DUKE-NUS Graduate Medical School 8 articles published in JoVE Immunology and Infection Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation Xiang Zhao1, Maryam Hamidinia1, Joanna Ai Ling Choo1, Chien Tei Too1,2, Zi Zong Ho3, Ee Chee Ren4, Antonio Bertoletti3, Paul A. MacAry1,2,5, Keith G. Gould6, Joanna Brzostek1, Nicholas R.J. Gascoigne1,2,5 1Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 2Immunology Programme, Life Sciences Institute, National University of Singapore, 3Emerging Infectious Diseases Program, Duke-NUS Graduate Medical School, 4Singapore Immunology Network, A*STAR, 5NUS Graduate School for Integrative Sciences and Engineering (NGS), National University of Singapore, 6Department of Immunology, Wright-Fleming Institute, Imperial College London This protocol describes the use of single chain MHC class I complexes to investigate molecular interactions in human CD8+ T cell activation: generation of engineered antigen presenting cells expressing single chain constructs, culture of human CD8+ T cell clone and T cell activation experiments. Biology Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions Xiaofeng Zheng1,2, Calvin Qing Wei Ho1, Xiaowei Zheng3, Kian Leong Lee4, Katarina Gradin5, Teresa S. Pereira3, Per-Olof Berggren1,2,3, Yusuf Ali1,2 1Lee Kong Chian School of Medicine, Nanyang Technological University, 2Singapore Eye Research Institute (SERI), Singapore General Hospital, 3The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Karolinska University Hospital, 4Cancer and Stem Cell Biology Program, Duke-NUS Medical School, 5Department of Cell and Molecular Biology, Karolinska Institutet Here we describe a co-immunoprecipitation protocol to study protein-protein interactions between endogenous nuclear proteins under hypoxic conditions. This method is suitable for demonstration of the interactions between transcription factors and transcriptional co-regulators at hypoxia. Immunology and Infection A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva Tanima Bose1, Aihua Hou2, Ryan Lee2, Louis Tong2,3,4,5, K. George Chandy1 1Lee Kong Chian School of Medicine, Nanyang Technological University, 2Singapore Eye Research Institute, 3Singapore National Eye Center, 4Duke-NUS Medical School, 5Yong Loo Lin School of Medicine The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva. Medicine Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation Rupesh Agrawal*1,2,3, Praveen Kumar Balne*2, Sai Bo Bo Tun2, Yeo Sia Wey2, Neha Khandelwal1, Veluchamy A. Barathi2,4,5 1National Healthcare Group Eye Institute, Tan Tock Seng Hospital, 2Singapore Eye Research Institute (SERI), Singapore National Eye Center, 3School of Material Science and Engineering, Nanyang Technological University, 4Department of Ophthalmology, Yong Loo Lin School of Medicine, National University Health Systems, National University of Singapore, 5Ophthalmology Academic Clinical Research Program, DUKE-NUS Graduate Medical School Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described. Immunology and Infection A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue Aihua Hou1,2, Tanima Bose3, K. George Chandy3, Louis Tong1,2,4,5 1Singapore Eye Research Insitute, A Member of SingHealth, 2DUKE-National University of Singapore Medical School, 3Lee Kong Chian School of Medicine, Nanyang Technological University, 4Singapore National Eye Center, 5Yong Loo Lin School of Medicine, National University of Singapore This report describes a method to induce chronic experimental autoimmune dry eye in Lewis rats through immunization with an emulsion of rat lacrimal gland extract, ovalbumin, and complete Freund's adjuvant, followed by the injection of lacrimal gland extract and ovalbumin into the forniceal subconjunctiva and lacrimal glands six weeks later. Engineering In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart Sindhu Ramesh*1, Anamika Singh*1, Dasan M. Cibi1, Derek J. Hausenloy1,2,3, Manvendra K. Singh1,2 1Program in Cardiovascular and Metabolic Disorders, Duke-NUS Graduate Medical School, 2National Heart Research Institute Singapore, National Heart Centre Singapore, 3The Hatter Cardiovascular Insititute, University College London The epicardium is an essential source of multipotent cardiovascular progenitor cells and paracrine factors that are required for cardiovascular development and regeneration. We describe here a method to culture mouse embryonic epicardial cells. Developmental Biology An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System Colin T. E. Su*1, Su-In Yoon*2, Guillaume Marcy*1, Eunice W. M. Chin1, George J. Augustine2, Eyleen L. K. Goh1 1Neuroscience & Behavioral Disorders, Duke-NUS Graduate Medical School, 2Lee Kong Chian School of Medicine, Nanyang Technological University A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2. Behavior Development of a Virtual Reality Assessment of Everyday Living Skills Stacy A. Ruse1, Vicki G. Davis1, Alexandra S. Atkins1, K. Ranga R. Krishnan2,3, Kolleen H. Fox4, Philip D. Harvey5, Richard S.E. Keefe1,3 1NeuroCog Trials, Inc., 2Duke-NUS Graduate Medical Center, 3Duke University Medical Center, 4Fox Evaluation and Consulting, PLLC, 5University of Miami Miller School of Medicine A challenge for proving treatment efficacy for cognitive impairments in schizophrenia is finding the optimizing measurement of skills related to everyday functioning. The Virtual Reality Functional Capacity Assessment Tool (VRFCAT) is an interactive gaming based computerized measure aimed at skills associated with everyday functioning, including baseline impairments and treatment related changes.
