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Nanyang Technological University

55 ARTICLES PUBLISHED IN JoVE

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Biology

Mouse Adrenal Chromaffin Cell Isolation
Aaron Kolski-Andreaco 1, Haijiang Cai 2,3, D. Spencer Currle 4, K. George Chandy 1, Robert H. Chow 2,3
1Department of Physiology and Biophysics, University of California, Irvine (UCI), 2Department of Physiology and Biophysics, University of Southern California, Keck School of Medicine, 3Zilkha Neurogenetic Institute, University of Southern California, Keck School of Medicine, 4Department of Developmental and Cell Biology, University of California, Irvine (UCI)

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting. This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex. The digestion of the medulla into single chromaffin cells is then demonstrated.

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Biology

Induction and Clinical Scoring of Chronic-Relapsing Experimental Autoimmune Encephalomyelitis
Christine Beeton 1, Adriana Garcia 1, K. George Chandy 1
1Department of Physiology and Biophysics, University of California, Irvine (UCI)

This video demonstrates the induction and clinical scoring of an animal model of multiple sclerosis: chronic-relapsing experimental autoimmune encephalomyelitis in DA rats. The disease, induced by immunizing rats with an emulsion containing whole rat spinal cord and complete Freund's adjuvant, presents clinical signs resembling the human disease.

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Biology

Induction and Monitoring of Active Delayed Type Hypersensitivity (DTH) in Rats
Christine Beeton 1, K. George Chandy 1
1Department of Physiology and Biophysics, University of California, Irvine (UCI)

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes. Here we demonstrate how to induce active DTH in Lewis rats and monitor the inflammatory response.

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Biology

Drawing Blood from Rats through the Saphenous Vein and by Cardiac Puncture
Christine Beeton 1, Adriana Garcia 1, K. George Chandy 1
1Department of Physiology and Biophysics, University of California, Irvine (UCI)

Blood draws are necessary in a large number of studies, for example to study the pharmacokinetics profile of a compound. Here, we demonstrate how to draw blood from rats using two techniques: blood draw from the saphenous vein or by cardiac puncture.

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Biology

Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs
Victor Chi 1, K. George Chandy 1
1Department of Physiology and Biophysics, University of California, Irvine (UCI)

Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs

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Biology

Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats
Christine Beeton 1, K. George Chandy 1
1Department of Physiology and Biophysics, University of California, Irvine (UCI)

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T (TEM) lymphocytes. Here we demonstrate how to activate antigen-specific TEM cells, induce adoptive DTH in Lewis rats and monitor the inflammatory response.

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Biology

Enrichment of NK Cells from Human Blood with the RosetteSep Kit from StemCell Technologies
Christine Beeton 1, K. George Chandy 1
1Department of Physiology and Biophysics, University of California, Irvine (UCI)

Natural killer cells are a small population of lymphocytes. Here we show how to isolate these cells from human blood by negative selection, using a kit from StemCell Technologies. The cells obtained are viable and untouched by antibodies, and therefore ready to be used for a number of procedures.

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Biology

Preparing T Cell Growth Factor from Rat Splenocytes
Christine Beeton 1, K. George Chandy 1
1Department of Physiology and Biophysics, University of California, Irvine (UCI)

We describe the preparation of T cell growth factor used for the in vitro expansion of antigen-specific rat T lymphocyte lines.

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Biology

Isolation of Mononuclear Cells from the Central Nervous System of Rats with EAE
Christine Beeton 1, K. George Chandy 1
1Department of Physiology and Biophysics, University of California, Irvine (UCI)

In this video we demonstrate how to isolate mononuclear cells from the central nervous system of rats with experimental autoimmune encephalomyelitis.

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Biology

Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH
Melanie P. Matheu 1, Christine Beeton 1, Ian Parker 2, K. George Chandy 1, Michael D. Cahalan 1
1Department of Physiology and Biophysics, University of California, Irvine (UCI), 2Department of Neurobiology and Behavior, University of California, Irvine (UCI)

Here we demonstrate a method for inducing and recording the progress of a delayed type-hypersensitivity (DTH) reaction in the rat ear. This is followed by a demonstration of the preparation of rat ear tissue for two-photon imaging of the effector / memory T cell response.

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Biology

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal
Xiaofeng Zheng 1, Guang Hu 1
1Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences

We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.

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Medicine

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time
Midhat H. Abdulreda 1,2, Alejandro Caicedo 1,3,4, Per-Olof Berggren 1,5
1Diabetes Research Institute, University of Miami Miller School of Medicine, 2Department of Surgery, University of Miami Miller School of Medicine, 3Department of Medicine, University of Miami Miller School of Medicine, 4Department of Physiology & Biophysics, University of Miami Miller School of Medicine, 5The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

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Bioengineering

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging
Artur Matysik 1, Rachel S. Kraut 1
1School of Biological Sciences, Nanyang Technological University

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

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JoVE Journal

Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability
Wahyu Surya 1, Jaume Torres 1
1School of Biological Sciences, Nanyang Technological University

Sedimentation equilibrium (SE) can be used to study protein-protein interactions in a physiological environment. This manuscript describes the use of this technique to determine the effect of pH on the stability of a homo-pentamer formed by the small hydrophobic (SH) protein encoded by the human syncytial respiratory virus (hRSV).

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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. Chin 1, George J. Augustine 2, Eyleen L. K. Goh 1
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.

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Bioengineering

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering
Pearlie S. J. Low 1, Monica S. Tjin 1, Eileen Fong 1
1School of Materials Science and Engineering, Nanyang Technological University

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

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Chemistry

Using Polystyrene-block-poly(acrylic acid)-coated Metal Nanoparticles as Monomers for Their Homo- and Co-polymerization
Yawen Wang 1, Xiohui Song 1, Hong Wang 1, Hongyu Chen 1
1Division of Chemistry and Biological Chemistry, Nanyang Technological University

We report protocols for “polymerizing” various types of polymer-encapsulated metal nanoparticles into long chains of “homo-“ and “co-polymers”.

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Bioengineering

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method
Seyed R. Tabaei 1,2, Joshua A. Jackman 1,2, Minchul Kim 1,2, Saziye Yorulmaz 1,2, Setareh Vafaei 1,2, Nam-Joon Cho 1,2,3
1School of Materials Science and Engineering, Nanyang Technological University, 2Centre for Biomimetic Sensor Science, Nanyang Technological University, 3School of Chemical and Biomedical Engineering, Nanyang Technological University

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

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Bioengineering

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology
Su C. Chew 1,2, Scott A. Rice 2,3,4,5, Staffan Kjelleberg 2,3,4,6, Liang Yang 2,3
1Interdisciplinary Graduate School, Nanyang Technological University, 2Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, 3School of Biological Sciences, Nanyang Technological University, 4Centre for Marine Bio-Innovation, University of New South Wales, 5School of Biological, Earth and Environmental Sciences, University of New South Wales, 6School of Biotechnology and Biomolecular Sciences Sciences, University of New South Wales

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

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Bioengineering

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening
Yong Yu 1, Siu Yee New 1, Jiaxian Lin 2, Xiaodi Su 1, Yen Nee Tan 1
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.

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Bioengineering

Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation
Malathi Mathiyazhakan 1, Weiwei Chan 2, Claus-Dieter Ohl 2, Chenjie Xu 1,3
1School of Chemical and Biomedical Engineering, Nanyang Technological University, 2Division of Physics, School of Physical and Mathematical Sciences, Nanyang Technological University, 3NTU-Northwestern Institute of Nanomedicine, Nanyang Technological University

This protocol describes a simple preparation method for gold nanoparticle integrated photo-responsive liposomes with the commercially available materials. It also shows how to measure the microbubble cavitation process of the synthesized liposomes upon the treatment of pulsed laser.

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Engineering

Compact Lens-less Digital Holographic Microscope for MEMS Inspection and Characterization
Thomas Bourgade 1, Sun Jianfei 1, Zhaomin Wang 1, Rosmin Elsa 2, Anand Asundi 1,2
1School of Mechanical and Aerospace Engineering, Center of Laser and Optical Engineering, Nanyang Technological University, 2d'Optron Pte Ltd

We present a compact reflection digital holographic system (CDHM) for inspection and characterization of MEMS devices. A lens-less design using a diverging input wave providing natural geometrical magnification is demonstrated. Both static and dynamic studies are presented.

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Biology

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture
Qianfan Bai *1, Zhiqiang Bai *1, Shaohai Xu 3, Lei Sun 1,2
1Cardiovascular and Metabolic Disorders Program, Duke-NUS Medical School, 2Institute of Molecular and Cell Biology, Singapore, 3Division of Bioengineering, School of Chemical & Biomedical Engineering, Nanyang Technological University

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

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Neuroscience

Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle (Mercynorrhina torquata)
T. Thang Vo Doan 1, Hirotaka Sato 1
1School of Mechanical and Aerospace Engineering, Nanyang Technological University

This protocol describes the process of constructing an insect-machine hybrid system and carrying out wireless electrical stimulation of the flight muscles required to control the turning motion of a flying insect.

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Bioengineering

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering
Hui Min Tay 1, David C. Yeo 2, Christian Wiraja 2, Chenjie Xu 2,3, Han Wei Hou 1
1Lee Kong Chian School of Medicine, Nanyang Technological University, 2School of Chemical and Biomedical Engineering, Nanyang Technological University, 3NTU-Northwestern Institute of Nanomedicine, Nanyang Technological University

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

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Bioengineering

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering
Hitomi Shirahama 1, Supriya K. Kumar 1, Won-Yong Jeon 1, Myung Hee Kim 1, Jae Ho Lee 1, Soon Seng Ng 1, Seyed R. Tabaei 1, Nam-Joon Cho 1,2
1School of Materials Science and Engineering, Nanyang Technological University, 2School of Chemical and Biomedical Engineering, Nanyang Technological University

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

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JoVE Core

1,3,5-Triphenylbenzene and Corannulene as Electron Receptors for Lithium Solvated Electron Solutions
Kim Seng Tan 1,2, Andrey V. Lunchev 2, Mihaiela C. Stuparu 2,3, Andrew C. Grimsdale 2, Rachid Yazami 1,2
1Energy Research Institute (ERI@N), Nanyang Technological University, 2School of Materials Science and Engineering, Nanyang Technological University, 3School of Physical and Mathematical Sciences, Nanyang Technological University

The authors report on conductivity studies carried out on lithium solvated electron solutions (LiSES) prepared using 1,3,5-triphenylbenzene (TPB) and corannulene as electron receptors.

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Immunology and Infection

Isolation and Activation of Murine Lymphocytes
Jun Feng Lim 1, Heidi Berger 1, I-hsin Su 1
1School of Biological Sciences, College of Science, Nanyang Technological University

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

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Bioengineering

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells
Ramya Bhuthalingam 1, Pei Q. Lim 1, Scott A. Irvine 1, Subbu S. Venkatraman 1
1School of Materials Science and Engineering, Nanyang Technological University

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

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Bioengineering

Extraction of Plant-based Capsules for Microencapsulation Applications
Michael G. Potroz 1,2, Raghavendra C. Mundargi 1,2, Jae Hyeon Park 1,2, Ee-Lin Tan 1,2, Nam-joon Cho 1,2,3
1School of Materials Science and Engineering, Nanyang Technological University, 2Centre for Biomimetic Sensor Science, Nanyang Technological University, 3School of Chemical and Biomedical Engineering, Nanyang Technological University

This protocol/manuscript describes a streamlined process for the production of sporopollenin exine capsules (SECs) from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs.

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Medicine

Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses
Chi Ching Goh 1,2, Jackson LiangYao Li 1, David Becker 3, Wolfgang Weninger 4,5,6, Veronique Angeli 2,7, Lai Guan Ng 1,2,5,8
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.

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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 Tun 2, Yeo Sia Wey 2, Neha Khandelwal 1, Veluchamy A. Barathi 2,4,5
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.

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Immunology and Infection

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva
Tanima Bose 1, Aihua Hou 2, Ryan Lee 2, Louis Tong 2,3,4,5, K. George Chandy 1
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.

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Immunology and Infection

A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue
Aihua Hou 1,2, Tanima Bose 3, K. George Chandy 3, Louis Tong 1,2,4,5
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.

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Biochemistry

LERLIC-MS/MS for In-depth Characterization and Quantification of Glutamine and Asparagine Deamidation in Shotgun Proteomics
Xavier Gallart-Palau *1, Aida Serra *1, Siu Kwan Sze 1
1Division of Structural Biology and Biochemistry, School of Biological Sciences, Nanyang Technological University

Here we present a step-by-step protocol of the long-length electrostatic repulsion-hydrophilic interaction chromatography-tandem mass spectrometry (LERLIC-MS/MS) method. This is a novel methodology that enables for the first time quantification and characterization of the glutamine and asparagine deamidation isoforms by shotgun proteomics.

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Bioengineering

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging
Mohesh Moothanchery 1, Arunima Sharma 1, Manojit Pramanik 1
1School of Chemical and Biomedical Engineering, Nanyang Technological University

Here a switchable acoustic resolution (AR) and optical resolution (OR) photoacoustic microscopy (AR-OR-PAM) system capable of both high resolution imaging at shallow depth and low resolution deep tissue imaging on the same sample in vivo is demonstrated.

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Bioengineering

A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging
Paul Kumar Upputuri 1, Vijitha Periyasamy 1, Sandeep Kumar Kalva 1, Manojit Pramanik 1
1School of Chemical and Biomedical Engineering, Nanyang Technological University

A compact pulsed-laser diode-based photoacoustic tomography (PLD-PAT) system for high-speed in vivo brain imaging in small animals is demonstrated.

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Immunology and Infection

Bead Based Multiplex Assay for Analysis of Tear Cytokine Profiles
Praveen Kumar Balne 1,2, Veonice Bijin AU 3, Louis Tong 2, Arkasubhra Ghosh 4, Mukesh Agrawal 5, John Connolly 3, Rupesh Agrawal 1
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.

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Biology

Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass
Hieng Chiong Tie 1, Bing Chen 1, Xiuping Sun 1, Li Cheng 2,3, Lei Lu 1
1School of Biological Sciences, Nanyang Technological University, 2Bioinformatics Institute, 3School of Computing, National University of Singapore

The precise localization of Golgi residents is essential for understanding the cellular functions of the Golgi. However, conventional optical microscopy is unable to resolve the sub-Golgi structure. Here we describe the protocol for a conventional microscopy based super-resolution method to quantitatively determine the sub-Golgi localization of a protein.

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Genetics

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids
Desiree-Faye Kaixin Toh *1, Kiran M. Patil *1, Gang Chen 1
1Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University

We report the protocols for the synthesis and purification of Peptide Nucleic Acid (PNA) oligomers incorporating modified residues. The biochemical and biophysical methods for the characterization of the recognition of RNA duplexes by the modified PNAs are described.

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Chemistry

Synthesis of Core-shell Lanthanide-doped Upconversion Nanocrystals for Cellular Applications
Xiangzhao Ai 1, Linna Lyu 1, Jing Mu 1, Ming Hu 1, Zhimin Wang 1, Bengang Xing 1,2
1Division of Chemistry and Biological Chemistry, School of Physical & Mathematical Sciences, Nanyang Technological University, 2Institute of Materials Research & Engineering, Agency for Science, Technology and Research (A*STAR)

A protocol is presented for the synthesis of core-shell lanthanide-doped upconversion nanocrystals (UCNs) and their cellular applications for channel protein regulation upon near-infrared (NIR) light illumination.

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Bioengineering

Hand-held Clinical Photoacoustic Imaging System for Real-time Non-invasive Small Animal Imaging
Kathyayini Sivasubramanian 1, Vijitha Periyasamy 1, Manojit Pramanik 1
1School of Chemical and Biomedical Engineering, Nanyang Technological University

A clinical handheld photoacoustic imaging system will be demonstrated for real-time non-invasive small animal imaging.

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Chemistry

Synthesis of Substrate-Bound Au Nanowires Via an Active Surface Growth Mechanism
Xinglong Wang *1, Xuesong Wu *1, Jiating He 2, Xiaolin Tao 1, Hongyan Li 1, Gui Zhao 1, Yawen Wang 1, Hongyu Chen 1,2
1Institute of Advanced Synthesis, School of Chemistry and Molecular Engineering, Jiangsu National Synergetic Innovation Center for Advanced Materials, Nanjing Tech University, 2Chemistry and Biological Chemistry, Nanyang Technological University

We report a solution-based method to synthesize substrate-bound Au nanowires. By tuning the molecular ligands used during the synthesis, the Au nanowires can be grown from various substrates with different surface properties. Au nanowire-based nanostructures can also be synthesized by adjusting the reaction parameters.

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Biology

Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions
Xiaofeng Zheng 1,2, Calvin Qing Wei Ho 1, Xiaowei Zheng 3, Kian Leong Lee 4, Katarina Gradin 5, Teresa S. Pereira 3, Per-Olof Berggren 1,2,3, Yusuf Ali 1,2
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.

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JoVE Journal

Microparticle Manipulation by Standing Surface Acoustic Waves with Dual-frequency Excitations
Yufeng Zhou 1, Yannapol Sriphutkiat 1
1School of Mechanical and Aerospace Engineering, Nanyang Technological University

A protocol for manipulating the microparticles in a microfluidic channel with a dual-frequency excitation is presented.

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JoVE Journal

Optimization, Test and Diagnostics of Miniaturized Hall Thrusters
Jian W. M. Lim 1, Igor Levchenko 1,2, Muhammad W. A. B. Rohaizat 1, Shiyong Huang 1, Luxiang Xu 1, Yu Fei Sun 1, George C. Potrivitu 1, Jen S. Yee 1, Roysmond Z. W. Sim 1, Youmei Wang 3, Svitlana Levchenko 1, Kateryna Bazaka 2,1, Shuyan Xu 1
1Plasma Sources and Applications Centre, National Institute of Education, Nanyang Technological University, 2School of Chemistry, Physics, Mechanical Engineering, Queensland University of Technology, 3Department of Physics, School of Science, Hangzhou Dianzi University

Here, we present a protocol to test and optimize space propulsion systems based on miniaturized Hall-type thrusters.

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Immunology and Infection

Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model
Ya-Wen Du *1, Qun Liu *1, Xiao-Cui Luo 1, Dong-Xiao Zhao 1, Jian-Bo Xue 1, Pu Feng 2, Robert F. Margolskee 2, Hong Wang 2, Liquan Huang 1,2
1College of Life Sciences, Zhejiang University, 2Monell Chemical Senses Center

Here we present a protocol to investigate the effect of the nullification of gustation-related genes on immune responses in a dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD) mouse model.

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Genetics

Genome Editing in Mammalian Cell Lines using CRISPR-Cas
Kaiwen Ivy Liu 1, Norfala-Aliah Binte Sutrisnoh 1, Yuanming Wang 1,2, Meng How Tan 1,2
1Genome Institute of Singapore, Agency for Science Technology and Research, 2School of Chemical and Biomedical Engineering, Nanyang Technological University

CRISPR-Cas is a powerful technology to engineer the complex genomes of plants and animals. Here, we detail a protocol to efficiently edit the human genome using different Cas endonucleases. We highlight important considerations and design parameters to optimize editing efficiency.

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Environment

Visualization of Flow Field Around a Vibrating Pipeline Within an Equilibrium Scour Hole
Dawei Guan 1,2, Yee-Meng Chiew 2, Maoxing Wei 3, Shih-Chun Hsieh 4
1Key Laboratory of Coastal Disaster and Defense, Ministry of Education, Hohai University, 2School of Civil and Environmental Engineering, Nanyang Technological University, 3Ocean College, Zhejiang University, 4AXESEA Information Technology Co. LTD

The goal of the protocol is to enable visualization of the detailed flow fields and determination of the near-boundary shear and normal stresses within an equilibrium scour hole induced by a vibrating pipeline.

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Bioengineering

Pulsed Laser Diode-Based Desktop Photoacoustic Tomography for Monitoring Wash-In and Wash-Out of Dye in Rat Cortical Vasculature
Sandeep Kumar Kalva 1, Paul Kumar Upputuri 1, Praveenbalaji Rajendran 1, Rhonnie Austria Dienzo 1, Manojit Pramanik 1
1School of Chemical and Biomedical Engineering, Nanyang Technological University

A compact pulsed laser diode-based desktop photoacoustic tomography (PLD-PAT) system is demonstrated for high-speed dynamic in vivo imaging of small animal cortical vasculature.

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Immunology and Infection

Screening Peptides that Activate MRGPRX2 using Engineered HEK Cells
Shammy Raj 1, Lei Lu 2, Larry D. Unsworth 1
1Department of Chemical and Materials Engineering, Donadeo Innovation Centre for Engineering, University of Alberta, 2School of Life Science and Engineering, Southwest Jiaotong University

Techniques for generating a library of short peptides that can activate mast cells via the MRGPRX2 receptor are described. Associated techniques are easy, inexpensive, and can be extended to other cell receptors.

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Bioengineering

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy
Su Yin Chaw 1,2, Tina Tzee Ling Wong 3,4, Subbu Venkatraman 2,5, Ann-Marie Chacko 1
1Laboratory for Translational and Molecular Imaging, Cancer and Stem Cell Biology Programme, Duke-NUS Medical School, 2School of Materials Science and Engineering, Nanyang Technological University, 3Singapore National Eye Centre, 4Singapore Eye Research Institute, 5Material Science & Engineering, National University of Singapore

We present a protocol for the use of fiberoptic confocal laser microendoscopy (CLM) to non-invasively study the spatio-temporal distribution of liposomes in the eye after subconjunctival injection.

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Biochemistry

A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro
Xinwen Miao *1, Jiawei Wu *1,2, Wenting Zhao 1,3
1School of Chemistry, Chemical Engineering, and Biotechnology, Nanyang Technological University, 2State Key Laboratory Breeding Base of Green Chemistry Synthesis Technology, College of Chemical Engineering, Zhejiang University of Technology, 3Institute for Digital Molecular Analytics and Science, Nanyang Technological University

Here, a nanobar-supported lipid bilayer system is developed to provide a synthetic membrane with a defined curvature that enables the characterization of proteins with curvature sensing ability in vitro.

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Biology

Optimized Protocol for Generating Functional Pancreatic Insulin-secreting Cells from Human Pluripotent Stem Cells
Ines Cherkaoui 1, Qian Du 2, Dieter Egli 2, Shivani Misra 1,3, Guy A. Rutter 1,4,5
1Department of Metabolism, Digestion and Reproduction, Faculty of Medicine, Cell Biology and Functional Genomics, 2Departments of Pediatrics, Naomi Berrie Diabetes Center, Obstetrics and Gynecology, Columbia Stem Cell Initiative, Columbia University Irving Medical Center, Columbia University, 3Department of Diabetes, Imperial College Healthcare NHS Trust, 4Faculté de Médicine, Université de Montréal, 5Lee Kong Chian Imperial Medical School, Nanyang Technological University

This article presents a protocol for directed differentiation and functional analysis of β-cell like cells. We describe optimal culture conditions and passages for human pluripotent stem cells before generating insulin-producing pancreatic cells. The six-stage differentiation progresses from definitive endoderm formation to functional β-cell like cells secreting insulin in response to glucose.

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Neuroscience

Home-Based EEG Hyperscanning for Infant-Caregiver Social Interactions
Vaidehi Ramanarayanan *1, Qian Chern Oon *1, Amritha Varshini Devarajan 1, Stanimira Georgieva 1,2, Vanessa Reindl 1,3
1Department of Psychology, School of Social Sciences, Nanyang Technological University, 2Department of Pediatrics, University of Cambridge, 3Child Neuropsychology Section, Department of Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy, Medical Faculty, RWTH Aachen University

This protocol describes how synchronized electroencephalography, electrocardiography, and behavioral recordings were captured from infant-caregiver dyads in a home setting.

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