74 ARTICLES PUBLISHED IN JoVE
BiologyiCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide ResolutionJulian Konig 1, Kathi Zarnack 2, Gregor Rot 3, Tomaz Curk 3, Melis Kayikci 1, Blaz Zupan 3, Daniel J. Turner 4, Nicholas M. Luscombe 2, Jernej Ule 11Laboratory of Molecular Biology, Medical Research Council - MRC, 2European Bioinformatics Institute, EMBL Heidelberg, 3Computer and Information Science, University of Ljubljana, 4Wellcome Trust Genome Campus, Wellcome Trust Sanger InstituteThe spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.BiologyPreparation of Quality Inositol PyrophosphatesOmar Loss 1, Cristina Azevedo 1, Zsolt Szijgyarto 1, Daniel Bosch 1, Adolfo Saiardi 11Medical Research Council (MRC), Cell Biology Unit and Laboratory for Molecular Cell Biology, University College LondonInositol pyrophosphates play an important role in human pathologies such cancer, diabetes and obesity; however, the exact mechanism of action is a matter of dispute. The lack of commercially available inositol pyrophosphates renders detailed studies problematic. Here we describe a simple protocol to produce and isolate milligrams of inositol pyrophosphates.NeuroscienceElectrophysiological Measurements and Analysis of Nociception in Human InfantsL. Fabrizi *1, A. Worley *2, D. Patten 1, S. Holdridge 1, L. Cornelissen 1, J. Meek 3, S. Boyd 2, R. Slater 1,41Neuroscience, Physiology and Pharmacology, University College London, 2Department of Clinical Neurophysiology, Great Ormond Street Hospital, 3Elizabeth Garrett Anderson Obstetric Hospital, University College Hospital, 4Nuffield Department of Anaesthetics, University of OxfordThe assessment and treatment of pain in infants is difficult because infants cannot verbally report their experience. In this video we describe quantitative electrophysiological methods and analysis techniques that can be used to measure the response to noxious events from the infant nervous system.MedicineHeterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and ProgressionKate Lawrenson 1, Barbara Grun 2, Simon A. Gayther 11Department of Preventive Medicine, University of Southern California, 2Institute for Women's Health, University College LondonWe describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.MedicineA Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse ModelMarianne S. Carlon *1, Jaan Toelen *2, Marina Mori da Cunha 2, Dragana Vidović 1, Anke Van der Perren 3, Steffi Mayer 2, Lourenço Sbragia 2, Johan Nuyts 4, Uwe Himmelreich 5, Zeger Debyser 1, Jan Deprest 21Molecular Virology and Gene Therapy, KU Leuven, 2Department of Woman and Child, KU Leuven, 3Neurobiology and Gene Therapy, KU Leuven, 4Division of Nuclear Medicine, KU Leuven, 5Biomedical NMR Unit/ MoSAIC, KU LeuvenWe developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.BiologyA High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression CloningJamie Freeman 1, Janos Kriston-Vizi 1, Brian Seed 2, Robin Ketteler 11MRC LMCB, University College London, 2Center for Computational and Integrative Biology, Massachusetts General HospitalA high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.NeuroscienceChicken Embryo Spinal Cord Slice Culture ProtocolKristina C. Tubby *1, Dee Norval *1, Stephen R. Price 11Research Department of Cell and Developmental Biology, University College LondonSlice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.MedicineFetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth RestrictionRyan Hodges 1,2, Masayuki Endo 1, Andre La Gerche 3, Elisenda Eixarch 4,5, Philip DeKoninck 1, Vessilina Ferferieva 3, Jan D'hooge 3, Euan M. Wallace 2, Jan Deprest 11Division Woman and Child, Department Women, University Hospitals Leuven, 2The Ritchie Centre, Monash Institute of Medical Research, Department of Obstetrics and Gynaecology, Monash University, Victoria, Australia, 3Department of Cardiovascular Sciences, Katholieke Universiteit Leuven, 4Fetal and Perinatal Medicine Research Group, Institut d'Investigacions Biomediques August Pi i Sunyer (IDIBAPS), 5Maternal-Fetal Medicine Department, ICGON, Hospital Clínic, Universitat de Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER)We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.EngineeringSetting Limits on Supersymmetry Using Simplified ModelsChristian Gütschow 1, Zachary Marshall 2,31Department of Physics and Astronomy, University College London, 2CERN, 3Physics Division, Lawrence Berkeley National LaboratoriesThis paper demonstrates a protocol for recasting experimental simplified model limits into conservative and aggressive limits on an arbitrary new physics model. Publicly available LHC experimental results can be recast in this manner into limits on almost any new physics model with a supersymmetry-like signature.BioengineeringTransplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle RegenerationMattia F. M. Gerli *1, Sara M. Maffioletti *1, Queensta Millet 1, Francesco Saverio Tedesco 1,21Department of Cell and Developmental Biology, University College London, 2Division of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele HospitalInduced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.BioengineeringA Decellularization Methodology for the Production of a Natural Acellular Intestinal MatrixPanagiotis Maghsoudlou *1, Giorgia Totonelli *1, Stavros P Loukogeorgakis 1, Simon Eaton 1, Paolo De Coppi 11Surgery Unit, Institute of Child Health and Great Ormond Street Hospital, University College LondonThe article describes a methodology for the production of an acellular matrix from rat intestine. The derivation of intestinal scaffolds is important for future applications in tissue engineering, stem cell biology and drug testing.Immunology and InfectionNanomechanics of Drug-target Interactions and Antibacterial Resistance DetectionJoseph W. Ndieyira 1, Moyu Watari 1, Rachel A. McKendry 11London Centre for Nanotechnology and Departments of Medicine, University College LondonAcquired resistance to antibiotics is a major public healthcare problem and is presently ranked by the WHO as one of the greatest threats to human life. Here we describe the use of cantilever technology to quantify antibacterial resistance, critical to the discovery of novel and powerful agents against multidrug resistant bacteria.Immunology and InfectionImaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenesAndreas Kühbacher 1,2,3, Edith Gouin 1,2,3, Jason Mercer 4, Mario Emmenlauer 5, Christoph Dehio 5, Pascale Cossart 1,2,3, Javier Pizarro-Cerdá 1,2,31Unité des Interactions Bactéries Cellules, Pasteur Institute, 2INSERM U604, 3Institut National de la Recherche Agronomique (INRA), USC2020, 4Institute of Biochemistry, ETH Zürich, 5Focal Area Infection Biology, Biozentrum, University of BaselListeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.NeurosciencePreparation of Neuronal Co-cultures with Single Cell PrecisionNgoc-Duy Dinh 1, Ya-Yu Chiang 1,2, Heike Hardelauf 1, Sarah Waide 1, Dirk Janasek 1, Jonathan West 1,31Leibniz-Institut für Analytische Wissenschaften, ISAS, 2Department of Biochemical Engineering, University College London, 3Institute for Life Sciences, University of SouthamptonProtocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.NeuroscienceMouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron MigrationMiguel Tillo 1, Quenten Schwarz 1,2, Christiana Ruhrberg 11UCL Institute of Ophthalmology, University College London, 2Department of Human Immunology, Centre for Cancer Biology, South AustraliaEmbryonic neurons are born in the ventricular zone of the neural tube, but migrate to reach appropriate targets. Facial branchiomotor (FBM) neurons are a useful model to study neuronal migration. This protocol describes the wholemount ex vivo culture of mouse embryo hindbrains to investigate mechanisms that regulate FBM migration.BioengineeringManufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as BinderKoon-Yang Lee 1,2, Siti Rosminah Shamsuddin 3, Marta Fortea-Verdejo 1, Alexander Bismarck 1,31Polymer and Composite Engineering (PaCE) Group, Institute of Materials Chemistry and Research, University of Vienna, 2Department of Chemical Engineering, University College London, 3Polymer and Composite Engineering (PaCE) Group, Department of Chemical Engineering, Imperial College LondonWe present a novel method of manufacturing rigid and robust short natural fiber preforms using a papermaking process. Bacterial cellulose acts simultaneously as the binder for the loose fibers and provides rigidity to the fiber preforms. These preforms can be infused with a resin to produce truly green hierarchical composites.Immunology and InfectionNon-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal RatFatma Dalgakiran 1, Luci A. Witcomb 1, Alex J. McCarthy 1, George M. H. Birchenough 2, Peter W. Taylor 11School of Pharmacy, University College London, 2Mucin Biology Group, University of GothenburgHere, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.NeuroscienceInhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA ReceptorsLaura E. Brown 1, Celine Fuchs 1, Martin W. Nicholson 1, F. Anne Stephenson 1, Alex M. Thomson 1, Jasmina N. Jovanovic 11UCL School of Pharmacy, University College LondonThe molecular mechanisms that co-ordinate the formation of inhibitory GABAergic synapses during ontogeny are largely unknown. To study these processes,we have developed a co-culture model system which incorporates embryonic medium spiny GABAergic neurons cultured together with stably transfected human embryonic kidney 293 (HEK293) cells expressing functional GABAA receptors.BiologyEvaluation of Zebrafish Kidney Function Using a Fluorescent Clearance AssaySonia Christou-Savina 1, Philip L. Beales 1, Daniel P. S. Osborn 21Genetics and Genomic Medicine, Institute of Child Health, University College London, 2Molecular Cell Science Research Centre, St. George's University of LondonThe zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay1 that allows quantitative analysis of zebrafish kidney function in CKD.NeuroscienceIntramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor NeuronsRahul Mohan *1, Andrew P. Tosolini *1, Renée Morris 11Translational Neuroscience Facility, School of Medical Sciences, University of New South WalesThe efficacy of intramuscular uptake and retrograde transport of molecules to corresponding motor neurons depends on the location of the injection sites with respect to the motor end plates (MEPs). Here, we describe how to locate MEPs on skeletal muscles to optimise retrograde transport of tracers into motor neurons.NeuroscienceSelective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl EsterJoseph Jebelli *1, Thomas Piers *2, Jennifer Pocock 31Department of Neurology, University of Washington, 2Therapeutic Innovation Group, School of Life and Medical Sciences, University College London, 3Department of Neuroinflammation, University College LondonMicroglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature. BehaviorUsing Fiberless, Wearable fNIRS to Monitor Brain Activity in Real-world Cognitive TasksPaola Pinti 1,2, Clarisse Aichelburg 3, Frida Lind 3, Sarah Power 1, Elizabeth Swingler 3, Arcangelo Merla 2, Antonia Hamilton 3, Sam Gilbert 3, Paul Burgess 3, Ilias Tachtsidis 11Department of Medical Physics and Biomedical Engineering, Malet Place Engineering Building, University College London, 2Infrared Imaging Lab, Institute for Advanced Biomedical Technology (ITAB), Department of Neuroscience, Imaging and Clinical Sciences, University of Chieti-Pescara, 3Institute of Cognitive Neuroscience, Alexandra House, University College LondonMonitoring brain activity outside the lab without physical constraints presents methodological challenges. A fiberless, wearable functional Near Infrared Spectroscopy (fNIRS) system was used to measure brain activity during an ecological prospective memory task. It was demonstrated that this system could be used to monitor brain activity during non-lab based experiments.BioengineeringHollow Fiber Bioreactors for In Vivo-like Mammalian Tissue CultureMichael P. Storm 1, Ian Sorrell 2, Rebecca Shipley 3, Sophie Regan 2, Kim A. Luetchford 1, Jean Sathish 2, Steven Webb 4, Marianne J. Ellis 11Department of Chemical Engineering and Centre for Regenerative Medicine, University of Bath, 2MRC Centre for Drug Safety Science and Institute of Translational Medicine, University of Liverpool, 3Mechanical Engineering, University College London, 4Department of Applied Mathematics, Liverpool John Moores UniversityThe functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.MedicineA Pipeline for 3D Multimodality Image Integration and Computer-assisted Planning in Epilepsy SurgeryMark Nowell 1, Roman Rodionov 1, Gergely Zombori 2, Rachel Sparks 2, Michele Rizzi 1, Sebastien Ourselin 2, Anna Miserocchi 3, Andrew McEvoy 3, John Duncan 11Department of Clinical and Experimental Epilepsy, UCL Institute of Neurology, 2Center of Medical Imaging and Computing, UCL, 3Department of Neurosurgery, National Hospital for Neurology and NeurosurgeryWe describe the steps to use our custom designed software for image integration, visualization and planning in epilepsy surgery.NeuroscienceEx Vivo Optogenetic Dissection of Fear Circuits in Brain SlicesDaniel Bosch 1, Douglas Asede 2, Ingrid Ehrlich 11Hertie Institute for Clinical Brain Research and Werner Reichardt Centre for Integrative Neuroscience, University of Tuebingen, 2Max Planck Florida Institute for NeuroscienceOptogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined thatupon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.NeuroscienceCombined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse AmygdalaSabine Schönherr 1, Anna Seewald 1, Yu Kasugai 1, Daniel Bosch 2, Ingrid Ehrlich 2, Francesco Ferraguti 11Department of Pharmacology, Medical University of Innsbruck, 2Hertie Institute for Clinical Brain Research and Centre for Integrative Neuroscience, University of TübingenThis article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.MedicineIntravenous Endotoxin Challenge in Healthy Humans: An Experimental Platform to Investigate and Modulate Systemic InflammationJames N. Fullerton 1, Elisabetta Segre 1, Roel P.H. De Maeyer 1, Alexander A.N. Maini 1, Derek W. Gilroy 11Centre for Clinical Pharmacology, Division of Medicine, University College LondonIntravenous administration of endotoxin reliably elicits dose-dependent physiological and immunological alterations consistent with several pathological states. This reductionist approach permits the investigation, modeling and experimental modification of systemic inflammation and its downstream effects in man.NeuroscienceCombining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain SectionsSarah Jolly *1, Alexander Fudge *1, Nigel Pringle 1, William D. Richardson 1, Huiliang Li 11Wolfson Institute for Biomedical Research, University College LondonLocalizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.EngineeringIn Vitro Culture of Epicardial Cells From Mouse Embryonic HeartSindhu Ramesh *1, Anamika Singh *1, Dasan M. Cibi 1, Derek J. Hausenloy 1,2,3, Manvendra K. Singh 1,21Program in Cardiovascular and Metabolic Disorders, Duke-NUS Graduate Medical School, 2National Heart Research Institute Singapore, National Heart Centre Singapore, 3The Hatter Cardiovascular Insititute, University College LondonThe epicardium is an essential source of multipotent cardiovascular progenitor cells and paracrine factors that are required for cardiovascular development and regeneration. We describe here a method to culture mouse embryonic epicardial cells. EngineeringAdvanced Compositional Analysis of Nanoparticle-polymer Composites Using Direct Fluorescence ImagingColin R. Crick *1, Sacha Noimark *2,3, William J. Peveler *3, Joseph C. Bear 3, Aleksandar P. Ivanov 1, Joshua B. Edel 1, Ivan P. Parkin 31Department of Chemistry, Imperial College London, 2Department of Medical Physics and Biomedical Engineering, University College London, 3Department of Chemistry, University College LondonHere we present a reliable method to monitor the incorporation of nanoparticles into a polymer host matrix via swell encapsulation. We show that the surface concentration of cadmium selenide quantum dots can be accurately visualized through cross-sectional fluorescence imaging.BehaviorThe 4 Mountains Test: A Short Test of Spatial Memory with High Sensitivity for the Diagnosis of Pre-dementia Alzheimer's DiseaseDennis Chan 1, Laura Marie Gallaher 2, Kuven Moodley 2, Ludovico Minati 3, Neil Burgess 4, Tom Hartley 51Department of Clinical Neurosciences, University of Cambridge, 2Clinical Imaging Sciences Centre, Brighton and Sussex Medical School, 3U.O. Direzione Scientifica, Fondazione IRCCS Istituto Neurologico Carlo Besta, 4Institute of Cognitive Neuroscience, University College London, 5Department of Psychology, University of YorkThis article describes the 4 Mountains Test (4MT), a hippocampus-dependent test of working allocentric spatial memory. The hippocampus is affected early in Alzheimer's disease (AD) and this article outlines the 4MT methodology and results of patient testing, which demonstrates the value of the 4MT in the diagnosis of pre-dementia AD. MedicineA High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms StatusWendy E. Heywood *1, Anna Baud *1, Emily Bliss 1, Ernestas Sirka 1, Jonathan M. Schott 2, Henrik Zetterberg 3, Daniela Galimberti 4, Neil J. Sebire 5, Kevin Mills 11Centre for Translational Omics, Genetics and Genomic Medicine Deptartment, Great Ormond Street Institute of Child Health, University College London, 2Dementia Research Centre, Institute of Neurology, University College London, 3Clinical Neurochemistry Laboratory, Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry, The Sahlgrenska Academy, University of Gothenburg, 4Neurology Unit, Department of Pathophysiology and Transplantation, University of Milan, 5Great Ormond Street Hospital for Children, University College LondonWe describe a high-throughput, multiplex, and targeted proteomic cerebrospinal fluid (CSF) assay developed with potential for clinical translation. The test can quantitate potential markers and risk factors for neurodegeneration, such as the apolipoprotein E variants (E2, E3 and E4), and measure their allelic expression.Developmental BiologyThe Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon SystemEnrica Bertin 1, Martina Piccoli 1, Chiara Franzin 1, Andras Nagy 2, Maria Mileikovsky 2, Paolo De Coppi 3, Michela Pozzobon 11Stem Cell and Regenerative Medicine Laboratory, Fondazione Istituto di Ricerca Pediatrica Citta della Speranza, 2Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 3Stem Cells and Regenerative Medicine Section, Developmental Biology and Cancer Programme, UCL Institute of Child Health and Great Ormond Street HospitalIn this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system. Developmental BiologyIsolation and Expansion of Adult Canine Hippocampal Neural PrecursorsThomas Duncan 1, Aileen Lowe 1, Marshall A. Dalton 2, Michael Valenzuela 11Regenerative Neuroscience Group, University of Sydney, 2Wellcome Trust Centre for Neuroimaging, Institute of Neurology, University College LondonThe canine brain is a valuable model in which to study adult neurogenesis. Presented here are protocols for isolating and expanding adult canine hippocampal neural precursor cells from primary brain tissue.Immunology and InfectionImaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH IndicatorJuliet R. Foote 1, Adam P. Levine 1, Philippe Behe 1, Michael R. Duchen 2, Anthony W. Segal 11Centre for Molecular Medicine, Division of Medicine, University College London, 2Cell and Developmental Biology, University College LondonThis manuscript describes a simple method to measure the phagosomal pH and area as well as the cytoplasmic pH of human and mouse neutrophils using the ratiometric indicator seminaphthorhodafluor (SNARF)-1, or S-1. This is achieved using live-cell confocal fluorescence microscopy and image analysis.BiologySimultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent MicroscopyMatthew McKenzie 1,2, Sze C. Lim 1, Michael R. Duchen 31Centre for Genetic Diseases, Hudson Institute of Medical Research, 2The Department of Molecular and Translational Sciences, Monash University, 3Department of Physiology, University College LondonMitochondria can utilize the electrochemical potential across their inner membrane (ΔΨm) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and ΔΨm in live cells using fluorescent dyes and confocal microscopy.BioengineeringMedical-grade Sterilizable Target for Fluid-immersed Fetoscope Optical Distortion CalibrationDaniil I. Nikitichev *1, Dzhoshkun I. Shakir *1, François Chadebecq 1, Marcel Tella 1, Jan Deprest 1,2, Danail Stoyanov 3, Sébastien Ourselin 1, Tom Vercauteren 11Translational Imaging Group, CMIC, University College London, 2Department of Obstetrics and Gynecology, University Hospitals Leuven, 3Surgical Robot Vision Group, CMIC, University College LondonThis article describes the design and development of a sterilizable custom camera optical distortion calibration target for the peri-operative, fluid-immersed calibration of endoscopes during endoscopic interventions.Developmental BiologyDefined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem CellsYu Wang 1, Sharmin Alhaque 1, Kate Cameron 1, Jose Meseguer-Ripolles 1, Baltasar Lucendo-Villarin 1, Hassan Rashidi 1, David C. Hay 11MRC Centre for Regenerative Medicine, University of EdinburghThe method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.BiologyOpen-source Single-particle Analysis for Super-resolution Microscopy with VirusMapperRobert D. M. Gray 1,2, Jason Mercer 1, Ricardo Henriques 1,31MRC Laboratory for Molecular Cell Biology, University College London, 2Centre for Mathematics and Physics in Life Sciences and Experimental Biology (CoMPLEX), University College London, 3Department of Cell and Developmental Biology, University College LondonThis manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.MedicineTransvaginal Mesh Insertion in the Ovine ModelIva Urbankova 1,2, Geertje Callewaert 1,3, Nikhil Sindhwani 1, Alice Turri 1, Lucie Hympanova 1,2, Andrew Feola 1, Jan Deprest 1,31Centrum for Surgical Technologies, Department of Development and Regeneration, Clinical Specialties Research Groups, Faculty of Medicine, KU Leuven, 2Institute for the Care of Mother and Child and Third Faculty of Medicine, Charles University, Prague, 3Pelvic Floor Unit, University Hospitals KU LeuvenThis protocol describes mesh implantation in the ovine rectovaginal septum using a single vaginal incision technique, with and without the trocar-guided insertion of anchoring arms. NeuroscienceA Prediction Error-driven Retrieval Procedure for Destabilizing and Rewriting Maladaptive Reward Memories in Hazardous DrinkersRavi K. Das 1, Grace Gale 1, Vanessa Hennessy 1, Sunjeev K. Kamboj 11Clinical Psychopharmacology Unit, University College LondonThis manuscript describes a memory retrieval procedure for destabilizing robust reward memories and rewriting them with counterconditioning prior to their reconsolidation.NeuroscienceEvaluation of Synapse Density in Hippocampal Rodent Brain SlicesFaye McLeod *1, Aude Marzo *1, Marina Podpolny 1, Soledad Galli 1, Patricia Salinas 11Department of Cell and Developmental Biology, University College LondonA protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.NeuroscienceThe Mouse Hindbrain As a Model for Studying Embryonic NeurogenesisMathew Tata 1, Christiana Ruhrberg 11UCL Institute of OphthalmologyThis article demonstrates how the mouse embryonic hindbrain can be used as a model for studying developmental neurogenesis in both whole organ and tissue section preparations.EngineeringThree-Dimensional Ultrasonic Needle Tip Tracking with a Fiber-Optic Ultrasound ReceiverWenfeng Xia 1,2, Simeon J. West 3, Malcolm C. Finlay 2,4, Rosalind Pratt 5,6, Sunish Mathews 1,2, Jean-Martial Mari 7, Sebastien Ourselin 1,2,6, Anna L. David 1,5,8,9, Adrien E. Desjardins 1,21Wellcome/EPSRC Centre for Interventional and Surgical Sciences, University College London, 2Department of Medical Physics and Biomedical Engineering, University College London, 3Department of Anaesthesia, University College Hospital, 4St Bartholomew's Hospital and Queen Mary University of London, 5Institute for Women's Health, University College London, 6Centre for Medical Imaging Computing, University College London, 7GePaSud, University of French Polynesia, 8Department of Development and Regeneration, KU Leuven (Katholieke Universiteit), 9NIHR University College London Hospitals Biomedical Research CentreAccurate and efficient visualization of invasive medical devices is extremely important in many ultrasound-guided minimally invasive procedures. Here, a method for localizing the spatial position of a needle tip relative to the ultrasound imaging probe is presented.BiologyEvaluating Vascular Hyperpermeability-inducing Agents in the Skin with the Miles AssayJames T. Brash 1, Christiana Ruhrberg 1, Alessandro Fantin 11UCL Institute of Ophthalmology, University College LondonHere, we present a protocol to measure the vascular leakage induced by intradermal administration of permeability promoting agents into the murine skin. This technique can be used to determine the ability of molecules to promote or inhibit vascular leakage or to study the molecular mechanisms that regulate vascular permeability.Immunology and InfectionA Suction Blister Protocol to Study Human T-cell Recall Responses In VivoLine L. Holm 1,2,3, Milica Vukmanovic-Stejic 4, Thomas Blauenfeldt 1, Thomas Benfield 2,3, Peter Andersen 1, Arne N. Akbar 4, Morten Ruhwald 11Department of Infectious Disease Immunology, Center for Vaccine Research, Statens Serum Institut, 2Department of Infectious Diseases, Hvidovre Hospital, 3Institute of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, 4Division of Infection and Immunity, University College LondonHere, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.JoVE CoreStress Distribution During Cold Compression of Rocks and Mineral Aggregates Using Synchrotron-based X-Ray DiffractionCecilia S.N. Cheung 1,2, Donald J. Weidner 1, Li Li 1, Philip G. Meredith 3, Haiyan Chen 1, Matthew Whitaker 1, Xianyin Chen 41Mineral Physics Institute, Department of Geoscience, Stony Brook University, 2Geological Engineering, Department of Civil and Environmental Engineering, University of Wisconsin-Madison, 3Rock and Ice Physics Laboratory, Department of Earth Sciences, University College London, 4Department of Chemistry, Stony Brook UniversityWe report detailed procedures for compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus coupled with synchrotron X-radiation. Such experiments allow quantification of the stress distribution within samples, that ultimately sheds light on compaction processes in geomaterials.BioengineeringA Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon DefectsPrasad Sawadkar 1,3, Jason Wong 2, Vivek Mudera 11Division of Surgery and Interventional Science, University College London, 2Blond McIndoe Laboratories, Division of Cell Matrix and Regenerative Medicine, MAHSC, University of Manchester, 3Department of Surgical Research, Northwick Park Institute for Medical Research, Northwick Park HospitalIn this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.JoVE CoreScalable Quantum Integrated Circuits on Superconducting Two-Dimensional Electron Gas PlatformKaveh Delfanazari 1,2, Pengcheng Ma 2, Reuben Puddy 2, Teng Yi 2, Moda Cao 2, Yilmaz Gul 3, Carly L. Richardson 4, Ian Farrer 2,5, David Ritchie 2, Hannah J. Joyce 1, Michael J. Kelly 1,2, Charles G. Smith 21Centre for Advanced Photonics and Electronics, Engineering Department, University of Cambridge, 2Department of Physics, Cavendish Laboratory, University of Cambridge, 3Department of Electronic and Electrical Engineering, University College London, 4Department of Materials Science and Metallurgy, University of Cambridge, 5Department of Electronic and Electrical Engineering, University of SheffieldQuantum integrated circuits (QICs) consisting of array of planar and ballistic Josephson junctions (JJs) based on In0.75Ga0.25As two-dimensional electron gas (2DEG) is demonstrated. Two different methods for fabrication of the two-dimensional (2D) JJs and QICs are discussed followed by the demonstration of quantum transport measurements in sub-Kelvin temperatures.GeneticsTissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ HybridizationFani Memi 1, Daniela Tirziu 2, Irinna Papangeli 31Department of Cell and Developmental Biology, University College London, 2Yale Cardiovascular Research Group, Section of Cardiovascular Medicine, Yale School of Medicine, 3Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Yale School of Medicinemicro-RNAs (miRNAs) are short and highly homologous RNA sequences, serving as post-transcriptional regulators of messenger RNAs (mRNAs). Current miRNA detection methods vary in sensitivity and specificity. We describe a protocol that combines in situ hybridization and immunostaining for concurrent detection of miRNA and protein molecules on mouse heart tissue sections.NeuroscienceBiocytin Recovery and 3D Reconstructions of Filled Hippocampal CA2 InterneuronsGeorgia Economides *1, Svenja Falk *1, Audrey Mercer 11Department of Pharmacology, University College LondonThe protocol outlined here describes the immunofluorescence analysis, biocytin recovery and high-quality reconstructions of hippocampal CA2 interneurons following the intracellular electrophysiological recordings in vitro, allowing neuronal characterization and ultimately fine neuronal anatomy to be studied. GeneticsGenetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on BacteriaPeggy Paschke 1, David A. Knecht 2, Thomas D. Williams 1, Peter A. Thomason 3, Robert H. Insall 3, Jonathan R. Chubb 4,5, Robert R. Kay 1, Douwe M. Veltman 11MRC Laboratory of Molecular Biology, 2Department of Molecular and Cell Biology, University of Connecticut, 3Cancer Research UK Beatson Institute Glasgow, 4MRC Laboratory for Molecular Cell Biology, University College London, 5Department of Cell and Developmental Biology, University College LondonDictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.Developmental BiologySerum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem CellsBalta Lucendo-Villarin 1, Hassan Rashidi 1,2, Sharmin Alhaque 1,3, Lena Fischer 1,4, Jose Meseguer-Ripolles 1, Yu Wang 1, Cliona O'Farrelly 4, Michael Themis 3, David C. Hay 11MRC Centre for Regenerative Medicine, University of Edinburgh, 2UCL Great Ormond Street Institute of Child Health, University College London, 3Division of Biosciences, Department of Life Sciences, College of Health and Life Sciences, Brunel University London, 4School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College DublinThis protocol describes an approach to produce hepatospheres from human pluripotent stem cells using a defined culture system and cell self-assembly. This protocol is reproducible in a number of cell lines, cost effective and allows the production of stable human hepatospheres for biomedical application.MedicineUse of Magnetic Resonance Imaging and Biopsy Data to Guide Sampling Procedures for Prostate Cancer BiobankingSusan Heavey 1, Aiman Haider 2, Ashwin Sridhar 3, Hayley Pye 1, Greg Shaw 1, Alex Freeman 2, Hayley Whitaker 11Molecular Diagnostics and Therapeutics Group, University College London, 2Research Department of Pathology, University College London, 3Department of Uro-Oncology, UCLH NHS Foundation TrustThis method involves utilization of clinical diagnostic data for prostate cancer patients in order to guide sampling procedures, when biobanking tissue following radical prostatectomy. This overcomes issues with previously published methods around efficiency and availability of fresh tissue for a wider range of downstream applications.NeuroscienceAn Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in NeuronsNicolás Stuardo 1,2, Guillermo Moya-Alvarado 1,3, Carolina Ramírez 1,3, Giampietro Schiavo 4, Francisca C. Bronfman 1,31Department of Physiology, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, 2Department of Molecular and Cellular Biology, Faculty of Biological Sciences, Center for Aging and Regeneration (CARE UC), Pontificia Universidad Católica de Chile, 3Institute of Biomedical Sciences. Faculty of Medicine and Faculty of Life Sciences, Universidad Andrés Bello, 4Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology and UK Dementia Research Institute at UCL, University College London CampusRecombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells in a cost-effective manner and is purified by affinity chromatography. BDNFavi is then directly mono-biotinylated with the enzyme BirA in a tube. BDNFavi and mono-biotinylated BDNFavi retain their biological activity when compared to commercially available BDNF.EnvironmentStreamlined Sampling and Cultivation of the Pelagic Cosmopolitan Larvacean, Oikopleura dioicaAki Masunaga 1, Andrew W. Liu 1, Yongkai Tan 1, Andrew Scott 1, Nicholas M. Luscombe 1,2,31Okinawa Institute of Science and Technology Graduate University, 2Francis Crick Institute, 3UCL Genetics Institute, Department of Genetics, Evolution and Environment, University College LondonOikopleura dioica is a tunicate model organism in various fields of biology. We describe sampling methods, species identification, culturing setup, and culturing protocols for the animals and algal feed. We highlight key factors that helped strengthen the culture system and discuss the possible problems and resolutions.Immunology and InfectionConstructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43Vanessa S. Terra 1, Charles D. Plumptre 2, Emma C. Wall 3, Jeremy S. Brown 2, Brendan W. Wren 11Faculty of Infections and Tropical Diseases, Department of Infection Biology, London School of Hygiene and Tropical Medicine, 2Division of Medicine, University College London, 3Division of Infection and Immunity, University College London UCLHere, we describe a S. pneumoniae serotype 1 strain 519/43 that can be genetically modified by using its ability to naturally acquire DNA and a suicide-plasmid. As proof of principle, an isogenic mutant in the pneumolysin (ply) gene was made.Immunology and InfectionExperimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse ModelChantelle E Bowers 1, Virginia L Calder 1, John Greenwood 1, Malihe Eskandarpour 11UCL Institute of Ophthalmology, University College LondonIn this report we present a protocol that allows the investigator to generate a mouse model of intraocular uveitis. More commonly referred to as experimental autoimmune uveitis (EAU), this established model captures many aspects of human disease. Herein, we will describe how to induce and monitor disease progression using several readouts. BiochemistryAbsolute Quantitation of Inositol Pyrophosphates by Capillary Electrophoresis Electrospray Ionization Mass SpectrometryDanye Qiu 1, Verena B. Eisenbeis 1, Adolfo Saiardi 2, Henning J. Jessen 11Institute of Organic Chemistry, University of Freiburg, 2Medical Research Council, Laboratory for Molecular Cell Biology, University College LondonA procedure for capillary electrophoresis electrospray ionization mass spectrometry for the absolute quantitation of inositol pyrophosphates from mammalian cell extracts is described.NeuroscienceExpanding the Toolkit for In Vivo Imaging of Axonal TransportAndrew P. Tosolini 1,2, David Villarroel-Campos 1,2,3, Giampietro Schiavo 1,2,3, James N. Sleigh 1,2,31Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, University College London, 2UCL Queen Square Motor Neuron Disease Centre, University College London, 3UK Dementia Research Institute, University College LondonUsing transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals. BiologyThree-dimensional Characterization of Interorganelle Contact Sites in Hepatocytes using Serial Section Electron MicroscopyGary Hong Chun Chung 1, Paul Gissen 1,2, Christopher J. Stefan 1, Jemima J. Burden 11MRC Laboratory for Molecular Cell Biology, University College London, 2NIHR Great Ormond Street Hospital Biomedical Research Centre, University College LondonA simple and comprehensive protocol to acquire three-dimensional details of membrane contact sites between organelles in hepatocytes from the liver or cells in other tissues.Developmental BiologyEye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual SystemOlivia L. Hagen 1, Yehyun Kim 1, Elaine Kushkowski 1,2, Hannah Rouse 3, Kara L. Cerveny 11Department of Biology, Reed College, 2Committee on Development, Regeneration, and Stem Cell Biology, University of Chicago, 3Department of Cell and Developmental Biology, University College LondonThe article explains how to surgically remove eyes from living zebrafish larvae as the first step toward investigating how retinal input influences optic tectum growth and development. In addition, the article provides information about larval anesthetization, fixation, and brain dissection, followed by immunohistochemistry and confocal imaging.NeuroscienceLong-Term Imaging of Identified Neural Populations using Microprisms in Freely Moving and Head-Fixed AnimalsRhys Burrows 1, Chi-Hsuan Ma 1, Yujiao Jennifer Sun 11Institute of Ophthalmology, University College LondonWhen integrated with a head-plate and an optical design compatible with both single- and two-photon microscopes, the microprism lens presents a significant advantage in measuring neural responses in a vertical column under diverse conditions, including well-controlled experiments in head-fixed states or natural behavioral tasks in freely moving animals.BiologyCell-Based Drug Screening for Inhibitors of Autophagy Related 4B Cysteine PeptidaseDenise R. B. Pilger 1, Christin Luft 1, Robin Ketteler 1,21Laboratory for Molecular Cell Biology, University College London, 2Department of Human Medicine, Medical School BerlinHere, we describe a detailed protocol for the use of a luciferase-based reporter assay in a semi-automated, high-throughput screening format.MedicineConsistent Delivery of Adeno-Associated Virus via Lateral Tail-Vein Injection in Adult MiceFady Guirguis 1,2, Véronique Bolduc 1, Matthew J. Slarve 3, Haiyan Zhou 4,5, Francesco Muntoni 2,4, Carsten G. Bönnemann 11Neuromuscular and Neurogenetic Disorders of Childhood Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 2The Dubowitz Neuromuscular Centre, Molecular Neurosciences Section, Developmental Neurosciences Research and Teaching Department, Great Ormond Street Institute of Child Health, University College London, 3Department of Molecular Microbiology and Immunology, University of Southern California, 4NIHR Great Ormond Street Hospital Biomedical Research Centre, 5Genetics and Genomic Medicine Research and Teaching Department, Great Ormond Street Institute of Child Health, University College LondonHere we detail an optimized protocol for mouse lateral tail-vein injection to systemically administer adeno-associated virus (AAV) in adult mice. Additionally, we describe protocols of commonly used assays to assess AAV transduction.BiologyOptimized and Simplified Technique for the Production and Culture of Precision-Cut Liver SlicesDany Perocheau 1, Claire Duff 1, Sonam Gurung 1, Loukia Touramanidou 1, Garima Sharma 1, Julien Baruteau 1,2,31Great Ormond Street Institute of Child Health, University College London, 2Great Ormond Street Hospital for Children NHS Foundation Trust, 3National Institute of Health Research Great Ormond Street Biomedical Research CentreA protocol for the production and culture of Precision-cut Liver Slices (PCLS) for the study of mouse livers. The article focuses on key aspects of the protocol, which only requires standard laboratory equipment with access to a vibratome and allows survival of PCLS for a minimum of 4 days.
