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University of Nottingham

17 ARTICLES PUBLISHED IN JoVE

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Bioengineering

Self-reporting Scaffolds for 3-Dimensional Cell Culture
Helen Harrington 1, Felicity R.A.J. Rose 2, Jonathan W. Aylott 3, Amir M. Ghaemmaghami 1
1School of Molecular Medical Sciences, University of Nottingham, 2Division of Drug Delivery and Tissue Engineering, University of Nottingham, 3Laboratory of Biophysics and Surface Analysis, University of Nottingham

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

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JoVE Journal

Methods to Assess Subcellular Compartments of Muscle in C. elegans
Christopher J. Gaffney 1, Joseph J. Bass 1, Thomas F. Barratt 1, Nathaniel J. Szewczyk 1
1MRC/ARUK Centre for Musculoskeletal Ageing Research, University of Nottingham

Skeletal muscle is essential for locomotion and is the bodies’ main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

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Chemistry

Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins
Jennifer T. Patel 1, Hannah R. Belsham 1, Alexandra J. Rathbone 1, Claire T. Friel 1
1School of Life Sciences, University of Nottingham

Kinesins are characterized by nucleotide-dependent interaction with microtubules: a cycle of ATP turnover coupled to a cycle of microtubule interaction. Here, we describe protocols to analyze the kinetics of individual nucleotide transitions in the ATP turnover cycle of a kinesin using fluorescently labeled nucleotides and stopped-flow fluorescence.

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Bioengineering

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall
Jack C. Bridge 1, Jonathan W. Aylott 2, Christopher E. Brightling 5, Amir M. Ghaemmaghami 3, Alan J. Knox 4, Mark P. Lewis 6, Felicity R.A.J. Rose 1, Gavin E. Morris 1
1Division of Drug Delivery and Tissue Engineering, University of Nottingham, 2Laboratory of Biophysics and Surface Analysis, School of Pharmacy, University of Nottingham, 3Division of Immunology and Allergy, School of Molecular Medical Sciences, University of Nottingham, 4Division of Respiratory Medicine, School of Clinical Sciences, University of Nottingham, 5NIHR Respiratory Biomedical Research Unit, University of Leicester, 6School of Sport, Exercise, and Health Sciences, Loughborough University

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

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Developmental Biology

Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression
Rabeea H. Mohammed 1, Dylan Sweetman 1
1Division of Animal Sciences, School of Biosciences, University of Nottingham

By grafting beads soaked in growth factors or specific inhibitors of signaling pathways into developing embryos it is possible to directly test their effects in vivo. In this protocol beads are grafted into the limb bud to determine the effects of these molecules on gene expression and signal transduction.

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Biology

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry
Abdulkadir Abakir 1, Lee Wheldon 2, Andrew D. Johnson 3, Patrick Laurent 1, Alexey Ruzov 4
1Laboratoire de Neurophysiologie (CP601), ULB Neuroscience Institute (UNI), Université Libre de Bruxelles, 2Medical Molecular Sciences, Centre for Biomolecular Sciences, University of Nottingham, 3School of Life Sciences, University of Nottingham, 4Division of Cancer and Stem Cells, Centre for Biomolecular Sciences, School of Medicine, University of Nottingham

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

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JoVE Journal

Magnetically Induced Rotating Rayleigh-Taylor Instability
Matthew M. Scase *1, Kyle A. Baldwin *2, Richard J. A. Hill *3
1School of Mathematical Sciences, University of Nottingham, 2Faculty of Engineering, University of Nottingham, 3School of Physics and Astronomy, University of Nottingham

We present a protocol for preparing a two-layer density-stratified liquid that can be spun-up into solid body rotation and subsequently induced into Rayleigh-Taylor instability by applying a gradient magnetic field.

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Chemistry

Microfluidic-based Synthesis of Covalent Organic Frameworks (COFs): A Tool for Continuous Production of COF Fibers and Direct Printing on a Surface
Afshin Abrishamkar 1, David Rodríguez-San-Miguel 2, Jorge Andrés Rodríguez Navarro 3, Romen Rodriguez-Trujillo 4, David B. Amabilino 5, Ruben Mas-Ballesté 2, Félix Zamora 2,6,7, Andrew J. deMello 1, Josep Puigmarti-Luis 1
1Institute of Chemical and Bioengineering, Department of Chemistry and Applied Bioscience, ETH Zurich, 2Departamento de Química Inorgánica, Universidad Autónoma de Madrid, 3Departamento de Química Inorgánica, Universidad de Granada, 4Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), 5School of Chemistry, University of Nottingham, 6Condensed Matter Physics Center (IFMAC), Universidad Autónoma de Madrid, 7Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA Nanociencia)

We present a novel microfluidic-based method for synthesis of covalent organic frameworks (COFs). We demonstrate how this approach can be used to produce continuous COF fibers, and also 2D or 3D COF structures on surfaces.

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Biology

Volume Segmentation and Analysis of Biological Materials Using SuRVoS (Super-region Volume Segmentation) Workbench
Michele C. Darrow 1, Imanol Luengo 1,2, Mark Basham 1, Matthew C. Spink 1, Sarah Irvine 1, Andrew P. French 2, Alun W. Ashton 1, Elizabeth M.H. Duke 1
1Science Division, Harwell Science and Innovation Campus, Diamond Light Source, 2School of Computer Science, University of Nottingham

Segmentation of three-dimensional data from many imaging techniques is a major bottleneck in analysis of complex biological systems. Here, we describe the use of SuRVoS Workbench to semi-automatically segment volumetric data at various length-scales using example datasets from cryo-electron tomography, cryo soft X-ray tomography, and phase contrast X-ray tomography techniques.

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Genetics

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images
Ashley H. Ramsawhook *1, Lara C. Lewis *1, Maria Eleftheriou 1, Abdulkadir Abakir 1, Paulina Durczak 1, Robert Markus 2, Seema Rajani 2, Nicholas R.F. Hannan 1, Beth Coyle 3, Alexey Ruzov 1
1Division of Cancer and Stem Cells, School of Medicine, Centre for Biomolecular Sciences, University of Nottingham, 2School of Life Sciences Imaging (SLIM), School of Life Sciences, University of Nottingham, 3Children's Brain Tumour Research Centre, School of Medicine, QMC, University of Nottingham

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

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Immunology and Infection

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection
Ola H. Negm 1,2, Mohamed Hamed 1,2, Tanya M. Monaghan 3
1Breast Surgery Group, Division of Medical Sciences and Graduate Entry Medicine, School of Medicine, Queen's Medical Centre, University of Nottingham, 2Medical Microbiology and Immunology Department, Faculty of Medicine, Mansoura University, 3Nottingham Digestive Diseases Centre and NIHR Nottingham Digestive Diseases Biomedical Research Centre, School of Medicine, Queen's Medical Centre, University of Nottingham

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

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Medicine

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice
Melissa Trotman-Lucas 1, Michael E. Kelly 2, Justyna Janus 2, Claire L. Gibson 1,3
1Department of Neuroscience, Psychology and Behaviour, University of Leicester, 2Preclinical Imaging Facility, Core Biotechnology Services, University of Leicester, 3School of Psychology, University of Nottingham

Intraluminal filament occlusion of the middle cerebral artery is the most frequently used in vivo model of experimental stroke in rodents. An alternative surgical approach to allow common carotid artery repair is performed here, which allows the reperfusion of the common carotid artery and a full reperfusion to the middle cerebral artery territory.

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Bioengineering

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells
Emma Pipó-Ollé 1, Prasad Walke 2, Martine K. Notabi 1, Rime B. El-Houri 1, Morten Østergaard Andersen 1, David Needham 2,3,4, Eva C. Arnspang 1
1Department of Chemical Engineering, Biotechnology and Environmental Technology, University of Southern Denmark, 2Center for Single Particle Science and Engineering (SPSE), Institute for Molecular Medicine, Health Sciences, University of Southern Denmark, 3Department of Mechanical Engineering and Material Science, Duke University, 4School of Pharmacy, University of Nottingham

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

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Generation of hiPSC-Derived Intestinal Organoids for Developmental and Disease Modelling Applications

Generation of hiPSC-Derived Intestinal Organoids for Developmental and Disease Modelling Applications
Paulina M. Durczak *1, Kathryn L. Fair *1, Nicholas Jinks 1, Sara Cuevas Ocaña 1, Carlos B. Sainz Zuñiga 1, Nicholas R.F. Hannan 1
1Biodiscovery Institute, Translational Medical Sciences, School of Medicine, University of Nottingham

This protocol allows for the differentiation of human pluripotent cells into intestinal organoids. The protocol mimics normal human development by differentiating cells into a population of definitive endoderm, hindgut endoderm and then intestinal epithelium. This makes the protocol suitable for studying both intestinal development as well as disease modelling applications.

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Biology

Preparation of a User-Defined Peptide Gel for Controlled 3D Culture Models of Cancer and Disease
Jennifer C. Ashworth 1, Rebecca L. Morgan 1, Kataryna Lis-Slimak 1, Kate A. Meade 1, Sal Jones 1, Katherine Spence 2, Charlotte E. Slater 1, Jamie L. Thompson 1, Anna M. Grabowska 1, Robert B. Clarke 2, Gilian Farnie 3, Cathy L. R. Merry 1
1Division of Cancer & Stem Cells, School of Medicine, Nottingham Biodiscovery Institute, University of Nottingham, 2Breast Biology Group, Manchester Breast Centre, Division of Cancer Sciences, Faculty of Biology, Medicine and Health, University of Manchester, 3SGC, Botnar Research Centre, NDORMS, University of Oxford

We present a method for creating a 3D cell culture environment, which can be used to investigate the importance of cell/matrix interactions in cancer progression. Using a simple self-assembling octapeptide, the matrix surrounding encapsulated cells can be controlled, with independent regulation of mechanical and biochemical cues.

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Behavior

Assessment of Spatial Lingual Tactile Sensitivity using a Gratings Orientation Test
Noemi Sofia Rabitti 1, Marta Appiani 1, Camilla Cattaneo 1, Rebecca Ford 2, Monica Laureati 1
1Department of Food, Environmental and Nutritional Sciences (DeFENS), University of Milan, 2Division of Food, Nutrition, and Dietetics, School of Biosciences, University of Nottingham

This work illustrates a standard procedure and threshold determination by the R-index to assess spatial lingual tactile sensitivity using a gratings orientation test.

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Immunology and Infection

Development of a Polymicrobial Colony Biofilm Model to Test Antimicrobials in Cystic Fibrosis
Brogan Richards 1, Shaun Robertson 1,2, Luisa Martinez Pomares 3, Miguel Cámara 1,4
1School of Life Sciences, Biodiscovery Institute, University of Nottingham, 2MiDx Ltd, Biodiscovery Institute, University of Nottingham, 3School of Life Sciences, Queen's Medical Centre, University of Nottingham, 4National Biofilms Innovation Centre, School of Life Sciences, Biodiscovery Institute, University of Nottingham

This methodology allows for the establishment of a polymicrobial biofilm model in cystic fibrosis for antimicrobial sensitivity testing in research and clinical laboratories. This model provides accurate and reliable results over a range of outputs.

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