A*STAR 10 articles published in JoVE Biology Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control Inn Chuan Ng*1, Li Zhang*2, Narelle Nichola Yi Ying Shen3, Yun Ting Soong4, Chan Way Ng5, Phoebe Kang Sheing Koh1, Yan Zhou3, Hanry Yu1,3,4,5,6 1Department of Physiology & The Institute for Digital Medicine (WisDM), National University of Singapore, 2College of Agriculture and Biology, Zhongkai University of Agriculture and Engineering, 3Mechanobiology Institute, National University of Singapore, 4Institute of Bioengineering and Nanotechnology, A*STAR, 5NUS Graduate School for Integrative Sciences and Engineering, 6CAMP, Singapore-MIT Alliance for Research and Technology This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes. 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. Immunology and Infection Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries Elijah W. Chen*1, Chyan Ying Ke*2,3, Joanna Brzostek*1, Nicholas R. J. Gascoigne1, Vasily Rybakin1,4 1Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 2Singapore Immunology Network, A*STAR, 3Curiox Biosystems, 4Department of Immunobiology, Rega Institute for Medical Research, Katholieke Universiteit (KU) Leuven This paper uses a flow-cytometry-based assay to screen libraries of chemical inhibitors for the identification of inhibitors and their targets that influence T-cell receptor signaling. The methods described here can also be expanded for high-throughput screenings. Immunology and Infection Bead Based Multiplex Assay for Analysis of Tear Cytokine Profiles Praveen Kumar Balne1,2, Veonice Bijin AU3, Louis Tong2, Arkasubhra Ghosh4, Mukesh Agrawal5, John Connolly3, Rupesh Agrawal1 1National Healthcare Group Eye Institute, Tan Tock Seng Hospital, 2Singapore Eye Research Institute (SERI), 3Institute of Molecular and Cell Biology, A*STAR, 4GROW Research Laboratory, Narayana Nethralaya Foundation, 5Vimta Labs Limited Analysis of tear film cytokines helps in studying various ocular diseases. Bead based multiplex assays are simple and sensitive and enable the testing of multiple targets in samples with small volumes. Here we describe a protocol for tear film cytokine profiling a using bead based multiplex assay. Genetics Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen Jong Ghut Ashley Aw1, Yang Shen2, Niranjan Nagarajan2, Yue Wan1 1Stem Cell and Regenerative Biology, Genome Institute of Singapore, A*STAR, 2Computational and Systems Biology, Genome Institute of Singapore, A*STAR Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans. Medicine Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses Chi Ching Goh1,2, Jackson LiangYao Li1, David Becker3, Wolfgang Weninger4,5,6, Veronique Angeli2,7, Lai Guan Ng1,2,5,8 1Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Biopolis, 2Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 3Lee Kong Chian School of Medicine, Nanyang Technological University, 4Centenary Institute for Cancer Medicine and Cell Biology, 5Discipline of Dermatology, University of Sydney, 6Department of Dermatology, Royal Prince Alfred Hospital, 7LSI Immunology Programme, National University of Singapore, 8School of Biological Sciences, Nanyang Technological University This protocol describes the induction of an ischemia-reperfusion (IR) model on mouse ear skin using magnet clamping. Using a custom-built intravital imaging model, we study in vivo inflammatory responses post-reperfusion. The rationale behind the development of this technique is to extend the understanding of how leukocytes respond to skin IR injury. Neuroscience Enumeration of Neural Stem Cells Using Clonal Assays Gunaseelan Narayanan1, Yuan Hong Yu1, Muly Tham1, Hui Theng Gan1, Srinivas Ramasamy1, Shvetha Sankaran1, Srivats Hariharan1, Sohail Ahmed1 1Neural Stem Cell Laboratory, Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR) Neural stem cells (NSCs) refer to cells which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions. Bioengineering Polymeric Microneedle Array Fabrication by Photolithography Himanshu Kathuria1, Jaspreet Singh Kochhar1, Michelle Hui Min Fong1, Michinao Hashimoto2, Ciprian Iliescu3, Hanry Yu3,4,5, Lifeng Kang1 1Department of Pharmacy, National University of Singapore, 2Singapore University of Technology and Design, 3Institute of Bioengineering and Nanotechnology, Agency for Science, Technology and Research (A*STAR), 4Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, 5Mechanobiology Institute, National University of Singapore Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography. Bioengineering A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening Yong Yu1, Siu Yee New1, Jiaxian Lin2, Xiaodi Su1, Yen Nee Tan1 1Institute of Materials Research and Engineering, Agency for Science, Technology and Research (A*STAR), 2School of Chemical & Biomedical Engineering, Nanyang Technological University A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs. Bioengineering Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion Yi-Zhen Ng1,2, Andrew P. South1 1Division of Cancer Research, Ninewells Hospital and Medical School, University of Dundee, 2Institute of Medical Biology, A*Star, Singapore Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.