University of Birmingham View Institution's Website 34 articles published in JoVE Biology Microarray Polymer Profiling (MAPP) for High-Throughput Glycan Analysis Cassie R. Bakshani1,2, Jiraporn Sangta3, Sarana Sommano3, William G. T. Willats1 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. Bioengineering Agarose Fluid Gels Formed by Shear Processing During Gelation for Suspended 3D Bioprinting Jessica J. Senior*1, Richard J. A. Moakes*2, Megan E. Cooke3, Samuel R. Moxon4, Alan M. Smith1, Liam M. Grover2 1Department of Pharmacy, University of Huddersfield, 2School of Chemical Engineering, University of Birmingham, 3Chemical and Biological Engineering, University of Colorado Boulder, 4School of Biological Sciences, University of Manchester Shear processing during hydrogel formation results in the production of microgel suspensions that shear-thin but rapidly restructure following the removal of shear forces. Such materials have been used as a supporting matrix for bioprinting complex, cell-laden structures. Here, methods used to manufacture the supporting bed and compatible bioinks are described. Biology Assessment of Chemical Toxicity in Adult Drosophila Melanogaster Jessica M. Holsopple*1,2, Shannon R. Smoot*1, Ellen M. Popodi1,2, John K. Colbourne3, Joseph R. Shaw4, Brian Oliver5, Thomas C. Kaufman1, Jason M. Tennessen1 1Department of Biology, Indiana University, 2Bloomington Drosophila Stock Center, Department of Biology, Indiana University, 3School of Biosciences, University of Birmingham, 4O'Neill School of Public and Environmental Affairs, Indiana University, 5Section of Developmental Genomics, Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Kidney and Digestive Diseases, National Institutes of Health This protocol describes an efficient and inexpensive method that uses liquid media to assess the effects of chemical toxicants on the viability of adult Drosophila melanogaster. Genetics Sample Preparation to Bioinformatics Analysis of DNA Methylation: Association Strategy for Obesity and Related Trait Studies Natália Yumi Noronha*1, Guilherme da Silva Rodrigues*1, Marcela Augusta de Souza Pinhel1, Jean-Baptiste Cazier2,3, Lígia Moriguchi Watanabe1, Albert Nobre Menezes4, Carlos Roberto Bueno5, Carolina Ferreira Nicoletti1, Bruno Affonso Parenti de Oliveira1, Isabelle Mello Schineider6, Isabella Harumi Yonehara Noma7, Igor Caetano Dias Alcarás8, Fernando Barbosa9, Carla Barbosa Nonino6 1Department of Internal Medicine, Ribeirão Preto Medical School, University of São Paulo, 2Centre for Computational Biology, University of Birmingham, 3Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, 4Cancer Genetics and Evolution Laboratory, Cancer Research UK, Institute of Genetics & Molecular Medicine, The University of Edinburgh, 5Ribeirão Preto School of Physical Education and Sport, University of São Paulo, 6Health Sciences Department, Ribeirao Preto Medical School, University of Sao Paulo, 7Faculty of Pharmaceutical Sciences, University of São Paulo, 8Department of Genetics, Ribeirão Preto Medical School, University of São Paulo, 9Department of Clinical, Bromatological and Toxicological Analysis, Faculty of Pharmaceutical Sciences of Ribeirao Preto, University of São Paulo The present study describes the workflow to manage DNA methylation data obtained by microarray technologies. The protocol demonstrates steps from sample preparation to data analysis. All procedures are described in detail, and the video shows the significant steps. Cancer Research Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids Using Extracellular Flux Analysis N. J. Coltman1, G. Rochford1, N. J. Hodges1, H. Ali-Boucetta2, J. P. Barlow3 1School of Biosciences, University of Birmingham, 2Nanomedicine, Drug Delivery & Nanotoxicology Lab, School of Pharmacy, University of Birmingham, 3Mitochondrial Profiling Centre, University of Birmingham These protocols will help users probe mitochondrial energy metabolism in 3D cancer cell-line-derived spheroids using Seahorse extracellular flux analysis. Engineering UV-Vis Spectroscopic Characterization of Nanomaterials in Aqueous Media Ana C. Quevedo1, Emily Guggenheim1, Sophie M. Briffa1, Jessica Adams2,3, Stephen Lofts2, Minjeong Kwak4, Tae Geol Lee4, Colin Johnston5, Stephan Wagner6, Timothy R. Holbrook6, Yves U. Hachenberger7, Jutta Tentschert7, Nicholas Davidson1, Eugenia Valsami-Jones1 1School of Geography, Earth and Environmental Sciences, University of Birmingham, 2UK Centre for Ecology and Hydrology, 3Natural England, 4Center for Nanosafety Metrology, Korea Research Institute of Standards and Science (KRISS), 5Department of Materials, University of Oxford, 6Department of Analytical Chemistry, Helmholtz-Centre for Environmental Research, 7Department of Chemical and Product Safety, German Federal Institute for Risk Assessment (BfR) This study presents the benchmarking results for an interlaboratory comparison (ILC) designed to test the standard operating procedure (SOP) developed for gold (Au) colloid dispersions characterized by ultraviolet-visible Spectroscopy (UV-Vis), amongst six partners from the H2020 ACEnano project for sample preparation, measurement, and analysis of the results. 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. Wilkinson2, Daniel A. Patten2, 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. Immunology and Infection Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung Niamh E. Harrington*1, Esther Sweeney*1, Ilyas Alav1,2, Freya Allen1, John Moat1,3, Freya Harrison1 1School of Life Science, University of Warwick, 2Institute of Microbiology and Infection, University of Birmingham, 3Warwick Antimicrobial Screening Facility, School of Life Science, University of Warwick This workflow can be used to perform antibiotic susceptibility testing using an established ex vivo model of bacterial biofilm in the lungs of individuals with cystic fibrosis. Use of this model could enhance the clinical validity of MBEC (minimal biofilm eradication concentration) assays. Chemistry Capillary Electrophoresis Mass Spectrometry Approaches for Characterization of the Protein and Metabolite Corona Acquired by Nanomaterials Andrew J. Chetwynd*1, Wei Zhang*2, Klaus Faserl*3, James A. Thorn4, Iseult Lynch1, Rawi Ramautar2, Herbert H. Lindner3 1School of Geography Earth and Environmental Sciences, University of Birmingham, 2Biomedical Microscale Analytics, Leiden University, 3Institute of Medical Biochemistry, Medical University of Innsbruck, 4AB Sciex UK Ltd Here we present a protocol to characterize the complete biomolecular corona, proteins, and metabolites, acquired by nanomaterials from biofluids using a capillary electrophoresis – mass spectrometry approach. Chemistry Nanoparticle Tracking Analysis of Gold Nanoparticles in Aqueous Media through an Inter-Laboratory Comparison Sophie M. Briffa1, Jo Sullivan2, Agnieszka Siupa2, Pauline Carnell-Morris2, Michele Carboni2, Kerstin Jurkschat3, Ruud J. B. Peters4, Carolin Schultz5, Kang Hee Seol6, Sook-Jin Kwon6, Sehee Park7, Tae Hyun Yoon6,7, Colin Johnston3, Stephen Lofts8, Eugenia Valsami-Jones1 1School of Geography, Earth and Environmental Sciences, University of Birmingham, 2Malvern Panalytical, 3Department of Materials, University of Oxford, 4Wageningen Food Safety Research, 5UK Centre for Ecology & Hydrology, 6Institute for Next Generation Material Design, Hanyang University, 7Department of Chemistry, College of Natural Sciences, Hanyang University, 8UK Centre for Ecology & Hydrology The protocol described here aims to measure the hydrodynamic diameter of spherical nanoparticles, more specifically gold nanoparticles, in aqueous media by means of Nanoparticle Tracking Analysis (NTA). The latter involves tracking the movement of particles due to Brownian motion and implementing the Stokes-Einstein equation to obtain the hydrodynamic diameter. Bioengineering Imaging and Quantification of the Area of Fast-Moving Microbubbles Using a High-Speed Camera and Image Analysis Nina Vyas1, Mehdi Mahmud2, Qianxi X Wang2, A. Damien Walmsley1 1School of Dentistry, University of Birmingham, 2Department of Mathematics, University of Birmingham Cavitation microbubbles are imaged using a high-speed camera attached to a zoom lens. The experimental setup is explained, and image analysis is used to calculate the area of the cavitation. Image analysis is done using ImageJ. Environment Using Tg(Vtg1:mcherry) Zebrafish Embryos to Test the Estrogenic Effects of Endocrine Disrupting Compounds Zsolt Csenki1, Ákos Horváth1, Illés Bock1, Edina Garai1, Flóra Kerekes1, Erna Vásárhelyi1, Balázs Kovács1, Béla Urbányi1, Ferenc Mueller2, Katalin Bakos1 1Department of Aquaculture, Institute of Aquaculture and Environmental Safety, Faculty of Agricultural and Environmental Sciences, Szent István University, 2Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham Present here is a detailed protocol for the use of zebrafish embryos Tg(vtg1: mCherry) for the detection of estrogenic effects. The protocol covers the propagation of the fish and treatment of embryos, and emphasizes the detection, documentation, and the evaluation of fluorescent signals induced by endocrine disrupting compounds (EDC). Bioengineering The Quantification of Injectability by Mechanical Testing Thomas E. Robinson1, Erik A. B. Hughes1, Neil M. Eisenstein1, Liam M. Grover1, Sophie C. Cox1 1School of Chemical Engineering, University of Birmingham Presented here is a protocol for quantitatively evaluating the injectability of a material through a syringe-needle system using a standard mechanical testing rig. Behavior Human Circadian Phenotyping and Diurnal Performance Testing in the Real World Elise R. Facer-Childs1,2,3, Benita Middleton1, Andrew P. Bagshaw2, Debra J. Skene1 1Chronobiology, Faculty of Health & Medical Sciences, University of Surrey, 2Centre for Human Brain Health, University of Birmingham, 3Turner Institute for Brain and Mental Health, School of Psychological Sciences, Monash University Here, we present a method to investigate diurnal rhythms in performance following accurate categorization of participants into circadian phenotype groups based on the Munich ChronoType Questionnaire, gold standard circadian phase biomarkers and actigraphic measures. Developmental Biology Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish Tomasz Dobrzycki*1,2, Monika Krecsmarik*1,2, Rui Monteiro1,2,3 1MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, 2BHF Centre of Research Excellence, 3Institute of Cancer and Genomic Sciences, University of Birmingham Gene editing technologies have enabled researchers to generate zebrafish mutants to investigate gene function with relative ease. Here, we provide a guide to perform parallel embryo genotyping and quantification of in situ hybridization signals in zebrafish. This unbiased approach provides greater accuracy in phenotypical analyses based on in situ hybridization. Developmental Biology DNAzyme-dependent Analysis of rRNA 2’-O-Methylation Kinga Winczura1, Pawel Grzechnik1 1School of Biosciences, University of Birmingham Here we present a protocol for DNAzyme-dependent cleavage of RNA. This enables fast and site-dependent analysis of RNA 2’-O-methylation. This approach can be used for the preliminary or major assessment of snoRNA activity. Chemistry Synthesis of Monodisperse Cylindrical Nanoparticles via Crystallization-driven Self-assembly of Biodegradable Block Copolymers Zachary Coe1, Andrew Weems1, Andrew P. Dove1, Rachel K. O'Reilly1 1Department of Chemistry, University of Birmingham Crystallization-driven self-assembly (CDSA) displays the unique ability to fabricate cylindrical nanostructures of narrow length distributions. The organocatalyzed ring-opening polymerization of ε-caprolactone and subsequent chain extensions of methyl methacrylate and N,N-dimethyl acrylamide are demonstrated. A living CDSA protocol that produces monodisperse cylinders up to 500 nm in length is outlined. Medicine High-Throughput Analysis of Optical Mapping Data Using ElectroMap Christopher O’Shea1,2,3, Andrew P. Holmes1,4, Ting Y. Yu1, James Winter1, Simon P. Wells1, Beth A. Parker1, Dannie Fobian1, Daniel M. Johnson1, Joao Correia5, Paulus Kirchhof1, Larissa Fabritz1, Kashif Rajpoot3, Davor Pavlovic1 1Institute of Cardiovascular Sciences, University of Birmingham, 2EPSRC Centre for Doctoral Training in Physical Sciences for Health, School of Chemistry, University of Birmingham, 3School of Computer Science, University of Birmingham, 4Institute of Clinical Sciences, University of Birmingham, 5Institute of Microbiology and Infection, School of Biosciences, University of Birmingham This protocol describes the setup and use of ElectroMap, a MATLAB-based open-source software platform for analysis of cardiac optical mapping data. ElectroMap provides a versatile high-throughput tool for analysis of optical mapping voltage and calcium datasets across a wide range of cardiac experimental models. Environment Resurrection of Dormant Daphnia magna: Protocol and Applications Maria Cuenca Cambronero1, Luisa Orsini1 1Environmental Genomics Group, School of Biosciences, University of Birmingham Long-term studies are essential to understanding the process of evolution and the mechanisms of adaptation. Generally, these studies require commitments beyond the life-time of researchers. Here, a powerful method is described that dramatically advances state-of-the-art data collection to generate longitudinal data in natural systems. Environment Dispersion of Nanomaterials in Aqueous Media: Towards Protocol Optimization Inder Kaur1, Laura-Jayne Ellis1, Isabella Romer1, Ratna Tantra2, Marie Carriere3,4, Soline Allard5, Martine Mayne-L'Hermite5, Caterina Minelli6, Wolfgang Unger7, Annegret Potthoff8, Steffi Rades7, Eugenia Valsami-Jones1 1School of Geography, Earth and Environmental Sciences, University of Birmingham, 2Analytical Science, National Physical Laboratory, 3INAC-LCIB, Université Grenoble Alpes, 4CEA, INAC-SyMMES, 5NIMBE, CEA, CNRS, Université Paris-Saclay, CEA Saclay, 6Chemical, Medical and Environmental Science, National Physical Laboratory, 7BAM Division 6.1 'Surface Analysis and Interfacial Chemistry', BAM Federal Institute for Materials Research and Testing, 8Fraunhofer Institute for Ceramic Technologies and Systems Here, we present a step-wise protocol for the dispersion of nanomaterials in aqueous media with real-time characterization to identify the optimal sonication conditions, intensity, and duration for improved stability and uniformity of nanoparticle dispersions without impacting the sample integrity. Immunology and Infection Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis Holly Payne1, Alexander Brill1 1Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham We describe here stenosis in the inferior vena cava as a murine model of deep vein thrombosis. This model recapitulates blood flow restriction, one of the major triggers of venous thrombosis in humans. Immunology and Infection Biomimetic Materials to Characterize Bacteria-host Interactions Daniel H. Stones*1, Fitua Al-Saedi*1, Diana Vaz1, Nicolas Perez-Soto1, Anne M. Krachler1 1Institute of Microbiology and Infection, School of Biosciences, University of Birmingham Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors. Behavior Modulating Cognition Using Transcranial Direct Current Stimulation of the Cerebellum Paul A. Pope1 1School of Psychology, University of Birmingham Transcranial direct current stimulation (tDCS) over the cerebellum exerts a remote effect on the prefrontal cortex, which can modulate cognition and performance. This was demonstrated using two information-processing tasks of varying complexity, whereby only cathodal tDCS improved performance when the task was difficult, but not easy. Immunology and Infection Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow Hafsa Munir1,2, G. Ed Rainger1,2, Gerard B. Nash1,2, Helen McGettrick2,3 1School of Clinical and Experimental Medicine, University of Birmingham, 2College of Medical and Dental Sciences, University of Birmingham, 3School of Immunity and Infection, University of Birmingham The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process. Immunology and Infection A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress Shishir Shetty1, Christopher J. Weston1, David H. Adams1, Patricia F. Lalor1 1NIHR Biomedical Research Unit, Centre for Liver Research, School of Immunity and Infection, University of Birmingham Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process. Immunology and Infection Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin Cecile Benezech1,2, Jorge H. Caamano1 1School of Immunity and Infection, IBR-MRC Centre for Immune Regulation, College of Medical and Dental Sciences, University of Birmingham, 2Centre for Cardiovascular Sciences, University of Edinburgh Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse. Engineering The Generation of Higher-order Laguerre-Gauss Optical Beams for High-precision Interferometry Ludovico Carbone1, Paul Fulda1, Charlotte Bond1, Frank Brueckner1, Daniel Brown1, Mengyao Wang1, Deepali Lodhia1, Rebecca Palmer1, Andreas Freise1 1School of Physics and Astronomy, University of Birmingham Large laser-interferometers are being constructed to create a new type of astronomy based on gravitational waves. Their sensitivities, as for many other high-precision experiments, are approaching fundamental noise limits such as the atomic vibration of their components. We are pioneering technologies to overcome these limits using novel laser beam shapes. Neuroscience An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila Kentaro Kato1, Alicia Hidalgo1 1Neurodevelopment Group, School of Biosciences, University of Birmingham An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair. Medicine Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers Kenneth M. Tichauer1, Robert W. Holt2, Kimberley S. Samkoe3, Fadi El-Ghussein1, Jason R. Gunn1, Michael Jermyn1, Hamid Dehghani4, Frederic Leblond1, Brian W. Pogue1,2 1Thayer School of Engineering, Dartmouth College, 2Department of Physics and Astronomy, Dartmouth College, 3Darmouth Medical School, Dartmouth College, 4School of Computer Science, University of Birmingham Diffuse fluorescence tomography offers a relatively low-cost and potentially high-throughout approach to preclinical in vivo tumor imaging. The methodology of optical data collection, calibration, and image reconstruction is presented for a computed tomography-guided non-contact time-domain system using fluorescent targeting of the tumor biomarker epidermal growth factor receptor in a mouse glioma model. Biology Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells Deborah Merrick1, Hung-Chih Chen1, Dean Larner1, Janet Smith1 1School of Biosciences, University of Birmingham The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation. Biology In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells Anyaporn Sawasdichai1, Hsin-Tien Chen1, Nazefah Abdul Hamid1, Padma-Sheela Jayaraman2, Kevin Gaston1 1Department of Biochemistry, School of Medical Sciences, University of Bristol, 2Division of Immunity and Infection, School of Medicine, University of Birmingham In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation. Biology Techniques for Imaging Ca2+ Signaling in Human Sperm Katherine Nash1, Linda Lefievre2, Ruben Peralta-Arias1, Jennifer Morris1, Aduen Morales-Garcia1, Tom Connolly2, Sarah Costello1, Jackson C. Kirkman-Brown3, Stephen J. Publicover1 1School of Biosciences, University of Birmingham, 2School of Medicine, University of Birmingham, 3Centre for Human Reproductive Science, Birmingham Women’s Hospital Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel. Biology Preparation of 2-dGuo-Treated Thymus Organ Cultures William Jenkinson1, Eric Jenkinson1, Graham Anderson1 1MRC Center for Immune Regulation, University of Birmingham This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus. Biology Reaggregate Thymus Cultures Andrea White1, Eric Jenkinson1, Graham Anderson1 1MRC Center for Immune Regulation, University of Birmingham In this video the preparation of 2-dGuo-treated reaggregate thymus cultures is demonstrated.
