University of California, San Francisco View Institution's Website 123 articles published in JoVE Medicine Erosion Identification in Metacarpophalangeal Joints in Rheumatoid Arthritis using High-Resolution Peripheral Quantitative Computed Tomography Yousif Al-Khoury1,2, Stephanie Finzel3, Camille Figueiredo4, Andrew J. Burghardt5, Kathryn S. Stok6, Lai-Shan Tam7, Isaac Cheng7, Justin J. Tse1, Sarah L. Manske1 1Department of Radiology, McCaig Institute for Bone and Joint Health, University of Calgary, 2Department of Biomedical Engineering, University of Calgary, 3University Medical Center Freiburg, 4Department of Rheumatology, University of São Paulo, 5Department of Radiology and Biomedical Imaging, University of California, San Francisco, 6Department of Biomedical Engineering, University of Melbourne, 7Department of Medicine and Therapeutics, The Chinese University of Hong Kong Bone erosions are an important pathological feature of rheumatoid arthritis. The purpose of this work is to introduce a training tool to provide users with guidance on identifying pathological cortical breaks on high resolution peripheral quantitative computed tomography images for erosion analysis. 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. Neuroscience Quantifying Pain Location and Intensity with Multimodal Pain Body Diagrams Jereen Kwong*1, Joanna Lin*2, Ryan Leriche*2, Thomas A. Wozny2, Ana Shaughnessy2, Ashlyn Schmitgen2, Prasad Shirvalkar3 1Anesthesiology (Division of Pain Management), University of California, San Francisco, 2Neurological Surgery and UCSF Weill Institute of Neurosciences, University of California, San Francisco, 3Anesthesiology (Division of Pain Management) and Neurological Surgery and UCSF Weill Institute of Neurosciences, University of California, San Francisco Current pain scales used to quantify pain severity, such as visual analog scales, fail to capture the complexity of subjective pain experiences. Pain body diagrams are qualitative but may be more informative. The goal of this method is to extract quantitative metrics from pain body diagrams using novel pressure-hue transformation. Neuroscience Closed-Loop Neurostimulation for Biomarker-Driven, Personalized Treatment of Major Depressive Disorder Kristin K. Sellers1,2, Ankit N. Khambhati1,2, Noah Stapper2,3, Joline M. Fan2,4, Vikram R. Rao2,4, Katherine W. Scangos2,3, Edward F. Chang1,2, Andrew D. Krystal2,3 1Department of Neurological Surgery, University of California, San Francisco, 2Weill Institute for Neurosciences, University of California, San Francisco, 3Department of Psychiatry and Behavioral Sciences, University of California, San Francisco, 4Department of Neurology, University of California, San Francisco Deep brain stimulation triggered by a patient-specific neural biomarker of a high-symptom state may better control symptoms of major depressive disorder compared to continuous, open-loop stimulation. This protocol provides a workflow for identifying a patient-specific neural biomarker and controlling the delivery of therapeutic stimulation based on the identified biomarker. 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. Cancer Research Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays Lillian van Biljon1, Bisiayo Fashemi1, Jeimmy Rodriguez1, Olivia Graham1, Amanda Compadre1, Katherine Fuh1,2, Dineo Khabele1, Mary Mullen1 1Washington University in St. Louis, 2University of California San Francisco The present protocol describes methods for evaluating DNA damage repair proteins in patient-derived ovarian cancer organoids. Included here are comprehensive plating and staining methods, as well as detailed, objective quantification procedures. Cancer Research Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer Irene Chicote1, Jordi Martínez-Quintanilla1, Juan Antonio Cámara2, Héctor G. Palmer1,3 1 This protocol describes the orthotopic implantation of patient-derived cancer cells in the cecum wall of immunodeficient mice. The model recapitulates advanced colorectal cancer metastatic disease and allows for the evaluation of new therapeutic drugs in a clinically relevant scenario of lung and liver metastases. Developmental Biology Antibody Uptake Assay for Tracking Notch/Delta Endocytosis During the Asymmetric Division of Zebrafish Radial Glia Progenitors Xiang Zhao1,2, Su Guo2,3 1Chan Zuckerberg Biohub, 2Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, 3Programs in Human Genetics and Biological Sciences, University of California San Francisco This work develops an antibody uptake assay for imaging intra-lineage Notch/DeltaD signaling in dividing radial glia progenitors of the embryonic zebrafish brain. Cancer Research Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids Olivia Graham*1, Jeimmy Rodriguez*1, Lillian van Biljon1, Bisiayo Fashemi1, Emily Graham1, Katherine Fuh1,2, Dineo Khabele1, Mary Mullen1 1Washington University in St. Louis, 2University of California San Francisco Patient-derived organoids (PDO) are a three-dimensional (3D) culture that can mimic the tumor environment in vitro. In high-grade serous ovarian cancer, PDOs represent a model to study novel biomarkers and therapeutics. Biology Studying Habituation in Stentor coeruleus Deepa Rajan1, Peter Chudinov1, Wallace Marshall1 1Department 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 Rabago1,2,3, Collin M. Blakely1,2, Franziska Haderk1,2,3, Trever G. Bivona1,2,3 1Department 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. Immunology and Infection Isolation of Primary Mouse Lung Endothelial Cells Erika Wong1,2, Nina Nguyen2, Judith Hellman2 1 In this article, primary lung endothelial cells were isolated and cultured from neonatal mice. Developmental Biology Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay Natasja G. J. Costermans1,2, Enrico M. Daldello1,2,3, Ria J. Marathe1,2, Marco Conti1,2 1Center 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. Marshall4 1W. 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. Abate1,2 1Department 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. Abate4,5,6, David V. Schaffer7,8, Lydia L. Sohn9, Zev J. Gartner2,6,10 1Graduate 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 Diaz1,2, Wallace Marshall2, Blake Riggs1 1Department 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. Zhang1,2, Kai-Chun Chang1, Leqian Liu1, Zev J. Gartner3,5, Adam R. Abate1,4,5 1Department 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 Gerona1 1Department 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. Hillel2 1Department 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. Noble1,2 1Department 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 Chen4,5, Kye Y Lee4, Jeanine A Pebbles4, Angela C Tooker4, Vanessa M Tolosa4,5, Loren M Frank2,6 1Medical 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. Muncie1,2, Roberto Falcón-Banchs1, Johnathon N. Lakins2, Lydia L. Sohn1,3, Valerie M. Weaver2,4,5,6 1Graduate 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 Hu1,2, Alessia Gravina2, Sivan G. Marcus1,2, Hao Zhang4, Jeffrey E Olgin4, Tobias Deuse1,2,5, Sonja Schrepfer1,2,3,5 1Transplant 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 Guan1 1Department 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 Iriondo1,2, Vinil Shah1, Emma Bahroos1, Vahid Ravanfar1,1, Melanie Regan1, Youngho Seo1, William P. Dillon1, Sharmila Majumdar1, Jason F. Talbott1,3 1Radiology 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. Clark1 1Department 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. Weiss1,2,3 1Department 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. Bonner1,2, Karim Saoud1, Sulgi Lee1,2, Eshini Panditharatna3, Madhuri Kambhampati1, Sabine Mueller4,5, Javad Nazarian1,2,5 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. Marmor1 1Department 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 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. Bioengineering Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies Ana C. Silva1,2,3,4, Maria J. Oliveira1,2,5, Todd C McDevitt4,6, Mário A. Barbosa1,2,3, Diana S. Nascimento1,2, Perpétua Pinto-do-Ó1,2,3 1i3S - 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 Wang1,3 1School 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 Albrecht1 1Department 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. Shokat2 1Division 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. Marshall1 1Department 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 Demaree1,2, Daniel Weisgerber1, Freeman Lan1,2, Adam R. Abate1,2,3 1Department 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 Tediashvili1,2,3, Dong Wang1,2,3,4, Hermann Reichenspurner4, Tobias Deuse1,2,3,4, Sonja Schrepfer1,2,3,4 1Transplant 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. Franck1,2, Monica R. McLemore1,2, Norlissa Cooper1, Baylee De Castro3, Anastasia Y. Gordon4, Schyneida Williams5, Shanell Williams2, Larry Rand2,3 1School 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. Wu1,4, Edward P. Amento5 1Plaxgen 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. Bush1 1Department 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 Shirtcliff3 1Center 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. Gonzalez1 1Department 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 Ansel1 1Department 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 Zou1,2, Diana S. Ren2, Qi Ou-yang1,3, Hao Li2, Jiashun Zheng2 1The 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 Wang1,2,3,4, Grigol Tediashvili1,2,3, Simon Pecha4, Hermann Reichenspurner4, Tobias Deuse1,2,3,4, Sonja Schrepfer1,2,3,4 1Transplant 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 Gouaux1,3 1Vollum 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 Jasmin1 1Department 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. Huang1 1Cardiovascular 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. Palmer1,2,3, Joshua J. Anzinger4, Tiffany R. Butterfield4, Joseph M. McCune5, Suzanne M. Crowe1,2,6 1Centre 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. Rizzuto1,2, Mirhan Kapidzic3, Matthew Gormley3, Anna I. Bakardjiev1,4,5 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. Abate2 1Department 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. Bush1,2, Zoe K. Caron1, Katherine S. Blackburn1, Abbey Alkon3 1Department 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 Hashizume2,3 1Department 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. Abate3,4 1Department 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 Brückner1,2,3 1Department 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 Saini1 1Department 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 Gazzaley2,3,4 1Department 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. Rubenstein1 1Department 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. Wilsbacher1,2, Shaun R. Coughlin2 1Feinberg 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 Norris1,2,3, Ali Danesh1,3 1Blood 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. Lustig1 1Department 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. Cahalan2 1Molecular 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. Young1,2,3, Hua Su1, Michael T. Lawton2 1Department 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 Miersch1,2, Zhijian Li1,2, Rachel Hanna1,2, Megan E. McLaughlin1,2, Michael Hornsby1,3, Tet Matsuguchi1,3, Marcin Paduch1,4, Annika Sääf1,4, Jim Wells1,3, Shohei Koide1,4, Anthony Kossiakoff1,4, Sachdev S. Sidhu1,2 1The 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 Reischauer2,3, Didier Y.R. Stainier2,3 1Cardiovascular 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. Knox1 1Craniofacial 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 Southard1,4,7, Young-wook Jun1,7, Zev J. Gartner4,5,6,7 1Department 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 Alley1,2,3, Christopher D. Owens1,2,3, Warren J. Gasper1,2,3, S. Marlene Grenon1,2,3 1Department 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 Roy1 1Department 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. Schneider1 1Department 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. Chavez1,2, Jimmy Hu1, Kerstin Seidel1, Chunying Li1,3, Andrew Jheon1, Adrien Naveau4,5,6, Orapin Horst1,7, Ophir D. Klein1,8 1Department 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 Abate1 1Department 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. Ho1 1Division 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 McMahon1 1Diller 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 Slabodnick1,2, Bram Prevo1,3, Peter Gross1,4, Janet Sheung1,5, Wallace Marshall1,2 1Physiology 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. Pannu1 1Materials 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 Farrar1,2, Judith Hellman1 1Department 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 Gupta1,2, Maya Debbaneh2,3, Daniel Butler2,4, Monica Huynh2,5, Ethan Levin2, Argentina Leon2, John Koo2, Wilson Liao2 1Keck 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. Cooke1 1Department 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 Aune1,2,3, Lauren Ruiz1, Shingo Kajimura1 1UCSF 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. Baraban1 1Epilepsy 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'Halloran1 1Department 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. Grova1,4, Min Lee5, Derrick C. Wan1, Michael T. Longaker1 1Department 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-Koffler1,2, Mohammad Naeemuddin1, Selena E. Bartlett1,3 1Medications 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. Simpson2,3, Phillip C. Yang1, Rajesh Dash1 1Division 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 Li1 1Department 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, III1 1Department 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 Akassoglou1,2 1Gladstone 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 Cheng2 1Graduate 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. Young1,2,3, Hua Su1 1Center 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. Feldstein4 1Division 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 Moltzahn1,2,3, Nathan Hunkapiller1,2,4, Alain A. Mir5, Tal Imbar1,2,6, Robert Blelloch1,2,3,7 1The 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. DeRisi1,2, Charles Y. Chiu1,2 1Department 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 Guo1 1Department 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. Marcucio1 1Department 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. Oishi1,2, Sompob Cholsiripunlert3, Wenhui Gong2, Anthony J. Baker4, Harold S. Bernstein1,2,5 1Cardiovascular 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 Chou1,2 1Department 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. Bresnahan1 1Department 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 James1 1Department 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. Bernstein1 1Cardiovascular 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 James1 1Department 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 Doetsch2,3, Kazunobu Sawamoto4, Hynek Wichterle2,5, Arturo Alvarez-Buylla1 1Department 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-Link1 1Department 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 McManus1 1Diabetes 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. Drake1 1Obstetrics, 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. Biology Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs Hui Zhang1,2, Niko G. Gubernator3,4, Minerva Yue1, Roland G. W. Staal1, Eugene V. Mosharov1, Daniela Pereira1, Vojtech Balsanek3, Paul A. Vadola3, Bipasha Mukherjee5, Robert H. Edwards5, David Sulzer1,2,6, Dalibor Sames3 1Departments 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. Biology Murine Model of Hindlimb Ischemia Hiroshi Niiyama1, Ngan F. Huang1, Mark D. Rollins2, John P. Cooke1 1Division 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. Biology Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging Sophie Boddington1, Tobias D. Henning1, Elizabeth J. Sutton1, Heike E. Daldrup-Link1 1Contrast 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. Biology 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-Link1 1Contrast 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 Barber1 1Department 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 Tseng1,2, Xianne Leong2, Edgar Engleman2 1Department 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. Biology Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice Gregory L. Szot1, Pavel Koudria1, Jeffrey A. Bluestone1 1Diabetes 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. Biology Murine Pancreatic Islet Isolation Gregory L. Szot1, Pavel Koudria1, Jeffrey A. Bluestone1 1Diabetes Center, University of California, San Francisco - UCSF Biology Investigating the Immunological Mechanisms Underlying Organ Transplant Rejection Sang Mo Kang1 1Department of Surgery, University of California, San Francisco - UCSF Biology Regulatory T cells: Therapeutic Potential for Treating Transplant Rejection and Type I Diabetes Jeffry A. Bluestone1 1Diabetes Center, University of California, San Francisco - UCSF Biology Small Bowel Transplantation In Mice Fengchun Liu1, Sang-Mo Kang1 1Department 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. Biology In Utero Intraventricular Injection and Electroporation of E16 Rat Embryos William Walantus1, Laura Elias1, Arnold Kriegstein1 1Institute for Regeneration Medicine, University of California, San Francisco - UCSF Biology Heterotopic Heart Transplantation in Mice Fengchun Liu1, Sang Mo Kang1 1Department 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. Biology In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos William Walantus1, David Castaneda1, Laura Elias1, Arnold Kriegstein1 1Institute for Regeneration Medicine, University of California, San Francisco - UCSF Biology Cortical Neurogenesis: Transitioning from Advances in the Laboratory to Cell-Based Therapies Arnold R. Kriegstein1 1Institute for Regeneration Medicine, University of California, San Francisco - UCSF Biology Organotypic Slice Culture of E18 Rat Brains Laura Elias1, Arnold Kriegstein1 1Institute for Regeneration Medicine, University of California, San Francisco - UCSF Biology Ex vivo Mechanical Loading of Tendon Krishna Asundi1, David Rempel2 1Department 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.
