu¹, Patrick Reilly¹, Shauna H. Yuan^{1 1}Department of Neurosciences, University of California, San Diego This protocol details a procedure in which human neuronal cultures are transduced with lentiviral constructs coding for mutant human tau. Transduced cultures display tau aggregates and associated pathologies.

• Engineering

  
  Synthesis, Functionalization, and Characterization of Fusogenic Porous Silicon Nanoparticles for Oligonucleotide Delivery Byungji Kim¹, Michael J. Sailor^{1,2 1}Materials Science and Engineering Program, University of California, San Diego, ²Department of Chemistry and Biochemistry, University of California, San Diego We demonstrate the synthesis of fusogenic porous silicon nanoparticles for effective in vitro and in vivo oligonucleotide delivery. Porous silicon nanoparticles are loaded with siRNA to form the core, which is coated by fusogenic lipids through extrusion to form the shell. Targeting moiety functionalization and particle characterization are included.

• Genetics

  
  ATAC-seq Assay with Low Mitochondrial DNA Contamination from Primary Human CD4+ T Lymphocytes Hannah D. Rickner¹, Sheng-Yong Niu^1,2, Christine S. Cheng^{1,3 1}Department of Biology, Boston University, ²Department of Computer Science and Engineering, University of California San Diego, ³Bioinformatics Program, Boston University Here, we present a protocol to perform an assay for transposase-accessible chromatin sequencing (ATAC-seq) on activated CD4+ human lymphocytes. The protocol has been modified to minimize contaminating mitochondrial DNA reads from 50% to 3% through the introduction of a new lysis buffer.

• Medicine

  
  Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer Shizuko Tachibana¹, Chao Chen¹, Oliver R. Zhang¹, Sarah V. Schurr¹, Cameron Hill¹, Ruixia Li¹, Ana M. Manso¹, Jianlin Zhang¹, Aleksander Andreyev², Anne N. Murphy², Robert S. Ross^1,3, Yoshitake Cho^{1 1}Division of Cardiology, Department of Medicine, University of California San Diego, ²Department of Pharmacology, University of California San Diego, ³Cardiology Section, Department of Medicine, Veterans Administration Healthcare, San Diego The goal of this protocol is to illustrate how to use mouse neonatal cardiomyocytes as a model system to examine how various factors can alter oxygen consumption in the heart.

• Neuroscience

  
  Disruption of Frontal Lobe Neural Synchrony During Cognitive Control by Alcohol Intoxication Ksenija Marinkovic^1,2, Lauren E. Beaton¹, Burke Q. Rosen^1,3, Joseph P. Happer⁴, Laura C. Wagner^{1 1}Department of Psychology, San Diego State University, ²Department of Radiology, University of California San Diego, ³Department of Neurosciences, University of California San Diego, ⁴San Diego State University/UC San Diego Joint Doctoral Program in Clinical Psychology This experiment uses an anatomically-constrained magnetoencephalography (aMEG) method to examine brain oscillatory dynamics and long-range functional synchrony during engagement of cognitive control as a function of acute alcohol intoxication.

• Cancer Research

  
  Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays Anagha Deshpande¹, Bo Rui Chen¹, Luyi Zhao¹, Kelsey Saddoris¹, Mayuri Kerr¹, Nan Zhu², Prashant Mali³, Aniruddha J. Deshpande^{1 1}Tumor Initiation and Maintenance Program, Sanford Burnham Prebys Medical Discovery Institute, ²Blood Research Institute, Blood Center of Wisconsin, ³Department of Bioengineering, University of California, San Diego This manuscript describes a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR-Cas9-based method for simple and expeditious investigation of the role of multiple candidate genes in Acute Myeloid Leukemia (AML) cell proliferation in parallel. This technique is scalable and can be applied in other cancer cell lines as well.

• Biochemistry

  
  A Guide to Production, Crystallization, and Structure Determination of Human IKK1/α Smarajit Polley^1,2, De-Bin Huang¹, Tapan Biswas¹, Gourisankar Ghosh^{1 1}Department of Chemistry & Biochemistry, University of California, San Diego, ²Department of Biophysics, Bose Institute IκB Kinase 1/α (IKK1/α CHUK) is a Ser/Thr protein kinase that is involved in a myriad of cellular activities primarily through activation of NF-κB transcription factors. Here, we describe the main steps necessary for the production and crystal structure determination of this protein.

• Behavior

  
  An Electrophysiology Protocol to Measure Reward Anticipation and Processing in Children Katherine K.M. Stavropoulos¹, Leslie J. Carver^{2 1}Graduate School of Education, University of California, Riverside, ²Psychology Department, University of California, San Diego This protocol is designed to measure reward anticipation and processing in young children with and without autism. Specifically, the protocol is designed to study the neural correlates of reward during social and nonsocial conditions while controlling for reward between conditions.

• Environment

  
  Individual Culturing of Tigriopus Copepods and Quantitative Analysis of Their Mate-guarding Behavior Satomi Tsuboko-Ishii¹, Ronald S. Burton^{1 1}Marine Biology Research Division, Scripps Institution of Oceanography, University of California, San Diego Mate-guarding behavior plays an important role in reproduction of intertidal copepods of the genus Tigriopus. However, methods for studying this behavior have not been well described. Here we describe methods for: 1) individual culture of virgin Tigriopus animals, and 2) quantitative analysis of their mate-guarding behavior.

• Immunology and Infection

  
  A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells Effie E Bastounis¹, Fabian E Ortega¹, Ricardo Serrano², Julie A Theriot^{3 1}Department of Biochemistry, Stanford University School of Medicine, ²Department of Mechanical and Aerospace Engineering, University of California San Diego, ³Departments of Biochemistry, Microbiology and Immunology and Howard Hughes Medical Institute, Stanford University School of Medicine We have developed a multi-well format polyacrylamide-based assay for probing the effect of extracellular matrix stiffness on bacterial infection of adherent cells. This assay is compatible with flow cytometry, immunostaining, and traction force microscopy, allowing for quantitative measurements of the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria.

• Developmental Biology

  
  Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells Michael S. Yu*^1,2, Sean Spiering*¹, Alexandre R. Colas^{1 1}Sanford Burnham Prebys Medical Discovery Institute, ²Department of Bioengineering, University of California at San Diego Here we describe a scalable method, using a simple combination of Activin A and lentivirus-mediated Id1-overexpression, to generate first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells.

• Cancer Research

  
  A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation Jayanth S. Shankara Narayanan¹, Partha Ray¹, Ibtehaj Naqvi², Rebekah White^{1 1}Moores Cancer Center, University California San Diego, ²Duke University School of Medicine Irreversible electroporation (IRE) is a non-thermal ablation technique used for the treatment of locally advanced pancreatic cancer. Being a relatively new technique, the effects of IRE on the tumor growth are poorly understood. We have developed a syngeneic mouse model that facilitates studying the effects of IRE on pancreatic cancer.

• Behavior

  
  Assessing Activity-based Anorexia in Mice Amanda C. Welch¹, William R. Katzka^1,2, Stephanie C. Dulawa^{1 1}Department of Psychiatry, University of California, San Diego, ²University of California, Los Angeles Mice individually housed with a running wheel while given limited access to food develop reductions in food consumption and increase activity on the running wheel. This experimental phenomenon is called activity-based anorexia. This paradigm provides an experimental tool for studying the neurobiology and behaviors underlying aspects of anorexia nervosa.

• Neuroscience

  
  Neurovascular Network Explorer 2.0: A Simple Tool for Exploring and Sharing a Database of Optogenetically-evoked Vasomotion in Mouse Cortex In Vivo Hana Uhlirova^1,2, Peifang Tian^3,4, Kıvılcım Kılıç^3,5, Martin Thunemann¹, Vishnu B. Sridhar⁶, Radim Chmelik^2,7, Hauke Bartsch¹, Anders M. Dale^1,3, Anna Devor^1,3,8, Payam A. Saisan^{3 1}Department of Radiology, University of California, San Diego, ²Central European Institute of Technology, Brno University of Technology, ³Department of Neurosciences, University of California, San Diego, ⁴Department of Physics, John Carroll University, ⁵Department of Biomedical Engineering, Boston University, ⁶Bioengineering Undergraduate Program, University of California, San Diego, ⁷Institute of Physical Engineering, Faculty of Mechanical Engineering, Brno University of Technology, ⁸Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Harvard Medical School A graphical user interface for exploring and sharing a database of optogenetically-induced vascular responses in mouse somatosensory cortex in vivo measured by 2-photon microscopy is presented. It allows browsing the data, criteria-based selection, averaging, localization of measurements within a 3D volume of vasculature and exporting the data.

• Cancer Research

  
  Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts Jorge A. Benitez*¹, Ciro Zanca*¹, Jianhui Ma¹, Webster K. Cavenee^1,2,3, Frank B. Furnari^{1,2,4 1}Ludwig Institute for Cancer Research, ²Moores Cancer Center, School of Medicine, University of California, San Diego, ³Department of Pathology, School of Medicine, University of California, San Diego, ⁴Department of Medicine, School of Medicine, University of California, San Diego Orthotopic intracranial injection of tumor cells has been used in cancer research to study brain tumor biology, progression, evolution, and therapeutic response. Here we present fluorescence molecular tomography of tumor xenografts, which provides real-time intravital imaging and quantification of a tumor mass in preclinical glioblastoma models.

• Engineering

  
  Focused Ion Beam Fabrication of LiPON-based Solid-state Lithium-ion Nanobatteries for In Situ Testing Jungwoo Z. Lee*¹, Thomas A. Wynn*², Ying Shirley Meng^1,2, Dhamodaran Santhanagopalan^{3 1}Department of NanoEngineering, University of California San Diego, ²Materials Science and Engineering Program, University of California San Diego, ³Amrita Centre for Nanosciences and Molecular Medicine, Amrita Vishwa Vidyapeetham University A protocol for the fabrication of electrochemically active LiPON-based solid-state lithium-ion nanobatteries using a focused ion beam is presented.

• Developmental Biology

  
  A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes Federica Cavalera¹, Mario Zanoni¹, Valeria Merico¹, Thi Thu Hien Bui^1,2, Martina Belli^1,3, Lorenzo Fassina⁴, Silvia Garagna¹, Maurizio Zuccotti^{1 1}Laboratorio di Biologia dello Sviluppo, Dipartimento di Biologia e Biotecnologie "Lazzaro Spallanzani", University of Pavia, ²Institute of Biotechnology, University of Helsinki, ³Department of Reproductive Medicine, University of California San Diego, ⁴Dipartimento di Ingegneria Industriale e dell'Informazione, University of Pavia Here, we present a protocol for non-invasive assessment of oocyte developmental competence performed during their in vitro maturation from the germinal vesicle to the metaphase II stage. This method combines time-lapse imaging with particle image velocimetry (PIV) and neural network analyses.

• Genetics

  
  Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation Ming Li^1,2, Michelle Bui^1,2, Omar S. Akbari^{1,2 1}Department of Entomology and Riverside Center of Disease Vector Research, Institute for Integrative Genome Biology, University of California, Riverside, ²Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego Microinjection of Nasonia vitripennis embryos is an essential method for generating heritable genome modifications. Described here is a detailed procedure for microinjection and transplantation of Nasonia vitripennis embryos, which will greatly facilitate future genome manipulation in this organism.

• Neuroscience

  
  Comprehensive Endovascular and Open Surgical Management of Cerebral Arteriovenous Malformations Robert C. Rennert¹, Jeffrey A. Steinberg¹, Vincent J. Cheung¹, David R. Santiago-Dieppa¹, Jeffrey Scott Pannell¹, Alexander A. Khalessi^{1 1}Department of Neurosurgery, University of California, San Diego Surgery is the gold standard for accessible arteriovenious malformations (AVMs), and pre-operative embolization can simplify this procedure. We describe our approach for staged endovascular embolization and open resection of AVMs, and provide a representative clinical example highlighting the advantages of a comprehensively trained neurovascular surgeon leading a multi-disciplinary clinical team.

• Bioengineering

  
  Pattern-based Search of Epigenomic Data Using GeNemo Alvin Zheng*¹, Xiaoyi Cao*¹, Sheng Zhong^{1 1}Department of Bioengineering, University of California San Diego Unlike DNA sequence data, epigenomic data are not readily subjected to text-based searches. Presented here are the procedures to use an upgraded version of GeNemo, a web-based bioinformatics tool, to conduct pattern-based searches for similarities in epigenomic data comparing available online databases including Encyclopedia of DNA Elements with user's data.

• Neuroscience

  
  Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility Renny Ng¹, Hui-Hao Lin¹, Jing W. Wang¹, Chih-Ying Su^{1 1}Division of Biological Sciences, University of California San Diego The overall goal of this protocol is to demonstrate how to present odorants of low volatility for single-sensillum recording from Drosophila olfactory receptor neurons that respond to long-chain cuticular pheromones.

• Biology

  
  Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin Fengwu Li¹, Christopher A. Adase¹, Ling-juan Zhang^{1 1}Department of Dermatology, School of Medicine, UC San Diego Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

• Neuroscience

  
  Subpial Adeno-associated Virus 9 (AAV9) Vector Delivery in Adult Mice Takahiro Tadokoro¹, Atsushi Miyanohara¹, Michael Navarro¹, Kota Kamizato¹, Stefan Juhas², Jana Juhasova², Silvia Marsala¹, Oleksandr Platoshyn¹, Erik Curtis³, Brandon Gabel³, Joseph Ciacci³, Nada Lukacova⁴, Katarina Bimbova⁴, Martin Marsala^{1,4 1}Neuroregeneration Laboratory, Department of Anesthesiology, University of California, San Diego, ²Institute of Animal Physiology and Genetics, Czech Academy of Sciences, ³Department of Neurosurgery, University of California, San Diego, ⁴Institute of Neurobiology, Slovak Academy of Sciences The goal of the present study was to develop and validate the potency and safety of spinal adeno-associated virus 9 (AAV9)-mediated gene delivery by using a novel subpial gene delivery technique in adult mice.

• Neuroscience

  
  Generation and Long-term Maintenance of Nerve-free Hydra Cassidy M. Tran¹, Sharon Fu¹, Trevor Rowe², Eva-Maria S. Collins^{1,2 1}Division of Biological Sciences, UC San Diego, ²Physics Department, UC San Diego Through a double treatment with colchicine, a plant-derived toxin that kills dividing cells, nerve-free Hydra vulgaris can be generated. These Hydra cannot feed or egest on their own. This paper describes an improved method for long-term maintenance of nerve free Hydra vulgaris in the laboratory.

• Developmental Biology

  
  Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments Liu Yang¹, Sayantanee Paul¹, Sarah DuBois-Coyne¹, Phillip Kyriakakis², Alexey Veraksa^{1 1}Biology Department, University of Massachusetts Boston, ²Department of Bioengineering, University of California, San Diego The goal of this protocol is to provide a straightforward and inexpensive approach to collecting Drosophila embryos at medium scale (0.5-1 g) and preparing protein extracts that can be used in downstream proteomic applications, such as affinity purification-mass spectrometry (AP-MS).

• Immunology and Infection

  
  The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections William J. Comstock¹, Edwin Huh², Reiley Weekes², Connor Watson², Tianyang Xu², Pieter C. Dorrestein¹, Robert A. Quinn^{1 1}Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, ²Department of Mechanical and Aerospace Engineering, University of California, San Diego The mucus plugged airways of cystic fibrosis (CF) patients are an ideal environment for microbial pathogens to thrive. The manuscript describes a novel method for studying the CF lung microbiome in an environment that mimics where they cause disease and how alterations of chemical conditions can drive microbial dynamics.

• Biochemistry

  
  A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing Irena Roci^1,2, Hector Gallart-Ayala³, Jeramie Watrous⁴, Mohit Jain⁴, Craig E. Wheelock³, Roland Nilsson^{1,2 1}Department of Medicine, Unit of Computational Medicine, Karolinska Institutet, ²Center for Molecular Medicine, Karolinska Institutet, ³Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry 2, Karolinska Institutet, ⁴Department of Medicine, University of California San Diego This article describes a method for studying cellular metabolism in complex communities of multiple cell types, using a combination of stable isotope tracing, cell sorting to isolate specific cell types, and mass spectrometry.

• Neuroscience

  
  Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice Simon H. Bayerl¹, Melina Nieminen-Kelhä¹, Thomas Broggini², Peter Vajkoczy¹, Vincent Prinz^{1 1}Department of Neurosurgery and Center for Stroke-research Berlin (CSB), Charité-Universitätsmedizin, ²Department of Physics, University of California San Diego Surgical occlusion of a distal middle cerebral artery branch (MCAo) is a frequently used model in experimental stroke research. This manuscript describes the basic technique of permanent MCAo, combined with the insertion of a lateral cranial window, which offers the opportunity for longitudinal intravital microscopy in mice.

• Genetics

  
  Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans Kentaro Noma¹, Yishi Jin^{1,2 1}Division of Biological Sciences, Section of Neurobiology, University of California in San Diego and Howard Hughes Medical Institute, ²Department of Cellular and Molecular Medicine, University of California in San Diego School of Medicine and Howard Hughes Medical Institute Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

• Behavior

  
  Treating Clinical Depression with Repetitive Deep Transcranial Magnetic Stimulation Using the Brainsway H1-coil David Feifel¹, Katherine Pappas^{1 1}Department of Psychiatry, University of California, San Diego Here we present a protocol outlining the methodology for treatment of Major Depressive Disorder using the deep Transcranial Magnetic Stimulation (dTMS) system.

• Cancer Research

  
  Fluorescent Orthotopic Mouse Model of Pancreatic Cancer Jonathan A. Moreno¹, Antonio Sanchez², Robert M. Hoffman^3,4, Saima Nur¹, Maria P. Lambros^{1 1}College of Pharmacy, Western University of Health Sciences, ²UVP, LLC, ³Department of Surgery, School of Medicine, University of California San Diego, ⁴Anticancer Inc. A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

• Neuroscience

  
  Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters (CNiFERs) for Optical Detection of Neurotransmitters In Vivo Emre Lacin¹, Arnaud Muller², Marian Fernando¹, David Kleinfeld^2,3, Paul A. Slesinger^{1 1}Department of Neuroscience, Icahn School of Medicine at Mount Sinai, ²Department of Physics, University of California, San Diego, ³Section of Neurobiology, University of California, San Diego We present a protocol to create cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) for the optical detection of volumetric neurotransmitter release.

• Bioengineering

  
  A Protocol for Bioinspired Design: A Ground Sampler Based on Sea Urchin Jaws Michael B. Frank¹, Steven E. Naleway¹, Taylor S. Wirth², Jae-Young Jung¹, Charlene L. Cheung², Faviola B. Loera², Sandra Medina², Kirk N. Sato³, Jennifer R. A. Taylor⁴, Joanna McKittrick^{1,2 1}Materials Science and Engineering Program, University of California, San Diego, ²Department of Mechanical and Aerospace Engineering, University of California, San Diego, ³Integrative Oceanography Division, Center for Marine Biodiversity and Conservation, Scripps Institution of Oceanography, ⁴Marine Biology Research Division, Scripps Institution of Oceanography A protocol for bioinspired design is described for a sampling device based on the jaws of a sea urchin. The bioinspiration process includes observing the sea urchins, characterizing the mouthpiece, 3D printing of the teeth and their assembly, and bioexploring the tooth structure.

• Developmental Biology

  
  Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres Miguel A. Lopez-Ramirez*^1,2, Charles-Félix Calvo*³, Emma Ristori¹, Jean-Léon Thomas^1,3, Stefania Nicoli^{1 1}Yale Cardiovascular Research Center, Internal Medicine, Yale University, ²Department of Medicine, University of California, San Diego, ³APHP Groupe Hospitalier Pitié-Salpètrière, Université Pierre and Marie Curie Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

• Developmental Biology

  
  Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis Jingting Li^1,2, George L. Sen^{1,2 1}Department of Dermatology, UCSD Stem Cell Program, University of California, San Diego, ²Department of Cellular and Molecular Medicine, UCSD Stem Cell Program, University of California, San Diego The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

• Neuroscience

  
  Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert, Head-restrained Rats Jeffrey D. Moore¹, Martin Deschênes², David Kleinfeld^{1 1}Department of Physics 0374, University of California, San Diego, ²Department of Psychiatry and Neuroscience, Centre de Recherche de l’Université Laval Robert-Giffard This protocol describes the design and surgical implantation of a head-restraining mechanism to monitor neuronal activity in sub-cortical brain structures in alert rats. It delineates procedures to isolate single neurons in the juxtacellular configuration and to efficiently identify their anatomical locations.

• Biology

  
  Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies Matthias Garten¹, Sophie Aimon², Patricia Bassereau¹, Gilman E. S. Toombes^{3 1}Institut Curie, Centre de Recherche, CNRS, UMR 168, PhysicoChimie Curie, Université Pierre et Marie Curie, ²Kavli Institute for Brain and Mind, University of California, San Diego, ³Molecular Physiology and Biophysics Section, National Institute for Neurological Disorders and Stroke, National Institute of Health The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods — electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

• Biology

  
  Methods for Skin Wounding and Assays for Wound Responses in C. elegans Suhong Xu¹, Andrew D. Chisholm^{1 1}Division of Biological Sciences, Section of Cell and Developmental Biology, University of California, San Diego The adult C. elegans skin is a tractable model for studies of epithelial wound responses, including wound closure, scar formation, and innate immunity.

• Immunology and Infection

  
  Generation of Human Alloantigen-specific T Cells from Peripheral Blood Burhan P Jama¹, Gerald P Morris^{1 1}Department of Pathology, University of California, San Diego This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

• Environment

  
  Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology Andreas Florian Haas¹, Ben Knowles¹, Yan Wei Lim¹, Tracey McDole Somera², Linda Wegley Kelly¹, Mark Hatay¹, Forest Rohwer^{1 1}Department of Biology, San Diego State University, ²Scripps Institution of Oceanography, University of California San Diego Here, we present a comprehensive protocol to assess the organic and inorganic nutrient availability and the abundance and structure of microbial and viral communities in remote marine environments.

• Neuroscience

  
  Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis Sylvia Neumann¹, George E. Campbell*¹, Lukasz Szpankowski*^2,3, Lawrence S.B. Goldstein^2,4, Sandra E. Encalada^{1 1}Department of Molecular and Experimental Medicine, Dorris Neuroscience Center, The Scripps Research Institute, ²Department of Cellular and Molecular Medicine, University of California San Diego, ³Department of Bioengineering, University of California San Diego, ⁴Department of Neurosciences, University of California San Diego School of Medicine Intracellular transport of cargoes, such as vesicles or organelles, is carried out by molecular motor proteins that track on polarized microtubules. This protocol describes the correlation of the directionality of transport of individual cargo particles moving inside neurons, to the relative amount and type of associated motor proteins.

• Medicine

  
  A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts Shaoping Hou¹, Armin Blesch², Paul Lu^{3,4 1}Spinal Cord Research Center, Department of Neurobiology & Anatomy, Drexel University College of Medicine, ²Spinal Cord Injury Center, Heidelberg University Hospital, ³Veterans Administration Medical Center, San Diego, CA, ⁴Department of Neurosciences, University of California, San Diego We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

• Neuroscience

  
  Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons Xiaobei Zhao¹, Yue Zhou², April M. Weissmiller¹, Matthew L. Pearn^3,4, William C. Mobley¹, Chengbiao Wu^{1 1}Department of Neurosciences, University of California, San Diego, ²School of Biomedical Engineering and Med-X Research Institute, Shanghai Jiao Tong University, ³Department of Anesthesiology, University of California, San Diego, ⁴VA San Diego Healthcare System Axonal transport of BDNF, a neurotrophic factor, is critical for the survival and function of several neuronal populations. Some degenerative disorders are marked by disruption of axonal structure and function. We demonstrated the techniques used to examine live trafficking of QD-BDNF in microfluidic chambers using primary neurons.

• Neuroscience

  
  Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury Paul Lu^1,2, Lori Graham², Yaozhi Wang², Di Wu², Mark Tuszynski^{1,2 1}Veterans Administration Medical Center, San Diego, ²Department of Neurosciences, University of California, San Diego Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances.

• Medicine

  
  Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters Ana D. Lopez¹, Ayse G. Kayali¹, Alberto Hayek¹, Charles C. King^{1 1}Pediatric Diabetes Research Center, Department of Pediatrics, University of California, San Diego A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.  The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

• Biology

  
  Cell Labeling and Injection in Developing Embryonic Mouse Hearts Emilye Hiriart*¹, Patrick van Vliet*², Ralf J. Dirschinger², Sylvia M. Evans², Michel Puceat^{1 1}INSERM UMR-910, Aix-Marseille University, ²Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development.

• Medicine

  
  Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope Joanna L. Thomas^1,2, Justin Dumouchel², Jinghong Li², Jenna Magat², Dana Balitzer³, Timothy D. Bigby^{1,2 1}Medical and Research Services, VA San Diego Healthcare System, ²Department of Medicine, University of California, San Diego, ³School of Medicine, University of California, San Diego We have developed a simple, reliable, and relatively inexpensive method for endotracheal intubation in mice via direct laryngoscopy using an otoscope with a 2.0 mm speculum. This technique is atraumatic and can be used for repeated measurements in chronic experiments. We find it superior to tracheostomy or previously reported nonsurgical techniques.

• Neuroscience

  
  Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography Sarah H. Shahmoradian¹, Mauricio R. Galiano², Chengbiao Wu³, Shurui Chen⁴, Matthew N. Rasband², William C. Mobley³, Wah Chiu^{4 1}Department of Molecular Physiology and Biophysics, Baylor College of Medicine, ²Department of Neuroscience, Baylor College of Medicine, ³Department of Neuroscience, University of California at San Diego, ⁴National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine To preserve neuronal processes for ultrastructural analysis, we describe a protocol for plating of primary neurons on electron microscopy grids followed by flash freezing, yielding samples suspended in a layer of vitreous ice. These samples can be examined with a cryo-electron microscope to visualize structures at the nanometer scale.

• Chemistry

  
  Conducting Miller-Urey Experiments Eric T. Parker¹, James H. Cleaves^2,3, Aaron S. Burton⁴, Daniel P. Glavin⁵, Jason P. Dworkin⁵, Manshui Zhou¹, Jeffrey L. Bada⁶, Facundo M. Fernández^{1 1}School of Chemistry and Biochemistry, Georgia Institute of Technology, ²Earth-Life Science Institute, Tokyo Institute of Technology, ³Institute for Advanced Study, ⁴Astromaterials Research and Exploration Science Directorate, NASA Johnson Space Center, ⁵Goddard Center for Astrobiology, NASA Goddard Space Flight Center, ⁶Geosciences Research Division, Scripps Institution of Oceanography, University of California at San Diego The Miller-Urey experiment was a pioneering study regarding the abiotic synthesis of organic compounds with possible relevance to the origins of life. Simple gases were introduced into a glass apparatus and subjected to an electric discharge, simulating the effects of lightning in the primordial Earth’s atmosphere-ocean system. The experiment was conducted for one week, after which, the samples collected from it were analyzed for the chemical building blocks of life.

• Behavior

  
  Handwriting Analysis Indicates Spontaneous Dyskinesias in Neuroleptic Naïve Adolescents at High Risk for Psychosis Derek J. Dean¹, Hans-Leo Teulings², Michael Caligiuri³, Vijay A. Mittal^{1 1}Department of Psychology and Neuroscience, Center for Neuroscience, University of Colorado Boulder, ²NeuroScript LLC, ³Department of Psychiatry, University of California, San Diego Handwriting analysis software significantly improves upon existing instruments measuring movement disorders. Individuals at risk for psychosis and healthy controls completed handwriting tasks to test for dyskinesia. Results suggest that youth at risk for psychosis exhibit dyskinesia and that handwriting analysis could significantly contribute to wider dissemination of early identification efforts

• Neuroscience

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

• Bioengineering

  
  Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos. Michelle T. Juarez¹, Rachel A. Patterson², Wilson Li², William McGinnis^{2 1}Sophie Davis School of Biomedical Education, The City College of New York, ²Cell & Developmental Biology, University of California, San Diego The embryonic epidermis of very late stage Drosophila embryos provides an in vivo system for rapid puncture wound response analysis and can be combined with genetic manipulations or chemical microinjection treatments to advance studies in wound healing for translation into mammalian models.

• Biology

  
  Isolation and Culture of Neonatal Mouse Cardiomyocytes Elisabeth Ehler¹, Thomas Moore-Morris², Stephan Lange^{2 1} Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

• Immunology and Infection

  
  Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria Tal Danino*¹, Arthur Prindle*², Jeff Hasty^2,3,4, Sangeeta Bhatia^{1,5,6,7,8 1}Health Sciences and Technology, Massachusetts Institute of Technology, ²Department of Bioengineering, University of California, San Diego, ³Biocircuits Institute, University of California, San Diego, ⁴Molecular Biology Section, Division of Biological Science, University of California, San Diego, ⁵Broad Institute of Harvard and MIT, ⁶Department of Medicine, Brigham and Women's Hospital, ⁷Electrical Engineering and Computer Science and David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, ⁸Howard Hughes Medical Institute The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. This video covers tumor cell preparation and implantation, bacteria preparation and injection, whole-animal luminescence imaging, tumor excision, and bacterial colony counting.

• Medicine

  
  Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells Charles Zhang¹, Arthur T. Suckow², Steven D. Chessler^{1,3 1}Pediatric Diabetes Research Center, University of California, San Diego, ²Janssen Research & Development, ³Department of Medicine, University of California, San Diego Transcellular protein interactions are important determinants of pancreatic beta-cell function. Detailed here is a method—adapted from a coculture model of synaptogenesis—for investigating how specific transmembrane proteins influence insulin secretion. Transfected HEK293 cells express proteins of interest; beta cells do not need to be transfected or otherwise directly perturbed.

• Biology

  
  A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice Timo Rieg^{1,2 1}Division of Nephrology-Hypertension, Department of Medicine, University of California, San Diego, ²San Diego VA Healthcare System Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

• Bioengineering

  
  Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering Shivanjali Joshi-Barr¹, Jerome V. Karpiak², Yogesh Ner¹, Jessica H. Wen³, Adam J. Engler³, Adah Almutairi^{1,2 1}Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, ²Biomedical Sciences Program, University of California, San Diego, ³Department of Bioengineering, University of California, San Diego Here we describe a unique strategy for creating biocompatible, layered matrices with continuous interfaces between distinct layers for tissue engineering. Such a scaffold could provide an ideal customizable environment to modulate cell behavior by various biological, chemical or mechanical cues

• Biology

  
  Using Unfixed, Frozen Tissues to Study Natural Mucin Distribution Miriam Cohen¹, Nissi M. Varki¹, Mark D. Jankowski², Pascal Gagneux^{1 1}Department of Cellular and Molecular Medicine, University of California, San Diego, ²Biosecurity and Public Health, Los Alamos National Laboratory Unfixed frozen tissue samples embedded in Optimal Cutting Temperature medium (OCT) can be used to study natural distribution and glycosylation of secreted mucus. In this approach tissue processing is minimal and the natural presentation of glycolipids, mucins and glycan-epitopes is preserved. Tissue sections can be analyzed by immunohistochemistry using fluorescence or chromogenic detection.

• Medicine

  
  Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry Wenhong Zhu¹, Richard L. Gallo^2,3, Chun-Ming Huang^{2,3,4 1}Sanford-Burnham Medical Research Institute, ²Division of Dermatology, University of California, San Diego, ³VA San Diego Healthcare Center, ⁴Moores Cancer Center, University of California, San Diego Considering saliva sampling for future clinical application, a lollipop-like ultrafiltration (LLUF) probe was fabricated to fit in the human oral cavity. Direct analysis of undigested saliva by NanoLC-LTQ mass spectrometry demonstrated the ability of LLUF probes to remove large proteins and high abundance proteins, and make low-abundant peptides more detectable.

• Medicine

  
  Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR Sonia Apaza¹, Susan Espetia¹, Robert H. Gilman^1,2, Sonia Montenegro³, Susana Pineda³, Fanny Herhold¹, Romeo Pomari¹, Margaret Kosek², Nancy Vu¹, Mayuko Saito^{1,2,4 1}Laboratorio de Investigación y Desarrollo (LID), Universidad Peruana Cayetano Heredia, ²Bloomberg School of Public Health, Johns Hopkins University, ³Laboratorio de Diagnostico Molecular, Facultad de Medicina, University of Concepcion,Chile, ⁴University of California San Diego School of Medicine A One-Step RT-PCR assay for detection and genogroup identification of Norovirus isolates from children’s stools, that utilizes primers and TaqMan probes specific to the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the Norovirus genome is described. A non-commercial, cost-effective RNA extraction method is detailed.

• Neuroscience

  
  A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain Andy Y. Shih¹, Celine Mateo¹, Patrick J. Drew^2,3, Philbert S. Tsai¹, David Kleinfeld^{1,4 1}Department of Physics, University of California, San Diego, ²Department of Engineering Science and Mechanics, Pennsylvania State University, ³Department of Neurosurgery, Pennsylvania State University, ⁴Section of Neurobiology, University of California, San Diego We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

• Neuroscience

  
  Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines Kenric J. Hoegler¹, Martin Distel², Reinhard W. Köster³, John H. Horne^{1 1}Department of Biology, Pace University, ²Cellular and Molecular Medicine, University of California, San Diego, ³Division of Cell Biology and Cell Physiology, Zoological Institute, Braunschweig University of Technology The temporal and spatial resolution of genetic manipulations determines the spectrum of biological phenomena that they can perturb. Here we use temporally and spatially discrete in vivo electroporation, combined with transgenic lines of zebrafish, to induce expression of a GFP transgene specifically in neurons of the developing olfactory bulb.

• Medicine

  
  Magnetic Resonance Imaging Quantification of Pulmonary Perfusion using Calibrated Arterial Spin Labeling Tatsuya J. Arai^1,2, G. Kim Prisk^1,3, Sebastiaan Holverda¹, Rui Carlos Sá¹, Rebecca J. Theilmann³, A. Cortney Henderson¹, Matthew V. Cronin³, Richard B. Buxton³, Susan R. Hopkins^{1,3 1}Medicine, University of California San Diego - UCSD, ²Bioengineering, University of California San Diego - UCSD, ³Radiology, University of California San Diego - UCSD A MR imaging method to study the distribution of pulmonary blood flow under a variety of physiological conditions, in this case exposure to three different inspired oxygen concentrations: hypoxia, normoxia, and hyperoxia, is described. This technique utilizes human pulmonary physiology research techniques in an MR scanning environment.

• Bioengineering

  
  Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy Diana E. Schlamadinger¹, Judy E. Kim^{1 1}Chemistry and Biochemistry, University of California San Diego - UCSD This video article details the experimental procedure for obtaining the Gibbs free energy of membrane protein folding by tryptophan fluorescence.

• Bioengineering

  
  Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering Sonya Seif-Naraghi¹, Jennifer Singelyn¹, Jessica DeQuach¹, Pamela Schup-Magoffin¹, Karen Christman^{1 1}University of California, San Diego Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

• Neuroscience

  
  Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window Pedro Cabrales¹, Leonardo J. M. Carvalho^{2 1}Bioengineering, University of California, San Diego, ²La Jolla Bioengineering Institute Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

• Neuroscience

  
  A Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia Stephan J. Guyenet¹, Stephanie A. Furrer², Vincent M. Damian¹, Travis D. Baughan², Albert R. La Spada*³, Gwenn A. Garden*^{2 1}Department of Biochemistry, University of Washington, ²Department of Neurology, University of Washington, ³Division of Genetics, Departments of Pediatrics and Cellular and Molecular Medicine, and the Institute for Genomic Medicine, University of California, San Diego - Rady Children’s Hospital We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebellar ataxia. Measures include hind limb clasping, ledge test, gait and kyphosis. This protocol effectively discriminates between affected and non-affected individuals, and detects the progression of affected individuals over time.

• Biology

  
  Spinal Cord Electrophysiology Allyn Meyer¹, Benjamin W. Gallarda^1,2, Samuel Pfaff¹, William Alaynick^{1 1}The Salk Institute for Biological Studies, Howard Hughes Medical Institute and Gene Expression Laboratory, ²Biology Graduate Program, University of California San Diego - UCSD A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

• Biology

  
  Localized RNAi and Ectopic Gene Expression in the Medicinal Leech Orit Shefi¹, Claire Simonnet², Alex Groisman², Eduardo R Macagno^{1 1}Division of Biological Sciences, University of California San Diego - UCSD, ²Department of Physics, University of California San Diego - UCSD In this video, we show a procedure for an accurate biolistic delivery of reagents into live tissue with a novel miniature gene gun. We are knocking down the expression of the axon guidance molecule Netrin in leech embryos by delivering molecules of dsRNA into the ventral body wall and ganglia of single segments.