University of Leicester View Institution's Website 8 articles published in JoVE Environment Application of the Intelligent High-Throughput Antimicrobial Sensitivity Testing/Phage Screening System and Lar Index of Antimicrobial Resistance Ming Hu1, Zhengjie Liu1,2, Ziang Song1,3, Lulu Li1, Xiaonan Zhao1, Yanbo Luo1, Qing Zhang1, Yibao Chen1, Xiaohui Xu1, Yahui Dong1, Nataliia Hrabchenko1, Wei Zhang3, Yuqing Liu1 1Shandong Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, P.R. China, Key Laboratory of Livestock and Poultry Multi-omics of MARA, 2Department of Genetic and Genomes Biology, University of Leicester, 3College of Veterinary Medicine, Nanjing Agricultural University Here we introduce the principle, structure, and instruction of the intelligent high-throughput antimicrobial sensitivity testing/phage screening system. Its application is illustrated by using Salmonella isolated from poultry in Shandong, China, as an example. The Lar index is calculated, and its significance in evaluating antimicrobial resistance is discussed comprehensively. Cancer Research Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients Giuditta Viticchié1, Ian Powley1, Constantinos Demetriou1, James Cooper1, Michael Butterworth1, Meeta Patel1, Naila Abid1, Gareth Miles1, Lynne Howells1, Howard Pringle1, Marion MacFarlane2, Catrin Pritchard1 1Leicester Cancer Research Center, University of Leicester, 2MRC Toxicology Unit This paper describes methods for the generation, drug treatment, and analysis of patient-derived explants for assessing tumor drug responses in a live, patient-relevant, preclinical model system. Medicine Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice Melissa Trotman-Lucas1, Michael E. Kelly2, Justyna Janus2, Claire L. Gibson1,3 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. Biology Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper (cTfh) Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry Elliot Byford1, Matthew Carr1, Lucia Piñon2, Matthew J. Ahearne1, Simon D. Wagner1 1Leicester Cancer Research Centre and Ernest and Helen Scott Haematology Research Institute, University of Leicester, 2MRC Toxicology Unit, University of Leicester Here, a protocol for the isolation and characterization of CD4+ T-cell subsets from human peripheral blood is described. Purified CD4+ T-cells are analyzed by flow cytometry to determine proportions of T-follicular helper cell subsets. Bioengineering Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall Jack C. Bridge1, Jonathan W. Aylott2, Christopher E. Brightling5, Amir M. Ghaemmaghami3, Alan J. Knox4, Mark P. Lewis6, Felicity R.A.J. Rose1, Gavin E. Morris1 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. Biology Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors Thimma R. Reddy1, Emma J. Kelsall1, Léna M.S. Fevat2, Sarah E. Munson*3, Shaun M. Cowley*1 1Department of Biochemistry, University of Leicester, 2Center for Fisheries, Environment and Aquaculture Sciences, 3ES Cell Facility, Centre for Core Biotechnology Services, University of Leicester Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term ‘subcloning plus insertion’, which can be used to generate custom gene targeting vectors rapidly. Biology Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach Nicola Portolano1, Peter J. Watson1, Louise Fairall1, Christopher J. Millard1, Charles P. Milano1, Yun Song1, Shaun M. Cowley1, John W.R. Schwabe1 1Department of Biochemistry, University of Leicester The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies. Medicine Experimental Human Pneumococcal Carriage Jenna F. Gritzfeld1, Angie D. Wright1,2,3, Andrea M. Collins1,2,4, Shaun H. Pennington1, Adam K.A. Wright5, Aras Kadioglu6, Daniela M. Ferreira1, Stephen B. Gordon1 1Respiratory Infection Group, Liverpool School of Tropical Medicine, 2Royal Liverpool and Broadgreen, University Hospital Trust, 3Comprehensive Local Research Network, 4NIHR Biomedical Research Centre in Microbial Diseases, Royal Liverpool and Broadgreen University Hospitals NHS Trust, 5Institute of Lung Health, Respiratory Biomedical Unit, University Hospitals of Leicester NHS Trust & University of Leicester, 6Department of Clinical Infection Microbiology & Immunology, Institute of Infection & Global Health, University of Liverpool Experimental human pneumococcal carriage offers a natural model of carriage and a potential model for use in vaccine development. This technique is valuable yet complex and involves clinical risk by introducing a pathogen into a human. We have developed a detailed protocol.