Stanford University

97 articles published in JoVE

• Engineering

  
  Triplet Fusion Upconversion Nanocapsule Synthesis Tracy H. Schloemer^1,2, Samuel N. Sanders¹, Qi Zhou², Pournima Narayanan³, Manchen Hu², Mahesh K. Gangishetty¹, Daniel Anderson¹, Michael Seitz^1,2, Arynn O. Gallegos², R. Christopher Stokes¹, Daniel N. Congreve^{1,2 1}Rowland 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 Kim^{1 1}Department 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 Amsallem^1,2, Jean-Baptiste Menager¹, Simon Dang Van¹, Peter Dorfmuller³, Andrew Connolly⁴, Alban Todesco⁵, Benoit Decante¹, Elie Fadel¹, Francois Haddad², Olaf Mercier^{1 1}Research 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.

• 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-Huerta^{1 1}Institute 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 Luo^{1 1}Department 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.

• Bioengineering

  
  Injectable Supramolecular Polymer-Nanoparticle Hydrogels for Cell and Drug Delivery Applications Catherine M. Meis*¹, Abigail K. Grosskopf*², Santiago Correa¹, Eric A. Appel^{1,3,4 1}Department 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 Woznak^1,4, Alexander Klibanov⁵, Erik Dumont⁶, Max Wintermark*², Kevin S. Lee*^{1,7,8 1}Department 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 Djurisic^{1 1}Department 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 Venkatraman^1,2,3, Adrija Darsha^1,2,3, Sean M. Wu^1,2,3, Joseph C. Wu^1,2,3, Timon Seeger^{1,2,3,4,5 1}Stanford 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 Morgado^{1 1}Stanford 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 Sharma^{1 1}Department 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 Ishak^1,3, Vivekananda Sunkari¹, Michelle S. Bach¹, Dan Liu¹, Robert Manasherob¹, Paul L. Bollyky^{1,2 1}Division 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-Eizagirre^1,2, Aleix Solanes^1,2, Miquel Angel Fullana^2,3, John P. A. Ioannidis⁴, Paolo Fusar-Poli^5,6,7, Carla Torrent^1,2,3,8, Brisa Solé^1,2,3,8, Caterina Mar Bonnín^1,2,3,8, Eduard Vieta^1,2,3,8, David Mataix-Cols⁹, Joaquim Radua^{1,2,5,9 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 Weisleitner^1,2, Lars Hunger³, Christoph Kohstall⁴, Albert Frisch⁵, Michael C. Storrie-Lombardi⁶, Birgit Sattler^{1,2 1}Institute 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. Wilson^{2 1}Apoptosis, 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. Weiss^1,2, Julie N. Chuong³, Rachel B. Brem^{1,3 1}Department of Plant and Microbial Biology, University of California Berkeley, ²Department of Biology, Stanford University, ³Buck Institute for Research on Aging Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait.

• Cancer Research

  
  Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels Steven M Alves¹, Tian Zhu¹, Anastasia Shostak¹, Ninna S. Rossen², Marjan Rafat^{1,3,4 1}Department 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 Tel^{1 1}Department 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 Jo^2,3,4,7, Young Seo Kim^3,4,5, Yeongjin Yu^2,3, Jiyeon Park⁶, Sumin Lee⁴, Wei Sun Park^2,3, YongKeun Park^{2,3,4 1}Department 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-Kruse^{1 1}Department 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-Eisenberg^{1 1}Department 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. James^{1,2 1}Department 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 Carmi^{1 1}Department 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. Heilshorn^{2 1}Department 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 Wu^1,2, Shawn Sallis^2,3, Ruimin Qiao², Qinghao Li^2,4, Zengqing Zhuo^2,5, Kehua Dai^2,6, Zixuan Guo^2,7, Wanli Yang^{2 1}Geballe 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 Beadle^{5,9 1}Department 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 Wang^{1 1}Rowland 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. Bohlen^{1 1}Department 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. Kim^1,2, Beth L. Pruitt^1,2,3, Miriam B. Goodman^1,2, Michael Krieg^{4 1}Department 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-Link^{1 1}Department 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 Feng^{1 1}Department 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 Velez^3,4, James E. DiCarlo^1,2, Anuradha M. Gore³, MaryAnn Mahajan³, Stephen H. Tsang^1,2, Alexander G. Bassuk^5,6, Vinit B. Mahajan^{3,9 1}Barbara & 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. Spencer^1,2, Peggy P. Ho³, Lawrence Steinman³, Ari J. Green¹, Raymond A. Sobel⁴, Scott S. Zamvil^{1,2 1}Department 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. Gosain^{2 1}Mechanical 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 Kong^{1 1}Anesthesiology, 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. Fordyce^{1,2,3,4 1}Department 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 Yuan^1,2, Mark E. Orcholski^1,2, Ngan F. Huang^2,3, Vinicio A. de Jesus Perez^{1,2 1}Division 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 1}Dept. 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 Lee^{1,2 1}Neurology 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 Yang^1,3, Nidhi Bhutani^{1 1}Orthopaedic 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 Hesselink^{2 1}Electrical 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. Snyder^{1,2 1}Department 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 Musacchia^1,2,3, Silvia Ortiz-Mantilla¹, Teresa Realpe-Bonilla¹, Cynthia P. Roesler¹, April A. Benasich^{1 1}Center 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. Su^{1 1}Department 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 Diesseroth^2,5, Colleen A. McClung^{1 1}Department 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. Longaker^1,2, Derrick C. Wan^{1 1}Hagey 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. Walmsley^1,2, Taylor Wearda^1,2, Owen Marecic^1,2, Michael S. Hu¹, Kevin J. Paik¹, Kshemendra Senarath-Yapa¹, David A. Atashroo¹, Elizabeth R. Zielins¹, Derrick C. Wan¹, Irving L. Weissman^1,2, Michael T. Longaker^{1,2 1}Hagey 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 1}Department 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 Huang^{1,4 1}Department 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. Garner^3,4, Kathleen R. Pritchett-Corning^{1 1}Research 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 Yang^{1,2 1}Department 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. Byer^{1 1}Edward 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. Oro^{1 1}Program 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 Clandinin^{1 1}Department 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. Chang^1,2, Jen-Jane Liu^1,2, Joseph C. Liao^{1,2 1}Department 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 Cheng^{1 1}Department 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. Maecker^{1 1}Human 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. Grova^1,4, Min Lee⁵, Derrick C. Wan¹, Michael T. Longaker^{1 1}Department of Surgery, Stanford University, ²Department of Surgery, Duke University, ³Department of Surgery, Saint Joseph Mercy Hospital, ⁴School of Medicine, University of California, San Francisco, ⁵School of Dentistry, University of California, Los Angeles This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

• Medicine

  
  Collecting And Measuring Wound Exudate Biochemical Mediators In Surgical Wounds Brendan Carvalho¹, David J Clark¹, David Yeomans¹, Martin S Angst^{1 1}Department 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. Simpson^2,3, Phillip C. Yang¹, Rajesh Dash^{1 1}Division of Cardiovascular Medicine, Department of Medicine, Stanford University Medical Center, ²Division of Cardiology, Department of Medicine, University of California, San Francisco, ³San Francisco VAMC Early detection of apoptosis may identify at-risk cell populations in a variety of diseases. Here we demonstrate a method to link an early apoptosis-detection protein (Annexin V) to a MRI-detectable iron oxide nanoparticle (SPIO). This method may be extended to other proteins of interest to generate MRI-detectable molecular imaging probes.

• Bioengineering

  
  Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers Arjun S. Adhikari¹, Jack Chai¹, Alexander R. Dunn^{1 1}Department 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 Johnson^{2 1}Department 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 Meier^2,3,4, Heather N. Cartwright¹, Davide Sosso¹, Stephen R. Quake^2,3, David W. Ehrhardt¹, Wolf B. Frommer^{1 1}Department 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 Hua^1,2, Tobias Deuse^1,2, Evangelos D. Michelakis³, Alois Haromy³, Phil S. Tsao⁴, Lars Maegdefessel⁴, Reinhold G. Erben⁵, Claudia Bergow⁵, Boris B. Behnisch⁶, Hermann Reichenspurner^1,2, Robert C. Robbins⁷, Sonja Schrepfer^{1,2,7 1}University 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. Chang^{1 1}Howard 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. Santiago^{1 1}Mechanical 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. Cheng^{1 1}Department 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. Castaneda^1,2, Aman Khurana^1,2, Ramsha Khan^1,2, Heike E. Daldrup-Link^{1 1}Department 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 Urbanus^1,2, Michael Proctor⁵, Lawrence E. Heisler^2,3, Guri Giaever^2,6, Corey Nislow^{1,2,3 1}Banting 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. Hsieh^{1 1}Department 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 Conradi^1,2, Christiane Pahrmann¹, Stephanie Schmidt¹, Tobias Deuse^1,3, Arne Hansen², Alexandra Eder², Hermann Reichenspurner¹, Robert C. Robbins³, Thomas Eschenhagen², Sonja Schrepfer^{1,3 1}Transplant 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. Gamse^{1 1}Department 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. Rasmussen^{3 1}Institute 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. Hoffman^{1 1}Medical 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-Vera^{1 1}Department 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 Deuse^1,2, Anna Hammel¹, Robert C. Robbins², Hermann Reichenspurner¹, Sonja Schrepfer^{1,2 1}University 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 Andreasson^{1 1}Department 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. Cooke^{1 1}Department 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. Hsieh^{2 1}Earth 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 Deuse^1,2, Karis R. Tang-Quan^1,2,3, Robert C. Robbins³, Hermann Reichenspurner^1,2, Sonja Schrepfer^{1,2,3 1}Transplant 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. Kolk^1,2, Danja Meyberg^1,2, Tobias Deuse^1,2, Karis R. Tang-Quan^1,2,3, Robert C. Robbins³, Hermann Reichenspurner^1,2, Sonja Schrepfer^{1,2,3 1}Transplant 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 Schrepfer^{1 1}Department 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. Tsao^{1 1}Department 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. Cooke^{1 1}Division 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. Cooke^{1 1}Division of Cardiovascular Medicine, Stanford University, ²Department of Anesthesiology, University of California, San Francisco The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

• Biology

  
  Calcium Imaging of Cortical Neurons using Fura-2 AM Odmara L Barreto-Chang¹, Ricardo E Dolmetsch^{2 1}Department 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 Carvalho^{1 1}Department 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 Schmelz^2,3, Brendan Carvalho¹, David C Yeomans^{1 1}Department 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 Andreasson^{1 1}Department 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. Angst^{1 1}Department 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. Goodman^{1 1}Department 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. Goodman^{2 1}Department 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. Goodman^{2 1}Department 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 Yang^{1 1}Division 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 Yang^{1 1}Division 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. Wu^{1 1}Departments 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 Tseng^1,2, Xianne Leong², Edgar Engleman^{2 1}Department of Surgery, University of California, San Francisco - UCSF, ²Department of Pathology, Stanford University School of Medicine Two techniques can be used to establish this model: injection of a cancer cell suspension into the cecal wall or transplantation of a piece of subcutaneous tumor onto the cecum. This model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.