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16 ARTICLES PUBLISHED IN JoVE

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Biology

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures
Maya Sieber-Blum 1,2, Yaofei Hu 2
1Institute of Human Genetics and Northeast England Stem Cell Institute, Newcastle University, 2Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

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Neuroscience

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes
Wolfgang Stein 1, Carola Städele 1, Peter Andras 2
1Institute of Neurobiology, Ulm University, 2School of Computing Science & Institute of Neuroscience, Newcastle University

Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.

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Bioengineering

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms
Derek S. Samarian 1, Nicholas S. Jakubovics 2, Ting L. Luo 1, Alexander H. Rickard 1
1Department of Epidemiology, School of Public Health, The University of Michigan, 2Centre for Oral Health Research, School of Dental Sciences, Newcastle University

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque. Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

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Medicine

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome
Alicja M. Baczynska 1,2, Sarah Shaw 3, Helen C. Roberts 1,2,3,5, Cyrus Cooper 2,3,4, Avan Aihie Sayer 1,2,3,5,6, Harnish P. Patel 1,2,3
1Academic Geriatric Medicine, University of Southampton, University Hospital Southampton, 2National Institute for Health Research Southampton Biomedical Research Center, University of Southampton and University Hospital Southampton NHS Foundation Trust, 3MRC Lifecourse Epidemiology Unit, University of Southampton, 4National Institute for Health Research Musculoskeletal Biomedical Research Unit, University of Oxford, 5National Institute for Health Research Collaboration for Leadership in Applied Health Research and Care, 6Newcastle University Institute of Ageing and Institute of Health and Society, Newcastle University

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

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Developmental Biology

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients
Simon W. Fellgett 1, Simon A. Ramsbottom 2, Richard J. Maguire 3, Stephen Cross 4, Peter O'Toole 5, Mary E. Pownall 5
1Department of Biomedical Science, The Bateson Centre, University of Sheffield, 2Institute of Genetic Medicine, Newcastle University, 3Department of Cardiovascular Science, The Bateson Centre, University of Sheffield, 4School of Biochemistry, University of Bristol, 5Biology Department, University of York

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

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Developmental Biology

Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover
Gavin D. Richardson 1
1Institute of Genetic Medicine, International Centre for Life, Newcastle University

Quantification of cardiomyocyte turnover is challenging. The protocol described here makes an important contribution to this challenge by enabling accurate and sensitive quantification of neo-cardiomyocyte nuclei generation and nuclei ploidy.

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Neuroscience

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees (Bombus terrestris)
Carolyn Ma 1, Sébastien Kessler 1, Alexander Simpson 1, Geraldine Wright 1
1Institute of Neuroscience, Newcastle University

A novel behavioral assay is described for investigating the short term gustatory responses of the mouthparts of freely-moving bumble bees (Bombus terrestris) toward nutrients and toxins in solution.

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Biochemistry

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits
Julia Schückel *1, Stjepan Krešimir Kračun *1, William G. T. Willats 2
1Department for Plant and Environmental Sciences, University of Copenhagen, 2School of Agriculture, Food and Rural Development, Newcastle University

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

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Behavior

A Real-world What-Where-When Memory Test
Tom V. Smulders 1, Amber Black-Dominique 2, Tahsina S. Choudhury 3, Simona E. Constantinescu 3, Kyriaki Foka 3, Tom J. Walker 3, Kevin Dick 4, Stephen Bradwel 4, R. Hamish McAllister-Williams 3, Peter Gallagher 3
1Institute of Neuroscience and Centre for Behavior & Evolution, Newcastle University, 2School of Psychology, Newcastle University, 3Institute of Neuroscience, Newcastle University, 4Digital Media Services, Newcastle University

The Real-World What-Where-When memory test is a novel episodic memory test, in which participants need to recall which objects have been hidden in which locations on which of two distinct occasions. It is easy to run and is sensitive to normal cognitive aging.

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Environment

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci
Simon Blaser 1,2,3, Hanspeter Diem 4, Andreas von Felten 4, Morgan Gueuning 1, Michael Andreou 5, Neil Boonham 6,7, Jennifer Tomlinson 6, Pie Müller 2,3, Jürg Utzinger 2,3, Beatrice Frey 1, Jürg E. Frey 1, Andreas Bühlmann 8
1Department of Method Development and Analytics, Agroscope, 2Swiss Tropical and Public Health Institute, 3University of Basel, 4Swiss Federal Plant Protection Service, Federal Office for Agriculture, 5OptiGene Limited, 6Fera Science Limited, 7School of Natural and Environmental Sciences, Newcastle University, 8Department of Plants and Plant Products, Agroscope

This paper reports the protocol for a rapid identification assay for Bemisia tabaci based on loop-mediated isothermal amplification (LAMP) technology. The protocol requires minimal laboratory training and can, therefore, be implemented on-site at points of entry for plant imports such as seaports and airports.

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Biochemistry

Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells
Vinciane Saint-Criq 1, Iram J. Haq 2,3, Aaron I. Gardner 2, James P. Garnett 2,4, Christopher Ward 1,2, Malcolm Brodlie 2,3, Michael A. Gray 1
1Epithelial Research Group, Institute for Cell and Molecular Biosciences, Faculty of Medical Sciences, Newcastle University, 2Respiratory Group, Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University, 3Paediatric Respiratory Medicine, Great North Children's Hospital, Newcastle upon Tyne Hospitals NHS Foundation Trust, 4Boehringer Ingelheim Pharma GmbH & Co

We present a protocol to make dynamic measurements of the airway surface liquid pH under thin film conditions using a plate-reader.

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Immunology and Infection

Isolation of L-form Bacteria from Urine using Filtration Method
Frances Davison 1, Jonathan Chapman 1, Katarzyna Mickiewicz 1
1Centre for Bacterial Cell Biology, Biosciences Institute, Medical School, Newcastle University

Here, we present a protocol for the isolation of L-form bacteria from urine, using a filtration method. Complementary methods for the preparation of L-form medium, observation of L-forms by phase-contrast microscopy and induction of L-forms under laboratory conditions are also described.

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Cancer Research

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma
Hiroki Kobayashi 1,2,3,4, Krystyna A. Gieniec 1,2, Jia Q. Ng 1,2, Jarrad Goyne 1,2, Tamsin R. M. Lannagan 1,2, Elaine M. Thomas 1,2, Georgette Radford 1,2, Tongtong Wang 1,2, Nobumi Suzuki 1,2,5, Mari Ichinose 1,2, Josephine A. Wright 2, Laura Vrbanac 1,2, Alastair D. Burt 6, Masahide Takahashi 3,4,7, Atsushi Enomoto 3, Daniel L. Worthley 2, Susan L. Woods 1,2
1Adelaide Medical School, University of Adelaide, 2South Australian Health and Medical Research Institute (SAHMRI), 3Department of Pathology, Nagoya University Graduate School of Medicine, 4Division of Molecular Pathology, Center for Neurological Disease and Cancer, Nagoya University Graduate School of Medicine, 5Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, 6Translational and Clinical Research Institute, Newcastle University, 7International Center for Cell and Gene Therapy, Fujita Health University

Portal vein injection of colorectal cancer (CRC) organoids generates stroma-rich liver metastasis. This mouse model of CRC hepatic metastasis represents a useful tool to study tumor-stroma interactions and develop novel stroma-directed therapeutics such as adeno-associated virus-mediated gene therapies.

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Cancer Research

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma
Marco Y. W. Zaki *1,2, Shishir Shetty *2, Alex L. Wilkinson 2, Daniel A. Patten 2, Fiona Oakley *3, Helen Reeves *4,5
1Department of Biochemistry, Faculty of Pharmacy, Minia University, 2National Institute for Health Research Birmingham Liver Biomedical Research Unit and Centre for Liver and Gastrointestinal Research, Institute of Immunology and Immunotherapy, University of Birmingham, 3Newcastle Fibrosis Research Group, Biosciences Institute, Faculty of Medical Sciences, Newcastle University, 4Newcastle University Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, 5The Liver Unit, Department of Medicine, Freeman Hospital, Newcastle-upon-Tyne Hospitals NHS Foundation Trust

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

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Biology

Microarray Polymer Profiling (MAPP) for High-Throughput Glycan Analysis
Cassie R. Bakshani 1,2, Jiraporn Sangta 3, Sarana Sommano 3, William G. T. Willats 1
1Department of Biology, School of Natural and Environmental Sciences, Newcastle University, 2Institute of Microbiology and Infection, College of Medical and Dental Sciences, University of Birmingham, 3Department of Plant and Soil Sciences, Faculty of Agriculture, Chiang Mai University

Microarray polymer profiling (MAPP) is a high-throughput technique for compositional analysis of glycans in biological samples.

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Biology

Methods for Embedding Cell-Free Protein Synthesis Reactions in Macro-Scale Hydrogels
Siji Kavil *1, Alex Laverick *1, Colette J. Whitfield 1, Alice M. Banks 2, Thomas P. Howard 1
1School of Natural and Environmental Sciences, Newcastle University, 2Department of Life Sciences, Imperial College London

Here, we present two protocols for embedding cell-free protein synthesis reactions in macro-scale hydrogel matrices without the need for an external liquid phase.

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