University of California, San Francisco

113 articles published in JoVE

• Biology

  
  Studying Habituation in Stentor coeruleus Deepa Rajan¹, Peter Chudinov¹, Wallace Marshall^{1 1}Department of Biochemistry and Biophysics, University of California San Francisco We introduce a method for quantifying Stentor habituation using a microcontroller board-linked apparatus that can deliver mechanical pulses at a specified force and frequency. We also include methods for assembling the apparatus and setting up the experiment in a way that minimizes external perturbations.

• Cancer Research

  
  Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids Dora Barbosa Rabago^1,2,3, Collin M. Blakely^1,2, Franziska Haderk^1,2,3, Trever G. Bivona^{1,2,3 1}Department of Medicine, University of California, San Francisco, ²Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, ³Department of Cellular and Molecular Pharmacology, University of California, San Francisco This protocol describes a standardized evaluation of drug sensitivities to targeted signaling inhibitors in NSCLC patient-derived organoid models.

• Developmental Biology

  
  Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay Natasja G. J. Costermans^1,2, Enrico M. Daldello^1,2,3, Ria J. Marathe^1,2, Marco Conti^{1,2 1}Center for Reproductive Sciences, University of California at San Francisco, ²Department of Obstetrics Gynecology and Reproductive Sciences, University of California at San Francisco, ³Present affiliation: Laboratoire de Biologie du Développement-Institut de Biologie Paris Seine, LBD-IBPS, Sorbonne Université This protocol describes a reporter assay to study the regulation of mRNA translation in single oocytes during in vitro maturation.

• Biology

  
  Analysis of Motility Patterns of Stentor During and After Oral Apparatus Regeneration Using Cell Tracking Janet Y. Sheung¹, Megan Otsuka², Gabriella Seifert³, Athena Lin⁴, Wallace F. Marshall^{4 1}W. M. Keck Science Department, Scripps, Pitzer, and Claremont McKenna of The Claremont Colleges, ²W. M. Keck Science Department, Pitzer College, ³W. M. Keck Science Department, Scripps College, ⁴Department of Biochemistry and Biophysics, School of Medicine, University of California at San Francisco We present a protocol for the characterization of motility and behavior of a population of hundred micron- to millimeter-sized cells using brightfield microscopy and cell tracking. This assay reveals that Stentor coeruleus transitions through four behaviorally distinct phases when regenerating a lost oral apparatus.

• Biology

  
  Particle Templated Emulsification enables Microfluidic-Free Droplet Assays Daniel W. Weisgerber¹, Makiko N. Hatori¹, Adam R. Abate^{1,2 1}Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California, ²Chan Zuckerberg Biohub Water-in-oil droplet assays are useful for analytical chemistry, enzyme evolution, and single cell analysis, but typically require microfluidics to form the droplets. Here, we describe particle templated emulsification, a microfluidic-free approach to perform droplet assays.

• 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.

• Developmental Biology

  
  Drosophila Embryo Preparation and Microinjection for Live Cell Microscopy Performed using an Automated High Content Analyzer Ulises Diaz^1,2, Wallace Marshall², Blake Riggs^{1 1}Department of Biology, San Francisco State University, ²Department of Biochemistry & Biophysics, UCSF Mission Bay Presented here is a protocol to microinject and simultaneously image multiple Drosophila embryos during embryonic development using a plate-based, high content imager.

• Bioengineering

  
  High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing Jesse Q. Zhang^1,2, Kai-Chun Chang¹, Leqian Liu¹, Zev J. Gartner^3,5, Adam R. Abate^{1,4,5 1}Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, ²University of California Berkeley-UCSF Graduate Program in Bioengineering, University of California San Francisco, ³Department of Pharmaceutical Chemistry, University of California San Francisco, ⁴California Institute for Quantitative Biosciences, University of California San Francisco, ⁵Chan Zuckerberg Biohub A bottleneck in the ‘design-build-test’ cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

• Medicine

  
  Validated LC-MS/MS Panel for Quantifying 11 Drug-Resistant TB Medications in Small Hair Samples Andrew Reckers¹, Anita Wen¹, David Aguilar¹, Peter Bacchetti², Monica Gandhi³, John Metcalfe⁴, Roy Gerona^{1 1}Department of Obstetrics, Gynecology and Reproductive Sciences, TB Hair Analysis Laboratory, University of California San Francisco, ²Department of Epidemiology and Biostatistics, University of California San Francisco, ³Department of Medicine, Division of HIV, Infectious Diseases, and Global Medicine, University of California San Francisco, ⁴Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of California San Francisco Current methods of analyzing patients’ adherence to complex drug resistant-tuberculosis (DR-TB) regimens can be inaccurate and resource-intensive. Our method analyzes hair, an easily collected and stored matrix, for concentrations of 11 DR-TB medications. Using LC-MS/MS, we can determine sub-nanogram drug levels that can be utilized to better understand drug adherence.

• Bioengineering

  
  Design of a Biocompatible Drug-Eluting Tracheal Stent in Mice with Laryngotracheal Stenosis Madhavi Duvvuri¹, Kevin Motz², Hsiu-Wen Tsai², Ioan Lina², Dacheng Ding², Andrew Lee², Alexander T. Hillel^{2 1}Department of General Surgery, University of California, San Francisco, ²Department of Otolaryngology Head and Neck Surgery, Johns Hopkins School of Medicine Laryngotracheal stenosis results from pathologic scar deposition that critically narrows the tracheal airway and lacks effective medical therapies. Using a PLLA-PCL (70% poly-L-lactide and 30% polycaprolactone) stent as a local drug delivery system, potential therapies aimed at decreasing scar proliferation in the trachea can be studied.

• Immunology and Infection

  
  Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization Jessica N. Witchley¹, Pallavi M. Penumetcha¹, Suzanne M. Noble^{1,2 1}Department of Microbiology and Immunology, University of California, San Francisco, ²Department of Medicine, Division of Infectious Disease, University of California, San Francisco The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

• Neuroscience

  
  Chronic Implantation of Multiple Flexible Polymer Electrode Arrays Jason E Chung*^1,2, Hannah R Joo*^1,2, Clay N Smyth², Jiang Lan Fan³, Charlotte Geaghan-Breiner², Hexin Liang², Daniel Fan Liu³, Demetris Roumis², Supin Chen^4,5, Kye Y Lee⁴, Jeanine A Pebbles⁴, Angela C Tooker⁴, Vanessa M Tolosa^4,5, Loren M Frank^{2,6 1}Medical Scientist Training Program and Neuroscience Graduate Program, University of California San Francisco, ²Kavli Institute for Fundamental Neuroscience, Center for Integrative Neuroscience, and Department of Physiology, University of California San Francisco, ³Bioengineering Graduate Program, University of California San Francisco, ⁴Center for Micro- and Nanotechnology, Lawrence Livermore National Laboratory, ⁵Neuralink Corp., ⁶Howard Hughes Medical Institute Described below is a method for implantation of multiple polymer electrode arrays across anatomically distant brain regions for chronic electrophysiological recording in freely moving rats. Preparation and surgical implantation are described in detail, with emphasis on design principles to guide adaptation of these methods for use in other species.

• 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.

• Medicine

  
  A Cryoinjury Model to Study Myocardial Infarction in the Mouse Dong Wang*^1,2, Grigol Tediashvili*^1,2, Xiaomeng Hu^1,2, Alessia Gravina², Sivan G. Marcus^1,2, Hao Zhang⁴, Jeffrey E Olgin⁴, Tobias Deuse^1,2,5, Sonja Schrepfer^{1,2,3,5 1}Transplant and Stem Cell Immunobiology Lab, University Heart Center, ²Department of Surgery, Transplant and Stem Cell Immunobiology Lab, University of California San Francisco, ³Cardiovascular Research Center (CVRC) and DZHK German Center for Cardiovascular Research, ⁴Division of Cardiology, Cardiovascular Research Institute, University of California San Francisco, ⁵Cardiovascular Surgery, University Heart Center This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

• Neuroscience

  
  Rapid Isolation of Dorsal Root Ganglion Macrophages Xiaobing Yu¹, Jacqueline Leff¹, Zhonghui Guan^{1 1}Department of Anesthesia and Perioperative Care, University of California San Francisco Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

• Medicine

  
  Quantitative [¹⁸F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain Nathaniel W. Jenkins¹, Claudia Iriondo^1,2, Vinil Shah¹, Emma Bahroos¹, Vahid Ravanfar^1,1, Melanie Regan¹, Youngho Seo¹, William P. Dillon¹, Sharmila Majumdar¹, Jason F. Talbott^{1,3 1}Radiology and Biomedical Imaging, University of California San Francisco, ²UCSF/UC Berkeley Graduate Program in Bioengineering, University of California San Francisco, ³Radiology and Biomedical Imaging, Zuckerberg San Francisco General Hospital Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. We present detailed methodologies for performing and analyzing dynamic [¹⁸F]-NaF-PET-MRI data in a patient with facetogenic low back pain using the lumbar facet joints as a prototypical region of interest.

• Medicine

  
  Cone Beam Intraoperative Computed Tomography-based Image Guidance for Minimally Invasive Transforaminal Interbody Fusion Michael Safaee¹, Taemin Oh¹, Murat Pekmezci², Aaron J. Clark^{1 1}Department of Neurological Surgery, University of California, San Francisco, ²Department of Orthopedic Surgery, University of California, San Francisco The purpose of this article is to provide image-guidance for minimally invasive transforaminal interbody fusion.

• Cancer Research

  
  In Vitro Assay to Study Tumor-macrophage Interaction Zhenyi An¹, William A. Weiss^{1,2,3 1}Department of Neurology, University of California, San Francisco, ²Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, ³Departments of Pediatrics and Neurological Surgery, University of California, San Francisco This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

• Cancer Research

  
  Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids Erin R. Bonner^1,2, Karim Saoud¹, Sulgi Lee^1,2, Eshini Panditharatna³, Madhuri Kambhampati¹, Sabine Mueller^4,5, Javad Nazarian^{1,2,5 1} Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

• Medicine

  
  A Novel Non-invasive Method for the Detection of Elevated Intra-compartmental Pressures of the Leg Matthew J. Herring¹, Erin Donohoe¹, Meir T. Marmor^{1 1}Department of Orthopedic Surgery, University of California, San Francisco An ultrasound probe coupled with a pressure sensor is used to assess the intra-compartmental pressure of the leg by directly measuring the compartment fascia flattening pressure (CFFP). This non-invasive protocol will provide reliable assessment of the pressure inside the anterior muscular compartment of the lower leg.

• Neuroscience

  
  Convection Enhanced Delivery of Optogenetic Adeno-associated Viral Vector to the Cortex of Rhesus Macaque Under Guidance of Online MRI Images Karam Khateeb^1,2, Devon J. Griggs^2,3, Philip N. Sabes⁴, Azadeh Yazdan-Shahmorad^{1,2,3,4 1}Departments 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.

• Bioengineering

  
  Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies Ana C. Silva^1,2,3,4, Maria J. Oliveira^1,2,5, Todd C McDevitt^4,6, Mário A. Barbosa^1,2,3, Diana S. Nascimento^1,2, Perpétua Pinto-do-Ó^{1,2,3 1}i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, ²INEB - Instituto de Engenharia Biomédica, Universidade do Porto, ³Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, ⁴Gladstone Institute of Cardiovascular Disease, ⁵Faculty of Medicine, University of Porto, ⁶University of California San Francisco The cardiac extracellular matrix (ECM) is a complex network of molecules that orchestrate key processes in tissues and organs while enduring physiological remodeling throughout life. Standardized decellularization of fetal and adult hearts permits comparative experimental studies of both tissues in a 3D context by capturing native architecture and biomechanical properties.

• Cancer Research

  
  Establishing Cell Lines Overexpressing DR3 to Assess the Apoptotic Response to Anti-mitotic Therapeutics Xin Wang *¹, Jiamin Zhou*², Chen Qi*¹, Gelin Wang^{1,3 1}School of Pharmaceutical Sciences, Tsinghua University, ²Department of Radiology, University of California, San Francisco, ³Beijing Advanced Innovation Center for Structural Biology Establishing a stable cell line overexpressing a gene of interest to study gene function can be done by stable transfection-picking single clones after transfecting them via retroviral infection. Here we show that HT29-DR3 cell lines generated in this way elucidate the mechanisms by which death receptor 3 (DR3) contributes to antimitotics-induced apoptosis.

• Neuroscience

  
  Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats Michael Dietrich¹, Christina Hecker¹, Alexander Hilla², Andrés Cruz-Herranz³, Hans-Peter Hartung¹, Dietmar Fischer², Ari Green³, Philipp Albrecht^{1 1}Department of Neurology, Heinrich-Heine-University Düsseldorf, ²Department of Cell Physiology, Faculty of Biology and Biotechnology, Ruhr-University Bochum, ³Division of Neuroinflammation and Glial Biology, Department of Neurology, University of California San Francisco A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

• Biochemistry

  
  A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9 John S. Chorba¹, Adri M. Galvan², Kevan M. Shokat^{2 1}Division of Cardiology, Department of Medicine, Zuckerberg San Francisco General and University of California San Francisco, ²Department of Cellular and Molecular Pharmacology and Howard Hughes Medical Institute, University of California San Francisco This protocol presents a method to evaluate the proteolytic activity of an intrinsically low-activity, single turnover protease in a cellular context. Specifically, this method is applied to evaluate the proteolytic activity of PCSK9, a key driver of lipid metabolism whose proteolytic activity is required for its ultimate hypercholesterolemic function.

• Developmental Biology

  
  Methods for the Study of Regeneration in Stentor Athena Lin¹, Tatyana Makushok¹, Ulises Diaz¹, Wallace F. Marshall^{1 1}Department of Biochemistry and Biophysics, University of California San Francisco The giant ciliate, Stentor coeruleus, is an excellent system to study regeneration and wound healing. We present procedures for establishing Stentor cell cultures from single cells or cell fragments, inducing regeneration by cutting cells, chemically inducing the regeneration of membranellar band and oral apparatus, imaging, and analysis of cell regeneration.

• Bioengineering

  
  An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing Benjamin Demaree^1,2, Daniel Weisgerber¹, Freeman Lan^1,2, Adam R. Abate^{1,2,3 1}Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California, San Francisco, ²UC Berkeley-UCSF Graduate Program in Bioengineering, University of California, San Francisco, ³Chan Zuckerberg Biohub Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing >50,000 single-cell genomes from diverse cell populations.

• Medicine

  
  Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta Grigol Tediashvili^1,2,3, Dong Wang^1,2,3,4, Hermann Reichenspurner⁴, Tobias Deuse^1,2,3,4, Sonja Schrepfer^{1,2,3,4 1}Transplant and Stem Cell Immunobiology Lab, University Heart Center, ²Department of Surgery, Transplant and Stem Cell Immunobiology Lab, University of California San Francisco (UCSF), ³Cardiovascular Research Center (CVRC) and DZHK German Center for Cardiovascular Research, ⁴Cardiovascular Surgery, University Heart Center This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

• Behavior

  
  A Novel Method for Involving Women of Color at High Risk for Preterm Birth in Research Priority Setting Linda S. Franck^1,2, Monica R. McLemore^1,2, Norlissa Cooper¹, Baylee De Castro³, Anastasia Y. Gordon⁴, Schyneida Williams⁵, Shanell Williams², Larry Rand^{2,3 1}School of Nursing, University of California, San Francisco, ²UCSF California Preterm Birth Initiative, University of California, San Francisco, ³School of Medicine, University of California, San Francisco, ⁴San Francisco Black Infant Health Program, ⁵Homeless Prenatal Program, San Francisco, CA This manuscript describes the Research Prioritization by Affected Communities (RPAC) protocol and findings from its use with women at risk for preterm birth. Using the protocol, women identified and prioritized their unanswered questions about pregnancy, birth and neonatal care aimed at influencing research priority setting by funders and researchers.

• Medicine

  
  Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles Shanmugavel Madasamy¹, David Liu¹, Jason Lundry¹, Benjamin Alderete², Raymond Kong², J. Paul Robinson³, Alan H.B. Wu^1,4, Edward P. Amento^{5 1}Plaxgen Inc, ²Millipore Sigma, ³Cytometry Laboratories, Purdue University, ⁴San Francisco General Hospital, 2M16 Clinical Chemistry, University of California, San Francisco, ⁵Molecular Medicine Research Institute The objective of this study was to evaluate in vitro lipid-lowering drug effects in modulating the morphology of cholesterol particles. Comparison of lipid-lowering drugs revealed variations in their effect in modulating the morphological features of cholesterol particles.

• Developmental Biology

  
  Live Imaging of Mouse Secondary Palate Fusion Seungil Kim¹, Jan Prochazka², Jeffrey O. Bush^{1 1}Department of Cell and Tissue Biology, Program in Craniofacial Biology and Institute for Human Genetics, University of California San Francisco, ²Laboratory of Transgenic Models Diseases, Czech Centre for Phenogenomics, Institute of Molecular Genetics of the Czech Academy of Sciences Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

• 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.

• Medicine

  
  Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats Amara Larpthaveesarp¹, Fernando F. Gonzalez^{1 1}Department of Pediatrics, University of California, San Francisco Neonatal stroke is a significant cause of early brain injury requiring a translational model with consistent focal injury patterns and high reproducibility in order to enable study. This study describes the detailed surgical procedure for creating a non-hemorrhagic, unilateral focal ischemia-reperfusion injury in full-term-equivalent rodents.

• Immunology and Infection

  
  Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation Misty M. Montoya¹, K. Mark Ansel^{1 1}Department of Microbiology & Immunology, Sandler Asthma Basic Research Center, University of California, San Francisco Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

• Biology

  
  Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells Ke Zou^1,2, Diana S. Ren², Qi Ou-yang^1,3, Hao Li², Jiashun Zheng^{2 1}The State Key Laboratory for Artificial Microstructures and Mesoscopic Physics, School of Physics, Peking University, ²Department of Biochemistry and Biophysics, University of California, San Francisco, ³Peking-Tsinghua Center for Life Sciences at Center for Quantitative Biology, Academy for Advanced Interdisciplinary Studies, Peking University This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

• Medicine

  
  Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency Dong Wang^1,2,3,4, Grigol Tediashvili^1,2,3, Simon Pecha⁴, Hermann Reichenspurner⁴, Tobias Deuse^1,2,3,4, Sonja Schrepfer^{1,2,3,4 1}Transplant and Stem Cell Immunobiology Lab, University Heart Center Hamburg, ²Department of Surgery, Transplant and Stem Cell Immunobiology Lab, University of California San Francisco (UCSF), ³Cardiovascular Research Center (CVRC) and DZHK German Center for Cardiovascular Research), partner site Hamburg/Kiel/Luebeck, ⁴Cardiovascular Surgery, University Heart Center Hamburg This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

• Biochemistry

  
  Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram Jonathan A. Coleman¹, Evan M. Green², Eric Gouaux^{1,3 1}Vollum Institute, Oregon Health & Science University, ²Graduate Group in Biophysics, University of California, San Francisco, ³Howard Hughes Medical Institute, Oregon Health & Science University This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

• Biochemistry

  
  Patch Clamp Recordings on Intact Dorsal Root Ganglia from Adult Rats Kerui Gong¹, Peter T. Ohara², Luc Jasmin^{1 1}Department of Oral and Maxillofacial Surgery, University of California, San Francisco, ²Department of Anatomy, University of California, San Francisco This manuscript describes how to prepare intact dorsal root ganglia for patch clamp recordings. This preparation maintains the microenvironment for neurons and satellite glial cells, thus avoiding the phenotypic and functional changes seen using dissociated DRG neurons.

• Biology

  
  Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes Justin Judd¹, Jonathan Lovas¹, Guo N. Huang^{1 1}Cardiovascular Research Institute, University of California, San Francisco This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

• Immunology and Infection

  
  A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood Clovis S. Palmer^1,2,3, Joshua J. Anzinger⁴, Tiffany R. Butterfield⁴, Joseph M. McCune⁵, Suzanne M. Crowe^{1,2,6 1}Centre for Biomedical Research, Macfarlane Burnet Institute for Medical Research and Public Health, ²Department of Infectious Diseases, Monash University, ³Department of Microbiology and Immunology, University of Melbourne, ⁴Department of Microbiology, The University of the West Indies, ⁵Division of Experimental Medicine, Department of Medicine, University of California, San Francisco, ⁶Department of Medicine, Monash University Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

• Immunology and Infection

  
  Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface Gabrielle A. Rizzuto^1,2, Mirhan Kapidzic³, Matthew Gormley³, Anna I. Bakardjiev^{1,4,5 1} A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

• Bioengineering

  
  Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers Russell H. Cole¹, Zev J. Gartner¹, Adam R. Abate^{2 1}Department of Pharmaceutical Chemistry, University of California, San Francisco, ²Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California, San Francisco Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

• Medicine

  
  Measuring Cardiac Autonomic Nervous System (ANS) Activity in Toddlers - Resting and Developmental Challenges Nicole R. Bush^1,2, Zoe K. Caron¹, Katherine S. Blackburn¹, Abbey Alkon^{3 1}Department of Psychiatry, University of California, San Francisco, ²Department of Pediatrics, University of California, San Francisco, ³School of Nursing, University of California, San Francisco We describe the methods for continuous monitoring of the autonomic nervous system under resting and challenge conditions with 18 month old children. Results revealed that this protocol can produce meaningful physiological responses in both branches of the autonomic nervous system and elicit significant individual variability in patterns of responses.

• Medicine

  
  Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma Aaron J. Clark¹, Shayan Fakurnejad², Quanhong Ma², Rintaro Hashizume^{2,3 1}Department of Neurological Surgery, University of California San Francisco, ²Department of Neurological Surgery, Northwestern University Feinberg School of Medicine, ³Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine GL261 glioma cells provide a useful immunocompetent animal model of glioblastoma. The goals of this protocol are to demonstrate proper techniques for monitoring intracranial tumor growth using in vivo bioluminescence imaging, and to verify the utility of luciferase-modified GL261 cells for studying tumor immunology and immunotherapeutic approaches for treating glioblastoma.

• Bioengineering

  
  Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device Russell H. Cole¹, Tuan M. Tran², Adam R. Abate^{3,4 1}Department of Pharmaceutical Chemistry, University of California, San Francisco, ²Joint UCSF/UCB Bioengineering Graduate Group, University of California, San Francisco, ³Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, ⁴California Institute for Quantitative Biosciences, University of California, San Francisco Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 µm.

• Developmental Biology

  
  Assaying Blood Cell Populations of the Drosophila melanogaster Larva Sophia Petraki¹, Brandy Alexander¹, Katja Brückner^{1,2,3 1}Department of Cell and Tissue Biology, University of California San Francisco, ²Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, ³Cardiovascular Research Institute, University of California San Francisco Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

• Medicine

  
  miRNA Expression Analyses in Prostate Cancer Clinical Tissues Nathan Bucay¹, Varahram Shahryari¹, Shahana Majid¹, Soichiro Yamamura¹, Yozo Mitsui¹, Z. Laura Tabatabai¹, Kirsten Greene¹, Guoren Deng¹, Rajvir Dahiya¹, Yuichiro Tanaka¹, Sharanjot Saini^{1 1}Department of Urology, Veterans Affairs Medical Center, San Francisco, University of California San Francisco Here we describe a simplified protocol for microRNA (miRNA) expression analyses in archived Formalin-Fixed, Paraffin-Embedded (FFPE) or fresh frozen prostate cancer (PCa) clinical tissues employing quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH).

• Behavior

  
  A Cognitive Paradigm to Investigate Interference in Working Memory by Distractions and Interruptions Jacki Janowich¹, Jyoti Mishra², Adam Gazzaley^{2,3,4 1}Department of Psychology, University of New Mexico, ²Department of Neurology, University of California, San Francisco, ³Department of Physiology, Center for Integrative Neuroscience, University of California, San Francisco, ⁴Department of Psychiatry, University of California, San Francisco A novel cognitive paradigm is developed to elucidate behavioral and neural correlates of interference by to-be-ignored distractors versus interference by to-be-attended interruptors during a working memory task. In this manuscript, several variants of this paradigm are detailed, and data obtained with this paradigm in younger/older adult participants is reviewed.

• Developmental Biology

  
  Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence (MGE) Cells for In Vivo Studies Daniel Vogt¹, Pei-Rung Wu¹, Shawn F. Sorrells², Christine Arnold², Arturo Alvarez-Buylla², John L. R. Rubenstein^{1 1}Department of Psychiatry, University of California San Francisco, ²Department of Neurological Surgery, University of California San Francisco GABAergic cortical interneuron progenitors disperse, develop and synaptically integrate into a host cortex after transplantation. These cells can be easily transduced before transplantation for in vivo studies of genetically modified GABAergic precursors. Here, we show viral labeling techniques to target specific interneuron subgroups using existing Cre lines and Cre-dependent reporters.

• Developmental Biology

  
  Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart Lisa D. Wilsbacher^1,2, Shaun R. Coughlin^{2 1}Feinberg Cardiovascular Research Institute, Northwestern University, ²Cardiovascular Research Institute, University of California, San Francisco Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

• Biology

  
  Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry Heather Inglis¹, Philip Norris^1,2,3, Ali Danesh^{1,3 1}Blood Systems Research Institute, ²Department of Medicine, University of California, San Francisco, ³Department of Laboratory Medicine, University of California, San Francisco Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

• Neuroscience

  
  Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane Omar Akil¹, Stephanie L. Rouse¹, Dylan K. Chan¹, Lawrence R. Lustig^{1 1}Department of Otolaryngology-Head and Neck Surgery, University of California, San Francisco The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

• Neuroscience

  
  Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord Jason G. Weinger¹, Milton L. Greenberg², Melanie P. Matheu⁴, Ian Parker³, Craig M. Walsh¹, Thomas E. Lane⁵, Michael D. Cahalan^{2 1}Molecular Biology and Biochemistry, University of California, Irvine, ²Physiology and Biophysics, University of California, Irvine, ³Neurobiology and Behavior, University of California, Irvine, ⁴University of California San Francisco Diabetes Center, University of California, San Francisco, ⁵Pathology, University of Utah A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

• Medicine

  
  Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis. Shun-Tai Yang¹, Ana Rodriguez-Hernandez², Espen J. Walker¹, William L. Young^1,2,3, Hua Su¹, Michael T. Lawton^{2 1}Department of Anesthesia and Perioperative Care and Center for Cerebrovascular Research, University of California, San Francisco, ²Department of Neurological Surgery, University of California, San Francisco, ³Department of Neurology, University of California, San Francisco We describe a method for creating a reliable model of cerebral venous hypertension in the adult mouse. This model has been widely described and tested in the rat. This new counterpart in the mice opens the possibility of using genetic modified animals and thereby broadens the applications of the model.

• Immunology and Infection

  
  Scalable High Throughput Selection From Phage-displayed Synthetic Antibody Libraries Shane Miersch^1,2, Zhijian Li^1,2, Rachel Hanna^1,2, Megan E. McLaughlin^1,2, Michael Hornsby^1,3, Tet Matsuguchi^1,3, Marcin Paduch^1,4, Annika Sääf^1,4, Jim Wells^1,3, Shohei Koide^1,4, Anthony Kossiakoff^1,4, Sachdev S. Sidhu^{1,2 1}The Recombinant Antibody Network, ²The Banting and Best Department of Medical Research, University of Toronto, ³Antibiome Center, University of California, San Francisco at Mission Bay, ⁴Department of Biochemistry and Molecular Biology, The University of Chicago A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

• Biology

  
  Recovery of Adult Zebrafish Hearts for High-throughput Applications Rima Arnaout¹, Sven Reischauer^2,3, Didier Y.R. Stainier^{2,3 1}Cardiovascular Research Institute and Division of Cardiology, University of California San Francisco, ²Department for Biochemistry and Biophysics, University of California San Francisco, ³Max-Planck Institute for Heart and Lung Research Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

• Biology

  
  Manipulating the Murine Lacrimal Gland Jennifer K. Finley¹, D'Juan Farmer¹, Elaine Emmerson¹, Noel Cruz Pacheco¹, Sarah M. Knox^{1 1}Craniofacial and Mesenchymal Biology, Cell and Tissue Biology, University of California San Francisco The lacrimal gland (LG) is a branching organ that produces the aqueous components of tears necessary for maintaining vision and ocular health. Here we describe murine LG dissection and ex vivo culture techniques to decipher signaling pathways involved in LG development.

• 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.

• Medicine

  
  Ultrasound Assessment of Endothelial-Dependent Flow-Mediated Vasodilation of the Brachial Artery in Clinical Research Hugh Alley^1,2,3, Christopher D. Owens^1,2,3, Warren J. Gasper^1,2,3, S. Marlene Grenon^{1,2,3 1}Department of Surgery, University of California, San Francisco, ²Department of Surgery, Veterans Affairs Medical Center, San Francisco, ³VipeRx Lab, Veterans Affairs Medical Center, San Francisco Endothelial dysfunction is associated with numerous disease states and is predictive of adverse cardiovascular events in humans. Flow-mediated vasodilation (FMD) is a non-invasive ultrasound method of evaluating endothelial function. Methodological choices and operator experience may affect results. A systematic approach to FMD in human studies is discussed here.

• Bioengineering

  
  Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding Philip Chung¹, J. Alex Heller¹, Mozziyar Etemadi¹, Paige E. Ottoson², Jonathan A. Liu³, Larry Rand², Shuvo Roy^{1 1}Department of Bioengineering & Therapeutic Sciences, University of California, San Francisco, ²Department of Obstetrics, Gynecology & Reproductive Sciences, University of California, San Francisco, ³Keck School of Medicine, University of Southern California We have devised a method for low-cost and rapid prototyping of liquid elastomer rubber injection molded devices by using fused deposition modeling 3D printers for mold design and a modified desiccator as a liquid injection system.

• Biology

  
  Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras Jennifer L. Fish¹, Richard A. Schneider^{1 1}Department of Orthopaedic Surgery, University of California at San Francisco This article describes a method to generate chimeric embryos that are designed to test the species-specific contributions of neural crest and/or other tissues to craniofacial development.

• Biology

  
  Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor Miquella G. Chavez^1,2, Jimmy Hu¹, Kerstin Seidel¹, Chunying Li^1,3, Andrew Jheon¹, Adrien Naveau^4,5,6, Orapin Horst^1,7, Ophir D. Klein^{1,8 1}Department of Orofacial Sciences and Program in Craniofacial and Mesenchymal Biology, University of California, San Francisco, ²Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, ³Department of Pathology and Research Center, Zhongshan Hospital of Dalian University, ⁴Université Paris Descartes, Sorbonne Paris Cite, UMR S872, ⁵Centre de Recherche des Cordeliers, Université Pierre et Marie Curie, UMR S872, ⁶INSERM U872, ⁷Division of Endodontics, Department of Preventive and Restorative Dental Sciences, University of California, San Francisco, ⁸Department of Pediatrics and Institute for Human Genetics, University of California, San Francisco The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

• Engineering

  
  Picoinjection of Microfluidic Drops Without Metal Electrodes Brian O'Donovan¹, Tuan Tran¹, Adam Sciambi¹, Adam Abate^{1 1}Department of Bioengineering and Therapeutic Sciences, Unversity of California, San Francisco We have developed a technique for picoinjecting microfluidic drops that does not require metal electrodes. As such, devices incorporating our technique are simpler to fabricate and to use.

• Bioengineering

  
  In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint Andrew T. Jang¹, Jeremy D. Lin¹, Youngho Seo², Sergey Etchin³, Arno Merkle³, Kevin Fahey³, Sunita P. Ho^{1 1}Division of Biomaterials and Bioengineering, Department of Preventive and Restorative Dental Sciences, University of California San Francisco, ²Department of Radiology and Biomedical Imaging, University of California San Francisco, ³Xradia Inc. In this study, the use of an in situ loading device coupled with micro-X-ray computed tomography for fibrous joint biomechanics will be discussed. Experimental readouts identifiable with an overall change in joint biomechanics will include: 1) reactionary force vs. displacement, i.e. tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics, i.e. geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. concentric or eccentric loads.

• Biology

  
  The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis Jillian M. Silva¹, Martin McMahon^{1 1}Diller Cancer Research Building, University of California, San Francisco This protocol explores the latest advancements in performing Western blot analyses. These novel modifications employ a Bis-Tris gel system with a 35 min electrophoresis run time, a 7 min dry blotting transfer system, and infrared fluorescent protein detection and imaging that generates higher resolution, quality, sensitivity, and improved accuracy of Western data.

• Biology

  
  Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus Mark Slabodnick^1,2, Bram Prevo^1,3, Peter Gross^1,4, Janet Sheung^1,5, Wallace Marshall^{1,2 1}Physiology Course, Marine Biological Laboratory, ²Department of Biochemistry & Biophysics, University of California San Francisco, ³Department of Physics and Astronomy, Vrije Universiteit Amsterdam, ⁴Max Planck Institute of Molecular Cell Biology and Genetics, ⁵Department of Physics, University of Illinois Urbana-Champaign The giant ciliate Stentor coeruleus is a classical system for studying regeneration and wound healing in single cells. By imaging Stentor cells simultaneously at low and high magnification it is possible to measure cytoplasmic flows before, during, and after wounding.

• Bioengineering

  
  Insertion of Flexible Neural Probes Using Rigid Stiffeners Attached with Biodissolvable Adhesive Sarah H. Felix¹, Kedar G. Shah¹, Vanessa M. Tolosa¹, Heeral J. Sheth¹, Angela C. Tooker¹, Terri L. Delima¹, Shantanu P. Jadhav², Loren M. Frank², Satinderpall S. Pannu^{1 1}Materials Engineering Division, Lawrence Livermore National Laboratory, ²UCSF Center for Integrative Neuroscience and the Department of Physiology, University of California, San Francisco Insertion of flexible neural microelectrode probes is enabled by attaching probes to rigid stiffeners with polyethylene glycol (PEG). A unique assembly process ensures uniform and repeatable attachment. After insertion into tissue, the PEG dissolves and the stiffener is extracted. An in vitro test method evaluates the technique in agarose gel.

• Immunology and Infection

  
  Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions Kevin Wilhelmsen¹, Katherine Farrar^1,2, Judith Hellman^{1 1}Department of Anesthesia and Perioperative Care, University of California, San Francisco, ²Graduate Program in Biomedical Sciences, University of California, San Francisco Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

• Medicine

  
  The Goeckerman Regimen for the Treatment of Moderate to Severe Psoriasis Rishu Gupta^1,2, Maya Debbaneh^2,3, Daniel Butler^2,4, Monica Huynh^2,5, Ethan Levin², Argentina Leon², John Koo², Wilson Liao^{2 1}Keck School of Medicine, University of Southern California, ²Psoriasis and Skin Treatment Center Dermatology, University of California, San Francisco, ³University of California Irvine School of Medicine, ⁴University of Arizona College of Medicine, ⁵Chicago College of Osteopathic Medicine Psoriasis is a chronic, immune-mediated inflammatory skin disease. The Goeckerman regimen, formulated for the treatment of psoriasis, consists of exposure to ultraviolet B (UVB) light and application of crude coal tar (CCT). The following protocol is for the administration of Goeckerman therapy for the treatment of moderate-to-severe psoriasis.

• Bioengineering

  
  Magnetically-Assisted Remote Controlled Microcatheter Tip Deflection under Magnetic Resonance Imaging Steven W. Hetts¹, Maythem Saeed¹, Alastair Martin¹, Prasheel Lillaney¹, Aaron Losey², Erin Jeannie Yee¹, Ryan Sincic³, Loi Do¹, Lee Evans¹, Vincent Malba¹, Anthony F. Bernhardt¹, Mark W. Wilson¹, Anand Patel¹, Ronald L. Arenson⁴, Curtis Caton⁵, Daniel L. Cooke^{1 1}Department of Radiology and Biomedical Imaging, University of California, San Francisco, ²School of Medicine, University of California, San Francisco, ³Department of Radiology and Biomedical Imaging, UCSF Medical Center, ⁴University of California, San Francisco, ⁵Hansen Medical, Mountain View, CA Current applied to an endovascular microcatheter with microcoil tip made by laser lathe lithography can achieve controllable deflections under magnetic resonance (MR) guidance, which may improve speed and efficacy of navigation of vasculature during various endovascular procedures.

• Biology

  
  Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells Ulrike Liisberg Aune^1,2,3, Lauren Ruiz¹, Shingo Kajimura^{1 1}UCSF Diabetes Center and Department of Cell and Tissue Biology, University of California, San Francisco, ²Department of Biology, University of Copenhagen, Denmark, ³National Institute of Nutrition and Seafood Research, Bergen, Norway Primary white preadipocytes isolated from white adipose tissues in mice can be differentiated into beige/brite cells. Presented here is a reliable cellular model system to study the molecular regulation of "browning" of white fat.

• Neuroscience

  
  Forebrain Electrophysiological Recording in Larval Zebrafish Scott C. Baraban^{1 1}Epilepsy Research Laboratory, Department of Neurological Surgery, University of California, San Francisco A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.

• Neuroscience

  
  A Molecular Readout of Long-term Olfactory Adaptation in C. elegans Chao He¹, Jin I. Lee², Noelle L'Etoile³, Damien O'Halloran^{1 1}Department of Biological Sciences and Institute for Neuroscience, George Washington University, ²Fred Hutchinson Cancer Research Center, ³Department of Cell and Tissue Biology, University of California San Francisco Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

• Medicine

  
  Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate David D. Lo*^1,2, Jeong S. Hyun*^1,3, Michael T. Chung¹, Daniel T. Montoro¹, Andrew Zimmermann¹, Monica M. Grova^1,4, Min Lee⁵, Derrick C. Wan¹, Michael T. Longaker^{1 1}Department of Surgery, Stanford University, ²Department of Surgery, Duke University, ³Department of Surgery, Saint Joseph Mercy Hospital, ⁴School of Medicine, University of California, San Francisco, ⁵School of Dentistry, University of California, Los Angeles This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

• Biology

  
  An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images Carolina L. Haass-Koffler^1,2, Mohammad Naeemuddin¹, Selena E. Bartlett^{1,3 1}Medications Development, Ernest Gallo Clinic and Research Center, University of California, San Francisco, ²Clinical Pharmacology and Experimental Therapeutics, University of California, San Francisco, ³Translational Research Institute and the Institute for Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Australia We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

• Biology

  
  Synthesis of an In vivo MRI-detectable Apoptosis Probe Justin Lam¹, Paul C. Simpson^2,3, Phillip C. Yang¹, Rajesh Dash^{1 1}Division of Cardiovascular Medicine, Department of Medicine, Stanford University Medical Center, ²Division of Cardiology, Department of Medicine, University of California, San Francisco, ³San Francisco VAMC Early detection of apoptosis may identify at-risk cell populations in a variety of diseases. Here we demonstrate a method to link an early apoptosis-detection protein (Annexin V) to a MRI-detectable iron oxide nanoparticle (SPIO). This method may be extended to other proteins of interest to generate MRI-detectable molecular imaging probes.

• Medicine

  
  An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment Moo Rim Kang*¹, Glen Yang*¹, Klaus Charisse², Hila Epstein-Barash², Muthiah Manoharan², Long-Cheng Li^{1 1}Department of Urology and Helen Diller Comprehensive Cancer Center, University of California, San Francisco, ²Alnylam Pharmaceuticals, Inc. Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

• Medicine

  
  Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification, Distraction Osteogenesis, or Healing of Critical Sized Defects Yan-yiu Yu¹, Chelsea Bahney¹, Diane Hu¹, Ralph S. Marcucio¹, Theodore Miclau, III^{1 1}Department of Orthopaedic Surgery, University of California, San Francisco This article describes a method for stabilizing long bone fractures that is based on the application of modified Ilizarov external fixators ^1-3. After application of the fixators and creation of the bone injury, healing can be assessed, distraction osteogenesis can be performed, or non-union or critical sized defect can be created and used to study therapeutic interventions.

• Neuroscience

  
  In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy Dimitrios Davalos¹, Katerina Akassoglou^{1,2 1}Gladstone Institute of Neurological Disease, University of California, San Francisco, ²Department of Neurology, University of California, San Francisco A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

• Bioengineering

  
  Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition David S. Booth¹, Agustin Avila-Sakar², Yifan Cheng^{2 1}Graduate Group in Biophysics, University of California San Francisco, ²Department of Biochemistry and Biophysics, University of California San Francisco Visualizing protein samples by negative stain electron microscopy (EM) has become a popular structural analysis method. It is useful for quantitative structural analysis, such as calculating a 3D reconstruction of the molecules being studied, and also for qualitative examination of the quality of protein preparations. In this article we present detailed protocols for preparing the EM grids, staining the sample and visualizing the sample in an electron microscope. Novice users can follow these protocols easily and to utilize negative stain EM as a routine assay, in addition to other biochemical assays, for evaluating their protein samples.

• Neuroscience

  
  Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis Espen J. Walker¹, Fanxia Shen¹, William L. Young^1,2,3, Hua Su^{1 1}Center for Cerebrovascular Research, Department of Anesthesia and Perioperative Care, University of California, San Francisco, ²Department of Neurological Surgery, University of California, San Francisco, ³Department of Neurology, University of California, San Francisco In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.

• Medicine

  
  Guide Wire Assisted Catheterization and Colored Dye Injection for Vascular Mapping of Monochorionic Twin Placentas Eric B. Jelin¹, Samuel C. Schecter¹, Kelly D. Gonzales¹, Shinjiro Hirose¹, Hanmin Lee¹, Geoffrey A. Machin², Larry Rand³, Vickie A. Feldstein^{4 1}Division of Pediatric and Fetal Surgery, Department of Surgery, University of California, San Francisco, ²Department of Pathology, University of Alberta, ³Department of Obstretics and Gynecology, University of California, San Francisco, ⁴Department of Radiology, University of California, San Francisco Vascular mapping of monochorionic (MC) twin placentas after birth provides a means for detailed demonstration of vascular connections between the twins’ circulations. Imbalance of these connections is thought to play a pivotal role in the development of complications of MC twinning including twin-to-twin transfusion syndrome.

• Bioengineering

  
  High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs Felix Moltzahn^1,2,3, Nathan Hunkapiller^1,2,4, Alain A. Mir⁵, Tal Imbar^1,2,6, Robert Blelloch^{1,2,3,7 1}The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, ²Center for Reproductive Sciences, University of California San Francisco, ³Department of Urology, University of California San Francisco, ⁴Department of Cell and Tissue Biology, University of California San Francisco, ⁵Fluidigm Corporation, Fluidigm Corporation, ⁶Department of Obstetrics and Gynecology, Hadassah-Hebrew University Medical Center, ⁷UCSF - Helen Diller Family Comprehensive Cancer Center, University of California San Francisco Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

• Immunology and Infection

  
  Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens Eunice C. Chen¹, Steve A. Miller¹, Joseph L. DeRisi^1,2, Charles Y. Chiu^{1,2 1}Department of Laboratory Medicine, University of California, San Francisco, ²Division of Infectious Diseases, University of California, San Francisco The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

• Neuroscience

  
  Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain Zhiqiang Dong¹, Mahendra Wagle¹, Su Guo^{1 1}Department of Bioengineering and Therapeutic Sciences, Programs in Human Genetics and Biological Sciences , University of California San Francisco The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

• Biology

  
  Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development Diane Hu¹, Ralph S. Marcucio^{1 1}Department of Orthopaedic Surgery, University of California San Francisco This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of surface cephalic ectoderm during craniofacial development.

• Medicine

  
  Myo-mechanical Analysis of Isolated Skeletal Muscle Peter E. Oishi^1,2, Sompob Cholsiripunlert³, Wenhui Gong², Anthony J. Baker⁴, Harold S. Bernstein^{1,2,5 1}Cardiovascular Research Institute, University of California San Francisco, ²Department of Pediatrics, University of California San Francisco, ³Department of Biology, San Francisco State University, ⁴Department of Medicine, University of California San Francisco, ⁵Eli and Edythe Broad Center of Regeneration Medicine & Stem Cell Research, University of California San Francisco To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

• Medicine

  
  Mouse Model of Middle Cerebral Artery Occlusion Terrance Chiang¹, Robert O. Messing¹, Wen-Hai Chou^{1,2 1}Department of Neurology, Ernest Gallo Clinic and Research Center, University of California, San Francisco, ²Department of Biological Sciences, Kent State University We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

• Neuroscience

  
  A Novel Method for Assessing Proximal and Distal Forelimb Function in the Rat: the Irvine, Beatties and Bresnahan (IBB) Forelimb Scale Karen-Amanda Irvine¹, Adam R. Ferguson¹, Kathleen D. Mitchell¹, Stephanie B. Beattie¹, Michael S. Beattie¹, Jacqueline C. Bresnahan^{1 1}Department of Neurological Surgery, University of California, San Francisco Here we will describe a rodent behavioral assay that can detect recovery of both proximal and distal forelimb function including digit movements during a naturally occurring behavior that does not require extensive training or deprivation to enhance motivation.

• Neuroscience

  
  Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models Laura Serwer¹, Rintaro Hashizume¹, Tomoko Ozawa¹, C. David James^{1 1}Department of Neurological Surgery, University of California, San Francisco - UCSF Thorough preclinical testing of drugs that act in the central nervous system often involves assessing and comparing drug biodistribution in association with specific routes of administration. Here, three commonly used methods of systemic delivery (intravenous, intraperitoneal, and oral) as well as a method for local delivery (convection-enhanced delivery) are demonstrated in mice.

• Biology

  
  Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation Carissa Ritner¹, Harold S. Bernstein^{1 1}Cardiovascular Research Institute, Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

• Neuroscience

  
  Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging Tomoko Ozawa¹, C. David James^{1 1}Department of Neurological Surgery, University of California, San Francisco - UCSF Luciferase-modified human brain tumor xenografts can be established intracranially in athymic mice, with subsequent monitoring of tumor growth and response to therapy using bioluminescence imaging. In combination with survival analysis, bioluminescence monitoring is an essential research tool for pre-clinical testing of therapies being considered for treating brain tumors.

• Neuroscience

  
  The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow Zaman Mirzadeh¹, Fiona Doetsch^2,3, Kazunobu Sawamoto⁴, Hynek Wichterle^2,5, Arturo Alvarez-Buylla^{1 1}Department of Neurosurgery, The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco - UCSF, ²Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, ³Department of Neuroscience and Neurology, College of Physicians and Surgeons, Columbia University, ⁴Department of Developmental and Regenerative Biology, Nagoya City University Graduate School of Medical Sciences, ⁵Center for Motor Neuron Biology and Disease, College of Physicians and Surgeons, Columbia University The lateral ventricle walls contain the largest germinal region in the adult mammalian brain. Traditionally, studies on neurogenesis in this region have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region.

• Biology

  
  Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects Alexander J. Nedopil¹, Lydia G. Mandrussow¹, Heike E. Daldrup-Link^{1 1}Department of Radiology and Biomedical Imaging, Medical Center, University of California San Francisco Goal of the presentation is to demonstrate a highly reproducible method to generate matrix associated stem cell implants in cartilage defects, which can be visualized with MR imaging. Stem cells are labeled with FDA-approved Ferumoxides, mixed with agarose, implanted into cartilage defects and imaged with a 7T MR scanner.

• Biology

  
  Lentivirus Production Xiaoyin Wang¹, Michael McManus^{1 1}Diabetes Center, University of California, San Francisco - UCSF To make lentiviruses, DNA vectors are transfected into human 293 cells. After harvest and concentrating the supernatant, virus titer is determined by fluorescence expression with a flow cytometer.

• Biology

  
  A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples Eric Johansen¹, Birgit Schilling², Michael Lerch¹, Richard K. Niles¹, Haichuan Liu¹, Bensheng Li², Simon Allen¹, Steven C. Hall¹, H. Ewa Witkowska¹, Fred E. Regnier³, Bradford W. Gibson², Susan J. Fisher¹, Penelope M. Drake^{1 1}Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco - UCSF, ²Buck Institute for Age Research, ³Department of Chemistry, Purdue University Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.

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  Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs Hui Zhang^1,2, Niko G. Gubernator^3,4, Minerva Yue¹, Roland G. W. Staal¹, Eugene V. Mosharov¹, Daniela Pereira¹, Vojtech Balsanek³, Paul A. Vadola³, Bipasha Mukherjee⁵, Robert H. Edwards⁵, David Sulzer^1,2,6, Dalibor Sames^{3 1}Departments of Neurology, Columbia University, ²Departments of Psychiatry and Pharmacology, Columbia University, ³Department of Chemistry, Columbia University, ⁴eMolecules, Inc., ⁵Departments of Neurology and Physiology, University of California School of Medicine, San Francisco, ⁶Division of Molecular Therapeutics, New York Psychiatric Institute A new means to measure neurotransmission optically using fluorescent dopamine analogs.

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  Murine Model of Hindlimb Ischemia Hiroshi Niiyama¹, Ngan F. Huang¹, Mark D. Rollins², John P. Cooke^{1 1}Division of Cardiovascular Medicine, Stanford University, ²Department of Anesthesiology, University of California, San Francisco The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

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  Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging Sophie Boddington¹, Tobias D. Henning¹, Elizabeth J. Sutton¹, Heike E. Daldrup-Link^{1 1}Contrast Agent Research Group at the Center for Molecular and Functional Imaging, Department of Radiology, University of California San Francisco This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

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  Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging Tobias D. Henning¹, Sophie Boddington¹, Heike E. Daldrup-Link^{1 1}Contrast Agent Research Group at the Center for Molecular and Functional Imaging, Department of Radiology, University of California San Francisco For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

• Biology

  
  Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin Jyoti Srivastava¹, Diane Barber^{1 1}Department of Cell and Tissue Biology, University of California, San Francisco - UCSF Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

• Biology

  
  Orthotopic Mouse Model of Colorectal Cancer William Tseng^1,2, Xianne Leong², Edgar Engleman^{2 1}Department of Surgery, University of California, San Francisco - UCSF, ²Department of Pathology, Stanford University School of Medicine Two techniques can be used to establish this model: injection of a cancer cell suspension into the cecal wall or transplantation of a piece of subcutaneous tumor onto the cecum. This model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

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  Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice Gregory L. Szot¹, Pavel Koudria¹, Jeffrey A. Bluestone^{1 1}Diabetes Center, University of California, San Francisco - UCSF Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of mice. Cells are concentrated into pellets in the final tubing used for transplanting the cells under the kidney capsule. The ease of this technique reduces stress to the cells and the mouse.

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  Murine Pancreatic Islet Isolation Gregory L. Szot¹, Pavel Koudria¹, Jeffrey A. Bluestone^{1 1}Diabetes Center, University of California, San Francisco - UCSF

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  Investigating the Immunological Mechanisms Underlying Organ Transplant Rejection Sang Mo Kang^{1 1}Department of Surgery, University of California, San Francisco - UCSF

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  Regulatory T cells: Therapeutic Potential for Treating Transplant Rejection and Type I Diabetes Jeffry A. Bluestone^{1 1}Diabetes Center, University of California, San Francisco - UCSF

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  Small Bowel Transplantation In Mice Fengchun Liu¹, Sang-Mo Kang^{1 1}Department of Surgery, University of California, San Francisco - UCSF The mouse small bowel transplantation model has been recognized as an important tool to study mechanismes of immune rejection and screen new immunosuppressive drugs. However, this model is limited to use because the techniques involved is an extremely technically challenge. Now we introduce the modified technique.

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  In Utero Intraventricular Injection and Electroporation of E16 Rat Embryos William Walantus¹, Laura Elias¹, Arnold Kriegstein^{1 1}Institute for Regeneration Medicine, University of California, San Francisco - UCSF

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  Heterotopic Heart Transplantation in Mice Fengchun Liu¹, Sang Mo Kang^{1 1}Department of Surgery, University of California, San Francisco - UCSF The mouse heterotopic heart transplantation model has been proven by many investigators to be an important method for studying mechanisms of rejection and immune response. However, the techniques involved are still challenging. By modifying standard techniques we have had success with more than 1000 transplants.

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  In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos William Walantus¹, David Castaneda¹, Laura Elias¹, Arnold Kriegstein^{1 1}Institute for Regeneration Medicine, University of California, San Francisco - UCSF

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  Cortical Neurogenesis: Transitioning from Advances in the Laboratory to Cell-Based Therapies Arnold R. Kriegstein^{1 1}Institute for Regeneration Medicine, University of California, San Francisco - UCSF

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  Organotypic Slice Culture of E18 Rat Brains Laura Elias¹, Arnold Kriegstein^{1 1}Institute for Regeneration Medicine, University of California, San Francisco - UCSF

• 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.