King's College London View Institution's Website 42 articles published in JoVE Biology Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease Anna Hadjihambi*1,2, Rallia-Iliana Velliou*3, Panagiotis Tsialios4, Aigli-Ioanna Legaki3, Antonios Chatzigeorgiou*3, Maritina G. Rouchota*4 1The Roger Williams Institute of Hepatology London, Foundation for Liver Research, 2 Using a diet-induced non-alcoholic fatty liver disease (NAFLD) mouse model, we describe the use of novel in vivo micro-computed tomography imaging techniques as a non-invasive method to assess the progression stages of NAFLD, focusing predominantly on the hepatic vascular network due to its significant involvement in NAFLD-related hepatic dysregulation. Developmental Biology Real Time and Repeated Measurement of Skeletal Muscle Growth in Individual Live Zebrafish Subjected to Altered Electrical Activity Michael Attwaters*1, Jeffrey J. Kelu*1, Tapan G. Pipalia1, Simon M. Hughes1 1 Optical clarity is a major advantage for cell biological and physiological work in zebrafish. Robust methods for measurement of cell growth in individual animals are described that permit novel insights into how growth of skeletal muscle and neighboring tissues are integrated with whole body growth. Immunology and Infection Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells Emily Read*1,2, Geraldine M. Jowett*1,2,3, Diana Coman1, Joana F. Neves1 1Wellcome Trust Cell Therapies and Regenerative Medicine Ph.D. Programme, King’s College London, 3Present address: Wellcome Trust/Cancer Research UK Gurdon Institute, Cambridge University This protocol offers detailed instructions for establishing murine small intestine organoids, isolating type-1 innate lymphoid cells from the murine small intestine lamina propria, and establishing 3-dimensional (3D) co-cultures between both cell types to study bi-directional interactions between intestinal epithelial cells and type-1 innate lymphoid cells. Biology Assessing Mineral Availability in Fish Feeds using Complementary Methods Demonstrated with the Example of Zinc in Atlantic Salmon Marta S. Silva1,2, Thea Stewart4, Heidi Amlund1,3, Jens J. Sloth1,3, Pedro Araujo1, Erik-Jan Lock1, Christer Hogstrand4, Robin Ørnsrud1, Rune Waagbø1,2, Antony Jesu Prabhu1 1Institute of Marine Research, 2Department of Biological Sciences, University of Bergen, 3National Food Institute, Technical University of Denmark, 4 This article explains in detail a systematic approach to assess micro-mineral availability in Atlantic salmon. The methodology includes tools and models with increasing biological complexity: (1) chemical speciation analysis, (2) in vitro solubility, (3) uptake studies in cell lines, and (4) in vivo fish studies. Biochemistry Preparing Lamellae from Vitreous Biological Samples Using a Dual-Beam Scanning Electron Microscope for Cryo-Electron Tomography Claudine Bisson1,2, Corey W. Hecksel3,4, James B. Gilchrist3, M. Alejandra Carbajal1, Roland A. Fleck1 1 Using focused ion beam milling to produce vitreous on-grid lamellae from plunge frozen biological samples for cryo-electron tomography. Bioengineering Patient-Specific Polyvinyl Alcohol Phantom Fabrication with Ultrasound and X-Ray Contrast for Brain Tumor Surgery Planning Eleanor C. Mackle*1,2, Jonathan Shapey*1,2,3,4, Efthymios Maneas1,2, Shakeel R. Saeed3,5,6, Robert Bradford3, Sebastien Ourselin4, Tom Vercauteren4, Adrien E. Desjardins1,2 1Wellcome / EPSRC Centre for Interventional and Surgical Sciences, University College London, 2Department of Medical Physics and Biomedical Engineering, University College London, 3Department of Neurosurgery, National Hospital for Neurology and Neurosurgery, 4 This protocol describes the fabrication of a patient specific skull, brain and tumor phantom. It uses 3D printing to create molds, and polyvinyl alcohol (PVA-c) is used as the tissue mimicking material. Behavior Implementation of a Real-Time Psychosis Risk Detection and Alerting System Based on Electronic Health Records using CogStack Tao Wang1, Dominic Oliver2, Yamiko Msosa1, Craig Colling3, Giulia Spada2, Łukasz Roguski4, Amos Folarin1, Robert Stewart3,5, Angus Roberts1,3, Richard J. B. Dobson1,3,4,6, Paolo Fusar-Poli2,3,7,8 1 We demonstrate how to deploy a real-time psychosis risk calculation and alerting system based on CogStack, an information retrieval and extraction platform for electronic health records. Neuroscience Meta-analysis of Voxel-Based Neuroimaging Studies using Seed-based d Mapping with Permutation of Subject Images (SDM-PSI) Anton Albajes-Eizagirre1,2, Aleix Solanes1,2, Miquel Angel Fullana2,3, John P. A. Ioannidis4, Paolo Fusar-Poli5,6,7, Carla Torrent1,2,3,8, Brisa Solé1,2,3,8, Caterina Mar Bonnín1,2,3,8, Eduard Vieta1,2,3,8, David Mataix-Cols9, Joaquim Radua1,2,5,9 1 We detail how to conduct a meta-analysis of voxel-based neuroimaging studies using Seed-based d Mapping with Permutation of Subject Images (SDM-PSI). Developmental Biology Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration Sandra Guadalupe Gonzalez Malagon*1,2, Lisa Dobson*1,3, Anna M Lopez Muñoz1, Marcus Dawson1, William Barrell1,3, Petros Marangos2,4, Matthias Krause3, Karen J Liu1 1 This protocol describes the dissection and culture of cranial neural crest cells from mouse models, primarily for the study of cell migration. We describe the live imaging techniques used and the analysis of speed and cell shape changes. Neuroscience Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains Myrto Denaxa1,2, Guilherme Neves3, Juan Burrone3, Vassilis Pachnis1 1Nervous System Development and Homeostasis Laboratory, The Francis Crick Institute, 2Neuroscience Centre, Biomedical Sciences Research Centre "Al. Fleming", 3 Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice. Genetics Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells Darja Pollpeter1, Andrew Sobala1, Michael H. Malim1 1 Here we present a deep sequencing approach that provides an unbiased determination of nascent 3'-termini as well as mutational profiles of single-stranded DNA molecules. The main application is the characterization of nascent retroviral complementary DNAs (cDNAs), the intermediates generated during the process of retroviral reverse transcription. Engineering Design and Implementation of a Bespoke Robotic Manipulator for Extra-corporeal Ultrasound Shuangyi Wang1, James Housden1, Yohan Noh1, Anisha Singh2, Junghwan Back3, Lukas Lindenroth3, Hongbin Liu3, Joseph Hajnal1, Kaspar Althoefer4, Davinder Singh2, Kawal Rhode1 1 This paper introduces the design and implementation of a bespoke robotic manipulator for extra-corporeal ultrasound examination. The system has five degrees of freedom with lightweight joints made by 3D printing and a mechanical clutch for safety management. Cancer Research Preparation of Exosomes for siRNA Delivery to Cancer Cells Farid N. Faruqu*1, Lizhou Xu*1, Khuloud T. Al-Jamal1 1 An exosome is a new generation of drug delivery carriers. We established an exosome isolation protocol with high yield and purity for siRNA delivery. We also encapsulated fluorescently labelled non-specific siRNA into exosomes and investigated the cellular uptake of siRNA-loaded exosomes in cancer cells. Immunology and Infection Quantitative Polymerase Chain Reaction-based Analyses of Murine Intestinal Microbiota After Oral Antibiotic Treatment Rebeca Jimeno1,2, Phillip M. Brailey1,2, Patricia Barral1,2 1 Here we provide detailed protocols for the oral administration of antibiotics to mice, collection of fecal samples, DNA extraction and quantification of fecal bacteria by qPCR. Chemistry Analyzing Protein Architectures and Protein-Ligand Complexes by Integrative Structural Mass Spectrometry Zainab Ahdash1, Andy M. Lau1, Chloe Martens1, Argyris Politis1 1 Mass spectrometry (MS) has emerged as an important tool for the investigation of structure and dynamics of macromolecular assemblies. Here, we integrate MS-based approaches to interrogate protein complex formation and ligand binding. Cancer Research Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models Alessia Volpe1, Francis Man1, Lindsay Lim1, Alex Khoshnevisan1, Julia Blower1, Philip J. Blower1, Gilbert O. Fruhwirth1 1 We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology. Medicine Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells Simone S. E. Nielsen1, Piotr Siupka1, Ana Georgian2, Jane E. Preston2, Andrea E. Tóth1, Siti R. Yusof2,3, N. Joan Abbott2, Morten S. Nielsen1 1Lundbeck Foundation Research Initiative on Brain Barriers and Drug Delivery, Department of Biomedicine, Aarhus University, 2 The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery. Genetics Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans Dhaval S Patel*1, Giovanni Diana*1, Eugeni V. Entchev1, Mei Zhan2,3,4, Hang Lu2,3,4, QueeLim Ch'ng1 1 Here, we present a framework to relate broad-range dietary restriction to gene expression and lifespan. We describe protocols for broad-range dietary restriction and for quantitative imaging of gene expression under this paradigm. We further outline computational analyses to reveal underlying information processing features of the genetic circuits involved in food-sensing. Developmental Biology Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin Lucia Salz1,2, Ryan R. Driskell1,2 1 This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types. Biochemistry Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry Javier Barallobre-Barreiro1, Ferheen Baig1, Marika Fava1, Xiaoke Yin1, Manuel Mayr1 1 This paper describes a methodology to prepare cardiovascular tissue samples for MS analysis that allows for (1) the analysis of ECM protein composition, (2) the identification of glycosylation sites, and (3) the compositional characterization of glycan forms. This methodology can be applied, with minor modifications, to the study of the ECM in other tissues. Developmental Biology Application of Impermeable Barriers Combined with Candidate Factor Soaked Beads to Study Inductive Signals in the Chick Susan Wilde1, Malcolm P. Logan1 1 Protocols using impermeable barriers to block induction events between tissues of the main body axis and flank in the chick embryo required for limb formation are described. Beads soaked in candidate inductive signals are used to overcome the effect of barrier placement and analysis of gene expression confirms this. Behavior Sit-to-stand-and-walk from 120% Knee Height: A Novel Approach to Assess Dynamic Postural Control Independent of Lead-limb Gareth D. Jones1,2, Darren C. James3, Michael Thacker1,2, David A. Green1 1 Here, we present a novel protocol to measure positional stability at key events during the sit-to-stand-to-walk using the center-of-pressure to the whole-body-center-of-mass distance. This was derived from the force platform and three-dimensional motion-capture technology. The paradigm is reliable and can be utilized for the assessment of neurologically compromised individuals. Medicine The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse Thomas Pitcher1, João Sousa-Valente1, Marzia Malcangio1 1 Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse. Neuroscience Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining Siwei Zhang*1,2, Jingjing Li*1,3, Robert Lea1, Enrique Amaya1 1The Healing Foundation Centre, Faculty of Life Sciences, University of Manchester, 2Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, 3 This article presents a convenient and rapid method for visualizing different neuronal cell populations in the central nervous system of Xenopus embryos using immunofluorescent staining on sections. Medicine Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability Christina Wayman*1,2, Denise A. Duricki*1,2, Lisa A. Roy3, Barbara Haenzi1, Shi-Yen Tsai4, Gwendolyn Kartje4,5,6, John S. Beech7, Diana Cash2, Lawrence Moon1 1Department of Neuroimaging, James Black Centre, Institute of Psychiatry, King's College London, University of London, 3Institute of Neuroscience and Psychology, Wellcome Surgical Institute, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, 4Research Service, Edward Hines Jr. VA Hospital, 5Neurology Service, Edward Hines Jr. VA Hospital, 6Department of Molecular Pharmacology and Therapeutics, Neuroscience Research Institute, Loyola University Chicago, 7Department of Oncology, The Gray Institute for Radiation, Oncology and Biology, University of Oxford Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke. Immunology and Infection Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data George Ashdown1, Elvis Pandžić3, Andrew Cope2, Paul Wiseman4, Dylan Owen1 1Academic Department of Rheumatology, Centre for Molecular and Cellular Biology of Inflammation, Division of Immunology, Infection and Inflammatory Disease, King's College London, 3ARC Centre for Advanced Molecular Imaging, Australian Centre for NanoMedicine, University of New South Wales Australia, 4Departments of Chemistry and Physic, McGill University To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy. Developmental Biology Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors Michalina Hanzel1, Richard J.T. Wingate1, Thomas Butts2 1 The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos. Immunology and Infection Kupffer Cell Isolation for Nanoparticle Toxicity Testing Maxime Bourgognon1, Rebecca Klippstein1, Khuloud T. Al-Jamal1 1 Liver macrophages, named Kupffer cells, are responsible for the capture of circulating nanoparticles. We describe here a method, of high cell purity and yield, for Kupffer cell isolation. The modified LDH assay is used here to measure the toxicity induced by carbon nanotubes in Kupffer cells. Neuroscience Investigating the Function of Deep Cortical and Subcortical Structures Using Stereotactic Electroencephalography: Lessons from the Anterior Cingulate Cortex Robert A. McGovern1,3, Tarini Ratneswaren4, Elliot H. Smith1,3, Jennifer F. Russo3, Amy C. Jongeling2,3, Lisa M. Bateman2,3, Catherine A. Schevon2,3, Neil A. Feldstein1,3, Guy M. McKhann, II1,3, Sameer Sheth1,3 1Department of Neurosurgery, Columbia University Medical Center, New York Presbyterian Hospital, 2Department of Neurology, Columbia University Medical Center, New York Presbyterian Hospital, 3Columbia University Medical Center, New York Presbyterian Hospital, 4 Stereotactic Electroencephalography (SEEG) is an operative technique used in epilepsy surgery to help localize seizure foci. It also affords a unique opportunity to investigate brain function. Here we describe how SEEG can be used to investigate cognitive processes in human subjects. Bioengineering Generation and Grafting of Tissue-engineered Vessels in a Mouse Model Mei M. Wong*1, Xuechong Hong*1, Eirini Karamariti1, Yanhua Hu1, Qingbo Xu1 1 Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor. Developmental Biology Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle Chibeza C. Agley1,2, Anthea M. Rowlerson1, Cristiana P. Velloso1, Norman L. Lazarus1, Stephen D. R. Harridge1 1 The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells. Neuroscience Unilateral Pyramidotomy of the Corticospinal Tract in Rats for Assessment of Neuroplasticity-inducing Therapies Claudia Kathe1, Thomas H. Hutson1, Qin Chen2, Harold D. Shine2, Stephen B. McMahon1, Lawrence D. F. Moon1 1 The corticospinal tract, one of the major sensorimotor tracts, can be lesioned unilaterally in the rodent brainstem in order to test neuroplasticity-inducing therapies for the central nervous system. This surgical procedure (“pyramidotomy”) and postoperative assessments are described in this protocol. Medicine From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia Benedetta Apollonio1,2, Maria Teresa Sabrina Bertilaccio1, Umberto Restuccia3, Pamela Ranghetti1, Federica Barbaglio1, Paolo Ghia1,4, Federico Caligaris-Cappio1,4, Cristina Scielzo1 1Division of Molecular Oncology, IRCCS, San Raffaele Scientific Institute, 2 Here we describe a protocol that couples two proteomic techniques, namely 2-dimensional Electrophoresis (2DE) and Mass Spectrometry (MS), to identify differentially expressed/post-translational modified proteins among two or more groups of primary samples. This approach, together with functional experiments, allows the identification and characterization of prognostic markers/therapeutic targets. Neuroscience In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices Martina Sonego*1, Ya Zhou*1, Madeleine Julie Oudin2, Patrick Doherty1, Giovanna Lalli1 1Wolfson Centre for Age-Related Diseases, King's College London, 2David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking. Biology The Slice Culture Method for Following Development of Tooth Germs In Explant Culture Sarah A. Alfaqeeh1,2, Abigail S. Tucker1 1 Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods. Neuroscience Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro Katarzyna Falenta*1, Sangeetha Gajendra*2, Martina Sonego1, Patrick Doherty1, Giovanna Lalli1 1Wolfson Centre for Age-Related Diseases, King's College London, 2MRC Centre for Developmental Neurobiology, King's College London Neuroblast migration is a crucial step in postnatal neurogenesis. The protocol described here can be used to investigate the role of candidate regulators of neuroblast migration by employing DNA/small hairpin RNA (shRNA) nucleofection and a 3D migration assay with neuroblasts isolated from the rodent postnatal rostral migratory stream. Biology Isolation and Culture of Neonatal Mouse Cardiomyocytes Elisabeth Ehler1, Thomas Moore-Morris2, Stephan Lange2 1 Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays. Bioengineering Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells Klaus Suhling1, James A. Levitt1, Pei- Hua Chung1, Marina. K. Kuimova2, Gokhan Yahioglu3 1Department of Physics, King's College London, 2Department of Chemistry, Imperial College London, 3PhotoBiotics Ltd Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells. Biology Electroporation of Craniofacial Mesenchyme Jacqueline M. Tabler1, Karen J. Liu1 1Department of Craniofacial Development, King's College London Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo. Neuroscience Single Drosophila Ommatidium Dissection and Imaging Vera Volpi1, Daniel Mackay1, Manolis Fanto1 1MRC Centre for Developmental Neurobiology, King's College London The limiting factor in the use of the adult Drosophila eye to study neurodegeneration and cell biology is the difficult imaging of intracellular processes. We describe the dissection of single ommatidia to generate a bona-fide primary neuronal cell culture, which can be subject to drug treatment and advanced imaging. Biology Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations Iwan R. Evans1, Jennifer Zanet2, Will Wood1, Brian M. Stramer2 1Department of Biology and Biochemistry, University of Bath, 2Randall Division of Cell and Molecular Biophysics, King's College London Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes. Biology A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila Aamna K. Kaul1, Joseph M. Bateman1 1Wolfson Centre for Age-Related Diseases, King's College London We describe a novel red fluorescent protein (RFP) reporter that is expressed specifically in the Drosophila eye. We detail a methodology for dissection of the eye imaginal disc and how this reporter can be used to aid in the dissection and identification of specific cell types in the developing eye.