Royal College of Surgeons in Ireland 5 articles published in JoVE Immunology and Infection An Automated Culture System for Use in Preclinical Testing of Host-Directed Therapies for Tuberculosis Seónadh O’Leary1, Ahmad Z. Bahlool1,2, Gemma O’Connor2, Sally-Ann Cryan2,3,4, Joseph M. Keane1, Mary P. O’Sullivan1 1Department of Clinical Medicine, Trinity Translational Medicine Institute, St. James's Hospital, Trinity College Dublin, The University of Dublin, 2School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland (RCSI), 3SFI Advanced Materials and Bioengineering Research (AMBER) Centre, RCSI & TCD, 4SFI Centre for Research in Medical Devices (CURAM), RCSI, Dublin and National University of Ireland Rapid and efficient quantification of intracellular M. tuberculosis growth is crucial for pursuing improved therapies against tuberculosis (TB). This protocol describes a broth-based colorimetric detection assay using an automated liquid culture system to quantify Mtb growth in macrophages treated with candidate host-directed therapies. Cancer Research Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds Ciara Gallagher*1,2,3, Catherine Murphy*1,2,3, Graeme Kelly4, Fergal J. O’Brien3,5,6, Olga Piskareva1,2,3,6,7 1Cancer Bioengineering Group, Department of Anatomy and Regenerative Medicine, RCSI University of Medicine and Health Sciences, Dublin, Ireland, 2School of Pharmacy and Biomolecular Sciences, RCSI University of Medicine and Health Sciences, Dublin, Ireland, 3Tissue Engineering Research Group, Department of Anatomy and Regenerative Medicine, RCSI University of Medicine and Health Sciences, Dublin, Ireland, 7Department of Chemistry, Royal College of Surgeons in Ireland (RCSI), 123 St. Stephen's Green, Dublin 2, Ireland, 5Trinity Centre for Bioengineering, Trinity College Dublin, Dublin, Ireland, 6Advanced Materials and Bioengineering Research Centre (AMBER), RCSI and TCD, Dublin, Ireland, 7National Children’s Research Centre, Our Lady's Children's Hospital Crumlin, Dublin, Ireland This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims. Immunology and Infection Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP) Jordan F. Hastings1, Jeremy Z.R. Han1, Robert F. Shearer1,2, Sean P. Kennedy1,3, Mary Iconomou1,4, Darren N. Saunders5, David R. Croucher1,6,7 1The Kinghorn Cancer Centre, Garvan Institute of Medical Research, 2Ubiquitin Signaling Group, Protein Signaling Program, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, 3RCSI Molecular Medicine, Royal College of Surgeons in Ireland, 4Department of Epigenetics, Max Planck Institute of Immunobiology and Epigenetics, 5School of Medical Sciences, University of New South Wales, 6St Vincent's Hospital Clinical School, University of New South Wales, 7School of Medicine and Medical Science, University College Dublin This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners. Immunology and Infection Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model Trevor Beaudoin1, Sarah Kennedy2, Yvonne Yau3, Valerie Waters4 1Physiology and Experimental Medicine, Hospital for Sick Children, 2Department of Clinical Microbiology, Royal College of Surgeons in Ireland, 3Department of Laboratory Medicine and Pathobiology, University of Toronto, 4Department of Pediatrics, University of Toronto This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs. Cancer Research Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion Salvatore Coniglio1, Ian Miller2, Marc Symons3, Jeffrey E. Segall4 1New Jersey Center for Science, Technology and Mathematics, Kean University, 2Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, 3The Feinstein Institute for Medical Research at North Shore-LIJ, 4Department of Anatomy and Structural Biology, Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.
