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A*STAR

6 ARTICLES PUBLISHED IN JoVE

Bioengineering

Polymeric Microneedle Array Fabrication by Photolithography

Himanshu Kathuria¹, Jaspreet Singh Kochhar¹, Michelle Hui Min Fong¹, Michinao Hashimoto², Ciprian Iliescu³, Hanry Yu^3,4,5, 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.

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.

Immunology and Infection

Bead Based Multiplex Assay for Analysis of Tear Cytokine Profiles

Praveen Kumar Balne^1,2, 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.

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 Rybakin^1,4

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

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 Too^1,2, Zi Zong Ho³, Ee Chee Ren⁴, Antonio Bertoletti³, Paul A. MacAry^1,2,5, Keith G. Gould⁶, Joanna Brzostek¹, Nicholas R.J. Gascoigne^1,2,5

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

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 Yu^1,3,4,5,6

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.