Stanford University View Institution's Website 104 articles published in JoVE Cancer Research Optimized Nuclei Isolation from Fresh and Frozen Solid Tumor Specimens for Multiome Sequencing Deshka S. Foster1, Michelle Griffin1, Michael Januszyk1, Daniel Delitto1, Jeffrey A. Norton1, Michael T. Longaker1 1Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Stanford University The protocol provides a reliable and optimized approach to the isolation of nuclei from solid tumor specimens for multiome sequencing using the 10x Genomics platform, including recommendations for tissue dissociation conditions, cryopreservation of single-cell suspensions, and assessment of isolated nuclei. Medicine Radiation Planning Assistant - A Web-based Tool to Support High-quality Radiotherapy in Clinics with Limited Resources Laurence Edward Court1, Ajay Aggarwal2, Hester Burger3, Carlos Cardenas4, Christine Chung1, Raphael Douglas1, Monique du Toit5, Anuja Jhingran1, Raymond Mumme1, Sikudhani Muya6, Komeela Naidoo5, Jerry Ndumbalo6, Tucker Netherton1, Callistus Nguyen1, Adenike Olanrewaju1, Jeannette Parkes3, Willie Shaw7, Christoph Trauernicht5, Melody Xu8, Jinzhong Yang1, Lifei Zhang1, Hannah Simonds9, Beth M. Beadle10 1The University of Texas MD Anderson Cancer Center, 2Guy’s and St. Thomas’ Hospital, 3Groote Schuur Hospital and University of Cape Town, 4University of Alabama at Birmingham, 5Tygerberg Hospital and Stellenbosch University, 6Ocean Road Cancer Institute, 7University of the Free State, 8University of California-San Francisco, 9University Hospitals Plymouth NHS Trust, 10Stanford University This protocol describes a series of automated tools designed for high-quality radiotherapy autocontouring and autoplanning that are being packaged into a web-based service to maximize robustness and scalability while minimizing operational costs. Medicine Biobanking of Human Aqueous and Vitreous Liquid Biopsies for Molecular Analyses Julian Wolf1,2, Teja Chemudupati1,2, Aarushi Kumar1,2, Ditte K. Rasmussen1,2, Karen M. Wai2, Robert T. Chang2, Artis A. Montague2, Peter H. Tang3,4, Alexander G. Bassuk5,6,7, Antoine Dufour8,9, Prithvi Mruthrunjaya2, Vinit B. Mahajan1,2,10 1Molecular Surgery Laboratory, Stanford University, 2Department of Ophthalmology, Byers Eye Institute, Stanford University, 3Department of Ophthalmology and Visual Neurosciences, University of Minnesota, 4Retina Consultants of Minnesota, 5Department of Pediatrics, University of Iowa, 6Department of Neurology, University of Iowa, 7The Iowa Neuroscience Institute (INI), University of Iowa, 8Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, 9Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, 10Veterans Affairs Palo Alto Health Care System This protocol presents an integrated biorepository platform for the standardized collection, annotation, and biobanking of high-quality human aqueous humor and vitreous liquid biopsies for molecular downstream analyses, including proteomics, metabolomics, and glycomics. Bioengineering Creation of a Knee Joint-on-a-Chip for Modeling Joint Diseases and Testing Drugs Meagan J. Makarcyzk1,2, Zhong Alan Li1,3, Ilhan Yu1, Haruyo Yagi1, Xiurui Zhang1, Lauren Yocum1, Eileen Li1, Madalyn R. Fritch1, Qi Gao4, Bruce A. Bunnell5, Stuart B. Goodman4,6, Rocky S. Tuan1,8, Peter G. Alexander1,7, Hang Lin1,2,7 1Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, 2Department of Bioengineering, University of Pittsburgh Swanson School of Engineering, 3Department of Neurobiology, University of Pittsburgh School of Medicine, 4Department of Orthopaedic Surgery, Stanford University, 5Department of Microbiology, Immunology, and Genetics, University of North Texas Health Science Center, 6Department of Bioengineering, Stanford University, 7McGowan Institute for Regenerative Medicine, University of Pittsburgh School of Medicine, 8The Chinese University of Hong Kong We provide detailed methods for generating four types of tissues from human mesenchymal stem cells, which are used to recapitulate the cartilage, bone, fat pad, and synovium in the human knee joint. These four tissues are integrated into a customized bioreactor and connected through microfluidics, thus generating a knee joint-on-a-chip. Neuroscience Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography Samantha T. Reyes*1, E. Carmen Azevedo*1, Haley C. Cropper1, Sydney Nagy1, Emily M. Deal1, Aisling M. Chaney1, Michelle L. James1,2 1Department of Radiology, Stanford University, 2Department of Neurology and Neurological Sciences, Stanford University This paper details methodology for radiolabeling a human-specific anti-CD19 monoclonal antibody and how to use it to quantify B cells in the central nervous system and peripheral tissues of a mouse model of multiple sclerosis using in vivo PET imaging, ex vivo gamma counting, and autoradiography approaches. Engineering Triplet Fusion Upconversion Nanocapsule Synthesis Tracy H. Schloemer1,2, Samuel N. Sanders1, Qi Zhou2, Pournima Narayanan3, Manchen Hu2, Mahesh K. Gangishetty1, Daniel Anderson1, Michael Seitz1,2, Arynn O. Gallegos2, R. Christopher Stokes1, Daniel N. Congreve1,2 1Rowland Institute, Harvard University, 2Department of Electrical Engineering, Stanford University, 3Department of Chemistry, Stanford University This protocol details the synthesis of upconversion nanocapsules for subsequent use in photopolymerizable resins for triplet fusion upconversion-facilitated volumetric 3D printing. Bioengineering Imaging-Guided Bioreactor for Generating Bioengineered Airway Tissue Seyed Mohammad Mir1, Jiawen Chen1, Meghan R. Pinezich1, John D. O’Neill3, Brandon A. Guenthart4, Gordana Vunjak-Novakovic2, Jinho Kim1 1Department of Biomedical Engineering, Stevens Institute of Technology, 2Department of Biomedical Engineering, Columbia University, 3Department of Cell Biology, State University of New York Downstate Medical Center, 4Department of Cardiothoracic Surgery, Stanford University The protocol describes an imaging-enabled bioreactor that allows the selective removal of the endogenous epithelium from the rat trachea and homogenous distribution of exogenous cells on the lumen surface, followed by long-term in vitro culture of the cell-tissue construct. Medicine Induction and Phenotyping of Acute Right Heart Failure in a Large Animal Model of Chronic Thromboembolic Pulmonary Hypertension David Boulate1, Myriam Amsallem1,2, Jean-Baptiste Menager1, Simon Dang Van1, Peter Dorfmuller3, Andrew Connolly4, Alban Todesco5, Benoit Decante1, Elie Fadel1, Francois Haddad2, Olaf Mercier1 1Research and Innovation Unit, RHU BioArt Lung 2020, Marie Lannelongue Hospital, 2Phenotypic and Biomarker Core Laboratory, Cardiovascular Institute, Stanford University, 3Department of pathology, Marie Lannelongue Hospital, 4Department of pathology, UCSF School of Medicine, 5Department of thoracic surgery, Hopital Nord, APHM, Aix-Marseille University We present a protocol to induce and phenotype an acute right heart failure in a large animal model with chronic pulmonary hypertension. This model can be used to test therapeutic interventions, to develop right heart metrics or to improve the understanding of acute right heart failure pathophysiology. Medicine 3D Imaging of the Liver Extracellular Matrix in a Mouse Model of Non-Alcoholic Steatohepatitis Weiguo Fan1, Yuan Li1, Koshi Kunimoto1, Natalie J. Török1 1Gastroenterology and Hepatology, Stanford University The present protocol optimizes the liver in situ perfusion/decellularization and two-photon microscopy methods to establish a reliable platform to visualize the dynamics of extracellular matrix (ECM) remodeling during non-alcoholic steatohepatitis (NASH). Neuroscience In Vivo Wireless Optogenetic Control of Skilled Motor Behavior Diana L. Rodriguez-Munoz1, Omar Jaidar2, Marcela Palomero-Rivero1, Mario A. Arias-Garcia3, Gordon W. Arbuthnott4, Violeta G. Lopez-Huerta1 1Institute of Cellular Physiology, National University of Mexico, 2Department of Neurosurgery, Stanford University, 3Facultad de Psicologia, National University of Mexico, 4Brain Mechanisms for Behaviour Unit, Okinawa Institute of Science and Technology Graduate University The present protocol describes how to use wireless optogenetics combined with high-speed videography in a single pellet reach-to-grasp task to characterize the neural circuits involved in the performance of skilled motor behavior in freely moving mice. Developmental Biology An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila Tongchao Li1, Liqun Luo1 1Department of Biology, Howard Hughes Medical Institute, Department of Biology, Stanford University This protocol describes the dissection procedure, culture condition, and live imaging of an antennae-brain explant system for the study of the olfactory circuit assembly. Biochemistry Preparing Lamellae from Vitreous Biological Samples Using a Dual-Beam Scanning Electron Microscope for Cryo-Electron Tomography Claudine Bisson1,2, Corey W. Hecksel3,4, James B. Gilchrist3, M. Alejandra Carbajal1, Roland A. Fleck1 1 Using focused ion beam milling to produce vitreous on-grid lamellae from plunge frozen biological samples for cryo-electron tomography. Bioengineering Injectable Supramolecular Polymer-Nanoparticle Hydrogels for Cell and Drug Delivery Applications Catherine M. Meis*1, Abigail K. Grosskopf*2, Santiago Correa1, Eric A. Appel1,3,4 1Department of Materials Science & Engineering, Stanford University, 2Department of Chemical Engineering, Stanford University, 3Department of Bioengineering, Stanford University, 4Department of Pediatrics - Endocrinology, Stanford University This protocol describes the synthesis and formulation of injectable, supramolecular polymer-nanoparticle (PNP) hydrogel biomaterials. Applications of these materials for drug delivery, biopharmaceutical stabilization, and cell encapsulation and delivery are demonstrated. Neuroscience Targeted Neuronal Injury for the Non-Invasive Disconnection of Brain Circuitry Wilson Wang*1, Yanrong Zhang*2, Matthew J. Anzivino1, Edward H. Bertram3, James Woznak1,4, Alexander Klibanov5, Erik Dumont6, Max Wintermark*2, Kevin S. Lee*1,7,8 1Department of Neuroscience, University of Virginia, 2Department of Radiology, Stanford University, 3Department of Neurology, University of Virginia, 4Global Internship Program, Focused Ultrasound Foundation, 5Department of Medicine, University of Virginia, 6Image Guided Therapy, 7Department of Neurosurgery, University of Virginia, 8Center for Brain, Immunology, and Glia, University of Virginia The goal of the protocol is to provide a method for producing non-invasive neuronal lesions in the brain. The method utilizes Magnetic Resonance-guided Focused Ultrasound (MRgFUS) to open the Blood Brain Barrier in a transient and focal manner, in order to deliver a circulating neurotoxin to the brain parenchyma. Neuroscience Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice Maja Djurisic1 1Department of Biology, Neurobiology, and Bio-X, Stanford University This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and field-recordings. Developmental Biology Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils Soah Lee*1,2,3, Huaxiao Yang*1,2,3, Caressa Chen*1,2,3, Sneha Venkatraman1,2,3, Adrija Darsha1,2,3, Sean M. Wu1,2,3, Joseph C. Wu1,2,3, Timon Seeger1,2,3,4,5 1Stanford Cardiovascular Institute, Stanford University School of Medicine, 2Department of Medicine, Division of Cardiovascular Medicine, Stanford University, 3Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, 4Department of Medicine III, University Hospital Heidelberg, 5German Centre for Cardiovascular Research (DZHK), Partner Site Heidelberg/Mannheim This protocol introduces a lithography-free micropatterning method that is simple and accessible to those with a limited bioengineering background. This method utilizes customized laser-cut stencils to micropattern extracellular matrix proteins in a shape of interest for modulating cell morphologies. The procedure for micropatterning is demonstrated using induced pluripotent stem cell derived cardiomyocytes. Developmental Biology High-Frequency Ultrasound Echocardiography to Assess Zebrafish Cardiac Function Alessandro Evangelisti1, Katharina Schimmel1, Shaurya Joshi2, Kavya Shah1, Sudeshna Fisch2, Kevin M. Alexander1, Ronglih Liao1, Isabel Morgado1 1Stanford Cardiovascular Institute, Stanford University, 2 We describe a protocol to assess heart morphology and function in adult zebrafish using high-frequency echocardiography. The method allows visualization of the heart and subsequent quantification of functional parameters, such as heart rate (HR), cardiac output (CO), fractional area change (FAC), ejection fraction (EF), and blood inflow and outflow velocities. Medicine In Vitro Model of Coronary Angiogenesis Colton L. Large1, Halie E. Vitali1, Jeffery D. Whatley1, Kristy Red-Horse2, Bikram Sharma1 1Department of Biology, Ball State University, 2Department of Biology, Stanford University In vitro models of coronary angiogenesis can be utilized for the discovery of the cellular and molecular mechanisms of coronary angiogenesis. In vitro explant cultures of sinus venosus and endocardium tissues show robust growth in response to VEGF-A and display a similar pattern of COUP-TFII expression as in vivo. Immunology and Infection A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection Christiaan R. de Vries*1, Johanna M. Sweere*1,2, Heather Ishak1,3, Vivekananda Sunkari1, Michelle S. Bach1, Dan Liu1, Robert Manasherob1, Paul L. Bollyky1,2 1Division of Infectious Diseases, School of Medicine, Stanford University, 2Stanford Immunology, Stanford University, 3Palo Alto Veterans Institute of Research We describe a delayed inoculation protocol for generating chronic wound infections in immunocompetent mice. Neuroscience Meta-analysis of Voxel-Based Neuroimaging Studies using Seed-based d Mapping with Permutation of Subject Images (SDM-PSI) Anton Albajes-Eizagirre1,2, Aleix Solanes1,2, Miquel Angel Fullana2,3, John P. A. Ioannidis4, Paolo Fusar-Poli5,6,7, Carla Torrent1,2,3,8, Brisa Solé1,2,3,8, Caterina Mar Bonnín1,2,3,8, Eduard Vieta1,2,3,8, David Mataix-Cols9, Joaquim Radua1,2,5,9 1 We detail how to conduct a meta-analysis of voxel-based neuroimaging studies using Seed-based d Mapping with Permutation of Subject Images (SDM-PSI). Environment Laser-Induced Fluorescence Emission (L.I.F.E.) as Novel Non-Invasive Tool for In-Situ Measurements of Biomarkers in Cryospheric Habitats Klemens Weisleitner1,2, Lars Hunger3, Christoph Kohstall4, Albert Frisch5, Michael C. Storrie-Lombardi6, Birgit Sattler1,2 1Institute of Ecology, University of Innsbruck, 2Austrian Polar Research Institute, University of Vienna, 3BrainLinks-BrainTools, Bernstein Center Freiburg, 4Atom Science, Kasevich Lab, Stanford University, 5Institute of Experimental Physics, University of Innsbruck, 6Department of Physics, Extraterrestrial Vehicle Instruments Laboratory, Harvey Mudd College Carbon fluxes in the cryosphere are hardly assessed yet but are crucial regarding climate change. Here we show a novel prototype device that captures the phototrophic potential in supraglacial environments based on laser-induced fluorescence emission (L.I.F.E.) technology offering high spectral and spatial resolution data under in situ conditions. Cancer Research In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research Jennifer C. Wischhusen1, Katheryne E. Wilson2 1Apoptosis, Cancer and Development Laboratory - Equipe labellisée 'La Ligue', LabEx DEVweCAN, Centre de Cancérologie de Lyon, INSERM U1052-CNRS UMR5286, Centre Léon Bérard, 2Department of Radiology/Molecular Imaging Program at Stanford, School of Medicine, Stanford University The in vivo immunofluorescence localization (IVIL) method can be used to examine in vivo biodistribution of antibodies and antibody conjugates for oncological purposes in living organisms using a combination of in vivo tumor targeting and ex vivo immunostaining methods. Genetics Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis Carly V. Weiss1,2, Julie N. Chuong3, Rachel B. Brem1,3 1Department of Plant and Microbial Biology, University of California Berkeley, 2Department of Biology, Stanford University, 3Buck Institute for Research on Aging Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait. Cancer Research Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels Steven M Alves1, Tian Zhu1, Anastasia Shostak1, Ninna S. Rossen2, Marjan Rafat1,3,4 1Department of Chemical and Biomolecular Engineering, Vanderbilt University, 2Department of Radiation Oncology, Stanford University, 3Depattment of Biomedical Engineering, Vanderbilt University, 4Department of Radiation Oncology, Vanderbilt University Medical Center This protocol presents a method for decellularization and subsequent hydrogel formation of murine mammary fat pads following ex vivo irradiation. Engineering A Pipette-Tip Based Method for Seeding Cells to Droplet Microfluidic Platforms Nidhi Sinha*1, Nikita Subedi*1, Florian Wimmers*2, Melf Soennichsen1, Jurjen Tel1 1Department of Biomedical Engineering and Institute for Complex Molecular Systems, Laboratory of Immunoengineering, Eindhoven University of Technology, 2Institute for Immunity, Transplantation, and Infection, Beckman Center, Stanford University This article presents a protocol for seeding scarce population of cells using pipette-tips to droplet microfluidic devices in order to provide higher encapsulation efficiency of cells in droplets. Immunology and Infection Label-Free Identification of Lymphocyte Subtypes Using Three-Dimensional Quantitative Phase Imaging and Machine Learning Jonghee Yoon1, YoungJu Jo2,3,4,7, Young Seo Kim3,4,5, Yeongjin Yu2,3, Jiyeon Park6, Sumin Lee4, Wei Sun Park2,3, YongKeun Park2,3,4 1Department of Physics, University of Cambridge, 2Department of Physics, Korea Advanced Institute of Science and Technology, 3KAIST Institute for Health Science and Technology, Korea Advanced Institute of Science and Technology, 4Tomocube, Inc., 5Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, 6Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 7Department of Applied Physics, Stanford University We describe a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and a machine learning algorithm. Measurements of 3D refractive index tomograms of lymphocytes present 3D morphological and biochemical information for individual cells, which is then analyzed with a machine-learning algorithm for identification of cell types. Bioengineering High-resolution Patterned Biofilm Deposition Using pDawn-Ag43 Xiaofan Jin1, Ingmar H. Riedel-Kruse1 1Department of Bioengineering, Stanford University We demonstrate a method for depositing Escherichia coli bacterial biofilms in arbitrary spatial patterns with a high resolution using optical stimulation of a genetically encoded surface-adhesion construct. Biology Imaging FITC-dextran as a Reporter for Regulated Exocytosis Ofir Klein1, Amit Roded1, Koret Hirschberg2, Mitsunori Fukuda3, Stephen J. Galli4, Ronit Sagi-Eisenberg1 1Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, 2Department of Pathology, Sackler Faculty of Medicine, Tel Aviv University, 3Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, 4Departments of Pathology and of Microbiology and Immunology and Sean N. Parker Center for Allergy and Asthma Research, School of Medicine, Stanford University Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically. Medicine PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke Aisling M. Chaney1, Emily M. Johnson1, Haley C. Cropper1, Michelle L. James1,2 1Department of Radiology, Stanford University, 2Department of Neurology and Neurological Sciences, Stanford University Positron Emission Tomography (PET) imaging of translocator protein 18 kDa (TSPO) provides a non-invasive means to visualize the dynamic role of neuroinflammation in the development and progression of brain diseases. This protocol describes TSPO-PET and ex vivo autoradiography to detect neuroinflammation in a mouse model of ischemic stroke. Cancer Research Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes Nadine Santana-Magal*1, Diana Rasoulouniriana*1, Corey Saperia1, Amit Gutwillig1, Peleg Rider1, Edgar G. Engleman2, Yaron Carmi1 1Department of Pathology, Sackler School of Medicine, Tel-Aviv University, 2Department of Pathology, School of Medicine, Stanford University Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation. Bioengineering Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D Bauer L. LeSavage*1, Nicholas A. Suhar*2, Christopher M. Madl1, Sarah C. Heilshorn2 1Department of Bioengineering, Stanford University, 2Department of Materials Science and Engineering, Stanford University Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation. Chemistry Elemental-sensitive Detection of the Chemistry in Batteries through Soft X-ray Absorption Spectroscopy and Resonant Inelastic X-ray Scattering Jinpeng Wu1,2, Shawn Sallis2,3, Ruimin Qiao2, Qinghao Li2,4, Zengqing Zhuo2,5, Kehua Dai2,6, Zixuan Guo2,7, Wanli Yang2 1Geballe Laboratory for Advanced Materials, Stanford University, 2Advanced Light Source, Lawrence Berkeley National Laboratory, 3Department of Materials Science and Engineering, Binghamton University, 4School of Physics, National Key Laboratory of Crystal Materials, Shandong University, 5School of Advanced Materials, Peking University Shenzhen Graduate School, 6School of Metallurgy, Northeastern University, 7Department of Chemical Engineering, University of California-Santa Barbara Here, we present a protocol for typical experiments of soft X-ray absorption spectroscopy (sXAS) and resonant inelastic X-ray scattering (RIXS) with applications in battery material studies. Medicine Radiation Planning Assistant - A Streamlined, Fully Automated Radiotherapy Treatment Planning System Laurence E. Court1, Kelly Kisling1, Rachel McCarroll1, Lifei Zhang1, Jinzhong Yang1, Hannah Simonds2, Monique du Toit2, Chris Trauernicht2, Hester Burger3, Jeannette Parkes3, Mike Mejia4, Maureen Bojador4, Peter Balter1, Daniela Branco1, Angela Steinmann1, Garrett Baltz1, Skylar Gay1, Brian Anderson1, Carlos Cardenas1, Anuja Jhingran5, Simona Shaitelman5, Oliver Bogler6, Kathleen Schmeller7, David Followill1, Rebecca Howell1, Christopher Nelson1, Christine Peterson8, Beth Beadle5,9 1Department of Radiation Physics, University of Texas MD Anderson Cancer Center, 2Department of Radiation Oncology, Stellenbosch University and Tygerberg Hospital, 3Departments of Radiation Oncology and Medical Physics, Groote Schuur Hospital and University of Cape Town, 4Department of Radiation Oncology, University of Santo Tomas Hospital, Benavides Cancer Institute, 5Department of Radiation Oncology, University of Texas MD Anderson Cancer Center, 6Academic Affairs, University of Texas MD Anderson Cancer Center, 7Department of Gynecological Oncology and Reproductive Medicine, University of Texas MD Anderson Cancer Center, 8Department of Biostatistics, University of Texas MD Anderson Cancer Center, 9Department of Radiation Oncology, Stanford University Radiation therapy is a highly complex cancer treatment that requires multiple specialists to create a treatment plan and provide quality assurance (QA) prior to delivery to a patient. This protocol describes the use of a fully automated system, the Radiation Planning Assistant (RPA), to create high-quality radiation treatment plans. Chemistry Synthesis and Performance Characterizations of Transition Metal Single Atom Catalyst for Electrochemical CO2 Reduction Kun Jiang1, Guangxu Chen2, Haotian Wang1 1Rowland Institute, Harvard University, 2Materials Science and Engineering, Stanford University Here, we present a protocol for the synthesis and electrochemical testing of transition metal single atoms coordinated in graphene vacancies as active centers for selective carbon dioxide reduction to carbon monoxide in aqueous solutions. Neuroscience Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium Hannah Y. Collins1, Christopher J. Bohlen1 1Department of Neurobiology, Stanford University Efforts to understand microglial function in detail have been hindered by the lack of microglial culture models that recapitulate the properties of mature in vivo microglia. This protocol describes an isolation and culture approach designed to maintain robust survival of highly ramified mature rat microglia under defined-medium conditions. Neuroscience Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans Holger Fehlauer*1, Adam L. Nekimken*1,2, Anna A. Kim1,2, Beth L. Pruitt1,2,3, Miriam B. Goodman1,2, Michael Krieg4 1Department of Molecular and Cellular Physiology, Stanford University, 2Department of Mechanical Engineering, Stanford University, 3Department of Bioengineering, Stanford University, 4Group of Neurophotonics and Mechanical Systems Biology, The Institute of Photonic Sciences (ICFO) New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses. Cancer Research Whole-body PET/MRI of Pediatric Patients: The Details That Matter Anuj Pareek1, Anne M. Muehe1, Ashok J. Theruvath1, Praveen K. Gulaka1, Sheri L. Spunt2, Heike E. Daldrup-Link1 1Department of Radiology, Pediatric Molecular Imaging Program at Stanford (PEDS-MIPS), Stanford University, 2Department of Pediatrics, Stanford University This article provides comprehensive step-by-step instructions for the acquisition of whole-body 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) PET/MRI scans for cancer staging of pediatric patients. The protocol was developed for children above 6 years, or old enough to comply with breath-hold instructions, but can be used for general anesthesia patients as well. Biology Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary Wei Hu*1, Amin Tamadon*1, Aaron J.W. Hsueh2, Yi Feng1 1Department of Integrative Medicine and Neurobiology, School of Basic Medical Sciences; Institutes of Brain Science, Brain Science Collaborative Innovation Center, State Key Laboratory of Medical Neurobiology, Institute of Acupuncture and Moxibustion, Fudan Institutes of Integrative Medicine, Fudan University, 2Program of Reproductive and Stem Cell Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford University Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries. Biochemistry Dissection of Human Retina and RPE-Choroid for Proteomic Analysis Thiago Cabral*1,2,7,8, Marcus A. Toral*3,4, Gabriel Velez3,4, James E. DiCarlo1,2, Anuradha M. Gore3, MaryAnn Mahajan3, Stephen H. Tsang1,2, Alexander G. Bassuk5,6, Vinit B. Mahajan3,9 1Barbara & Donald Jonas Stem Cell Laboratory, and Bernard & Shirlee Brown Glaucoma Laboratory, Department of Pathology & Cell Biology, Institute of Human Nutrition, College of Physicians and Surgeons, Columbia University, 2Edward S. Harkness Eye Institute, New York-Presbyterian Hospital, 3Omics Laboratory, Byers Eye Institute, Department of Ophthalmology, Stanford University, 4Medical Scientist Training Program, University of Iowa, 5Department of Pediatrics, University of Iowa, 6Department of Neurology, University of Iowa, 7Department of Ophthalmology, Federal University of Sao Paulo (UNIFESP), 8Department of Ophthalmology, Federal University of EspÍrito Santo (UFES), 9Palo Alto Veterans Administration, Palo Alto, CA The human retina is composed of functionally and molecularly distinct regions, including the fovea, macula, and peripheral retina. Here, we describe a method using punch biopsies and manual removal of tissue layers from a human eye to dissect and collect these distinct retinal regions for downstream proteomic analysis. Immunology and Infection Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4 Sharon A. Sagan*1,2, Andrés Cruz-Herranz*1, Collin M. Spencer1,2, Peggy P. Ho3, Lawrence Steinman3, Ari J. Green1, Raymond A. Sobel4, Scott S. Zamvil1,2 1Department of Neurology, University of California, 2Program in Immunology, University of California, 3Department of Neurology and Neurological Sciences, Stanford University, 4Department of Pathology, Stanford University Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction. Bioengineering Quantification of Strain in a Porcine Model of Skin Expansion Using Multi-View Stereo and Isogeometric Kinematics Adrian Buganza Tepole1, Elbert E. Vaca2, Chad A. Purnell2, Michael Gart2, Jennifer McGrath2, Ellen Kuhl3, Arun K. Gosain2 1Mechanical Engineering, Purdue University, 2Division of Plastic Surgery, Ann and Robert H. Lurie Children's Hospital of Chicago, Northwestern University Feinberg School of Medicine, 3Mechanical Engineering, Bioengineering, Cardiothoracic Surgery, Stanford University This protocol uses multi-view stereo to generate three-dimensional (3D) models out of uncalibrated sequences of photographs, making it affordable and adjustable to a surgical setting. Strain maps between the 3D models are quantified with spline-based isogeometric kinematics, which facilitate representation of smooth surfaces over coarse meshes sharing the same parameterization. Neuroscience Dynamic Quantitative Sensory Testing to Characterize Central Pain Processing Ian G. Mackey1, Eric A. Dixon1, Kevin Johnson1, Jiang-Ti Kong1 1Anesthesiology, Perioperative and Pain Medicine, Stanford University School of Medicine By assessing pain in response to repetitive or different types of standardized stimuli, dynamic quantitative sensory testing (QST) can reveal changes in the central processing of pain. We present methods to optimize and individualize two dynamic QST measures: temporal summation (TS) and conditioned pain modulation (CPM). Bioengineering Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices Kara Brower*1,2,4, Adam K. White*1,2, Polly M. Fordyce1,2,3,4 1Department of Bioengineering, Stanford University, 2Microfluidic Foundry, Stanford University, 3Department of Genetics, Stanford University, 4Chem-H Institute, Stanford University Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads. Biology In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse Ke Yuan1,2, Mark E. Orcholski1,2, Ngan F. Huang2,3, Vinicio A. de Jesus Perez1,2 1Division of Pulmonary and Critical Care Medicine, School of Medicine, Stanford University, 2Stanford Cardiovascular Institute, School of Medicine, Stanford University, 3VA Palo Alto Health Care System, Department of Cardiothoracic Surgery, School of Medicine, Stanford University We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay. Developmental Biology An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery Martin H. M. Sailer1, Durga Sarvepalli2, Catherine Brégère3, Urs Fisch3, Marin Guentchev4, Michael Weller6, Raphael Guzman3, Bernhard Bettler1, Arkasubhra Ghosh*2, Gregor Hutter*5 1Dept. of Biomedicine, Pharmacenter, University of Basel, 2Molecular Signalling and Gene Therapy, Narayana Nethralaya Foundation, Narayana Health City, 3Brain Ischemia and Regeneration, Department of Biomedicine, University Hospital Basel, 4Department of Neurosurgery, Klinikum Idar-Oberstein, 5Department of Neurosurgery and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, 6Department of Neurology, Laboratory of Molecular Neuro Oncology, University Hospital of Zurich Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery. Neuroscience Optogenetic Functional MRI Peter Lin1, Zhongnan Fang2, Jia Liu1, Jin Hyung Lee1,2 1Neurology and Neurological Sciences, Stanford University, 2Electrical Engineering, Neurology and Neurological Sciences, Stanford University This protocol describes the steps and data analysis required to successfully perform optogenetic functional magnetic resonance imaging (ofMRI). ofMRI is a novel technique that combines high-field fMRI readout with optogenetic stimulation, allowing for cell type-specific mapping of functional neural circuits and their dynamics across the whole living brain. Bioengineering 3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation Piera Smeriglio*1, Janice H. Lai*1,2, Fan Yang1,3, Nidhi Bhutani1 1Orthopaedic Surgery Department, Stanford University, 2Mechanical Engineering Department, Stanford University, 3Bioengineering Department, Stanford University Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes. Engineering Fabrication and Operation of a Nano-Optical Conveyor Belt Jason Ryan1, Yuxin Zheng1, Paul Hansen1, Lambertus Hesselink2 1Electrical Engineering, Stanford University, 2Applied Physics, Stanford University The scalability and resolution of conventional optical manipulation techniques are limited by diffraction. We circumvent the diffraction limit and describe a method of optically transporting nanoparticles across a chip using a gold surface patterned with a path of closely spaced C-shaped plasmonic resonators. Biology Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions Joseph Fasolo1, Hogune Im1, Michael P. Snyder1,2 1Department of Genetics, Stanford University, 2Stanford Center for Genomics and Personalized Medicine, Stanford University Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput. Behavior Infant Auditory Processing and Event-related Brain Oscillations Gabriella Musacchia1,2,3, Silvia Ortiz-Mantilla1, Teresa Realpe-Bonilla1, Cynthia P. Roesler1, April A. Benasich1 1Center for Molecular & Behavioral Neuroscience, Rutgers University, State University of New Jersey, Newark, 2Department of Audiology, University of the Pacific, 3Department of Otolaryngology, Head & Neck Surgery, Stanford University High-density electroencephalography (dEEG) is being used increasingly to study brain development and plasticity in the early years of life. Here we present an application of sophisticated analysis techniques that builds on traditional EEG recording to understand the oscillatory dynamics of rapid auditory processing in the infant brain. Medicine Reduction of Iatrogenic Atrial Septal Defects with an Anterior and Inferior Transseptal Puncture Site when Operating the Cryoballoon Ablation Catheter Michael E. Rich1, Andrew Tseng2, Hae W. Lim3, Paul J. Wang4, Wilber W. Su1 1Department of Cardiovascular Medicine, Cavanagh Heart Center, Banner-University Medical Center, 2Mayo Medical School, Mayo Clinic, 3AF Solutions, Medtronic plc, 4Cardiology Division, Stanford University School of Medicine, Stanford University The goal of this study is to demonstrate the preferential location of transseptal puncture during a cryoballoon catheter ablation procedure for the treatment of atrial fibrillation. Neuroscience In vivo Optogenetic Stimulation of the Rodent Central Nervous System Michelle M. Sidor1, Thomas J. Davidson2, Kay M. Tye3, Melissa R. Warden4, Karl Diesseroth2,5, Colleen A. McClung1 1Department of Psychiatry, University of Pittsburgh Medical Center, 2Department of Bioengineering, Stanford University, 3Department of Brain and Cognitive Sciences, Picower Institute for Learning and Memory, Massachusetts Institute of Technology, 4Department of Neurobiology and Behavior, Cornell University, 5Department of Psychiatry and Behavioral Sciences, Stanford University Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms. Developmental Biology Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis Elizabeth R. Zielins*1, Ruth Tevlin*1, Michael S. Hu1, Michael T. Chung1, Adrian McArdle1, Kevin J. Paik1, David Atashroo1, Christopher R. Duldulao1, Anna Luan1, Kshemendra Senarath-Yapa1, Graham G. Walmsley1, Taylor Wearda1, Michael T. Longaker1,2, Derrick C. Wan1 1Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, 2Institute for Stem Cell Biology and Regenerative Medicine, Stanford University The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction. Biology Osteoclast Derivation from Mouse Bone Marrow Ruth Tevlin*1, Adrian McArdle*1,2, Charles K.F. Chan2, John Pluvinage2, Graham G. Walmsley1,2, Taylor Wearda1,2, Owen Marecic1,2, Michael S. Hu1, Kevin J. Paik1, Kshemendra Senarath-Yapa1, David A. Atashroo1, Elizabeth R. Zielins1, Derrick C. Wan1, Irving L. Weissman1,2, Michael T. Longaker1,2 1Hagey Laboratory for Pediatric Regenerative Medicine, Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University School of Medicine, 2Institute for Stem Cell Biology and Regenerative Medicine, Stanford University Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro. Neuroscience Paired Whole Cell Recordings in Organotypic Hippocampal Slices Chantelle Fourie1, Marianna Kiraly2, Daniel V. Madison*2, Johanna M. Montgomery*1 1Department of Physiology and Centre for Brain Research, University of Auckland, 2Department of Molecular and Cellular Physiology, Stanford University Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology. Chemistry Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography Samantha M. Desmarais1, Felipe Cava2, Miguel A. de Pedro3, Kerwyn Casey Huang1,4 1Department of Bioengineering, Stanford University, 2Department of Molecular Biology and Laboratory for Molecular Infection Medicine Sweden, Umeå Centre for Microbial Research, Umeå University, 3Campus de Cantoblanco, Universidad Autonoma de Madrid, 4Department of Microbiology and Immunology, Stanford University School of Medicine The bacterial cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by peptides. Ultra Performance Liquid Chromatography provides high resolution and throughput for novel discoveries of peptidoglycan composition. We present a procedure for the isolation of cell walls (sacculi) and their subsequent preparation for analysis via UPLC. Behavior Nest Building as an Indicator of Health and Welfare in Laboratory Mice Brianna N. Gaskill1, Alicia Z. Karas2, Joseph P. Garner3,4, Kathleen R. Pritchett-Corning1 1Research Models and Services, Charles River, 2Department of Clinical Sciences, Tufts University, 3Department of Comparative Medicine, Stanford University, 4Department of Psychiatry and Behavioral Sciences, Stanford University We demonstrate the utility of nest building behavior in laboratory mice as an indicator of welfare. Nest scoring is a sensitive technique that is altered by temperature, illness, and aggression. The time to integrate into nest test (TINT) is a simple cage-side assessment that can detect postoperative pain. Bioengineering Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles Michael Keeney*1, Lorenzo Deveza*1,2, Fan Yang1,2 1Department of Orthopaedic Surgery, Stanford University, 2Department of Bioengineering, Stanford University We describe the method of programming stem cells to overexpress therapeutic factors for angiogenesis using biodegradable polymeric nanoparticles. Processes described include polymer synthesis, transfecting adipose-derived stem cells in vitro, and validating the efficacy of programmed stem cells to promote angiogenesis in a murine hindlimb ischemia model. Engineering In-situ Tapering of Chalcogenide Fiber for Mid-infrared Supercontinuum Generation Charles W. Rudy1, Alireza Marandi1, Konstantin L. Vodopyanov1, Robert L. Byer1 1Edward L. Ginzton Laboratory, Stanford University We describe a method for in-situ tapering of As2S3 fibers to achieve efficient mid-infrared supercontinuum generation. By tapering while monitoring the supercontinuum’s spectrum, the spectral width can be maximized for a fiber taper. In-situ fiber tapering can be applied to optimize the performance of other fiber-based devices. Biology Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration Wei-Meng Woo*1, Scott X. Atwood*1, Hanson H. Zhen1, Anthony E. Oro1 1Program in Epithelial Biology, Stanford University School of Medicine Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function. Neuroscience Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster Saskia E.J. de Vries1, Tom Clandinin1 1Department of Neurobiology, Stanford University Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies. Medicine Probe-based Confocal Laser Endomicroscopy of the Urinary Tract: The Technique Timothy C. Chang1,2, Jen-Jane Liu1,2, Joseph C. Liao1,2 1Department of Urology, Stanford University School of Medicine, 2Veterans Affairs Palo Alto Health Care System Probe-based confocal laser endomicroscopy enables real-time microscopy of the human urinary tract during cystoscopy, providing dynamic, intravital imaging of pathological states such as bladder cancer with cellular resolution. Endomicroscopy may augment the diagnostic accuracy of standard white light endoscopy and provide intraoperative image guidance to improve surgical resection. Medicine Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring Yingding Xu1, Hongguang Liu1, Edwin Chang1, Han Jiang1, Zhen Cheng1 1Department of Radiology and Bio-X Program Canary Cancer at Stanford for Cancer Early Detection, Stanford University Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening. Bioengineering Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer Michael D. Leipold1, Holden T. Maecker1 1Human Immune Monitoring Center, Institute for Immunity, Transplantation, and Infection, Stanford University The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed 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. Medicine Collecting And Measuring Wound Exudate Biochemical Mediators In Surgical Wounds Brendan Carvalho1, David J Clark1, David Yeomans1, Martin S Angst1 1Department of Anesthesia, Stanford University School of Medicine This article provides a detailed and visual description of a methodology for collecting and measuring biochemical inflammatory and nociceptive mediators at the surgical wound site following cesarean delivery. This human bioassay has been used to determine correlations between wound and serum cytokine concentrations and drug-mediated changes in wound cytokines, chemokines and neuropetides. 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. Bioengineering Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers Arjun S. Adhikari1, Jack Chai1, Alexander R. Dunn1 1Department of Chemical Engineering, Stanford University In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner. Neuroscience Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI Gabriel P. Howles1, Yi Qi2, Stephen J. Rosenzweig3, Kathryn R. Nightingale3, G. Allan Johnson2 1Department of Radiology, Stanford University, 2Center for In Vivo Microscopy, Duke University Medical Center, 3Department of Biomedical Engineering, Duke University A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity. Bioengineering Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip Guido Grossmann1, Matthias Meier2,3,4, Heather N. Cartwright1, Davide Sosso1, Stephen R. Quake2,3, David W. Ehrhardt1, Wolf B. Frommer1 1Department of Plant Biology, Carnegie Institution for Science, 2Howard Hughes Medical Institute, 3Departments of Applied Physics and Bioengineering, Stanford University, 4Department of Microsystems Engineering (IMTEK) and Center for Biological Signaling Studies (BIOSS), University of Freiburg This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels. Medicine Human Internal Mammary Artery (IMA) Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis Xiaoqin Hua1,2, Tobias Deuse1,2, Evangelos D. Michelakis3, Alois Haromy3, Phil S. Tsao4, Lars Maegdefessel4, Reinhold G. Erben5, Claudia Bergow5, Boris B. Behnisch6, Hermann Reichenspurner1,2, Robert C. Robbins7, Sonja Schrepfer1,2,7 1University Heart Center Hamburg, TSI-Lab, Germany, 2Cardiovascular Research Center, University of Hamburg, 3Department of Medicine, Cardiology Division, Pulmonary Hypertension Program, University of Alberta, 4Department of Medicine, Stanford University School of Medicine, 5Department of Biomedical Sciences, Institute of Physiology, Pathophysiology, and Biophysics, University of Veterinary Medicine, Vienna, 6Translumina GmbH, Hechingen, 7Department of Cardiothoracic Surgery, Stanford University School of Medicine This video shows a model to study the development of intimal hyperplasia after stent deployment using a human vessel (IMA) in an immunodeficient rat model. Biology Chromatin Isolation by RNA Purification (ChIRP) Ci Chu1, Jeffrey Quinn1, Howard Y. Chang1 1Howard Hughes Medical Institute and Program in Epithelial Biology, Stanford University School of Medicine ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites. Bioengineering On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids Giancarlo Garcia-Schwarz1, Anita Rogacs1, Supreet S. Bahga1, Juan G. Santiago1 1Mechanical Engineering, Stanford University Isotachophoresis (ITP) is a robust electrokinetic separation and preconcentration technique with applications ranging from toxin detection to sample preparation. We review the physical principles of ITP and the methodology of applying this technique to two specific example applications: separation and detection of small molecules and purification of nucleic acids from cell culture lysate. Neuroscience Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry Taha A. Jan1, Renjie Chai1, Zahra N. Sayyid1, Alan G. Cheng1 1Department of Otolaryngology-Head and Neck Surgery, Stanford University School of Medicine Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of β-galactosidase prior to separation via flow cytometry. Medicine Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle Rosalinda T. Castaneda1,2, Aman Khurana1,2, Ramsha Khan1,2, Heike E. Daldrup-Link1 1Department of Radiology, Molecular Imaging Program at Stanford (MIPS), 2Stanford School of Medicine, Stanford University We describe a technique for labeling and tracking stem cells with FDA-approved, superparamagnetic iron oxide (SPIO), ferumoxytol (Feraheme). This cellular imaging technique that utilizes magnetic resonance (MR) imaging for visualization, is readily accessible for long-term monitoring and diagnosis of successful or unsuccessful stem cell engraftments in patients. Biology Competitive Genomic Screens of Barcoded Yeast Libraries Andrew M. Smith*1,2, Tanja Durbic*2,3, Julia Oh*4, Malene Urbanus1,2, Michael Proctor5, Lawrence E. Heisler2,3, Guri Giaever2,6, Corey Nislow1,2,3 1Banting and Best Department of Medical Research and Department of Molecular Genetics, University of Toronto, 2Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 3Donnelly Sequencing Centre, University of Toronto, 4Genetics and Molecular Biology Branch, National Human Genome Research Institute, NIH, 5Stanford Genome Technology Center, Stanford School of Medicine, Stanford University, 6Department of Pharmaceutical Sciences, University of Toronto We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented. Medicine Mouse Bladder Wall Injection Chi-Ling Fu1, Charity A. Apelo*1, Baldemar Torres*1, Kim H. Thai1, Michael H. Hsieh1 1Department of Urology, Stanford University School of Medicine Mouse bladder wall injection is a useful approach to orthotopically study bladder stem cell and cancer biology. This delicate microsurgical method can be mastered with careful technique and practice. Bioengineering Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT) Lenard Conradi1,2, Christiane Pahrmann1, Stephanie Schmidt1, Tobias Deuse1,3, Arne Hansen2, Alexandra Eder2, Hermann Reichenspurner1, Robert C. Robbins3, Thomas Eschenhagen2, Sonja Schrepfer1,3 1Transplant and Stem Cell Immunobiology Lab (TSI) and CVRC, University Hospital Hamburg, University Heart Center Hamburg, 2Department of Experimental and Clinical Pharmacology and Toxicology, University Heart Center Hamburg, 3CT Surgery, Stanford University School of Medicine This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats. Biology Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran Joshua A. Clanton1, Ilya A. Shestopalov2, James K. Chen2, Joshua T. Gamse1 1Department of Biological Sciences, Vanderbilt University, 2Department of Chemical and Systems Biology, Stanford University This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein. Biology Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA Bo Dai1, Farida Dahmani2, Joseph A. Cichocki3, Lindsey C. Swanson2, Theodore P. Rasmussen3 1Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, 2Department of Molecular and Cell Biology, University of Connecticut, 3Department of Pharmaceutical Sciences, University of Connecticut Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types. Biology Associated Chromosome Trap for Identifying Long-range DNA Interactions Jianqun Ling1, Andrew R. Hoffman1 1Medical Service, VA Palo Alto Health Care System , Stanford University School of Medicine The associated chromosome trap (ACT) assay is a novel unbiased method for identifying long-range DNA interactions. The characterization of long range DNA interactions will allow us to determine the relationship of nuclear architecture to gene expression in both normal physiology and in diseased states. Biology Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses Nitin Shirole1, Sreeram Balasubramanian1, Charles Yanofsky2, Luis Cruz-Vera1 1Department of Biological Sciences, University of Alabama Huntsville, 2Department of Biology, Stanford University A major impediment to biochemical analyses of ribosomes containing nascent peptidyl-tRNAs has been the presence of other ribosomes in the same samples, ribosomes not involved in the translation of the specific mRNA sequence being analyzed. We developed a simple methodology to purify, exclusively, the ribosomes containing the nascent peptidyl-tRNA of interest. Medicine Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy Mandy Stubbendorff1, Tobias Deuse1,2, Anna Hammel1, Robert C. Robbins2, Hermann Reichenspurner1, Sonja Schrepfer1,2 1University Heart Center Hamburg, Transplant and Stem Cell Immunobiology Lab (TSI), University Hospital Hamburg, 2Stanford University School of Medicine This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats. Neuroscience The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury Qian Wang1, Katrin Andreasson1 1Department of Neurology and Neurological Sciences, Stanford University School of Medicine The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury. Medicine Assessing Endothelial Vasodilator Function with the Endo-PAT 2000 Andrea L. Axtell1, Fatemeh A. Gomari1, John P. Cooke1 1Department of Cardiovascular Medicine, Stanford University A noninvasive procedure to assess endothelial function is demonstrated using the Endo-PAT 2000. Immunology and Infection Transurethral Induction of Mouse Urinary Tract Infection Kim H. Thai1, Anuradha Thathireddy2, Michael H. Hsieh2 1Earth Systems Program, School of Earth Sciences, Stanford University, 2Department of Urology, Stanford University School of Medicine This video will demonstrate methods to transurethrally induce mouse urinary tract infections and quantify the extent of resulting infections. Biology Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of Obliterative Airway Disease Xiaoqin Hua1, Tobias Deuse1,2, Karis R. Tang-Quan1,2,3, Robert C. Robbins3, Hermann Reichenspurner1,2, Sonja Schrepfer1,2,3 1Transplant and Stem Cell Immunobiology Lab (TSI), University Heart Center Hamburg, 2CVRC, University Hospital Hamburg, 3Department of CT Surgery, Stanford University School of Medicine This video shows and compares two experimental models to study the development of obliterative airway disease (OAD) in mice, the heterotopic and orthotopic tracheal transplantation model. Biology LAD-Ligation: A Murine Model of Myocardial Infarction Mandy V.V. Kolk1,2, Danja Meyberg1,2, Tobias Deuse1,2, Karis R. Tang-Quan1,2,3, Robert C. Robbins3, Hermann Reichenspurner1,2, Sonja Schrepfer1,2,3 1Transplant and Stem Cell Immunobiology Lab (TSI), University Heart Center Hamburg, 2CVRC, University Hospital Hamburg, 3Department of CT Surgery, Stanford University School of Medicine This video demonstrates how to use a fast and reliable model to study pathobiological and pathophysiological processes of myocardial ischemia. Biology Imaging In-Stent Restenosis: An Inexpensive, Reliable, and Rapid Preclinical Model Tobias Deuse1, Fumiaki Ikeno2, Robert C. Robbins1, Sonja Schrepfer1 1Department of Cardiothoracic Surgery, Stanford University School of Medicine, 2Stanford University School of Medicine This video demonstrates how to use a preclinical inexpensive and reliable model to study pathobiological and pathophysiological processes of in-stent restenosis development. Longitudinal in vivo monitoring using OCT (Optical Coherence Tomography) and analysis of OCT images are also demonstrated. Biology Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase Junya Azuma1, Tomoko Asagami2, Ronald Dalman2, Philip S. Tsao1 1Department of Cardiovascular Medicine, Stanford University School of Medicine, 2Department of Vascular Surgery, Stanford University School of Medicine This video shows how to induce abdominal aortic aneurysms (AAA) in mice via transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta. The model has the ability to add broad insight into the pathobiology of AAA due to the emergence of numerous transgenic and gene knockout mice. Biology Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia Ngan F. Huang1, Hiroshi Niiyama1, Abhijit De2, Sanjiv S. Gambhir2, John P. Cooke1 1Division of Cardiovascular Medicine, Stanford University, 2Department of Radiology, Stanford University The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging. 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 Calcium Imaging of Cortical Neurons using Fura-2 AM Odmara L Barreto-Chang1, Ricardo E Dolmetsch2 1Department of Neurobiology, Stanford University, 2Department of Neurobiology, Stanford University School of Medicine Calcium signals play a key role in many cellular processes including gene expression, survival and differentiation. Here we demonstrate how to perform calcium imaging using Fura-2 AM. Calcium imaging is a valuable tool to study the regulation of intracellular calcium in real time and its regulation of signaling cascades. Biology Determining heat and mechanical pain threshold in inflamed skin of human subjects Martin S Angst1, Martha Tingle1, Nicholas G Phillips1, Brendan Carvalho1 1Department of Anesthesia, Stanford University School of Medicine Algorithms assessing heat and mechanical pain thresholds in experimentally inflamed skin of human study subjects are shown. The two pain testing paradigms independently examine nociceptive processing by the two major peripheral nerve fiber populations transmitting pain, i.e., non-myelinated C fibers and small myelinated A-delta fibers. Biology Human In-Vivo Bioassay for the Tissue-Specific Measurement of Nociceptive and Inflammatory Mediators Martin S Angst1, Martha Tingle1, Martin Schmelz2,3, Brendan Carvalho1, David C Yeomans1 1Department of Anesthesia, Stanford University School of Medicine, 2Department of Anaesthesiology, University of Mannheim, 3Department of Anaesthesiology, University of Heidelberg A technique is presented for the in-vivo collection of interstitial fluid samples from pertinent tissue sites (here, experimentally inflamed skin) for the measurement of biochemicals mediating pain and inflammation. Biology The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia Hidetoshi Taniguchi1, Katrin Andreasson1 1Department of Neurology and Neurological Sciences, Stanford University School of Medicine The postnatal rat model for hypoxic-ischemic brain injury is a well-established model of human neonatal hypoxic ischemic encephalopathy (HIE). In this article, we describe the model of HIE in post-natal rat pups. Biology Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds Brendan Carvalho1, David J. Clark1, David Yeomans1, Martin S. Angst1 1Department of Anesthesiology, Stanford University School of Medicine A technique to collect and measure surgical wound biochemical mediators at specific time points. Biology Pressure-polishing Pipettes for Improved Patch-clamp Recording Brandon E. Johnson1, Austin L. Brown1, Miriam B. Goodman1 1Department of Molecular and Cellular Physiology, Stanford University School of Medicine This is a guide to modifying the shape of glass micropipettes. Specifically, by using heat and air pressure the taper is widened without increasing the tip opening, leading to lower pipette resistance. This is critical to obtain low noise recordings of small cells but is useful in many applications. Biology Patch Clamp Recording of Ion Channels Expressed in Xenopus Oocytes Austin L Brown1, Brandon E. Johnson2, Miriam B. Goodman2 1Department of Molecular and Cellular Physiology, Stanford University, 2Department of Molecular and Cellular Physiology, Stanford University School of Medicine This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology. Biology Making Patch-pipettes and Sharp Electrodes with a Programmable Puller Austin L. Brown1, Brandon E. Johnson2, Miriam B. Goodman2 1Department of Molecular and Cellular Physiology, Stanford University, 2Department of Molecular and Cellular Physiology, Stanford University School of Medicine This video shows how to use a programmable puller to make patch pipettes and sharp electrodes for electrophysiology. The same procedure can be used to make a variety of glass tools, including injection needles. Biology In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging Mayumi Yamada1, Phillip Yang1 1Division of Cardiovascular Medicine, Stanford University In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC. Biology In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method Xiang Wang1, Phillip Yang1 1Division of Cardiovascular Medicine, Stanford University This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method. Biology In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells Kitchener Wilson1, Jin Yu1, Andrew Lee1, Joseph C. Wu1 1Departments of Radiology and Medicine (Cardiology), Stanford University School of Medicine With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse. 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.
