University of California, Berkeley

42 articles published in JoVE

• Biology

  
  Isolation of Retinal Pigment Epithelial Cells from Guinea Pig Eyes So Goto^1,2,3, Michael Frost⁴, Christine Wildsoet^{1 1}Herbert Wertheim School Optometry and Vision Science, University of California, Berkeley, ²Department of Ophthalmology, Osaka University Graduate School of Medicine, ³Department of Ophthalmology, Tokyo Medical Center, National Hospital Organization, ⁴Department of Optometry and Vision Science, School of Optometry, University of Alabama at Birmingham We describe a simple and efficient method for isolating cells of the retinal pigment epithelium (RPE) cells from the eyes of young pigmented guinea pigs. This procedure allows for follow-up molecular biology studies on the isolated RPE, including gene expression analyses.

• Environment

  
  Visualizing Field Data Collection Procedures of Exposure and Biomarker Assessments for the Household Air Pollution Intervention Network Trial in India Karthikeyan D. Rajamani¹, Sankar Sambandam¹, Krishnendu Mukhopadhyay¹, Naveen Puttaswamy¹, Gurusamy Thangavel¹, Durairaj Natesan¹, Rengaraj Ramasamy¹, Saritha Sendhil¹, Amudha Natarajan¹, Vigneswari Aravindalochan¹, Ajay Pillarisetti², Michael Johnson³, Joshua Rosenthal*⁴, Kyle Steenland⁵, Ricardo Piedhrahita³, Jennifer Peel⁶, Maggie L. Clark⁶, Dana Boyd Barr⁵, Sarah Rajkumar⁶, Bonnie Young⁶, Shirin Jabbarzadeh⁷, Ghislaine Rosa⁸, Miles Kirby⁹, Lindsay J. Underhill¹⁰, Anaite Diaz-Artiga¹¹, Amy Lovvorn⁵, William Checkley¹², Thomas Clasen⁵, Kalpana Balakrishnan^{1 1}Department of Environmental Health Engineering, ICMR Center for Advanced Research on Air Quality, Climate and Health, Faculty of Public Health, Sri Ramachandra Institute of Higher Education and Research (Deemed University), ²Division of Environmental Health Sciences, University of California, Berkeley, ³Berkeley Air Monitoring Group, ⁴Division of International Epidemiology and Population Studies, National Institutes of Health, ⁵Gangarosa Department of Environmental Health, Rollins School of Public Health, Emory University, ⁶Department of Environmental and Radiological Health Sciences, Colorado State University, ⁷Department of Biostatistics and Informatics, Rollins School of Public Health, Emory University, ⁸Department of Disease Control, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, ⁹Department of Global Health & Population, Harvard, T.H. Chan School of Public Health, ¹⁰Cardiovascular Division, Washington University School of Medicine, Washington University, ¹¹Centro de Estudios en Salud, Universidad del Valle de Guatemala, ¹²Division of Pulmonary and Critical Care, School of Medicine, Johns Hopkins University We detail the consistent, high-quality procedures used throughout air and biological sampling processes at Indian field sites during a large randomized controlled trial. Insights gathered from the oversight of applications of innovative technologies, adapted for exposure assessment in rural regions, enable better field data collection practices with more reliable outcomes.

• Biology

  
  Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans Kenneth Trang¹, Rahul Bodkhe¹, Michael Shapira^{1 1}Department of Integrative Biology, University of California, Berkeley Compost microcosms bring the microbial diversity found in nature into the laboratory to facilitate microbiome research in Caenorhabditis elegans. Provided here are protocols for setting up microcosm experiments, with the experiments demonstrating the ability to modulate environmental microbial diversity to explore the relationships between environmental microbial diversity and worm gut microbiome composition.

• Biology

  
  Methods for Rearing the Parasitoid Ganaspis brasiliensis, a Promising Biological Control Agent for the Invasive Drosophila suzukii Marco Valerio Rossi-Stacconi¹, Xingeng Wang², Amanda Stout², Lorenzo Fellin^1,3, Kent M. Daane⁴, Antonio Biondi⁵, Judith M. Stahl⁴, Matthew L. Buffington⁶, Gianfranco Anfora^1,3, Kim A. Hoelmer^{2 1}Research and Innovation Centre, Fondazione Edmund Mach, ²Beneficial Insects Introduction Research Unit, Agricultural Research Service, United States Department of Agriculture, ³Center for Agriculture, Food and Environment, University of Trento, ⁴Department of Environmental Science, Policy and Management, University of California Berkeley, ⁵Department of Agriculture, Food and Environment, University of Catania, ⁶Systematic Entomology Laboratory, Agricultural Research Service, United States Department of Agriculture Ganaspis brasiliensis-a larval parasitoid of Drosophila suzukii (a global invasive fruit crop pest)-has been approved or is considered for introduction into Europe and the United States for biological control of this pest. This article provides protocols for both small-scale and large-scale rearing of this parasitoid.

• 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.

• Bioengineering

  
  Simple, Affordable, and Modular Patterning of Cells using DNA Katelyn A. Cabral¹, David M. Patterson², Olivia J. Scheideler¹, Russell Cole³, Adam R. Abate^4,5,6, David V. Schaffer^7,8, Lydia L. Sohn⁹, Zev J. Gartner^{2,6,10 1}Graduate Program in Bioengineering, University of California San Francisco and University of California Berkeley, ²Department of Pharmaceutical Chemistry, University of California San Francisco, ³Scribe Biosciences, ⁴Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, ⁵California Institute for Quantitative Biosciences, University of California San Francisco, ⁶Chan Zuckerberg Biohub, University of California San Francisco, ⁷Department of Chemical & Biomolecular Engineering, University of California Berkeley, ⁸Helen Wills Neuroscience Institute, University of California Berkeley, ⁹Department of Mechanical Engineering, University of California Berkeley, ¹⁰Center for Cellular Construction, University of California San Francisco Here we present a protocol to micropattern cells at single-cell resolution using DNA-programmed adhesion. This protocol uses a benchtop photolithography platform to create patterns of DNA oligonucleotides on a glass slide and then labels cell membranes with commercially available complementary oligonucleotides. Hybridization of the oligos results in programmed cell adhesion.

• Biology

  
  Measurements of Physiological Stress Responses in C. Elegans Raz Bar-Ziv*¹, Ashley E. Frakes*¹, Ryo Higuchi-Sanabria*¹, Theodore Bolas¹, Phillip A. Frankino¹, Holly K. Gildea¹, Melissa G. Metcalf¹, Andrew Dillin^{1 1}Department of Molecular and Cell Biology, University of California, Berkeley Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

• Bioengineering

  
  High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay Jana A. Hassan¹, Ilea J. Chau-Ly¹, Jennifer D. Lewis^{1,2 1}Department of Plant and Microbial Biology, University of California, Berkeley, ²Plant Gene Expression Center, United States Department of Agriculture The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

• Bioengineering

  
  Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics Jonathon M. Muncie^1,2, Roberto Falcón-Banchs¹, Johnathon N. Lakins², Lydia L. Sohn^1,3, Valerie M. Weaver^{2,4,5,6 1}Graduate Program in Bioengineering, University of California San Francisco and University of California Berkeley, ²Center for Bioengineering and Tissue Regeneration, Department of Surgery, University of California San Francisco, ³Department of Mechanical Engineering, University of California Berkeley, ⁴Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, ⁵UCSF Comprehensive Cancer Center, Helen Diller Family Cancer Research Center, University of California San Francisco, ⁶Department of Anatomy, Department of Bioengineering and Therapeutic Sciences, and Department of Radiation Oncology, University of California San Francisco Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

• Genetics

  
  Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis Carly V. Weiss^1,2, Julie N. Chuong³, Rachel B. Brem^{1,3 1}Department of Plant and Microbial Biology, University of California Berkeley, ²Department of Biology, Stanford University, ³Buck Institute for Research on Aging Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait.

• Genetics

  
  Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis Jingxun Chen¹, David McSwiggen², Elçin Ünal^{1 1}Department of Molecular and Cell Biology, Barker Hall, University of California, Berkeley, ²Department of Molecular and Cell Biology, Li Ka Shing Center, University of California, Berkeley This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

• 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.

• Chemistry

  
  Using Graphene Liquid Cell Transmission Electron Microscopy to Study in Situ Nanocrystal Etching Matthew R. Hauwiller¹, Justin C. Ondry¹, A. Paul Alivisatos^{1,2,3,4 1}Department of Chemistry, University of California-Berkeley, ²Department of Material Science and Engineering, University of California-Berkeley, ³Kavli Energy NanoScience Institute, ⁴Materials Sciences Division, Lawrence Berkeley National Laboratory Graphene liquid cell electron microscopy can be used to observe nanocrystal dynamics in a liquid environment with greater spatial resolution than other liquid cell electron microscopy techniques. Etching premade nanocrystals and following their shape using graphene liquid cell Transmission Electron Microscopy can yield important mechanistic information about nanoparticle transformations.

• Genetics

  
  Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere Tuesday Simmons¹, Daniel F. Caddell¹, Siwen Deng¹, Devin Coleman-Derr^{1,2 1}Department of Plant and Microbial Biology, University of California, Berkeley, ²Plant Gene Expression Center, USDA ARS Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

• Environment

  
  Ecosystem Fabrication (EcoFAB) Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions Jian Gao^1,2, Joelle Sasse^1,2, Kyle M. Lewald^1,2, Kateryna Zhalnina^1,2, Lloyd T. Cornmesser^1,2, Todd A. Duncombe³, Yasuo Yoshikuni², John P. Vogel², Mary K. Firestone⁴, Trent R. Northen^{1,2 1}Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, ²Joint Genome Institute, Department of Energy, ³Joint BioEnergy Institute, ⁴Department of Environmental Science Policy and Management, University of California This article describes detailed protocols for ecosystem fabrication of devices (EcoFABs) that enable the studies of plants and plant-microbe interactions in highly controlled laboratory conditions.

• Chemistry

  
  Low Pressure Vapor-assisted Solution Process for Tunable Band Gap Pinhole-free Methylammonium Lead Halide Perovskite Films Carolin M. Sutter-Fella*^1,2,3, Yanbo Li*^1,4, Nicola Cefarin^1,5,6, Aya Buckley^1,7, Quynh Phuong Ngo^8,9, Ali Javey^2,3, Ian D. Sharp¹, Francesca M. Toma^{1 1}Joint Center for Artificial Photosynthesis, Chemical Sciences Division, Lawrence Berkeley National Laboratory, ²Electrical Engineering and Computer Sciences, University of California, Berkeley, ³Materials Science Division, Lawrence Berkeley National Laboratory, ⁴Institute of Fundamental and Frontier Sciences, University of Electronic Science and Technology of China, ⁵Department of Physics, Graduate School of Nanotechnology, University of Trieste, ⁶TASC Laboratory, IOM-CNR - Istituto Officina dei Materiali, ⁷Department of Chemistry, University of California, Berkeley, ⁸Materials Science and Engineering, University of California, Berkeley, ⁹Joint Center for Artificial Photosynthesis, Lawrence Berkeley National Laboratory Here, we present a protocol for the synthesis of CH3NH3I and CH3NH3Br precursors and the subsequent formation of pinhole-free, continuous CH3NH3PbI3-xBrx thin films for the application in high efficiency solar cells and other optoelectronic devices.

• Developmental Biology

  
  A Modified Trier Social Stress Test for Vulnerable Mexican American Adolescents Megan M. Johnson¹, Julianna Deardorff¹, Kimberly Parra¹, Abbey Alkon², Brenda Eskenazi¹, Elizabeth Shirtcliff^{3 1}Center for Environmental Research and Children's Health (CERCH), Berkley School of Public Health, University of California, Berkeley, ²San Francisco (UCSF) School of Nursing, University of California, San Francisco, ³Human Development and Family Studies, Iowa State University Here, we present a protocol that provoked cortisol reactivity in a vulnerable adolescent Mexican American sample utilizing a modified version of the Trier Social Stress Test (TSST). Saliva samples were collected at baseline, 15, 30, and 45 min post-TSST onset. Future research could utilize this modified TSST with vulnerable youth.

• Genetics

  
  Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing Joanna M. Feehan*^1,2, Katherine E. Scheibel*¹, Salim Bourras³, William Underwood⁴, Beat Keller³, Shauna C. Somerville^{1 1}Department of Plant and Microbial Biology, University of California Berkeley, ²John Innes Centre, Norwich Research Park, ³Department of Plant and Microbial Biology, University of Zürich, ⁴USDA-ARS Sunflower and Plant Biology Research Unit Described here is a method for the extraction, purification, and quality control of genomic DNA from the obligate biotrophic fungal pathogen, powdery mildew, for use in long-read genome sequencing.

• Bioengineering

  
  Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes Jackson T. Del Bonis-O’Donnell¹, Abraham Beyene¹, Linda Chio¹, Gözde Demirer¹, Darwin Yang¹, Markita P. Landry^{1,2 1}Department of Chemical and Biomolecular Engineering, University of California Berkeley, ²California Institute for Quantitative Biosciences (QB3), University of California Berkeley We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

• Chemistry

  
  Combining Solid-state and Solution-based Techniques: Synthesis and Reactivity of Chalcogenidoplumbates(II or IV) Günther Thiele¹, Carsten Donsbach², Isabell Nußbruch², Stefanie Dehnen^{2 1}Department of Chemistry, University of California, Berkeley, ²Fachbereich Chemie, Philipps-Universität Marburg and Wissenschaftliches Zentrum für Materialwissenschaften The synthesis of chalcogenidoplumbates(II,IV) via the in situ reduction of nominal "PbCh2" (Ch = Chalcogen) and via a solid-state reaction and subsequent solvothermal reactions is presented. Additionally, reactivities of plumbate(II) solutions are portrayed, which yield the heaviest-known CO homolog known to date: the µ-PbSe ligand.

• Biology

  
  Visualizing Stromule Frequency with Fluorescence Microscopy Jacob O. Brunkard¹, Anne M. Runkel², Patricia Zambryski^{2 1}Plant Gene Expression Center, Agricultural Research Service, USDA, ²Department of Plant and Microbial Biology, University of California, Berkeley Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

• Chemistry

  
  Synthesis of a Water-soluble Metal–Organic Complex Array Purnandhu Bose¹, Pradip K. Sukul¹, Omar M. Yaghi^2,3,4, Kentaro Tashiro^{1 1}International Center for Materials Nanoarchitectonics, National Institute for Materials Science, ²Department of Chemistry, University of California–Berkeley, ³Materials Sciences Division, Lawrence Berkeley National Laboratory, ⁴Kavli Energy NanoSciences Institute at Berkeley, University of California–Berkeley A potential general method for the synthesis of water-soluble multimetallic peptidic arrays containing a predetermined sequence of metal centers is presented.

• Developmental Biology

  
  Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks Priscilla A. Erickson¹, Nicholas A. Ellis¹, Craig T. Miller^{1 1}Department of Molecular and Cell Biology, University of California, Berkeley Transgenic manipulations and genome editing are critical for functionally testing the roles of genes and cis-regulatory elements. Here a detailed microinjection protocol for the generation of genomic modifications (including Tol2-mediated fluorescent reporter transgene constructs, TALENs, and CRISPRs) is presented for the emergent model fish, the threespine stickleback.

• Biology

  
  Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton Nicholas A. Ellis¹, Craig T. Miller^{1 1}Department of Molecular and Cell Biology, University of California, Berkeley The branchial skeleton, including gill rakers, pharyngeal teeth, and branchial bones, serves as the primary site of food processing in most fish. Here we describe a protocol to dissect and flat-mount this internal skeleton in threespine sticklebacks. This method is also applicable to a variety of other fish species.

• Developmental Biology

  
  Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation John D. Blair¹, Helen S. Bateup¹, Dirk F. Hockemeyer^{1 1}Department of Molecular and Cell Biology, University of California, Berkeley Genome editing of human pluripotent stem cells (hPSCs) can be done quickly and efficiently. Presented here is a robust experimental procedure to genetically engineer hPSCs as exemplified by editing the AAVS1 safe harbor locus to express EGFP and introduce antibiotic resistance.

• Immunology and Infection

  
  Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells Meghan Zuck^1,2, Caroline Feng², Kevin Hybiske^{1 1}Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, ²Program in Infectious Diseases, School of Public Health, University of California at Berkeley A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

• Engineering

  
  Failure Analysis of Batteries Using Synchrotron-based Hard X-ray Microtomography Katherine J. Harry^1,2, Dilworth Y. Parkinson³, Nitash P. Balsara^{2,4,5 1}Department of Materials Science and Engineering, University of California Berkeley, ²Materials Science Division, Lawrence Berkeley National Laboratory, ³Advanced Light Source Division, Lawrence Berkeley National Laboratory, ⁴Department of Chemical and Biomolecular Engineering, University of California Berkeley, ⁵Environmental Energy Technology Division, Lawrence Berkeley National Laboratory Synchrotron-based hard X-ray microtomography is used to image the electrochemical growth of dendrites from a lithium metal electrode through a solid polymer electrolyte membrane.

• Engineering

  
  Fabrication of Gate-tunable Graphene Devices for Scanning Tunneling Microscopy Studies with Coulomb Impurities Han Sae Jung^1,2, Hsin-Zon Tsai¹, Dillon Wong¹, Chad Germany¹, Salman Kahn¹, Youngkyou Kim^1,3, Andrew S. Aikawa¹, Dhruv K. Desai¹, Griffin F. Rodgers¹, Aaron J. Bradley¹, Jairo Velasco Jr.¹, Kenji Watanabe⁴, Takashi Taniguchi⁴, Feng Wang^1,5,6, Alex Zettl^1,5,6, Michael F. Crommie^{1,5,6 1}Department of Physics, University of California at Berkeley, ²Department of Chemistry, University of California at Berkeley, ³Department of Chemical and Biomolecular Engineering, University of California at Berkeley, ⁴National Institute for Materials Science (Japan), ⁵Materials Sciences Division, Lawrence Berkeley National Laboratory, ⁶Kavli Energy NanoSciences Institute, University of California at Berkeley and Lawrence Berkeley National Laboratory This paper details the fabrication process of a gate-tunable graphene device, decorated with Coulomb impurities for scanning tunneling microscopy studies. Mapping the spatially dependent electronic structure of graphene in the presence of charged impurities unveils the unique behavior of its relativistic charge carriers in response to a local Coulomb potential.

• Bioengineering

  
  Production and Targeting of Monovalent Quantum Dots Daeha Seo*^1,2,3, Justin Farlow*^4,5,6, Kade Southard^1,4,7, Young-wook Jun^1,7, Zev J. Gartner^{4,5,6,7 1}Department of Otolaryngology, University of California, San Francisco, ²Department of Chemistry, University of California, Berkeley, ³Materials Science Division, Lawrence Berkeley National Laboratory, ⁴Department of Pharmaceutical Chemistry, University of California, San Francisco, ⁵Tetrad Graduate Program, University of California, San Francisco, ⁶Center for Systems and Synthetic Biology, University of California, San Francisco, ⁷Chemistry and Chemical Biology Graduate Program, University of California, San Francisco We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

• Immunology and Infection

  
  Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen Kwame Twumasi-Boateng¹, Maureen Berg¹, Michael Shapira^{1 1}Department of Integrative Biology, University of California, Berkeley The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage: sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.

• Bioengineering

  
  Lipid Bilayer Vesicle Generation Using Microfluidic Jetting Christopher W. Coyne¹, Karan Patel¹, Johanna Heureaux¹, Jeanne Stachowiak³, Daniel A. Fletcher^4,5, Allen P. Liu^{1,2 1}Department of Mechanical Engineering, University of Michigan, ²Department of Biomedical Engineering, University of Michigan, ³Department of Biomedical Engineering, Institute for Cellular and Molecular Biology, The University of Texas at Austin, ⁴Department of Bioengineering, University of California, Berkeley, ⁵Physical Biosciences Division, Lawrence Berkeley National Laboratory Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

• Chemistry

  
  Seeded Synthesis of CdSe/CdS Rod and Tetrapod Nanocrystals Karthish Manthiram*^1,4, Brandon J. Beberwyck*^2,4, Dmitri V. Talapin^5,6, A. Paul Alivisatos^{2,3,4 1}Department of Chemical Engineering, UC Berkeley, ²Department of Materials Science and Engineering, UC Berkeley, ³Department of Chemistry, UC Berkeley, ⁴Materials Sciences Division, Lawrence Berkeley National Laboratory, ⁵Department of Chemistry, University of Chicago, ⁶Center for Nanoscale Materials, Argonne National Laboratory A protocol for the seeded synthesis of rod-shaped and tetrapod-shaped multicomponent nanostructures consisting of CdS and CdSe is presented.

• Engineering

  
  Nanomoulding of Functional Materials, a Versatile Complementary Pattern Replication Method to Nanoimprinting Corsin Battaglia^1,2, Karin Söderström¹, Jordi Escarré¹, Franz-Josef Haug¹, Matthieu Despeisse¹, Christophe Ballif^{1 1}Institute of Microengineering (IMT), Photovoltaics and Thin Film Electronics Laboratory, Ecole Polytechnique Fédérale de Lausanne (EPFL), ²Department of Electrical Engineering and Computer Sciences, University of California, Berkeley We describe a nanomoulding technique which allows low-cost nanoscale patterning of functional materials, materials stacks and full devices. Nanomoulding can be performed on any nanoimprinting setup and can be applied to a wide range of materials and deposition processes.

• Biology

  
  Fabrication and Use of MicroEnvironment microArrays (MEArrays) Chun-Han Lin^1,2, Jonathan K. Lee¹, Mark A. LaBarge^{1 1}Life Science Division, Lawrence Berkeley National Laboratory, ²Department of Comparative Biochemistry, University of California, Berkeley A combinatorial functional screening method for gaining insights into the impacts of the molecular composition of microenvironments on cellular functions is described. The method takes advantage of existing microarray-based technologies to generate arrays of defined combinatorial microenvironments that support cell adhesion and functional analysis.

• Neuroscience

  
  Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat Elizabeth D. Kirby¹, Kelly Jensen², Ki A. Goosens³, Daniela Kaufer^{1,4 1}Helen Wills Neuroscience Institute, University of California Berkeley, ²Office of Laboratory Animal Care, University of California Berkeley, ³McGovern Institute for Brain Research & The Department of Brain and Cognitive Science, Massachusetts Institute of Technology, ⁴Integrative Biology Department, University of California Berkeley Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

• Biology

  
  Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay Zhiying Ji¹, Luoping Zhang^{1 1}Genes and Environment Laboratory, University of California, Berkeley A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

• Biology

  
  Label-free in situ Imaging of Lignification in Plant Cell Walls Martin Schmidt¹, Pradeep Perera¹, Adam M. Schwartzberg², Paul D. Adams³, P. James Schuck^{2 1}Energy Biosciences Institute, University of California, Berkeley, ²Molecular Foundry, Lawrence Berkeley National Laboratory, ³Physical Biosciences Division, Lawrence Berkeley National Laboratory A method based on confocal Raman microscopy is presented that affords label-free visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species.

• Biology

  
  OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides Markus Günl¹, Sascha Gille¹, Markus Pauly^{1,2 1}Energy Biosciences Institute, University of California, Berkeley, ²Department of Plant and Microbial Biology, University of California, Berkeley A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

• Biology

  
  Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila Iris C. Howlett¹, Mark A. Tanouye^{1,2 1}Department of Molecular and Cell Biology, University of California, Berkeley, ²Department of Environmental Science, Policy Management, University of California, Berkeley Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

• Biology

  
  In situ Imaging of the Mouse Thymus Using 2-Photon Microscopy Ena Ladi¹, Paul Herzmark¹, Ellen Robey^{1 1}Department of Molecular and Cell Biology, University of California, Berkeley We present step-by-step instructions for the generation of neonatal chimeras as well as the dissection and preparation of the thymus for ex vivo imaging by 2-Photon Microscopy.

• Biology

  
  Mechanical Stimulation of Stem Cells Using Cyclic Uniaxial Strain Kyle Kurpinski¹, Song Li^{1 1}Department of Bioengineering, University of California, Berkeley It is widely understood that mechanical forces in the body can influence cell differentiation and proliferation. Here we present a video protocol demonstrating the use of a custom-built bioreactor for delivering uniaxial cyclic tensile strain to stem cells cultured on flexible micropatterned substrates.

• Biology

  
  Ex vivo Mechanical Loading of Tendon Krishna Asundi¹, David Rempel^{2 1}Department of Bioengineering, University of California, Berkeley, ²Department of Bioengineering, University of California, Berkeley; Division of Occupational Medicine, University of California, San Francisco A new in vitro system for simultaneously loading four tendons in culture is described.