University of Washington View Institution's Website 81 articles published in JoVE Developmental Biology Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells Alexander Blümke1,2, Jessica Simon1, Elizabeth Leber1, Marta Scatena1, Cecilia M. Giachelli1 1Department of Bioengineering, Department of Medicine, University of Washington, 2Department of Orthopedics and Trauma Surgery, Medical Faculty Mannheim, Heidelberg University This protocol presents the differentiation of human osteoclasts from induced pluripotent stem cells (iPSCs) and describes methods for the characterization of osteoclasts and osteoclast precursors. Neuroscience Bringing the Clinic Home: An At-Home Multi-Modal Data Collection Ecosystem to Support Adaptive Deep Brain Stimulation Gabrielle Strandquist1, Tomasz Frączek2, Tanner Dixon3, Shravanan Ravi3, Raphael Bechtold4, Daryl Lawrence5, Alicia Zeng6, Jack Gallant7, Simon Little3, Jeffrey Herron8 1Computer Science and Engineering, University of Washington, 2Neuroscience, University of Washington, 3Neurology, University of California, San Francisco, 4Bioengineering, University of Washington, 5Bioengineering, University of California, Berkeley, 6Biophysics, University of California, Berkeley, 7Psychology, University of California, Berkeley, 8Neurological Surgery, University of Washington The protocol shows a prototype of the at-home multi-modal data collection platform that supports research optimizing adaptive deep brain stimulation (aDBS) for people with neurological movement disorders. We also present key findings from deploying the platform for over a year to the home of an individual with Parkinson's disease. Bioengineering Maintaining and Assessing Various Tissue and Cell Types of the Eye Using a Novel Pumpless Fluidics System Matthew K. Grumbine1, Varun Kamat2, Khang Bao1, Trevor Crupi1, Kedar Mokate2, Rayne Lim3, Jennifer R. Chao3, Brian M. Robbings4, Daniel T. Hass4, James B. Hurley4, Ian R. Sweet1,2 1EnTox Sciences, Inc, 2UW Medicine Diabetes Institute, University of Washington, 3Department of Ophthalmology, University of Washington, 4Department of Biochemistry, University of Washington Real-time analysis of live tissue yields important functional and mechanistic data. This paper describes the protocols and critical variables to ensure accurate and reproducible generation of data by a novel and pump-free multi-channel fluidics system that maintains and assesses a wide range of tissue and cell models. Developmental Biology Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brains Roberto Carlos Segura1, Clemens Cabernard1 1Department of Biology, University of Washington Here, we discuss a workflow to prepare, dissect, mount, and image live explant brains from Drosophila melanogaster third instar larvae to observe the cellular and subcellular dynamics under physiological conditions. Biology Scalable Transfection of Maize Mesophyll Protoplasts Jackson Tonnies1, Nicholas A. Mueth1, Sayeh Gorjifard1, Jonah Chu1, Christine Queitsch1 1Genome Sciences Department, University of Washington Here, we present a high-throughput protocol to transiently transfect millions of maize protoplasts for testing large libraries of plasmids in maize mesophyll cells. Neuroscience A Procedure for Mouse Dorsal Root Ganglion Cryosectioning Liangliang He*1,2, Wenxing Zhao*1, Lingyi Zhang2,3, Maalveka Ilango2,4, Na Zhao2, Liqiang Yang1, Zhonghui Guan2 1Department of Pain Management, Xuanwu Hospital, Capital Medical University, 2Department of Anesthesia and Perioperative Care, University of California San Francisco, 3Department of Anesthesiology, Sun Yat-Sen University, 4University of Washington Presented here is the development for consistently acquiring high-quality dorsal root ganglion cryostat sections. Engineering A Coregistered Ultrasound and Photoacoustic Imaging Protocol for the Transvaginal Imaging of Ovarian Lesions Haolin Nie1, Hongbo Luo2, Lin Chen1, Quing Zhu1,3 1Department of Biomedical Engineering, Washington University, 2Department of Electrical & Systems Engineering, Washington University, 3Department of Radiology, Washington University School of Medicine We report a coregistered ultrasound and photoacoustic imaging protocol for the transvaginal imaging of ovarian/adnexal lesions. The protocol may be valuable to other translational photoacoustic imaging studies, especially those using commercial ultrasound arrays for the detection of photoacoustic signals and standard delay-and-sum beamforming algorithms for imaging. Cancer Research Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor (CAR) T Cell Infusions in Mice Kyra Harvey1, Peter J. Madsen2,3, Tiffany Smith2, Crystal Griffin1,2, Luke Patterson1,2, Nicholas A. Vitanza4,5, Phillip B. Storm2,3, Adam C. Resnick2,3, Jessica B. Foster1,2 1Center for Data Driven Discovery in Biomedicine, Children’s Hospital of Philadelphia, 3Ben Towne Center for Childhood Cancer Research, Seattle Children’s Research Institute, 5Department of Pediatrics, Seattle Children’s Hospital, University of Washington Central nervous system (CNS) tumors are the leading cause of cancer-related death in children, and locoregional immune-based therapies are increasingly being tested for patients in clinical trials. This protocol describes methods for locoregional cannula implantation in mice for the preclinical evaluation of immunotherapeutic infusions targeting CNS tumors. 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 Balakrishnan1 1Department 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. Bioengineering Real-Time, Two-Color Stimulated Raman Scattering Imaging of Mouse Brain for Tissue Diagnosis Robert Espinoza*1, Brian Wong*1, Dan Fu1 1Department of Chemistry, University of Washington Stimulated Raman scattering (SRS) microscopy is a powerful, nondestructive, and label-free imaging technique. One emerging application is stimulated Raman histology, where two-color SRS imaging at the protein and lipid Raman transitions are used to generate pseudo-hematoxylin and eosin images. Here, we demonstrate a protocol for real-time, two-color SRS imaging for tissue diagnosis. Immunology and Infection Primed Mycobacterial Uveitis (PMU) as a Model for Post-Infectious Uveitis Sarah John1, Oliver H. Bell2, Leslie Wilson1, David A. Copland2, Kathryn L. Pepple1 1Department of Ophthalmology, University of Washington, 2Academic Unit of Ophthalmology, Translational Health Sciences, University of Bristol This protocol outlines the steps for inducing Primed Mycobacterial Uveitis (PMU) in mice. This method outlines the steps to help produce reliable and robust ocular inflammation in the mouse model system. Using this protocol, we generated uveitic eyes and uninflamed fellow eyes from single animals for further evaluation with immunologic, transcriptomic, and proteomic assays. Neuroscience Evaluation of Motor Impairment in C. elegans Models of Amyotrophic Lateral Sclerosis Heather N. Currey1, Nicole F. Liachko1,2 1Geriatrics Research Education and Clinical Center, Veterans Affairs Puget Sound Health Care System, 2Division of Gerontology and Geriatric Medicine, Department of Medicine, University of Washington This protocol describes two sensitive assays for discriminating among mild, moderate, and severe motor impairment in C. elegans models of amyotrophic lateral sclerosis, with general utility for C. elegans strains, with altered motility. Neuroscience Injections of AAV Vectors for Optogenetics in Anesthetized and Awake Behaving Non-Human Primate Brain Yoshiko Kojima1,2, Jonathan T. Ting2,3,4, Robijanto Soetedjo2,4, Shane D. Gibson2,4, Gregory D. Horwitz2,4 1Dept. of Otolaryngology - Head and Neck Surgery, University of Washington, 2Washington National Primate Research Center, University of Washington, 3Allen Institute for Brain Science, 4Dept. of Physiology & Biophysics, University of Washington As currently implemented, optogenetics in non-human primates requires injection of viral vectors into the brain. An optimal injection method should be reliable and, for many applications, capable of targeting individual sites of arbitrary depth that are readily and unambiguously identified in postmortem histology. An injection method with these properties is presented. Medicine Laser Doppler Perfusion Imaging in the Mouse Hindlimb Gale L. Tang1,2, Kevin J. Kim2 1Division of Vascular Surgery, University of Washington, 2VA Puget Sound Health Care System Here, we present a protocol that demonstrates the technique and necessary controls for Laser Doppler perfusion imaging to measure blood flow in the mouse hindlimb. Medicine Segmentation and Linear Measurement for Body Composition Analysis using Slice-O-Matic and Horos Sean Steele1, Fangyi Lin1, Thien-Linh Le1, Alexandra Medline1, Michelle Higgins1, Alex Sandberg1, Sean Evans1, Gordon Hong2, Milton A. Williams3, Mehmet A. Bilen4, Sarah Psutka5, Kenneth Ogan1, Viraj A. Master1 1Department of Urology, Emory University School of Medicine, 2Northeast Ohio Medical University, 3Department of Urology, University of Alabama at Birmingham, 4Department of Medical Oncology, Winship Cancer Institute of Emory University, 5Department of Urology, University of Washington Segmentation and linear measurements quantify skeletal muscle mass and adipose tissues using Computed Tomography and/or Magnetic Resonance Imaging images. Here, we outline the use of Slice-O-Matic software and Horos image viewer for rapid and accurate analysis of body composition. These methods can provide important information for prognosis and risk stratification. Neuroscience Low-intensity Blast Wave Model for Preclinical Assessment of Closed-head Mild Traumatic Brain Injury in Rodents Aric F. Logsdon*1,2, Brandon P. Lucke-Wold*3, Ryan C. Turner*4, Sean M. Collins5, Evan L. Reeder5, Jason D. Huber4, Charles L. Rosen6, Matthew J. Robson*5, Florian Plattner*7 1Geriatrics Research Education and Clinical Center, Veterans Affairs, 2Division of Gerontology and Geriatric Medicine, University of Washington, 3Department of Neurosurgery, University of Florida, 4Department of Neurosurgery, West Virginia University, 5Division of Pharmaceutical Sciences, University of Cincinnati, 6Central Illinois Neuro Health Sciences, Bloomington, IL, 7Neuro-research, Dallas, TX We present here a protocol of a blast wave model for rodents to investigate neurobiological and pathophysiological effects of mild to moderate traumatic brain injury. We established a gas-driven, bench-top setup equipped with pressure sensors allowing for reliable and reproducible generation of blast-induced mild to moderate traumatic brain injury. Bioengineering A MRI-Based Toolbox for Neurosurgical Planning in Nonhuman Primates William K.S. Ojemann1,2, Devon J. Griggs2,3, Zachary Ip1,2, Olivya Caballero4, Hesamoddin Jahanian5, Susana Martinez-Conde4, Stephen Macknik4, Azadeh Yazdan-Shahmorad1,2,3 1Bioengineering Department, University of Washington, 2Washington National Primate Research Center, University of Washington, 3Electrical and Computer Engineering Department, University of Washington, 4Department of Ophthalmology, SUNY Downstate Health Sciences University, 5Radiology Department, University of Washington The method outlined below aims to provide a comprehensive protocol for the preparation of nonhuman primate (NHP) neurosurgery using a novel combination of three-dimensional (3D) printing methods and MRI data extraction. Neuroscience Adaptable Angled Stereotactic Approach for Versatile Neuroscience Techniques Chelsea L. Faber1, Miles E. Matsen1, Thomas H. Meek1, Jordan E. Krull1, Gregory J. Morton1 1Department of Medicine, University of Washington Described here is a stereotactic procedure that can target challenging and difficult-to-reach brain regions (due to spatial limitations) using an angled coronal approach. This protocol is adaptable to both mouse and rat models and can be applied to diverse neuroscientific applications, including cannula implantation and microinjections of viral constructs. Environment In Situ Chemotaxis Assay to Examine Microbial Behavior in Aquatic Ecosystems Estelle E. Clerc1, Jean-Baptiste Raina2, Bennett S. Lambert3, Justin Seymour2, Roman Stocker1 1Institute of Environmental Engineering, Department of Civil, Environmental and Geomatic Engineering, ETH Zürich, 2Climate Change Cluster, University of Technology Sydney, 3School of Oceanography, University of Washington Presented here is the protocol for an in situ chemotaxis assay, a recently developed microfluidic device that enables studies of microbial behavior directly in the environment. Cancer Research Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices Nao Nishida-Aoki1, Andrew J. Bondesson1,2, Taranjit S. Gujral1,2,3 1Division of Human Biology, Fred Hutchinson Cancer Research Center, 2Department of Molecular and Cellular Biology, University of Washington, 3Department of Pharmacology, University of Washington We introduce a protocol for measuring real-time drug response in organotypic tumor tissue slices. The experimental strategy outlined here provides a platform to carry out medium-high throughput drug screens on tissue slices derived from clinical or mouse tumors in ex vivo conditions. Biology Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles Joshua C. Russell1, Nadia Postupna1, Alexandra Golubeva1, C. Dirk Keene1, Matt Kaeberlein1 1Department of Pathology, University of Washington This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles. Biochemistry Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library Luis Gomez-Castillo*1, Kurumi Watanabe*1, Huayi Jiang*1, Shoukai Kang1, Liangcai Gu1 1Department of Biochemistry and Institute for Protein Design, University of Washington Creating chemically induced protein dimerization systems with desired affinity and specificity for any given small molecule ligand would have many biological sensing and actuation applications. Here, we describe an efficient, generalizable method for de novo engineering of chemically induced dimerization systems via the stepwise selection of a phage-displayed combinatorial single-domain antibody library. Neuroscience A Ferret Model of Inflammation-sensitized Late Preterm Hypoxic-ischemic Brain Injury Thomas Wood1, Daniel Moralejo1, Kylie Corry1, Cole Fisher1, Jessica M. Snyder2, Vivienne Acuna1, Alair Holden-Hunt1, Simar Virk1, Olivia White1, Janessa Law1, Pratik Parikh1, Sandra E. Juul1 1Division of Neonatology, Department of Pediatrics, University of Washington, 2Department of Comparative Medicine, University of Washington The method describes inflammation-sensitized hypoxic-ischemic and hyperoxic brain injury in the P17 ferret to model the complex interaction between prolonged inflammation and oxidative brain injury experienced in a number of late preterm infants. Biochemistry Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation Emily A. Brown1,2, Steven C. Neier3,4, Claudia Neuhauser5, Adam G. Schrum6,7,8, Stephen E.P. Smith1,2,9 1 Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network. Medicine Ultrasonography of the Adult Male Urinary Tract for Urinary Functional Testing Teresa T. Liu*1,2, Allison C. Rodgers*3, Tristan M. Nicholson4, Jill A. Macoska5,6, Paul C. Marker6,7, Chad M. Vezina6,8, Dale E. Bjorling6,8, Alejandro Roldan-Alzate2,9,10, Diego Hernando10,11, Granville L. Lloyd12, Timothy A. Hacker3, William A. Ricke1,6 1Department of Urology, University of Wisconsin-Madison, 2K12 Kure, University of Wisconsin-Madison, 3Cardiovascular Research Center, Department of Medicine, University of Wisconsin-Madison, 4Department of Urology, University of Washington, 5University of Massachusetts Boston, 6U54 George M. O'Brien Center, University of Wisconsin-Madison, 7College of Pharmacy, University of Wisconsin-Madison, 8School of Veterinary Medicine, University of Wisconsin-Madison, 9Department of Mechanical Engineering, University of Wisconsin-Madison, 10Department of Radiology, University of Wisconsin-Madison, 11Department of Medical Physics, University of Wisconsin-Madison, 12Rocky Mountain Regional VA Medical Center, University of Colorado We describe the use of high frequency ultrasound with contrast imaging as a method to measure bladder volume, bladder wall thickness, urine velocity, void volume, void duration, and urethral diameter. This strategy can be used to assess voiding dysfunction and treatment efficacy in various mouse models of lower urinary tract dysfunction (LUTD). Behavior Noninvasive, In-pen Approach Test for Laboratory-housed Pigs Lindsey E. Hulbert1, Eduarda M. Bortoluzzi1, Yunzhi Luo1, Jared M. Mumm1, Morgan J. Coffin1, Gina Y. Becker2, Pamela J. Vandevord3, Elizabeth M. McNeil3, Tim Walilko4, Zin Z. Khaing5,6, Laila Zai4,5 1Animal Sciences and Industry, Kansas State University, 2DynaSim Technical Services, INC, 3Center for Injury Biomechanics, Virginia Polytechnic Institute and State University, 4Applied Research Associates, Inc., 5Lucent Research, LLC, 6Department of Neurological Surgery, University of Washington This protocol describes a new behavioral test—the human approach test in the pigs' home pen—to detect functional deficits in laboratory pigs after subconcussive traumatic brain injury. Neuroscience Convection Enhanced Delivery of Optogenetic Adeno-associated Viral Vector to the Cortex of Rhesus Macaque Under Guidance of Online MRI Images Karam Khateeb1,2, Devon J. Griggs2,3, Philip N. Sabes4, Azadeh Yazdan-Shahmorad1,2,3,4 1Departments of Bioengineering, University of Washington, 2Washington National Primate Research Center, 3Department of Electrical and Computer Engineering, University of Washington, 4Department of Physiology and Center for Integrative Neuroscience, University of California, San Francisco Here, we demonstrate magnetic resonance (MR)-guided convection enhanced delivery (CED) of viral vectors into the cortex as an efficient and simplified approach for achieving optogenetic expression across large cortical areas in the macaque brain. Chemistry Synthesis of In37P20(O2CR)51 Clusters and Their Conversion to InP Quantum Dots Nayon Park1, Madison Monahan1, Andrew Ritchhart1, Max R. Friedfeld1, Brandi M. Cossairt1 1Department of Chemistry, University of Washington A protocol for the synthesis of In37P20(O2C14H27)51 clusters and their conversion to indium phosphide quantum dots is presented. Genetics Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells Stefan Radtke1, Anai M. Perez1, Rasika Venkataraman1, Sowmya Reddy1, Kevin G. Haworth1, Olivier Humbert1, Hans-Peter Kiem1,2,3, Christopher W. Peterson1,2 1Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center, 2Department of Medicine, University of Washington, 3Department of Pathology, University of Washington The goal of this protocol is to isolate nonhuman primate CD34+ cells from primed bone marrow, to gene-modify these cells with lentiviral vectors, and to prepare a product for infusion into the autologous host. The total protocol length is approximately 48 h. Bioengineering Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel Harrison L. Hiraki1, Ryan J. Nagao2, Jonathan Himmelfarb3, Ying Zheng2 1Department of Bioengineering, University of Washington, 2Department of Bioengineering, Center for Cardiovascular Biology, Institute for Stem Cell and Regenerative Medicine, University of Washington, 3Department of Medicine, Kidney Research Institute, University of Washington Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed. Biochemistry An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity Antonio E. Chambers*1, Adam E. Richardson*1, David F. Read2, Thomas J. Waller3, Douglas A. Bernstein1, Philip J. Smaldino1 1Department of Biology, Ball State University, 2Department of Genome Sciences, University of Washington, 3Department of Molecular, Cellular, and Developmental Biology, University of Michigan Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes. Immunology and Infection Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages Andreas B. den Hartigh1, Susan L. Fink1 1Department of Laboratory Medicine, University of Washington We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology. Neuroscience Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments Michelle M. Giarmarco1, Whitney M. Cleghorn1, James B. Hurley1,2, Susan E. Brockerhoff1,2 1Department of Biochemistry, University of Washington, 2Department of Ophthalmology, University of Washington Imaging retinal tissue can provide single-cell information that cannot be gathered from traditional biochemical methods. This protocol describes preparation of retinal slices from zebrafish for confocal imaging. Fluorescent genetically encoded sensors or indicator dyes allow visualization of numerous biological processes in distinct retinal cell types. Medicine In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium Bradley K. Wacker1, Lianxiang Bi1, David A. Dichek1 1Department of Medicine, University of Washington This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences. Genetics G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome Eric J. Foss1, Uyen Lao1, Antonio Bedalov1,2 1Division of Clinical Research, Fred Hutchinson Cancer Research Center, 2Departments of Medicine and Biochemistry, University of Washington We describe a technique for combining flow cytometry and high throughput sequencing to identify late replicating regions of the genome. Genetics Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome Vid Leko1,2, Smitha Sripathy1, Robin L. Adrianse1, Taylor Loe1, Angela Park1, Uyen Lao1, Eric J. Foss1, Marisa S. Bartolomei3, Antonio Bedalov1,4 1Clinical Research Division, Fred Hutchinson Cancer Research Center, 2Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, 3Epigenetics Program, Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, 4Departments of Medicine and Biochemistry, University of Washington We report a small hairpin RNA (shRNA) and next generation sequencing-based protocol for identifying regulators of X-chromosome inactivation in a murine cell line with firefly luciferase and hygromycin resistance genes fused to the methyl CpG binding protein 2 (MeCP2) gene on the inactive X chromosome. Behavior Assessing Spatial Memory Impairment in a Mouse Model of Traumatic Brain Injury Using a Radial Water Tread Maze Marcella M. Cline1, Megan A. Ostlie2, Chloe G. Cross3, Gregory G. Garwin2, Satoshi Minoshima2, Donna J. Cross2 1Department of Radiology, University of Washington, 2Department of Radiology, University of Utah, 3Geriatric Research Education and Clinical Center (GRECC), VA Puget Sound Here we present a protocol for a mouse-specific test of cognition that does not require swimming. This test can be used to successfully distinguish controlled cortical impact-induced traumatic brain injury mice from sham controls. Genetics Flow-sorting and Exome Sequencing of the Reed-Sternberg Cells of Classical Hodgkin Lymphoma Jonathan B. Reichel1, Jason McCormick2, Jonathan R. Fromm3, Olivier Elemento4, Ethel Cesarman5, Mikhail Roshal6 1Innovation Laboratory, Center for Molecular Oncology, Memorial Sloan Kettering Cancer Center, 2Flow-Sorting Core Facility, Weill Cornell Medical College, 3Department of Laboratory Medicine, University of Washington, 4Department of Physiology and Biophysics, Institute for Computational Biomedicine, Weill Cornell Medical College, 5Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, 6Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center Here, we describe a combined flow cytometric cell sorting and low-input, next-generation library construction protocol designed to produce high-quality, whole-exome data from the Hodgkin Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (CHL). Medicine A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers Catherine E. Lang1,2,3, Kimberly J. Waddell1, Joseph W. Klaesner1,4,5, Marghuretta D. Bland1,2,3 1Program in Physical Therapy, Washington University School of Medicine, 2Program in Occupational Therapy, Washington University School of Medicine, 3Department of Neurology, Washington University School of Medicine, 4Mallinckrodt Institute of Radiology, Washington University School of Medicine, 5Department of Biomedical Engineering, Washington University This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers. Bioengineering Micropatterning and Assembly of 3D Microvessels Meredith A. Roberts*1, Surya S. Kotha*1, Kiet T. Phong2, Ying Zheng1,2 1Department of Bioengineering, University of Washington, 2Center for Cardiovascular Biology, Institute for Stem Cell and Regenerative Medicine, University of Washington This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described. Medicine Assessment of Cardiac Morphological and Functional Changes in Mouse Model of Transverse Aortic Constriction by Echocardiographic Imaging Lei Li*1, Xiaoyun Guo*1, Yi Chen1, Haifeng Yin1, Jing Li1, Jessica Doan1, Qinghang Liu1 1Department of Physiology and Biophysics, University of Washington The goal of this protocol is to noninvasively assess cardiac structural and functional changes in a mouse model of heart disease created by transverse aortic constriction, using B- and M-mode echocardiography and color/pulse wave Doppler imaging. Chemistry Synthesis of Ligand-free CdS Nanoparticles within a Sulfur Copolymer Matrix Trevor R. Martin1,2,3, Katherine A. Mazzio4, Hugh W. Hillhouse2,3,5, Christine K. Luscombe1,2,3,6 1Department of Materials Science and Engineering, University of Washington, 2Molecular Engineering and Sciences Institute, University of Washington, 3Clean Energy Institute, University of Washington, 4Institut für Nanospektroskopie, Helmholtz-Zentrum Berlin für Materialien und Energie GmbH, 5Department of Chemical Engineering, University of Washington, 6Department of Chemistry, University of Washington Herein we present a method to synthesize ligand-free cadmium sulfide (CdS) nanoparticles based on a unique sulfur copolymer. The sulfur copolymer operates as a high temperature solvent and a sulfur source during the nanoparticle synthesis and stabilizes the nanoparticles after the reaction. Medicine An Improved Method for Rapid Intubation of the Trachea in Mice Tyler C. Vandivort1,2, Dowon An3, William C. Parks2 1Department of Environmental and Occupational Health Sciences, University of Washington, 2Department of Medicine, Cedars-Sinai Medical Center, 3Division of Pulmonary and Critical Care Medicine, University of Washington This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times. Biology Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks Yuan-Jyue Chen1, Sundipta D. Rao1, Georg Seelig1,2 1Department of Electrical Engineering, University of Washington, 2Department of Computer Science & Engineering, University of Washington This protocol describes a method for deriving DNA strand displacement gates from plasmids and testing them using fluorescence kinetics measurements. Gates can be modularly composed into multi-component systems to approximate the behavior of formal chemical reaction networks (CRN), demonstrating a new use for CRNs as a molecular programming language. Immunology and Infection Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells Meghan Zuck1,2, Caroline Feng2, Kevin Hybiske1 1Division 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. Neuroscience Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester Joseph Jebelli*1, Thomas Piers*2, Jennifer Pocock3 1Department of Neurology, University of Washington, 2Therapeutic Innovation Group, School of Life and Medical Sciences, University College London, 3Department of Neuroinflammation, University College London Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature. Immunology and Infection Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses Siriphan Manocheewa1, Erinn C. Lanxon-Cookson1, Yi Liu1, J. Victor Swain1, Jan McClure1, Ushnal Rao1, Brandon Maust1, Wenjie Deng1, Justine E. Sunshine1, Moon Kim1, Morgane Rolland3,4, James I. Mullins1,2 1Department of Microbiology, University of Washington, 2Departments of Medicine and Laboratory Medicine, University of Washington, 3U.S Military HIV Research Program, Walter Reed Army Institute of Research, 4Henry M. Jackson Foundation Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results. Behavior Assessment of Murine Exercise Endurance Without the Use of a Shock Grid: An Alternative to Forced Exercise Jennifer D. Conner1,2, Tami Wolden-Hanson1, LeBris S. Quinn1,3,4 1Research Service, VA Puget Sound Health Care System, 2Seattle Institute for Biomedical and Clinical Research, 3Division of Gerontology and Geriatric Medicine, Department of Medicine, University of Washington, 4Geriatric Research, Education, and Clinical Center, VA Puget Sound Health Care System A method to assess exercise endurance in laboratory mice without the use of a shock grid is demonstrated. This method is a humane refinement that can decrease the confounding effects of stress on experimental parameters. Biology Tissue Triage and Freezing for Models of Skeletal Muscle Disease Hui Meng1, Paul M.L. Janssen2, Robert W. Grange3, Lin Yang4, Alan H. Beggs5, Lindsay C. Swanson5, Stacy A. Cossette1,6, Alison Frase7, Martin K. Childers8, Henk Granzier9, Emanuela Gussoni5, Michael W. Lawlor1 1Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, 2Department of Physiology and Cell Biology, The Ohio State University, 3Department of Human Nutrition, Foods and Exercise, Virginia Tech, 4Division of Biomedical Informatics, Department of Biostatistics, Department of Computer Science, University of Kentucky, 5 The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies. Bioengineering Capillary Force Lithography for Cardiac Tissue Engineering Jesse Macadangdang1, Hyun Jung Lee1, Daniel Carson1, Alex Jiao1, James Fugate2, Lil Pabon2, Michael Regnier1, Charles Murry2, Deok-Ho Kim1 1Department of Bioengineering, University of Washington, 2Department of Pathology, University of Washington In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications. Medicine EEG Mu Rhythm in Typical and Atypical Development Raphael Bernier1,2, Benjamin Aaronson1,2, Anna Kresse1 1Department of Psychiatry, University of Washington, 2Department of Educational Psychology, University of Washington Assessment of the EEG mu rhythm provides a unique methodology for examining brain activity and when combined with behaviorally based assays, can be a powerful tool for elucidating aspects of social cognition, such as imitation, in clinical populations. Neuroscience Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition Daniel A. Lee1,2, Juan Salvatierra2, Esteban Velarde3, John Wong3, Eric C. Ford4, Seth Blackshaw2,5 1Division of Biology, California Institute of Technology, 2Solomon H. Snyder Department of Neuroscience, Neurology, and Ophthalamology, Johns Hopkins University School of Medicine, 3Department of Radiation Oncology & Molecular Radiation Sciences, Johns Hopkins University School of Medicine, 4Department of Radiation Oncology, University Of Washington Medical Center, 5Institute for Cell Engineering and High-Throughput Biology Center, Johns Hopkins University School of Medicine The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis. Biology Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo Guohua Gong1, Wang Wang1 1Mitochondria and Metabolism Center, Department of Anesthesiology and Pain Medicine, University of Washington Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations. Biology Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology Marcus D. Collins1, Sharona E. Gordon1 1Department of Physiology and Biophysics, University of Washington Reconstituting functional membrane proteins into giant liposomes of defined composition is a powerful approach when combined with patch-clamp electrophysiology. However, conventional giant liposome production may be incompatible with protein stability. We describe protocols for producing giant liposomes from pure lipids or small liposomes containing ion channels. Neuroscience Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe Kelsey J. R. P. Byers1, Elischa Sanders1, Jeffrey A. Riffell1 1Department of Biology, University of Washington Olfactory cues mediate many different behaviors in insects, and are often complex mixtures comprised of tens to hundreds of volatile compounds. Using gas chromatography with multi-channel recording in the insect antennal lobe, we describe a method for the identification of bioactive compounds. Biology Design and Use of Multiplexed Chemostat Arrays Aaron W. Miller1, Corrie Befort1, Emily O. Kerr1, Maitreya J. Dunham1 1Department of Genome Sciences, University of Washington We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications. Biology Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination Kylee M. Peterson1, Keiko U. Torii1,2,3 1Department of Biology, University of Washington, 2Howard Hughes Medical Institute, University of Washington, 3PRESTO, Japan Science and Technology Agency We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation. Biology Spatial Multiobjective Optimization of Agricultural Conservation Practices using a SWAT Model and an Evolutionary Algorithm Sergey Rabotyagov1, Todd Campbell2, Adriana Valcu2, Philip Gassman2, Manoj Jha3, Keith Schilling4, Calvin Wolter4, Catherine Kling2 1School of Environmental and Forest Sciences, University of Washington, 2Center for Agricultural and Rural Development, Department of Economics, Iowa State University, 3Department of Civil, Architectural, and Environmental Engineering, North Carolina A&T University, 4Iowa Geological and Water Survey This work demonstrates an integration of a water quality model with an optimization component utilizing evolutionary algorithms to solve for optimal (lowest-cost) placement of agricultural conservation practices for a specified set of water quality improvement objectives. The solutions are generated using a multi-objective approach, allowing for explicit quantification of tradeoffs. Neuroscience Mapping Cortical Dynamics Using Simultaneous MEG/EEG and Anatomically-constrained Minimum-norm Estimates: an Auditory Attention Example Adrian K.C. Lee1, Eric Larson1, Ross K. Maddox1 1Department of Speech & Hearing Sciences, Institute for Learning and Brain Sciences, University of Washington We use magneto- and electroencephalography (MEG/EEG), combined with anatomical information captured by magnetic resonance imaging (MRI), to map the dynamics of the cortical network associated with auditory attention. Biology Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria Raluca Marcu1, Chris K. Neeley1, Georgios Karamanlidis1, Brian J. Hawkins1 1Department of Anesthesiology & Pain Medicine, Mitochondria and Metabolism Center, University of Washington, Seattle A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described. Neuroscience Whole Animal Perfusion Fixation for Rodents Gregory J. Gage1, Daryl R. Kipke1, William Shain2 1Biomedical Engineering, University of Michigan, 2Department of Neurological Surgery, University of Washington School of Medicine Here we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain. Biology Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans Emily M. Fawcett1,2, Joseph W. Horsman1, Dana L. Miller1 1Department of Biochemistry, University of Washington, 2Molecular and Cellular Biology Program, University of Washington This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems Medicine Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging Jill J. Weyers1, Dara D. Carlson1, Charles E. Murry1,2, Stephen M. Schwartz1, William M. Mahoney, Jr.1 1Department of Pathology, Center for Cardiovascular Biology, and Institute for Stem Cell and Regenerative Medicine, University of Washington, 2Departments of Bioengineering and Medicine/Cardiology, University of Washington Visualization of the coronary vessels is critical to advancing our understanding of cardiovascular diseases. Here we describe a method for perfusing murine coronary vasculature with a radiopaque silicone rubber (Microfil), in preparation for micro-Computed Tomography (μCT) imaging. Biology Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes James M. Flynn1, Luis F. Santana2, Simon Melov1 1Buck Institute for Research on Aging, 2Department of Physiology & Biophysics, University of Washington Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets. Biology Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS Karthik Mallilankaraman1, Rajesh Kumar Gandhirajan1, Brian J. Hawkins2, Muniswamy Madesh1 1Department of Biochemistry, Temple University, 2Department of Anesthesiology and Pain Medicine, University of Washington An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model. Bioengineering Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation Cameron S. Simmons1, Emily Christine Knouf2,3, Muneesh Tewari2,4,5, Lih Y. Lin1 1Electrical Engineering Department, University of Washington, 2Division of Human Biology, Fred Hutchinson Cancer Research Center, 3Molecular and Cellular Biology Program, University of Washington, 4Clinical Research, Fred Hutchinson Cancer Research Center, 5Public Health Sciences, Fred Hutchinson Cancer Research Center Plasmonic tweezers and photonic crystal nanostructures are shown to produce useful enhancements in the efficiency and orientation control of optically trapping micro- and nano-particles. Biology Biochemical Reconstitution of Steroid Receptor•Hsp90 Protein Complexes and Reactivation of Ligand Binding Patrick J. M. Murphy1, Hannah R. Franklin2, Nathan W. Furukawa3 1College of Nursing, Interdisciplinary Life Sciences Research Laboratory, Seattle University, 2College of Science and Engineering, Interdisciplinary Life Sciences Research Laboratory, Seattle University, 3School of Medicine, University of Washington An in vitro method for preparing functional glucocorticoid receptor (GR)•hsp90 protein complexes from purified proteins and cellular lysates is described. The method utilizes immunoadsorption of recombinant GR followed by salt-stripping and protein complex reconstitution. The importance of cofactors and buffer conditions are discussed, as are potential method applications. Immunology and Infection Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice Christina M. Parkinson1, Alexandra O'Brien1, Theresa M. Albers1, Meredith A. Simon1, Charles B. Clifford1, Kathleen R. Pritchett-Corning2,3 1Research Animal Diagnostic Services, Charles River, 2Research Models and Services, Charles River, 3Department of Comparative Medicine, University of Washington This article describes various procedures for screening rats and mice to detect endo- or ectoparasitism. Several diagnostic assays will be demonstrated, both those suitable for use on live animals and those used after euthanasia of the animal. Photographs to aid in identification of rat and mouse parasites will be included. Biology Diagnostic Necropsy and Selected Tissue and Sample Collection in Rats and Mice Christina M. Parkinson1, Alexandra O'Brien1, Theresa M. Albers1, Meredith A. Simon1, Charles B. Clifford2, Kathleen R. Pritchett-Corning2,3 1Research Animal Diagnostic Services, Charles River, 2Research Models and Services, Charles River, 3Department of Comparative Medicine, University of Washington This article describes the procedures for conducting a basic postmortem examination of a mouse or rat, and the collection of basic organs, as well as more challenging sample types from for histological, microbiological, and PCR evaluation. Neuroscience Preparation and Culture of Chicken Auditory Brainstem Slices Jason T. Sanchez*1, Armin H. Seidl*1, Edwin W. Rubel1,2, Andres Barria2 1Department of Otolaryngology-Head and Neck Surgery, Virginia Merrill Bloedel Hearing Research Center, University of Washington, 2Department of Physiology and Biophysics, Virginia Merrill Bloedel Hearing Research Center, University of Washington The chicken auditory brainstem is comprised of nuclei responsible for binaural sound processing. A single coronal slice preparation maintains the entire circuitry while the cultured approach provides a unique preparation to study the development of neuronal structure and auditory function at the molecular, cellular and network levels. Neuroscience Organotypic Hippocampal Slice Cultures Ximena Opitz-Araya1, Andres Barria1 1Department of Physiology and Biophysics, University of Washington School of Medicine We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture. Medicine Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy Stephen C. Kolwicz Jr.1, Rong Tian1 1Department of Anesthesiology & Pain Medicine, University of Washington School of Medicine Langendorff-mode isolated heart perfusion, in conjunction with 31P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment. The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function. When performed correctly, this is a valuable technique in the assessment of cardiac energetics. 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 1Department 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. Medicine An Experimental Paradigm for the Prediction of Post-Operative Pain (PPOP) Ruth Landau1, John C. Kraft1, Lisa Y. Flint1, Brendan Carvalho1, Philippe Richebé1, Monica Cardoso1, Patricia Lavand'homme1, Michal Granot1, David Yarnitsky1, Alex Cahana1 1Department of Anesthesiology and Pain Medicine, University of Washington School of Medicine Diffuse noxious inhibitory control, temporal summation and wound hyperalgesia testing are demonstrated in the obstetric patient. These tests evaluate inhibitory and excitatory mechanisms of pain processing and are here utilized to evaluate endogenous analgesia at different time-points during pregnancy and the peripartum period to help reveal individual s risk for persistent pain. Biology Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures Donghoon Lee1, David Marcinek1,2 1Department of Radiology, University of Washington, 2Department of Bioengineering, University of Washington This work describes basic procedures of noninvasive small animal MRI and MRS in vivo. Biology Measuring Replicative Life Span in the Budding Yeast Kristan K. Steffen1, Brian K. Kennedy1, Matt Kaeberlein2 1Department of Biochemistry, University of Washington, 2Department of Pathology, University of Washington In this article we present a general protocol for measuring the replicative life span of yeast mother cells. Biology Measuring Caenorhabditis elegans Life Span on Solid Media George L. Sutphin1,2, Matt Kaeberlein1 1Department of Pathology, University of Washington, 2Molecular and Cellular Biology Program, University of Washington In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food. Biology Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells Christopher Murakami1, Matt Kaeberlein1 1Department of Pathology, University of Washington Chronological aging in yeast refers to the loss of cell viability associated with time in stationary phase. Here we describe a high-throughput method for quantitatively determining yeast chronological life span. Biology Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces Ravi A. Desai1, Michael T. Yang1, Nathan J. Sniadecki2, Wesley R. Legant1, Christopher S. Chen1 1Department of Bioengineering, University of Pennsylvania, 2University of Washington In this video, we demonstrate how to fabricate and utilize microfabricated post array detectors (mPADs) to assess modulations of cellular contractility. Biology Microfluidic Chips Controlled with Elastomeric Microvalve Arrays Nianzhen Li1, Chris Sip1, Albert Folch1 1Dept. of Bioengineering, University of Washington We demonstrate protocols for manufacturing and automating elastomeric polydimethylsiloxane (PDMS)-based microvalve arrays that need no extra energy to close and feature photolithographically defined precise volumes. A parallel subnanoliter-volume mixer and an integrated microfluidic perfusion system are presented. Biology BioMEMS and Cellular Biology: Perspectives and Applications Albert Folch1 1Department of Biomedical Engineering, University of Washington
