University of California

56 articles published in JoVE

• Biochemistry

  
  "Cell Surface Capture" Workflow for Label-Free Quantification of the Cell Surface Proteome Akul Naik¹, Sanjeeva Srivastava^1,2, Arun P. Wiita^{1,3,4 1}Department of Laboratory Medicine, University of California, San Francisco, ²Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, ³Deptartment of Bioengineering and Therapeutic Sciences, University of California, San Francisco, ⁴Chan Zuckerberg Biohub Here, we describe a proteomics workflow for characterization of the cell surface proteome of various cell types. This workflow includes cell surface protein enrichment, subsequent sample preparation, analysis using an LC-MS/MS platform, and data processing with specialized software.

• Neuroscience

  
  Measuring the Influence of Magnetic Vestibular Stimulation on Nystagmus, Self-Motion Perception, and Cognitive Performance in a 7T MRT Gerda Wyssen^1,6, Miranda Morrison², Athanasia Korda², Wilhelm Wimmer^2,3, Jorge Otero-Millan⁴, Matthias Ertl¹, Andreas A. Szukics^1,6, Thomas Wyss², Franca Wagner^5,6, Marco D. Caversaccio^2,3, Georgios Mantokoudis^2,6, Fred W. Mast^{1,6 1}Department of Psychology, University of Bern, ²Department of Otorhinolaryngology, Head and Neck Surgery, lnselspital, University Hospital Bern and University of Bern, ³Hearing Research Laboratory, ARTORG Center for Biomedical Engineering Research, University of Bern, ⁴Herbert Wertheim School of Optometry and Vision Science, University of California, ⁵University Department of Diagnostic and Interventional Neuroradiology, Inselspital, Bern University Hospital, University of Bern, ⁶Translational Imaging Center (TIC), Swiss Institute for Translational and Entrepreneurial Medicine In this article, we describe the experimental setup, material, and procedures to assess reflexive eye movements, self-motion perception, and cognitive tasks under magnetic vestibular stimulation, as well as the anatomical orientation of the vestibular organs, in a 7 Tesla Magnetic resonance tomography (7T-MRT) scanner.

• Biology

  
  Real-Time Quantification of the Effects of IS200/IS605 Family-Associated TnpB on Transposon Activity Michael Worcester*¹, Femila Manoj*^1,2, Thomas E. Kuhlman^{1,2,3 1}Department of Physics and Astronomy, University of California, ²Microbiology Program, University of California, ³Biophysics Program, University of California A protocol is outlined to perform live real-time imaging to quantify how the accessory protein TnpB affects the dynamics of transposition in individual live Escherichia coli cells.

• Biology

  
  Forming, Confining, and Observing Microtubule-Based Active Nematics Fereshteh L. Memarian¹, Dimitrius A. Khaladj¹, Derek Hammar¹, Linda S. Hirst^{1 1}Department of Physics, University of California Presented here are methods for preparing active nematics from microtubules and kinesin motors, including protein preparation and construction and the use of wells for active nematic confinement.

• Cancer Research

  
  Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens Jonathan Weitz¹, Kevin Christian Montecillo Gulay¹, Tatiana Hurtado de Mendoza¹, Herve Tiriac¹, Joel Baumgartner¹, Kaitlyn Kelly¹, Jula Veerapong¹, Andrew M. Lowy^{1 1}Department of Surgery, University of California We describe a protocol for the production, culture, and visualization of human cancers, which have metastasized to the peritoneal surfaces. Resected tumor specimens are cut using a vibratome and cultured on permeable inserts for increased oxygenation and viability, followed by imaging and downstream analyses using confocal microscopy and flow cytometry.

• Bioengineering

  
  Mechano-Node-Pore Sensing: A Rapid, Label-Free Platform for Multi-Parameter Single-Cell Viscoelastic Measurements Andre Lai*¹, Rachel Rex*², Kristen L. Cotner¹, Alan Dong³, Michael Lustig^1,3, Lydia L. Sohn^{1,2 1}Graduate Program in Bioengineering, University of California, Berkeley and University of California, San Francisco, ²Department of Mechanical Engineering, University of California, Berkeley, ³Department of Electrical Engineering and Computer Sciences, University of California, Berkeley Presented here is a method to mechanically phenotype single cells using an electronics-based microfluidic platform called mechano-node-pore sensing (mechano-NPS). This platform maintains moderate throughput of 1-10 cells/s while measuring both the elastic and viscous biophysical properties of cells.

• Developmental Biology

  
  Rapid and Efficient Spatiotemporal Monitoring of Normal and Aberrant Cytosine Methylation within Intact Zebrafish Embryos Sarah Avila-Barnard¹, David C. Volz^{1 1}Department of Environmental Sciences, University of California, Riverside This paper describes a protocol for the rapid and efficient spatiotemporal monitoring of normal and aberrant cytosine methylation within intact zebrafish embryos.

• Biochemistry

  
  Capturing Actively Produced Microbial Volatile Organic Compounds from Human-Associated Samples with Vacuum-Assisted Sorbent Extraction Joann Phan¹, Joseph Kapcia III¹, Cynthia I. Rodriguez², Victoria L. Vogel³, Daniel B Cardin³, Sage J. B. Dunham³, Katrine Whiteson^{1 1}Department of Molecular Biology and Biochemistry, University of California Irvine, ²Department of Ecology and Evolutionary Biology, University of California Irvine, ³Entech Instruments Inc. This protocol describes the extraction of volatile organic compounds from a biological sample with the vacuum-assisted sorbent extraction method, gas chromatography coupled with mass spectrometry using the Entech Sample Preparation Rail, and data analysis. It also describes culture of biological samples and stable isotope probing.

• Behavior

  
  Large-Scale Gravitaxis Assay of Caenorhabditis Dauer Larvae Caroline Ackley¹, Lindsey Washiashi¹, Ruchira Krishnamurthy¹, Joel H. Rothman^{1 1}Molecular Cellular and Developmental Biology, University of California The present protocol outlines methods for conducting a large-scale gravitaxis assay with Caenorhabditis dauer larvae. This protocol allows for better detection of gravitaxis behavior compared with a plate-based assay.

• Immunology and Infection

  
  Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria Sydney R. Morrill^1,2,3, Kavita Agarwal^2,3, Sudeshna Saha^2,3, Warren G. Lewis^2,3, Nicole M. Gilbert⁴, Amanda L. Lewis^{2,3 1}Division of Biology and Biomedical Sciences, Washington University School of Medicine, ²Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, ³Glycobiology Research and Training Center, UCSD, ⁴Department of Pediatrics, Washington University School of Medicine The protocol presents a mouse model of vaginal colonization with anaerobically cultured human vaginal bacteria. We focus on Gardnerella vaginalis, while including suggestions for Prevotella bivia and Fusobacterium nucleatum. This protocol can also be used as a guide for vaginal inoculations and viable recovery of other anaerobically grown bacteria.

• Biochemistry

  
  Quantifying the Binding Interactions Between Cu(II) and Peptide Residues in the Presence and Absence of Chromophores Sohee Choi¹, Jessica A. San Juan², Marie C. Heffern², Michael J. Stevenson^{1 1}Department of Chemistry, University of San Francisco, ²Department of Chemistry, University of California, Davis This article focuses on the use of electronic absorption spectroscopy and isothermal titration calorimetry to probe and quantify the thermodynamics of Cu(II) binding to peptides and proteins.

• Biochemistry

  
  Manual Blot-and-Plunge Freezing of Biological Specimens for Single-Particle Cryogenic Electron Microscopy Hoang P. M. Nguyen*¹, Kelly L. McGuire*¹, Brian D. Cook*¹, Mark A. Herzik, Jr.^{1 1}Department of Chemistry and Biochemistry, University of California, San Diego This manuscript outlines the blot-and-plunge method to manually freeze biological specimens for single-particle cryogenic electron microscopy.

• Bioengineering

  
  All-optical Mechanobiology Interrogation of Yes-associated Protein in Human Cancer and Normal Cells using a Multi-functional System Qin Luo*¹, Miao Huang*², Chenyu Liang², Justin Zhang³, Gaoming Lin¹, Sydney Yu¹, Mai Tanaka^4,5, Sharon Lepler^4,5, Juan Guan^5,6,7, Dietmar Siemann^4,5, Xin Tang^{2,5 1}Department of Electrical and Computer Engineering, Herbert Wertheim College of Engineering, University of Florida, ²Department of Mechanical and Aerospace Engineering, Herbert Wertheim College of Engineering, University of Florida, ³Department of Electrical and Computer Engineering, University of California, ⁴Department of Radiation Oncology, College of Medicine, University of Florida, ⁵UF Health Cancer Center, University of Florida, ⁶Department of Physics, College of Liberal Arts and Sciences, University of Florida, ⁷Department of Anatomy and Cell Biology, College of Medicine, University of Florida This paper presents a detailed stepwise protocol on how to utilize an integrated multi-functional and user-programmable system that enables automatic multi-channel imaging and mechanobiological analysis to elucidate the mechano-sensitivity of Yes-associated protein (YAP).

• Developmental Biology

  
  Microdissection and Dissociation of the Murine Oviduct: Individual Segment Identification and Single Cell Isolation Kelly C. Radecki¹, Mary Y. Lorenson¹, David G. Carter², Ameae M. Walker^{1 1}Division of Biomedical Sciences, School of Medicine, University of California, Riverside, ²Microscopy and Imaging Core Facility, University of California, Riverside A method for microdissection of the mouse oviduct that allows collection of the individual segments while maintaining RNA integrity is presented. In addition, non-enzymatic oviductal cell dissociation procedure is described. The methods are appropriate for subsequent gene and protein analysis of the functionally different oviductal segments and dissociated oviductal cells.

• Biology

  
  Rapid, Affordable, and Uncomplicated Production of Bacterial Cell-free Lysate Robert M. Cooper*¹, Taishi Tonooka*^1,2, Andriy Didovyk*^1,3, Jeff Hasty^{1,4,5 1}Biocircuits Institute, University of California, San Diego, ²Present address, Kyoto Institute of Technology, ³Present address, Vertex Pharmaceuticals, ⁴Department of Bioengineering, University of California, San Diego, ⁵Molecular Biology Section, University of California, San Diego This protocol describes a rapid and simple method to produce bacterial lysate for cell-free gene expression, using an engineered strain of Escherichia coli and requiring only standard laboratory equipment.

• Immunology and Infection

  
  Generating Transgenics and Knockouts in Strongyloides Species by Microinjection Michelle L. Castelletto¹, Elissa A. Hallem^{1,2 1}Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, ²Molecular Biology Institute, University of California, Los Angeles The functional genomic toolkit for the parasitic nematodes Strongyloides stercoralis and Strongyloides ratti includes transgenesis, CRISPR/Cas9-mediated mutagenesis, and RNAi. This protocol will demonstrate how to use intragonadal microinjection to introduce transgenes and CRISPR components into S. stercoralis and S. ratti.

• Developmental Biology

  
  Light-Induced GFP Expression in Zebrafish Embryos using the Optogenetic TAEL/C120 System Jesselynn LaBelle¹, Stephanie Woo^{1 1}Department of Molecular Cell Biology, University of California Optogenetics is a powerful tool with wide-ranging applications. This protocol demonstrates how to achieve light-inducible gene expression in zebrafish embryos using the blue light-responsive TAEL/C120 system.

• Medicine

  
  Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome Jacob Vieira¹, Tara Gallagher², Hui-Yu Sui¹, Sirus Jesudasen³, Katrine Whiteson², George A. O'Toole⁴, Kurt Hanselmann⁵, Peggy S. Lai^{1 1}Division of Pulmonary and Critical Care Medicine, Massachusetts General Hospital, ²Department of Molecular Biology & Biochemistry, University of California, Irvine, ³Department of Medicine, Massachusetts General Hospital, ⁴Department of Microbiology & Immunology, Geisel School of Medicine at Dartmouth, ⁵Swiss i-research and teaching institute The goal of this protocol is to develop a model system for the effect of hyperoxia on cystic fibrosis airway microbial communities. Artificial sputum medium emulates the composition of sputum, and hyperoxic culture conditions model the effects of supplemental oxygen on lung microbial communities.

• Neuroscience

  
  A Behavioral Screen for Heat-Induced Seizures in Mouse Models of Epilepsy Antara Das¹, Martin A. Smith², Diane K. O'Dowd^{1 1}Department of Developmental and Cell Biology, University of California, ²Department of Anatomy and Neurobiology, University of California The goal of the method is to screen for hyperthermia or heat-induced seizures in mouse models. The protocol describes the use of a custom-built chamber with continuous monitoring of the body temperature to determine whether elevated body temperature leads to seizures.

• Chemistry

  
  Gold Nanoparticle Synthesis Jonathan Marrs¹, Taher Ghomian², Lucas Domulevicz¹, Cliff McCold³, Joshua Hihath^{1 1}Department of Electrical and Computer Engineering, University of California, Davis, ²Department of Computer Sciences and Electrical Engineering, Marshall University, ³Department of Materials Science and Engineering, University of California, Davis A protocol for synthesizing ~12 nm diameter gold nanoparticles (Au nanoparticles) in an organic solvent is presented. The gold nanoparticles are capped with oleylamine ligands to prevent agglomeration. The gold nanoparticles are soluble in organic solvents such as toluene.

• Genetics

  
  Microinjection Method for Anopheles gambiae Embryos Rebeca Carballar-Lejarazú¹, Taylor Tushar¹, Thai Binh Pham², Anthony A. James^{1,2 1}Department of Microbiology & Molecular Genetics, University of California, Irvine, ²Department of Molecular Biology & Biochemistry, University of California, Irvine Microinjection techniques are essential to introduce exogenous genes into the genomes of mosquitoes. This protocol explains a method used by the James laboratory to microinject DNA constructs into Anopheles gambiae embryos to generate transformed mosquitoes.

• Genetics

  
  Digital-Droplet PCR to Detect Indels Mutations in Genetically Modified Anopheline Mosquito Populations Rebeca Carballar-Lejarazú¹, Thai Binh Pham², Adam Kelsey¹, Taylor Tushar¹, Anthony A. James^{1,2 1}Department of Microbiology & Molecular Genetics, University of California, Irvine, ²Department of Molecular Biology & Biochemistry, University of California, Irvine This protocol provides the steps from DNA extraction to experimental set-up for digital droplet PCR (ddPCR), including analysis for the identification and quantification of non-homologous end-joining (NHEJ) events at target sites following gRNA-induced Cas9 cleavage and DNA repair. Other uses of this method include applications such as polymorphism detection and gene-editing variant verification.

• Genetics

  
  Small-Cage Laboratory Trials of Genetically-Engineered Anopheline Mosquitoes Rebeca Carballar-Lejarazú¹, Thai Binh Pham², Vanessa Bottino-Rojas¹, Adriana Adolfi^1,3, Anthony A. James^{1,2 1}Department of Microbiology & Molecular Genetics, University of California, Irvine, ²Department of Molecular Biology & Biochemistry, University of California, Irvine, ³Vector Biology Department, Liverpool School of Tropical Medicine The protocols reported here illustrate three alternative ways to assess the performance of genetically-engineered mosquitoes destined for vector control in laboratory-contained small cage trials. Each protocol is tailored to the specific modification the mosquito strain bears (gene drive or non-gene drive) and the types of parameters measured.

• Biology

  
  Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning Nathan Grainger^1,2, Kenton M. Sanders¹, Bernard T. Drumm^{1,3 1}Department of Physiology & Cell Biology, University of Nevada, Reno School of Medicine, ²Department of Physiology & Membrane Biology, University of California School of Medicine, ³Department of Life & Health Sciences, Dundalk Institute of Technology The goal of this protocol is to isolate intact pacemaker regions of the mouse renal pelvis using vibratome sectioning. These sections can then be used for in situ Ca²⁺ imaging to elucidate Ca²⁺ transient properties of pacemaker cells and other interstitial cells in vibratome slices.

• Biochemistry

  
  Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers Mowei Zhou¹, Shadan H. Abdali¹, David Dilworth², Lifeng Liu², Benjamin Cole², Neha Malhan¹, Amir H. Ahkami¹, Tanya E. Winkler¹, Joy Hollingsworth³, Julie Sievert³, Jeff Dahlberg³, Robert Hutmacher^4,5, Mary Madera⁶, Judith A. Owiti⁶, Kim K. Hixson¹, Peggy G. Lemaux⁶, Christer Jansson¹, Ljiljana Paša-Tolić^{1 1}Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, ²DOE-Joint Genome Institute, Lawrence Berkeley Laboratory, ³Kearney Agricultural Research and Extension Center, University of California Agriculture and Natural Resources, ⁴West Side Research and Extension Center, University of California, ⁵Department of Plant Sciences, University of California, Davis, ⁶Department of Plant and Microbial Biology, University of California, Berkeley The protocol has been developed to effectively extract intact histones from sorghum leaf materials for profiling of histone post-translational modifications that can serve as potential epigenetic markers to aid engineering drought resistant crops.

• Developmental Biology

  
  Creating Avian Forebrain Chimeras to Assess Facial Development Diane Hu¹, Ralph S. Marcucio^{1 1}Department of Orthopaedic Surgery, Orthopaedic Trauma Institute, University of California This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of basal forebrain during craniofacial development.

• Bioengineering

  
  Generation of Self-assembled Vascularized Human Skin Equivalents Martina M. Sanchez¹, Joshua T. Morgan^{1 1}Departments of Bioengineering, University of California The goal of this protocol is to describe the generation and volumetric analysis of vascularized human skin equivalents using accessible and simple techniques for long term culture. To the extent possible, the rationale for steps is described to allow researchers the ability to customize based on their research needs.

• Immunology and Infection

  
  Ubiquitous and Tissue-specific RNA Targeting in Drosophila Melanogaster using CRISPR/CasRx Ruichen Sun¹, Daniel Brogan¹, Anna Buchman¹, Ting Yang¹, Omar S. Akbari^{1 1}Division of Biological Sciences, Section of Cell and Developmental Biology, University of California This article outlines a detailed protocol for using the RNA-targeting Cas13D enzyme (RfxCas13D) in flies.

• Genetics

  
  Site-Directed φC31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria Adriana Adolfi^1,2, Amy Lynd¹, Gareth J. Lycett¹, Anthony A. James^{2,3 1}Vector Biology Department, Liverpool School of Tropical Medicine, ²Department of Microbiology & Molecular Genetics, University of California, ³Department of Molecular Biology & Biochemistry, University of California The protocol describes how to achieve site-directed modifications in the genome of Anopheles malaria mosquitoes using the φC31 system. Modifications described include both the integration and the exchange of transgenic cassettes in the genome of attP-bearing docking lines.

• 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.

• Medicine

  
  Implantation of an Isoproterenol Mini-Pump to Induce Heart Failure in Mice Shuxun Ren¹, Sunny Chang², Alex Tran³, Arianna Mandelli⁴, Yibin Wang¹, Jessica J. Wang^{2 1}Department of Anesthesiology, University of California, ²Department of Medicine, University of California, ³Department of Microbiology, Immunology & Molecular Genetics, University of California, ⁴Department of Molecular, Cell, and Developmental Biology, University of California The chronic administration of isoproterenol via an implanted osmotic pump has been used widely to mimic advanced heart failure in mice. Here, we describe detailed methods in surgical mini-pump implantation for the continuous isoproterenol administration over 3 weeks, as well as, echocardiographic assessment for the successful model creation.

• Bioengineering

  
  Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles Gregory Michael Newkirk*^1,2, Honghong Wu*¹, Israel Santana¹, Juan Pablo Giraldo^{1,2 1}Department of Botany and Plant Sciences, University of California, ²Department of Microbiology and Plant Pathology, University of California Here, we present a protocol for the synthesis and characterization of cerium oxide nanoparticles (nanoceria) for ROS (reactive oxygen species) scavenging in vivo, nanoceria imaging in plant tissues by confocal microscopy, and in vivo monitoring of nanoceria ROS scavenging by confocal microscopy.

• Bioengineering

  
  A Rapid Image-based Bacterial Virulence Assay Using Amoeba Kumar Perinbam¹, Albert Siryaporn^{1,2 1}Department of Physics and Astronomy, University of California, ²Department of Molecular Biology and Biochemistry, University of California Here, we present a protocol to measure the virulence of planktonic or surface-attached bacteria using D. discoideum (amoeba) as a host. Virulence is measured over a period of 1 h and host killing is quantified using fluorescence microscopy and image analysis. We demonstrate this protocol using the bacterium P. aeruginosa.

• Genetics

  
  Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms Behnom Farboud*^1,2, Erin Jarvis*¹, Theodore L. Roth*^3,4,5,6, Jiyung Shin*^1,3, Jacob E. Corn^1,3, Alexander Marson^3,5,6,7,8,9, Barbara J. Meyer^1,2, Nipam H. Patel^1,10, Megan L. Hochstrasser^{3 1}Department of Molecular Cell Biology, University of California, Berkeley, ²Howard Hughes Medical Institute, University of California, Berkeley, ³Innovative Genomics Institute, University of California, Berkeley, ⁴Biomedical Sciences Graduate Program, University of California, San Francisco, ⁵Department of Microbiology and Immunology, University of California, San Francisco, ⁶Diabetes Center, University of California, San Francisco, ⁷Chan Zuckerberg Biohub, ⁸Department of Medicine, University of California, San Francisco, ⁹UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, ¹⁰Department of Integrative Biology, University of California, Berkeley Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

• Genetics

  
  Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus Ira L. Blitz^{1 1}Department of Developmental and Cell Biology, University of California, Irvine Genes essential for survival pose technical hurdles for creating mutant lines. Leapfrogging circumvents lethality by combining genome editing with primordial germ cell transplantation to create wild-type animals carrying germline mutations. Leapfrogging also permits the efficient generation of homozygous null mutants in the F1 generation. Here, the transplantation step is demonstrated.

• 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

  
  Generalized Psychophysiological Interaction (PPI) Analysis of Memory Related Connectivity in Individuals at Genetic Risk for Alzheimer's Disease Theresa M. Harrison¹, Donald G. McLaren², Teena D. Moody¹, Jamie D. Feusner¹, Susan Y. Bookheimer^{1 1}Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, ²Biospective, Inc. This manuscript describes how to implement a psychophysiological interaction analysis to reveal task-dependent changes in functional connectivity between a selected seed region and voxels in other regions of the brain. Psychophysiological interaction analysis is a popular method to examine task effects on brain connectivity, distinct from traditional univariate activation effects.

• Immunology and Infection

  
  Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein Shubhendu Sen Roy¹, Huy Cong Xuan Nguyen¹, Thomas A. Angelovich^2,3, Anna C. Hearps², Diana Huynh^1,4, Anthony Jaworowski^2,4, Theodoros Kelesidis^{1 1}University of California, Los Angeles, ²Centre for Biomedical Research, Burnet Institute, ³School of Health and Biomedical Sciences, RMIT University, ⁴Department of Infectious Diseases, Monash University We describe here a fluorometric cell-free biochemical assay for determination of HDL-lipid peroxidation. This rapid and reproducible assay can be used to determine HDL function in large scale studies and can contribute to our understanding of HDL function in human disease.

• Developmental Biology

  
  Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue Sumitra Tatapudy¹, Marimar Benitez¹, Todd Nystul^{1 1}Departments of Anatomy and OB/GYN-RS, University of California, San Francisco We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values.

• Immunology and Infection

  
  Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4 Sharon A. Sagan*^1,2, Andrés Cruz-Herranz*¹, Collin M. Spencer^1,2, Peggy P. Ho³, Lawrence Steinman³, Ari J. Green¹, Raymond A. Sobel⁴, Scott S. Zamvil^{1,2 1}Department of Neurology, University of California, ²Program in Immunology, University of California, ³Department of Neurology and Neurological Sciences, Stanford University, ⁴Department of Pathology, Stanford University Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4^-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

• 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.

• Behavior

  
  Examining Recall Memory in Infancy and Early Childhood Using the Elicited Imitation Paradigm Angela F. Lukowski¹, Helen M. Milojevich^{1 1}Department of Psychology and Social Behavior, University of California-Irvine The elicited imitation procedure was established to examine the development of recall memory in infancy and early childhood. This procedure has been widely used to establish a solid foundation of the nature of recall memory in infancy and early childhood.

• Neuroscience

  
  Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons Ji-Yong Kang¹, Daichi Kawaguchi², Lei Wang^{3 1}Department of Neuroscience, School of Medicine, Tufts University, ²Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, ³Department of Pharmaceutical Chemistry and the Cardiovascular Research Institute, University of California, San Francisco Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

• Medicine

  
  Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment Renske J.E. van den Bijgaart*¹, Niwen Kong*¹, Carrie Maynard^1,2, Vicki Plaks^{1,3 1}Department of Anatomy, University of California, ²Hubrecht Institute-Royal Dutch Academy of Science and University Medical Center Utrecht, ³Department of Cell and Developmental Biology, Sackler Faculty of Medicine We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

• Medicine

  
  Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy Yan Wang^1,3,4, Dale P. Brown¹, Brant D. Watson², Jeffrey L. Goldberg^{1,3 1}Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, ²Departments of Neurology and Biomedical Engineering, University of Miami Miller School of Medicine, ³Shiley Eye Center, University of California, ⁴Department of Ophthalmology and Vision Science, Fudan University The goal of this protocol is to photochemically induce ischemic injury to the posterior optic nerve in rat. This model is critical to studies of the pathophysiology of posterior ischemic optic neuropathy, and therapeutic approaches for this and other optic neuropathies, as well as of other CNS ischemic diseases.

• Developmental Biology

  
  Mechanical Vessel Injury in Zebrafish Embryos Hilary Clay¹, Shaun R. Coughlin^{1 1}Cardiovascular Research Institute, University of California This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

• Immunology and Infection

  
  Real-time Imaging of Myeloid Cells Dynamics in Apc^Min/+ Intestinal Tumors by Spinning Disk Confocal Microscopy Caroline Bonnans*^1,2, Marja Lohela*², Zena Werb^{2 1}Department of Oncology, INSERM U661, Functional Genomic Institute, ²Department of Anatomy, University of California By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the Apc^Min/+ colorectal cancer model.

• Medicine

  
  Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice: A Model for Immunotherapy Development Daniel P. Vang¹, Gregory T. Wurz¹, Stephen M. Griffey², Chiao-Jung Kao¹, Audrey M. Gutierrez¹, Gregory K. Hanson¹, Michael Wolf³, Michael W. DeGregorio^{1 1}Department of Internal Medicine, Division of Hematology and Oncology, University of California, Davis, ²Comparative Pathology Laboratory, UC Davis School of Veterinary Medicine, University of California, Davis, ³Merck Serono Research, Merck KGaA, Darmstadt, Germany An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

• Biology

  
  Quantifying Agonist Activity at G Protein-coupled Receptors Frederick J. Ehlert¹, Hinako Suga², Michael T. Griffin^{3 1}Department of Pharmacology, University of California, Irvine, ²Department of Pharmacology, University of California, ³Schmid College of Science, Chapman University A method for estimating the affinity constant of an agonist for the active state (Kb) of a G protein-coupled receptor is described. The analysis provides absolute or relative measures of Kb depending on whether constitutive receptor activation is measurable. Our method applies to various responses downstream from receptor activation.

• Medicine

  
  Teratoma Generation in the Testis Capsule Suzanne E. Peterson¹, Ha T. Tran¹, Ibon Garitaonandia¹, Sangyoon Han¹, Kyle S. Nickey¹, Trevor Leonardo², Louise C. Laurent³, Jeanne F. Loring^{1 1}Department of Chemical Physiology, Scripps Research Institute, ²Department of Chemical Physiology, Scripps Research Institute, ³Department of Reproductive Medicine, University of California Human pluripotent stem cells (hPSCs) have the potential to treat a myriad of different diseases. The utility of these cells lies in the fact that they can differentiate into any cell type in the body. Here we describe the teratoma assay, which is used to demonstrate the pluripotence of hPSCs.

• Biology

  
  Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA Srikumar Sengupta¹, Jennifer M. Bolin¹, Victor Ruotti¹, Bao Kim Nguyen¹, James A. Thomson^1,2,3, Angela L. Elwell¹, Ron Stewart^{1 1}Regenerative Biology, Morgridge Institute for Research, ²Department of Cell & Regenerative Biology, University of Wisconsin, ³Department of Molecular, Cellular, & Regenerative Biology, University of California Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.

• Bioengineering

  
  Adaptation of a Haptic Robot in a 3T fMRI Joseph Snider¹, Markus Plank¹, Larry May², Thomas T. Liu², Howard Poizner^{3 1}Institute for Neural Computation, University of California, ²Department of Radiology, University of California, ³Department of Cognitive Science and Program in Neurosciences, University of California The adaptation and use of a haptic robot in a 3T fMRI is described.

• Neuroscience

  
  Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence Sava Sakadžić¹, Emmanuel Roussakis², Mohammad A. Yaseen¹, Emiri T. Mandeville³, Vivek J. Srinivasan¹, Ken Arai³, Svetlana Ruvinskaya¹, Weicheng Wu¹, Anna Devor^1,4, Eng H. Lo³, Sergei A. Vinogradov², David A. Boas^{1 1}Optics Division, Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital and Harvard Medical School, ²Department of Biochemistry and Biophysics, University of Pennsylvania, ³Neuroprotection Research Laboratory, Departments of Radiology and Neurology, Massachusetts General Hospital and Harvard Medical School, ⁴Departments of Neurosciences and Radiology, University of California We present an experimental procedure for measuring the partial pressure of oxygen (pO2) in cerebral vasculature based on oxygen-dependent quenching of phosphorescence. Animal preparation and imaging procedures were outlined for both large field of view CCD-based imaging of pO2 in rats and 2-photon excitation based imaging of pO2 in mice.

• Immunology and Infection

  
  Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry: a Novel Use for a Cell Purification Method Hal X. Nguyen^1,2,3,4, Kevin D. Beck⁵, Aileen J. Anderson^{1,2,3,4,6 1}Institute for Memory Impairments and Neurological Disorders, University of California, ²Physical Medicine & Rehabilitation, University of California, ³Anatomy & Neurobiology, University of California, ⁴Sue and Bill Gross Stem Cell Research Center, University of California, ⁵Section of Molecular Biology, University of California, ⁶Reeve-Irvine Research Center, University of California Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

• Medicine

  
  A Mouse Model of in Utero Transplantation Amar Nijagal^1,2, Tom Le^1,2, Marta Wegorzewska^1,2,3, Tippi C. MacKenzie^{1,2,3 1}Department of Surgery, University of California, ²Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, ³Biomedical Sciences Program, University of California The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

• Biology

  
  Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth Sabrina Lin^1,2,3, Shawn Fonteno¹, Shruthi Satish^1,3, Bir Bhanu⁴, Prue Talbot^{1,2 1}UCR Stem Cell Center, University of California, ²Department of Cell Biology and Neuroscience, University of California, ³Cell, Molecular, and Developmental Biology Graduate Program, University of California, ⁴Center for Research in Intelligent Systems, University of California Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.