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64 ARTICLES PUBLISHED IN JoVE

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Biology

Monitoring Acupuncture Effects on Human Brain by fMRI
Kathleen K. S. Hui 1, Vitaly Napadow 1, Jing Liu 1, Ming Li 1, Ovidiu Marina 1,2, Erika E. Nixon 1, Joshua D. Claunch 1, Lauren LaCount 1, Tara Sporko 1, Kenneth K. Kwong 1
1Department of Radiology, Massachusetts General Hospital and Harvard Medical School, 2William Beaumont Hospital

FMRI and physiological monitoring is used to study the effects of Acupuncture on the central and peripheral nervous systems. Acupuncture mobilizes a limbic-paralimbic-neocortical network, with great overlap with the default mode network, to modulate neurological activity, possibly related to its autonomic effect in the peripheral nervous system.

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Immunology and Infection

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT
Oktay Kirak 1, Eva-Maria Frickel 1, Gijsbert M. Grotenbreg 1,2, Heikyung Suh 1, Rudolf Jaenisch 1,3, Hidde L. Ploegh 1,3
1Whitehead Institute for Biomedical Research, 2Departments of Microbiology and Biological Sciences, National University of Singapore, 3Department of Biology, Massachusetts Institute of Technology

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

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Bioengineering

Plasma Lithography Surface Patterning for Creation of Cell Networks
Michael Junkin 1, Siu Ling Leung 1, Yongliang Yang 1, Yi Lu 1, Justin Volmering 1, Pak Kin Wong 1,2
1Aerospace and Mechanical Engineering, University of Arizona , 2Biomedical Engineering IDP and BIO5 Institute, University of Arizona

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

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JoVE Journal

Fabrication and Testing of Microfluidic Optomechanical Oscillators
Kewen Han 1, Kyu Hyun Kim 2, Junhwan Kim 1, Wonsuk Lee 2,3, Jing Liu 3, Xudong Fan 3, Tal Carmon 2, Gaurav Bahl 1
1Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, 2Electrical Engineering and Computer Science, University of Michigan, 3Biomedical Engineering, University of Michigan

Parametric optomechanical excitations have recently been experimentally demonstrated in microfluidic optomechanical resonators by means of optical radiation pressure and stimulated Brillouin scattering. This paper describes the fabrication of these microfluidic resonators along with methodologies for generating and verifying optomechanical oscillations.

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Bioengineering

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration
Nicholas Agung Kurniawan 1, Parthiv Kant Chaudhuri 2, Chwee Teck Lim 2,3
1Systems Biophysics Department, FOM Institute AMOLF, 2Mechanobiology Institute, National University of Singapore, 3Department of Biomedical Engineering, National University of Singapore

The mechanical properties and microstructure of the extracellular matrix strongly affect 3D migration of cells. An in vitro method to study the spatiotemporal cell migration behavior in biophysically variable environments, at both population and individual cell levels, is described.

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Bioengineering

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5
Eric Ehrke-Schulz 1, Wenli Zhang 1, Maren Schiwon 1, Thorsten Bergmann 1, Manish Solanki 1, Jing Liu 1, Philip Boehme 1, Theo Leitner 1, Anja Ehrhardt 1
1Institute of Virology and Microbiology, Center for Biomedical Education and Research (ZBAF), Department of Human Medicine, Faculty of Health, University Witten/Herdecke

A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.

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Bioengineering

Polymeric Microneedle Array Fabrication by Photolithography
Himanshu Kathuria 1, Jaspreet Singh Kochhar 1, Michelle Hui Min Fong 1, Michinao Hashimoto 2, Ciprian Iliescu 3, Hanry Yu 3,4,5, Lifeng Kang 1
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.

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Neuroscience

Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents
Mahesh Shivarama Shetty 1,2, Mahima Sharma 1,2, Neo Sin Hui 1,2, Ananya Dasgupta 1,2, Suma Gopinadhan 1,2, Sreedharan Sajikumar 1,2
1Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, 2Neurobiology/Aging Programme, Life Sciences Institute, National University of Singapore

This video article describes experimental procedures to study long-term plasticity and its associative processes such as synaptic tagging, capture and cross-tagging in the CA1 pyramidal neurons using acute hippocampal slices from rodents.

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Bioengineering

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules
Marie Francene A. Cutiongco 1, Benjamin Kim Kiat Teo 2, Evelyn King Fai Yim 1,2,3
1Department of Biomedical Engineering, National University of Singapore, 2Mechanobiology Institute, Singapore, National University of Singapore, 3Department of Surgery, National University of Singapore

Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

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Immunology and Infection

Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases
Song Buck Tay 1,3, Jeng Yeong Chow 2, Maybelle Kho Go 1,3, Wen Shan Yew 1,3
1Department of Biochemistry, Yong Loo Lin School of Medicine, 2Department of Chemistry, University of Utah, 3Synthetic Biology Research Consortium, National University of Singapore

Quorum-quenching enzymes are anti-virulent and anti-bacterial options that can mitigate pathogenesis without risk of incurring resistance, by preventing the expression of virulence factors and genes associated with antibiotic resistance and biofilm formation. In this study, we report a method that demonstrates the efficacy of quorum-quenching enzymes in bacterial biofilm disruption.

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Immunology and Infection

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging
Shruti Shankar 1,2, Meredith E.K. Calvert 3,4, Joanne Y. Yew 1,2,5
1Temasek Life Sciences Laboratory, 2Department of Biological Science, National University of Singapore, 3Bioimaging and Biocomputing Facility, Temasek Life Sciences Laboratory, 4Histology and Light Microscopy Core, Gladstone Institutes, 5Pacific Biosciences Research Center, University of Hawai'i at Mānoa

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

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Developmental Biology

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues
Muznah Khan 1, Stephan Gasser 1,2
1Immunology Program, Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, 2NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore

We describe a simple and quick experimental procedure for generating primary fibroblasts from the ears and tails of mice. The procedure does not require special animal training and can be used for the generation of fibroblast cultures from ears stored at RT for up to 10 days.

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Bioengineering

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding
Paula Benny 1,2, Cedric Badowski 2, E. Birgitte Lane 2, Michael Raghunath 1,2,3
1Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 2Epithelial Biology Laboratory, Institute of Medical Biology, A*STAR, 3Department of Biomedical Engineering, Faculty of Engineering, National University of Singapore

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

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Medicine

A Step by Step Protocol for Subretinal Surgery in Rabbits
Sami Al-Nawaiseh 1, Fabian Thieltges 1, Zengping Liu 1,2, Claudine Strack 1, Ralf Brinken 1, Norbert Braun 3, Marc Wolschendorf 3, Arvydas Maminishkis 5, Nicole Eter 4, Boris V. Stanzel 1,6
1Department of Ophthalmology, University of Bonn, 2Department of Ophthalmology, National University of Singapore, 3Geuder AG, 4Department of Ophthalmology, University of Münster, 5Section on Epithelial and Retinal Physiology and Disease, National Eye Institute/National Institutes of Health, 6Surgical Retina Department, Singapore National Eye Centre

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

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Developmental Biology

Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates
Geetika Sahni *1, Jun Yuan *1, Yi-Chin Toh 1,2
1Department of Biomedical Engineering, National University of Singapore, 2Singapore Institute of Neurotechnology (SINAPSE), National University of Singapore

Human pluripotent stem cells (hPSCs) have the intrinsic ability to differentiate and self-organize into distinct tissue patterns; although this requires the presentation of spatial environmental gradients. We present stencil micropatterning as a simple and robust method to generate biochemical and mechanical gradients for controlling hPSC differentiation patterns.

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Bioengineering

Rod-based Fabrication of Customizable Soft Robotic Pneumatic Gripper Devices for Delicate Tissue Manipulation
Jin-Huat Low 1, Chen-Hua Yeow 1
1Department of Biomedical Engineering, Advanced Robotics Centre, Singapore Institute for Neurotechnology, National University of Singapore

This protocol describes a rod-based approach, combining 3D-printing and soft lithography techniques for fabricating the soft gripper devices. This approach eliminates the need for an external air source by incorporating a chamber component and reduces the chance of occlusion during the sealing process, particularly for miniaturized pneumatic channels.

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Bioengineering

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery
Raymond Wing Moon Lam 1, Sunny Akogwu Abbah 2, Wang Ming 1, Mathanapriya Naidu 1, Felly Ng 1, Hu Tao 1, James Goh Cho Hong 3,4, Kang Ting 5, Wong Hee Kit 1,4
1Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, 2Centre for Research in Medical Devices (CÚRAM), National University of Ireland Galway, 3Department of Bioengineering, Faculty of Engineering, National University of Singapore, 4Tissue Engineering Program, National University of Singapore, 5Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, University of California, Los Angeles

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

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Genetics

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production
Ai-Qun Yu 1,2, Nina Pratomo 1,2, Tee-Kheang Ng 1,2, Hua Ling 1,2, Han-Saem Cho 1,2, Susanna Su Jan Leong 1,2,3, Matthew Wook Chang 1,2
1Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 2NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of Singapore, 3Food Science and Chemical Engineering, Singapore Institute of Technology

We herein report methods on the molecular genetic manipulation of the Yarrowia lipolytica Po1g strain for improved gene deletion efficiency. The resulting engineered Y. lipolytica strains have potential applications in biofuel and biochemical production.

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Biology

Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle
Yan Cheng Ng 1,2, Liam K. Fisher 2, Veena Salim 2, Sangho Kim 1,2,3, Bumseok Namgung 2
1NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, 2Department of Biomedical Engineering, National University of Singapore, 3Department of Surgery, National University of Singapore

This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

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Biology

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)
Yilin Wang 1, Pakorn Kanchanawong 2,3
1Department of Biology, South University of Science and Technology of China, Shenzhen, 2Mechanobiology Institute, Singapore, 3Department of Biomedical Engineering, National University of Singapore

We present a protocol for the application of interferometric PhotoActivated Localization Microscopy (iPALM), a 3-dimensional single-molecule localization super resolution microscopy method, to the imaging of the actin cytoskeleton in adherent mammalian cells. This approach allows light-based visualization of nanoscale structural features that would otherwise remain unresolved by conventional diffraction-limited optical microscopy.

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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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Developmental Biology

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model
Tingsheng Yu 1,2, Christoph Winkler 1,2
1Department of Biological Sciences, National University of Singapore, 2NUS Centre for Bioimaging Sciences (CBIS), National University of Singapore

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

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Genetics

Detection of Copy Number Alterations Using Single Cell Sequencing
Kristin A. Knouse 1,2,3, Jie Wu 4, Austin Hendricks 5
1Koch Institute for Integrative Cancer Research, Department of Biology, Massachusetts Institute of Technology, 2Howard Hughes Medical Institute, 3Division of Health Sciences and Technology, Harvard Medical School, 4The Barbara K. Ostrom (1978) Bioinformatics and Computing Facility in the Swanson Biotechnology Center, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 5BioMicro Center, Department of Biology, Massachusetts Institute of Technology

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

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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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Bioengineering

Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum
Abhijeet Patra 1, Tao Ding 2, Minghui Hong 3, Arthur Mark Richards 2, Ten It Wong 4, Xiaodong Zhou 4, Chester Lee Drum 2
1NUS Nanoscience and Nanotechnology Initiative, National University of Singapore, 2Cardiovascular Research Institute, Yong Loo Lin School of Medicine, National University of Singapore, 3Department of Electrical and Computer Engineering, National University of Singapore, 4Institute of Materials Research Engineering, A*STAR (Agency for Science, Technology and Research)

This work describes a nanoimprinting lithography method to fabricate high-quality sensing arrays that work on the principle of extraordinary optical transmission. The biosensor is low-cost, robust, easy to use, and can detect cardiac troponin I in serum at clinically relevant concentrations (99th percentile cutoff ∼10-400 pg/mL, depending on the assay).

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Bioengineering

Soft Lithographic Procedure for Producing Plastic Microfluidic Devices with View-ports Transparent to Visible and Infrared Light
Mona Suryana 1, Jegan V. Shanmugarajah 1, Sivakumar M. Maniam 1, Gianluca Grenci 1
1Mechanobiology Institute (MBI), National University of Singapore

A protocol for the fabrication of plastic microfluidic devices with transparent view-ports for visible and infrared light imaging is described.

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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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Chemistry

Facet-to-facet Linking of Shape-anisotropic Colloidal Cadmium Chalcogenide Nanostructures
Xuanwei Ong *1, Shashank Gupta *1, Wen-Ya Wu 2, Sabyasachi Chakrabortty 1, Yinthai Chan 1,3
1Department of Chemistry, National University of Singapore, 2Materials Processing and Characterisation Department, A*STAR, Institute of Materials Research and Engineering, 3Ceramics Department, A*STAR, Institute of Materials Research and Engineering

A protocol detailing how shape-anisotropic colloidal cadmium chalcogenide nanocrystals can be covalently linked via their end facets is presented here.

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Medicine

Using Retinal Imaging to Study Dementia
Victor T.T. Chan 1, Tiffany H.K. Tso 1, Fangyao Tang 1, Clement Tham 1, Vincent Mok 2,3,4, Christopher Chen 5,6, Tien Y. Wong 7,8, Carol Y. Cheung 1
1Department of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, 2Department of Medicine & Therapeutics, The Chinese University of Hong Kong, 3Therese Pei Fong Chow Research Centre for Prevention of Dementia, The Chinese University of Hong Kong, 4Gerald Choa Neuroscience Centre, The Chinese University of Hong Kong, 5Memory Aging and Cognition Centre, National University Health System, 6Department of Pharmacology, National University of Singapore, 7Singapore Eye Research Institute, Singapore National Eye Centre, 8Duke-NUS Medical School, National University of Singapore

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

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Distinguishing Allosteric Effects from Orthosteric Binding in Protein-Ligand Interactions
Arun Chandramohan 1, Srinath Krishnamurthy 1,2, Ganesh S Anand 1
1Department of Biological Sciences, National University of Singapore, 2Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven

Application of amide hydrogen-deuterium exchange mass spectrometry to map interactions of low affinity fragment and ligands is demonstrated. This protocol describes a method for distinguishing orthosteric binding from allosteric changes accompanying high affinity ligand and low-affinity fragment binding to target protein, Hsp90, and finds important applications in fragment-based drug design.

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Cancer Research

Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60
Yanzhou Zhang 1, Grace SuShin Chia 1, Cheng Yong Tham 1, Sudhakar Jha 1,2
1Cancer Science Institute of Singapore, National University of Singapore, 2Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore

Here, we present the real-time monitoring of cell migration in a wound-healing assay using TIP60-depleted MCF10A breast epithelial cells. The implementation of live-cell imaging techniques in our protocol allows us to analyze and visualize single-cell movement in real time and across time.

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Biochemistry

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers
Huijuan You *1, Shimin Le *2,3, Hu Chen 2,4, Linyan Qin 1, Jie Yan 2,4,5
1School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, 2Mechanobiology Institute, National University of Singapore, 3Department of Physics, National University of Singapore, 4Research Institute for Biomimetics and Soft Matter, Fujian Provincial Key Lab for Soft Functional Materials Research, Department of Physics, Xiamen University, 5Center for Bioimaging Sciences, National University of Singapore

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

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Chemistry

Utilization of Stop-flow Micro-tubing Reactors for the Development of Organic Transformations
Ren Wei Toh 1, Jie Sheng Li 1, Jie Wu 1
1Department of Chemistry, National University of Singapore

A protocol for organic reaction screening using stop-flow micro-tubing (SFMT) reactors employing gaseous reactants and/or visible-light mediated reactions is presented.

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Engineering

Atmospheric Pressure Fabrication of Large-Sized Single-Layer Rectangular SnSe Flakes
Jizhou Jiang 1,2, Calvin Pei Yu Wong 2,3, Wenjing Zhang 1, Andrew Thye Shen Wee 2,4
1SZU-NUS Collaborative Innovation Center for Optoelectronic Science & Technology, Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Optoelectronic Engineering, Shenzhen University, 2Department of Physics, National University of Singapore, 3NUS Graduate School for Integrative Sciences and Engineering, Centre for Life Sciences, 4Centre for Advanced 2D Materials and Graphene Research Centre, National University of Singapore

A protocol is presented demonstrating a two-step fabrication technique to grow large-sized single-layer rectangular shaped SnSe flakes on low-cost SiO2/Si dielectrics wafers in an atmospheric pressure quartz tube furnace system.

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JoVE Journal

Model Surgical Training: Skills Acquisition in Fetoscopic Laser Photocoagulation of Monochorionic Diamniotic Twin Placenta Using Realistic Simulators
Tuangsit Wataganara 1, Arundhati Gosavi 2, Katika Nawapun 1, Pradip D. Vijayakumar 2, Nisarat Phithakwatchara 1, Mahesh Choolani 2,3, Lin Lin Su 2,3, Arijit Biswas 2,3, Citra N. Z. Mattar 2,3
1Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Faculty of Medicine, Siriraj Hospital, 2Department of Obstetrics and Gynaecology, National University Health Systems, 3Experimental Fetal Medicine Group, Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore

Practicing the specific skills required for fetoscopic laser coagulation of monochorionic placental anastomoses on realistic models can aid less experienced surgeons in overcoming the steep learning curve associated with this procedure that is now regarded as the standard of care for twin-twin transfusion syndrome.

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Medicine

Intraoperative Ultrasound in Spinal Surgery
Melissa M.J. Chua 1, Viren S. Vasudeva 1, Yi Lu 1
1Brigham and Women's Hospital, Harvard Medical School

Here, we present a protocol on the use of intraoperative ultrasound in spinal surgery, particularly in cases of intradural lesions and lesions in the ventral spinal canal when using a posterior approach.

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Immunology and Infection

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells
Yi Lu 1,2,3, Xin-Yuan Fu 3, Yongliang Zhang 1,2
1Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 2Immunology Programme, Life Sciences Institute, National University of Singapore, 3Cancer Science Institute of Singapore, Yong Loo Lin School of Medicine, National University of Singapore

Here, we present a protocol to differentiate murine granulocyte-macrophage-colony-stimulating-factor-producing T helper (THGM) cells from naive CD4+ T cells, including isolation of naive CD4+ T cells, differentiation of THGM, and analysis of differentiated THGM cells. This method can be applied to studies of the regulation and function of THGM cells.

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Immunology and Infection

Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma
Guo Hong *1,2, Shuangyi Fan *3, The Phyu 3, Priyanka Maheshwari 2, Michal Marek Hoppe 2, Hoang Mai Phuong 2, Sanjay de Mel 4, Michelle Poon 4, Siok-Bian Ng 2,3, Anand D. Jeyasekharan 2,4
1Department of Laboratory Medicine, AnSteel Group General Hospital, 2Cancer Science Institute of Singapore, National University of Singapore, 3Department of Pathology, Yong Loo Lin School of Medicine, National University of Singapore, 4Department of Haematology-Oncology, National University Health System

Here we describe a protocol for multiplex fluorescent immunohistochemical staining and imaging for the simultaneous localization of multiple cancer-associated antigens in lymphoma. This protocol can be extended to the colocalization analysis of biomarkers within all tissue sections.

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Immunology and Infection

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis
EnJun Yang 1, Jacqueline L.Y. Chee 1, Suhanya Duraiswamy 2, Siyi Chen 3, Kristin Lees 3, Swaine L. Chen 1,3
1Infectious Diseases Group, Genome Institute of Singapore, 2Department of Chemical and Biomolecular Engineering, National University of Singapore, 3Division of Infectious Diseases, Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore

This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection.

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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. Gascoigne 1, 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.

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Medicine

Protocol and Guidelines for Point-of-Care Lung Ultrasound in Diagnosing Neonatal Pulmonary Diseases Based on International Expert Consensus
Jing Liu 1,2, Roberto Copetti 3, Erich Sorantin 4, Jovan Lovrenski 5, Javier Rodriguez-Fanjul 6, Dalibor Kurepa 7, Xing Feng 8, Luigi Cattaross 9, Huayan Zhang 10,11, Misun Hwang 12, Tsu F. Yeh 13,14, Yisrael Lipener 7, Abhay Lodha 15, Jia-Qin Wang 16, Hai-Ying Cao 2,17, Cai-Bao Hu 2,18, Guo-Rong Lyu 19, Xin-Ru Qiu 1,2, Li-Qun Jia 20, Xiao-Man Wang 20, Xiao-Ling Ren 1,2, Jiu-Ye Guo 1,2, Yue-Qiao Gao 1,2, Jian-Jun Li 1,2, Ying Liu 1,2, Wei Fu 1,2, Yan Wang 21, Zu-Lin Lu 1,2, Hua-Wei Wang 8, Li-Li Shang 22
1Department of Neonatology and NICU, Beijing Chaoyang District Maternal and Child Healthcare Hospital, 2The Neonatal Lung Ultrasound Training Base, Chinese College of Critical Ultrasound, 3Emergency Department, Cattinara University Hospital, 4Division of Pediatric Radiology, Department of Radiology, Medical University Graz, 5Faculty of Medicine, University of Novi Sad, Radiology Department, Institute for Children and Adolescents Health Care of Vojvodina, 6Pediatric Intensive Care Unit, Pediatric Service Hospital Joan XXIII Tarragona, University Rovira i Virgil, 7Division of Neonatal-Perinatal Medicine, Cohen Children's Medical Center, 8Department of Neonatology, Children's Hospital of Soochow University, 9Department of Neonatology, Udine University Hospital, 10Center for Newborn Care, Guangzhou Women and Children's Medical Center, 11Division of Neonatology, Children's Hospital of Philadelphia, 12Department of Radiology, Children's Hospital of Philadelphia, 13Division of Neonatology and NICU, Cook County Children's Hospital, University of Illinois, 14Division of Neonatology, Department of Pediatrics, Taipei Medical University, 15Department of Pediatrics and Community Health Sciences, University of Calgary, 16Department of Pediatrics, The Third Affiliated Hospital of Xinxiang Medical University, 17Department of Ultrasound, GE Healthcare, 18Intensive Care Unit, Zhejiang Hospital, 19Collaborative Innovation Center for Maternal and Infant Health Service Application Technology, Quanzhou Medical College, 20Department of Ultrasound, Beijing Children's Hospital Affiliated with Capital Medical University, 21Department of Neonatology and NICU, Tai'an City Central Hospital of Shandong Province, 22Department of Intensive Care Unit, The Second Affiliated Hospital of Heilongjiang University of Chinese Medicine

Lung ultrasound is a noninvasive and valuable tool for bedside evaluation of neonatal lung diseases. However, a relative lack of reference standards, protocols and guidelines may limit its application. Here, we aim to develop a standardized neonatal lung ultrasound diagnostic protocol to be used in clinical decision-making.

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Immunology and Infection

Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation
Xiang Zhao 1, Maryam Hamidinia 1, Joanna Ai Ling Choo 1, Chien Tei Too 1,2, Zi Zong Ho 3, Ee Chee Ren 4, Antonio Bertoletti 3, Paul A. MacAry 1,2,5, Keith G. Gould 6, Joanna Brzostek 1, 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.

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Behavior

Modified Blood Collection from Tail Veins of Non-anesthetized Mice with a Vacuum Blood Collection System and Eyeglass Magnifier
Xiaoyan Liu *1, Huanhuan Li *1, Jie Wu 1, Yijun Yu 1, Mingjing Zhang 1, Wusong Zou 1, Ye Gu 1
1Heart Center, Wuhan Fourth Hospital, Puai Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology

This study reports blood sampling from tail vein in mice using a vacuum extraction tube system with eyeglass magnifier. Our method is easy to practice and could be used for repeat blood sampling in mice.

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Immunology and Infection

Toxicity Study of Zinc Oxide Nanoparticles in Cell Culture and in Drosophila melanogaster
Cheng Teng Ng 1, Choon Nam Ong 2, Liya E. Yu 3, Boon Huat Bay 4, Gyeong Hun Baeg 4
1NUS Environmental Research Institute & Department of Anatomy, National University of Singapore, 2NUS Environmental Research Institute & School of Public Health, National University of Singapore, 3Department of Civil and Environmental Engineering, National University of Singapore, 4Department of Anatomy, National University of Singapore

We describe a detailed protocol for evaluating the toxicological profiles of zinc oxide nanoparticles (ZnO NPs) in particular, the type of cell death in human MRC5 lung fibroblasts and ROS formation in the fruit fly Drosophila.

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Immunology and Infection

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex
Masanori Asai *1, Yanwen Li *1, Jasmeet Singh Khara 1,2, Camilla A. Gladstone 1, Brian D. Robertson 3, Paul R. Langford *1, Sandra M. Newton *1
1Section of Pediatric Infectious Diseases and Allergy, Department of Medicine, Imperial College London, 2Department of Pharmacy, National University of Singapore, 3MRC Centre for Molecular Bacteriology and Infection, Department of Medicine, Imperial College London

Galleria mellonella was recently established as a reproducible, cheap, and ethically acceptable infection model for the Mycobacterium tuberculosis complex. Here we describe and demonstrate the steps taken to establish successful infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux.

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Developmental Biology

Injecting Gryllus bimaculatus Eggs
Samantha K. Barry 1, Taro Nakamura 2, Yuji Matsuoka 3, Christoph Straub 4, Hadley W. Horch 4, Cassandra G. Extavour 5
1Colby College, 2Division of Evolutionary Developmental Biology, National Institute for Basic Biology, 3Department of Biological Sciences, National University of Singapore, 4Department Biology and Department of Neuroscience, Bowdoin College, 5Department of Organismic and Evolutionary Biology and Department of Molecular and Cellular Biology, Harvard University

Here we present a protocol to inject cricket eggs, a technique which serves as a foundational method in many experiments in the cricket, including, but not limited to, RNA interference and genomic manipulation.

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Immunology and Infection

TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks
Yongliang Zhang *1, Yuanyuan Li *2,3, Xinxin Yang 1, Zhiyan Wen 1, Ugrappa Nagalakshmi 2, Savithramma P. Dinesh-Kumar 2,3
1State Key Laboratory of Agro-Biotechnology and Ministry of Agriculture Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, 2Department of Plant Biology, College of Biological Sciences, University of California, Davis, 3Genome Center, College of Biological Sciences, University of California, Davis

Described here is a proximity labeling method for identification of interaction partners of the TIR domain of the NLR immune receptor in Nicotiana benthamiana leaf tissue. Also provided is a detailed protocol for the identification of interactions between other proteins of interest using this technique in Nicotiana and other plant species.

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JoVE Core

International Expert Consensus and Recommendations for Neonatal Pneumothorax Ultrasound Diagnosis and Ultrasound-guided Thoracentesis Procedure
Jing Liu 1,2, Dalibor Kurepa 3, Francesco Feletti 4,5, Almudena Alonso-Ojembarrena 6, Jovan Lovrenski 7, Roberto Copetti 8, Erich Sorantin 9, Javier Rodriguez-Fanjul 10, Karishma Katti 3, Andrea Aliverti 4, Huayan Zhang 11,12, Misun Hwang 13, Tsu F. Yeh 14, Cai-Bao Hu 15, Xing Feng 16, Ru-Xin Qiu 1,2, Jing-Han Chi 17, Li-Li Shang 18, Guo-Rong Lyu 19, Shao-Zheng He 20, Yan-Fen Chai 21, Zhan-Jun Qiu 22, Hai-Ying Cao 2,23, Yue-Qiao Gao 1,2, Xiao-Ling Ren 1,2, Guo Guo 1,24, Li Zhang 1,2, Ying Liu 1,2, Wei Fu 1,2, Zu-Lin Lu 1,2, Hong-Lei Li 1,2
1Department of Neonatology and NICU, Beijing Chaoyang District Maternal and Child Healthcare Hospital, 2The National Neonatal Lung Ultrasound Training Base, 3Division of Neonatal-Perinatal Medicine, Cohen Children's Medical Center, 4Department of Electronics, Information and Bioengineering, Politecnico di Milano, 5Dipartimento di Diagnostica per Immagini, Ausl della Romagna, S. Maria delle Croci Hospital, 6Neonatal Intensive Care Unit, Puerta del Mar University Hospital, 7Faculty of Medicine, University of Novi Sad, Serbia, Institute for Children and Adolescents Health Care of Vojvodina, 8Emergency Department, University Hospital of Cattinara, 9Division of Pediatric Radiology, Department of Radiology, Medical University Graz, 10Pediatric Intensive Care Unit, Pediatric Service Hospital Joan XXIII Tarragona, University Rovira i Virgil, 11Center for Newborn Care, Guangzhou Women and Children's Medical Center, 12Division of Neonatology, Children's Hospital of Philadelphia, 13Section of Neonatal Imaging, Department of Radiology, Children's Hospital of Philadelphia, 14Maternal Child Health Research institute, Taipei Medical University and China Medical University, 15Intensive Care Unit, Zhejiang Hospital, 16Department of Neonatology, Children's Hospital of Soochow University, 17Department of Neonatology and NICU, Bayi Children's Hospital Affiliated to the Seventh Medical Center of Chinese PLA General Hospital, 18Intensive Care Unit, Second Affiliated Hospital of Heilongjiang University of Chinese Medicine, 19Collaborative Innovation Center for Maternal and Infant Health Service Application Technology, Quanzhou Medical College, 20Department of Ultrasound, Second Affiliated Hospital of Fujian Medical University, 21Department of Emergency Medicine, Tianjin Medical University General Hospital, 22Department of Emergency and Critical Care Medicine, Affiliated Hospital of Traditional Chinese Medicine, 23Department of Ultrasound, GE Healthcare, 24The Neonatal Intensive Care Unit, Fifth Medical Center of Chinese PLA General Hospital

Pneumothorax is a common emergency and critical disease in newborn infants that needs rapid, clear diagnosis and timely treatment. Diagnosis and treatment based on chest X-rays are associated with delayed management and radiation damage. Lung ultrasound (US) provides useful guidance for rapid, accurate diagnosis and the precise thoracentesis of pneumothorax.

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Behavior

Fully Automated Leg Tracking in Freely Moving Insects using Feature Learning Leg Segmentation and Tracking (FLLIT)
Animesh Banerjee 1, Shuang Wu 2, Li Cheng 3, Sherry Shiying Aw 1
1Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, 2Bioinformatics Institute, Agency for Science, Technology and Research, 3Department of Electrical and Computer Engineering, University of Alberta

We describe detailed protocols for using FLLIT, a fully automated machine learning method for leg claw movement tracking in freely moving Drosophila melanogaster and other insects. These protocols can be used to quantitatively measure subtle walking gait movements in wild type flies, mutant flies and fly models of neurodegeneration.

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Medicine

Applying a Three-dimensional Uniaxial Mechanical Stimulation Bioreactor System to Induce Tenogenic Differentiation of Tendon-Derived Stem Cells
Ziming Chen *1, Peilin Chen *1, Rui Ruan *1, Lianzhi Chen 1, Jun Yuan 1, David Wood 1, Tao Wang 1, Ming Hao Zheng 1
1Centre of Orthopaedic Translational Research, Medical School, University of Western Australia

A three-dimensional uniaxial mechanical stimulation bioreactor system is an ideal bioreactor for tenogenic-specific differentiation of tendon-derived stem cells and neo-tendon formation.

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Medicine

A Preclinical Human-Derived Autologous Gastric Cancer Organoid/Immune Cell Co-Culture Model to Predict the Efficacy of Targeted Therapies
Jayati Chakrabarti *1, Vivien Koh *2,3, Jimmy Bok Yan So 2,4, Wei Peng Yong 2,3, Yana Zavros 1
1Department of Cellular and Molecular Medicine, University of Arizona, Tucson, 2National University Cancer Institute Singapore, National University Health System, Singapore, 3Cancer Science Institute of Singapore, National University of Singapore, 4Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore

The goals of the protocol are to use this approach to 1) understand the role of the immunosuppressive gastric tumor microenvironment and 2) predict the efficacy of patient response, thus increasing the survival rate of patients.

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Medicine

OP-IVM: Combining In vitro Maturation after Oocyte Retrieval with Gynecological Surgery
Tao Liu *1,2,3,4,5, Xueling Song *1,2,3,4,5, Xiaoying Zheng *1,2,3,4,5, Liying Yan 1,2,3,4,5, Caihong Ma 1,2,3,5, Rong Li 1,2,3,4,5, Jie Yan 1,2,3,4,5, Jie Qiao 1,2,3,4,5
1Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, 2National Clinical Research Center for Obstetrics and Gynecology, 3Key Laboratory of Assisted Reproduction, Ministry of Education, 4Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproduction, 5Research Units of Comprehensive Diagnosis and Treatment of Oocyte Maturation Arrest, Chinese Academy of Medical Sciences

In vitro maturation (IVM) before gynecological operation (OP-IVM) combines IVM following oocyte retrieval with routine gynecological surgery and serves as an extension of conventional IVM applications for fertility preservation.

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Immunology and Infection

Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection
Zhe Zhang Ryan Lew *1, Jing Liu *1, Hsiao Hui Ong 1, Vivian Jiayi Tan 2, Annika Luukkainen 3, Yew Kwang Ong 1,4, Mark Thong 1,4, Kia Joo Puan 5, Vincent Tak Kwong Chow 2,6, Kai Sen Tan 1,2,6, De Yun Wang 1,6
1Department of Otolaryngology, Yong Loo Lin School of Medicine, National University of Singapore, 2Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 3Haartman Institute, Medicum, University of Helsinki, 4Department of Otolaryngology, Head and Neck Surgery, National University Hospital, National University Health System, 5Agency for Science, Technology and Research (A*STAR), Singapore Immunology Network (SIgN), 6NUHS Infectious Diseases Translational Research Program, Yong Loo Lin School of Medicine, National University of Singapore

This protocol details an investigation of the early interactions between virally infected nasal epithelial cells and innate cell activation. Individual subsets of immune cells can be distinguished based on their activation in response to viral infections. They can then be further investigated to determine their effects on early antiviral responses.

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Biology

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control
Inn Chuan Ng *1, Li Zhang *2, Narelle Nichola Yi Ying Shen 3, Yun Ting Soong 4, Chan Way Ng 5, Phoebe Kang Sheing Koh 1, Yan Zhou 3, 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.

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Medicine

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases
Ivan Seah *1,2, Zengping Liu *1,3,4, Daniel Soo Lin Wong 1, Wendy Wong 2, Graham E. Holder 1,2,5, Veluchamy Amutha Barathi 1,4,6, Gopal Lingam 1,2,4, Xinyi Su 1,2,3,4, Boris V. Stanzel 1,7,8
1Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, 2Department of Ophthalmology, National University Hospital, Singapore, 3Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), 4Singapore Eye Research Institute (SERI), 5UCL Institute of Ophthalmology, 6Academic Clinical Program in Ophthalmology, Duke-NUS Medical School, 7Macula Center Saar, Eye Clinic Sulzbach, Knappschaft Hospital Saar, 8Department of Ophthalmology, University of Bonn

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

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Neuroscience

Three-dimensional Navigation-guided, Prone, Single-position, Lateral Lumbar Interbody Fusion Technique
Samantha E. Hoffman 1, Saksham Gupta 1, Kevin Huang 1, Neil Klinger 1, Yi Lu 1
1Department of Neurosurgery, Brigham and Women’s Hospital, Harvard Medical School

The single-position, prone, lateral approach allows for both lateral lumbar interbody placement and direct posterior decompression with pedicle screw placement in one position.

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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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Bioengineering

Isolation, Expansion, and Differentiation of Mesenchymal Stem Cells from the Infrapatellar Fat Pad of the Goat Stifle Joint
Aman Mahajan *1,2, Sugata Hazra *1,2, Aditya Arora 1,3, Dhirendra S. Katti 1,2
1Department of Biological Sciences and Bioengineering, Indian Institute of Technology-Kanpur, 2The Mehta Family Centre for Engineering in Medicine, Indian Institute of Technology-Kanpur, 3Mechanobiology Institute, National University of Singapore

Infrapatellar fat pad mesenchymal stem cells (IFP-MSCs) can be isolated easily from the infrapatellar fat pad of the knee joint. They proliferate well in vitro, form CFU-F colonies, and differentiate into adipogenic, chondrogenic, and osteogenic lineages. Herein, the methodology for the isolation, expansion, and differentiation of IFP-MSCs from goat stifle joint is provided.

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Biology

A High-Throughput Platform for Culture and 3D Imaging of Organoids
Gianluca Grenci 1,2, Florian Dilasser 1, Saburnisha Binte Mohamad Raffi 1, Marion Marchand 1, Mona Suryana 1, Geetika Sahni 1, Virgile Viasnoff 1,3,4, Anne Beghin 1,5
1Mechanobiology Institute (MBI), National University of Singapore, 2Biomedical Engineering Department, National University of Singapore, 3Department of Biological Sciences, National University of Singapore, 4IRL 3639 CNRS, 5Department of Microbiology & Immunology, Immunology Translational Research Program, Yong Loo Lin School of Medicine, National University of Singapore

This paper presents a fabrication protocol for a new type of culture substrate with hundreds of microcontainers per mm2, in which organoids can be cultured and observed using high-resolution microscopy. The cell seeding and immunostaining protocols are also detailed.

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Medicine

Application of Stand-Alone Oblique Lateral Interbody Fusion in L4-5 Lumbar Diseases
Ruoyu Zhao 1, Yachong Huo 1, Trifanny Yeo 2,3, Felicity Y. Han 4, Lei Ma 1, Dalong Yang 1, Wenyuan Ding 1,5, Sidong Yang 1,5
1Department of Spine Surgery, The Third Hospital of Hebei Medical University, 2Institute for Health Innovation and Technology (iHealthtech), National University of Singapore, 3Department of Biomedical Engineering, National University of Singapore, 4Australian Institute for Bioengineering and Nanotechnology, The University of Queensland. Brisbane, 5Hebei Joint International Research Center for Spinal Diseases

This study describes the application of oblique lateral interbody fusion in lumbar spinal surgeries.

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Medicine

Sub-Retinal Delivery of Human Embryonic Stem Cell Derived Photoreceptor Progenitors in rd10 Mice
Sai Bo Bo Tun *1, Edwin Shepherdson *2, Hwee Goon Tay 2,3, Veluchamy Amutha Barathi 1,3,4
1Singapore National Eye Centre, Singapore Eye Research Institute, 2Centre for Vision Research, Duke-NUS Medical School, 3Ophthalmology and Visual Sciences Academic Clinical Program, DUKE-NUS Medical School, 4Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore

We describe a detailed protocol for the preparation of post-cryopreserved hESC-derived photoreceptor progenitor cells and the sub-retinal delivery of these cells in rd10 mice.

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