Cardiff University View Institution's Website 8 articles published in JoVE Neuroscience In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages Hazel Hall-Roberts1,2,3, Elena Di Daniel2, William S. James1, John B. Davis2, Sally A. Cowley1 1James Martin Stem Cell Facility, Sir William Dunn School of Pathology, University of Oxford, 2Alzheimer’s Research UK Oxford Drug Discovery Institute, Nuffield Department of Medicine Research Building, University of Oxford, 3UK Dementia Research Institute, Cardiff University Neurodegenerative diseases are associated with dysregulated microglia functions. This article outlines an in vitro assay of phagocytosis of neuroblastoma cells by iPSC-macrophages. Quantitative microscopy readouts are described for both live-cell time-lapse imaging and fixed-cell high-content imaging. Engineering Method for Recording Broadband High Resolution Emission Spectra of Laboratory Lightning Arcs D. Mitchard1, D. Clark1, D. Carr1, C. Stone1, A. Haddad1 1Morgan-Botti Lightning Laboratory, Advanced High Voltage Research Centre, School of Engineering, Cardiff University Emission spectroscopy techniques have traditionally been used to analyze inherently random lightning arcs occurring in nature. In this paper, a method developed to obtain the emission spectroscopy from reproducible lightning arcs generated within a laboratory environment is described. Immunology and Infection Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens Emily A. Lees1,2, Jessica L. Forbester1,3, Sally Forrest2, Leanne Kane1, David Goulding1, Gordon Dougan1,2 1Wellcome Trust Sanger Institute, 2Department of Medicine, University of Cambridge, 3University of Cardiff Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids offer exciting opportunities to model enteric diseases in vitro. We demonstrate the differentiation of hiPSCs into intestinal organoids (iHOs), the stimulation of these iHOs with cytokines, and the microinjection of Salmonella Typhimurium into the iHO lumen, enabling the study of an epithelial invasion by this pathogen. Immunology and Infection Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens Bruce J. MacLachlan*1, Alexander Greenshields-Watson*1, Georgina H Mason*1, Andrea J Schauenburg1, Valentina Bianchi1,2,3, Pierre J Rizkallah1, Andrew K Sewell1, Anna Fuller1, David K Cole1 1Division of Infection and Immunity and Systems Immunity Research Institute, Cardiff University, 2Department of Oncology, University Hospital of Lausanne (CHUV), 3Ludwig Insitutue for Cancer Research, Lausanne Branch, University of Lausanne Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between the T-cell receptor and tumor antigens, presented by human leukocyte antigens, governs T-cell functionality; these methods include protein production, X-ray crystallography, biophysics, and functional T-cell experiments. Biology Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study Paulina J. Stanczyk1, F. Anthony Lai1, Spyros Zissimopoulos1 1Wales Heart Research Institute, Cardiff University School of Medicine, College of Biomedical and Life Sciences Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry. Developmental Biology Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models Lee Parry1, Madeleine Young1, Fatima El Marjou2, Alan Richard Clarke1 1European Cancer Stem Cell Research Institute, Cardiff University, 2Institut Curie Intestinal epithelial stem cells (ISCs) are intermingled with Paneth cells. These cells are differentiated progeny of the ISC, which support the ISCs and provide antibacterial protection. Here we demonstrate how we used transgenic conditional mouse models to establish that Paneth cells play a crucial role in maintaining the intestinal epithelia. Biology Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond Eleftherios Karanasios1, Eloise Stapleton2, Simon A. Walker1, Maria Manifava1, Nicholas T. Ktistakis1 1Signalling Programme, The Babraham Institute, 2MRC Group, Cardiff School of Biosciences, Cardiff University Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context. Bioengineering Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments Xiaoting Meng*1, Wenfei Li*2,3, Fraser Young1, Runchi Gao3, Laura Chalmers3, Min Zhao3, Bing Song1 1School of Dentistry, Cardiff Institute of Tissue Engineering & Repair, Cardiff University, 2Shandong Qianfoshan Hospital, Shandong University School of Medicine, 3Dermatology and Ophthalmology Research, Institute for Regenerative Cures, University of California at Davis This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).