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King's College London

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41 articles published in JoVE

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. Silva^1,2, Thea Stewart⁴, Heidi Amlund^1,3, Jens J. Sloth^1,3, Pedro Araujo¹, Erik-Jan Lock¹, Christer Hogstrand⁴, Robin Ørnsrud¹, Rune Waagbø^1,2, 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 Bisson^1,2, Corey W. Hecksel^3,4, 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 Maneas^1,2, Shakeel R. Saeed^3,5,6, Robert Bradford³, Sebastien Ourselin⁴, Tom Vercauteren⁴, Adrien E. Desjardins^1,2 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 Stewart^3,5, Angus Roberts^1,3, Richard J. B. Dobson^1,3,4,6, Paolo Fusar-Poli^2,3,7,8 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-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).
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 Barrell^1,3, Petros Marangos^2,4, 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 Denaxa^1,2, 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 Jimeno^1,2, Phillip M. Brailey^1,2, Patricia Barral^1,2 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. Yusof^2,3, 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 Zhan^2,3,4, Hang Lu^2,3,4, 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 Salz^1,2, Ryan R. Driskell^1,2 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. Jones^1,2, Darren C. James³, Michael Thacker^1,2, 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 Kartje^4,5,6, 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. McGovern^1,3, Tarini Ratneswaren⁴, Elliot H. Smith^1,3, Jennifer F. Russo³, Amy C. Jongeling^2,3, Lisa M. Bateman^2,3, Catherine A. Schevon^2,3, Neil A. Feldstein^1,3, Guy M. McKhann, II^1,3, Sameer Sheth^1,3 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. Agley^1,2, 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 Apollonio^1,2, Maria Teresa Sabrina Bertilaccio¹, Umberto Restuccia³, Pamela Ranghetti¹, Federica Barbaglio¹, Paolo Ghia^1,4, Federico Caligaris-Cappio^1,4, 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. Alfaqeeh^1,2, 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.

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