University of Sydney View Institution's Website 61 articles published in JoVE Biology High-Throughput Contractile Measurements of Hydrogel-Embedded Intact Mouse Muscle Fibers Using an Optics-Based System Leander A. Vonk*1, Osman Esen*1, Michaela Yuen1,4, Tyler J. Kirby1,2,3 1Department of Physiology, Amsterdam UMC, 2Amsterdam Cardiovascular Sciences, Heart Failures and Arrhythmias, Amsterdam UMC, 3Amsterdam Movement Sciences, Tissue Function and Regeneration, Amsterdam UMC, 4Discipline of Child and Adolescent Health, Faculty of Health and Medicine, University of Sydney Skeletal muscle function can be assessed by quantifying the contractility of isolated muscle fibers, traditionally using laborious, low-throughput approaches. Here, we describe an optics-based, high-throughput method to quantify the contractility of hydrogel-embedded muscle fibers. This approach has applications for drug screening and therapeutic development. Medicine Delivery of Cardioactive Therapeutics in a Porcine Myocardial Infarction Model Dinesh Selvakumar*1,2, Emma Wilkie*1, Tejas Deshmukh1,2, Dhanya Ravindran1, Yasuhito Kotake2, Juntang Lu2, Tony Barry2, Vu Tran2, Hugh Paterson3, Alfred Hing4, Timothy Campbell2, Saurabh Kumar2, Eddy Kizana1,2, James J. H. Chong1,2 1Centre for Heart Research, The Westmead Institute for Medical Research, The University of Sydney, 2Department of Cardiology, Westmead Hospital, 3Sydney Imaging, Core Research Facility, The University of Sydney, 4Department of Cardiothoracic Surgery, Liverpool Hospital The present protocol describes three methods of administering cardioactive therapeutic agents in a porcine model. Female landrace swine received treatment through either: (1) thoracotomy and transepicardial injection, (2) catheter-based transendocardial injection, or (3) intravenous infusion via jugular vein osmotic minipump. Bioengineering Ceramic Omnidirectional Bioprinting in Cell-Laden Suspensions for the Generation of Bone Analogs Gagan Jalandhra1, Sara Romanazzo2, Stephanie Nemec1, Iman Roohani3, Kristopher A. Kilian1,2 1School of Materials Science and Engineering, University of New South Wales, 2School of Chemistry, Australian Centre for NanoMedicine, University of New South Wales, 3School of Biomedical Engineering, University of Sydney This protocol describes a 3D printing technique to fabricate bone-like structures by depositing a calcium phosphate ink in a gelatin-based granular support. Printed bone analogs are deposited in freeform, with flexibility for direct harvesting of the print or crosslinking within a living cell matrix for multiphasic constructs. Neuroscience Computational Modeling of Retinal Neurons for Visual Prosthesis Research - Fundamental Approaches Ariastity Pratiwi1, Orsolya Kekesi1, Gregg Suaning1 1The University of Sydney We summarize a workflow to computationally model a retinal neuron's behaviors in response to electrical stimulation. The computational model is versatile and includes automation steps that are useful in simulating a range of physiological scenarios and anticipating the outcomes of future in vivo/in vitro studies. Biology Anesthesia and Intubation of Preadolescent Mouse Pups for Cardiothoracic Surgery Jianxin Wu*1, Amy M. Nicks1,2, Justin J. Skowno3,4, Michael P. Feneley1,2, Robert M. Graham1,2, Siiri E. Iismaa*1,2 1Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, 2St Vincent’s Clinical School, University of New South Wales Sydney, 3Department of Anaesthesia, The Children’s Hospital at Westmead, 4School of Child and Adolescent Health, University of Sydney Cardiothoracic surgical models in mice >7 days old require intubation, but this is challenging for preadolescent (8-14-day-old) mouse pups and there is little information on anesthetic regimens for intubation. Here, we present dosage regimens of ketamine/xylazine/atropine in 10-day-old C57BL/6J mouse pups that allow endotracheal intubation, while minimizing animal mortality. Bioengineering Molecular Spring Constant Analysis by Biomembrane Force Probe Spectroscopy Peyman Obeidy1, Haoqing Wang1,2,3, Mingqin Du1, Huiqian Hu1,4, Fang Zhou1, Haoruo Zhou5, Hao Huang5, Yunduo Charles Zhao1,2, Lining Arnold Ju1,2,3 1School of Biomedical Engineering, Faculty of Engineering, The University of Sydney, 2Charles Perkins Centre, The University of Sydney, 3Heart Research Institute, 4Department of Chemistry, The Hong Kong University of Science and Technology, 5School of Aerospace, Mechanical and Mechatronic Engineering, Faculty of Engineering, The University of Sydney A biomembrane force probe (BFP) is an in situ dynamic force spectroscopy (DFS) technique. BFP can be used to measure the spring constant of molecular interactions on living cells. This protocol presents spring constant analysis for molecular bonds detected by BFP. Neuroscience Conventional and Threshold-Tracking Transcranial Magnetic Stimulation Tests for Single-handed Operation Hatice Tankisi1, James Howells2, Bülent Cengiz3, Gintaute Samusyte4, Martin Koltzenburg5, Hugh Bostock6 1Department of Clinical Neurophysiology, Aarhus University Hospital, 2Central Clinical School, Faculty of Medicine and Health, University of Sydney, 3Department of Neurology, Gazi University Faculty of Medicine, 4Department of Neurology, Medical Academy, Lithuanian University of Health Sciences, 5Department of Clinical Neurophysiology, National Hospital for Neurology and Neurosurgery; Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, 6Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology We present a suite of standardized single- and paired-pulse transcranial magnetic stimulation (TMS) recording protocols, with options for conventional amplitude measurements and threshold-tracking. This program can control three different types of magnetic stimulators and is designed to enable all tests to be performed conveniently by a single operator. Neuroscience Objectively Assessing Sports Concussion Utilizing Visual Evoked Potentials Daryl H. Fong1, Adrian J. Cohen2, Dylan E. Mahony2, Neil G. Simon4, Joseph E. Herrera3, Rebecca B. Baron3, David Putrino3 1School of Aerospace, Mechanical and Mechatronic Engineering, Faculty of Engineering and Information Technologies, University of Sydney, 2HeadsafeIP, 3Department of Rehabilitation Medicine, Icahn School of Medicine at Mount Sinai, 4St Vincent's Clinical School, Faculty of Medicine, University of New South Wales A portable system capable of measuring steady-state visual-evoked potentials was developed and trialed on 65 amateur rugby players over 18 weeks to investigate SSVEP as a potential electrophysiological biomarker for concussion. Players' baselines were measured pre-season, with retesting for reliability, concussion, and recovery assessment being conducted within controlled time-periods, respectively. Bioengineering Cardiac Spheroids as in vitro Bioengineered Heart Tissues to Study Human Heart Pathophysiology Poonam Sharma1,2,3,4, Carmine Gentile2,3,4 1University of Newcastle, 2University of Sydney, 3Kolling Institute of Medical Research, Royal North Shore Hospital, 4University of Technology, Sydney This protocol aims to fabricate 3D cardiac spheroids (CSs) by co-culturing cells in hanging drops. Collagen-embedded CSs are treated with doxorubicin (DOX, a cardiotoxic agent) at physiological concentrations to model heart failure. In vitro testing using DOX-treated CSs may be used to identify novel therapies for heart failure patients. Bioengineering Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction Christopher D. Roche1,2,3,4, Carmine Gentile1,2,3 1The University of Sydney, 2University of Technology Sydney (UTS), 3The Royal North Shore Hospital, 4University Hospital of Wales This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart. Cancer Research An Orthotopic Resectional Mouse Model of Pancreatic Cancer Tony C. Y. Pang1,2,4,5, Zhihong Xu1,2, Alpha Raj Mekapogu1,2, Srinivasa Pothula1,2, Therese M. Becker3, David Goldstein1, Romano C. Pirola1,2, Jeremy S. Wilson1,2, Minoti V. Apte1,2 1Pancreatic Research Group, South Western Sydney Clinical School, University of New South Wales, 2Ingham Institute for Applied Medical Research, 3Centre for Circulating Tumour Cell Diagnostics and Research, Ingham Institute for Applied Medical Research, 4Surgical Innovations Unit, Westmead Hospital, 5Westmead Clinical School, University of Sydney In the clinical context, patients with localized pancreatic cancer will undergo pancreatectomy followed by adjuvant treatment. This protocol reported here aims to establish a safe and effective method of modelling this clinical scenario in nude mice, through orthotopic implantation of pancreatic cancer followed by distal pancreatectomy and splenectomy. Medicine Intratracheal Administration of Dry Powder Formulation in Mice Yingshan Qiu1, Qiuying Liao1, Michael Y.T. Chow1,2, Jenny K.W. Lam1 1Department of Pharmacology and Pharmacy, LKS Faculty of Medicine, The University of Hong Kong, 2Advanced Drug Delivery Group, Sydney Pharmacy School, Faculty of Medicine and Health, The University of Sydney Dry powder formulations for inhalation have great potential in treating respiratory diseases. Before entering human studies, it is necessary to evaluate the efficacy of the dry powder formulation in preclinical studies. A simple and noninvasive method of the administration of dry powder in mice through the intratracheal route is presented. Medicine Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model Patrick Haubruck1,2, Aimee C. Colbath1,3, Yolanda Liu1, Shihani Stoner1, Cindy Shu1, Christopher B. Little1 1Raymond Purves Bone and Joint Research Laboratory, Institute of Bone and Joint Research, Kolling Institute, Royal North Shore Hospital, University of Sydney, 2HTRG - Heidelberg Trauma Research Group, Center for Orthopedics, Trauma Surgery and Spinal Cord Injury, Trauma and Reconstructive Surgery, Heidelberg University Hospital, 3Department of Large Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis. Behavior Assessing the Autonomic and Behavioral Effects of Passive Motion in Rats using Elevator Vertical Motion and Ferris-Wheel Rotation Francis A. M. Manno1,2, Leilei Pan3, Yuqi Mao3, Yang Su3, Sinai H. C. Manno2,4,5, Shuk Han Cheng4,5,6, Condon Lau2, Yiling Cai3 1School of Biomedical Engineering, Faculty of Engineering, University of Sydney, 2Department of Physics, City University of Hong Kong, 3Department of Nautical Injury Prevention, Faculty of Navy Medicine, Second Military Medical University, 4State Key Laboratory of Marine Pollution (SKLMP), City University of Hong Kong, 5Department of Biomedical Sciences, College of Veterinary Medicine and Life Sciences, City University of Hong Kong, 6Department of Materials Science and Engineering, City University of Hong Kong Protocols are presented to assess the autonomic and behavioral effects of passive motion in rodents using elevator vertical motion and Ferris-wheel rotation. Immunology and Infection In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages Yunjia Zhang1,2, Benjamin S. Rayner1,2, Mathias Jensen3, Clare L. Hawkins1,2,3 1Heart Research Institute, 2Sydney Medical School, University of Sydney, 3Department of Biomedical Sciences, University of Copenhagen Presented here is a protocol to detect macrophage extracellular trap (MET) production in live cell culture using microscopy and fluorescence staining. This protocol can be further extended to examine specific MET protein markers by immunofluorescence staining. Neuroscience Stochastic Noise Application for the Assessment of Medial Vestibular Nucleus Neuron Sensitivity In Vitro Sebastian P. Stefani1,2, Paul P. Breen3, Jorge M. Serrador4, Aaron J. Camp1,2 1Discipline of Physiology, Sydney Medical School, University of Sydney, 2Bosch Institute, Sydney Medical School, University of Sydney, 3The MARCS Institute, Western Sydney University, 4Department of Pharmacology, Physiology & Neuroscience, Rutgers Biomedical and Health Sciences Galvanic vestibular stimulation in humans exhibits improvements in vestibular function. However, it is unknown how these effects occur. Here, we describe how to apply sinusoidal and stochastic electrical noise and evaluate appropriate stimulus amplitudes in individual medial vestibular nucleus neurons in the C57BL/6 mouse. Environment PARbars: Cheap, Easy to Build Ceptometers for Continuous Measurement of Light Interception in Plant Canopies William T. Salter1, Andrew M. Merchant1, Matthew E. Gilbert2, Thomas N. Buckley2 1School of Life and Environmental Sciences, Sydney Institute of Agriculture, University of Sydney, 2Department of Plant Sciences, University of California, Davis Here, we present detailed instructions on how to build and calibrate research quality ceptometers (light sensors that integrate light intensity across many sensors arrayed linearly along a horizontal bar). Developmental Biology Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells Hannah C. Leeson1,2, Tailoi Chan-Ling3,4, Michael D. Lovelace3,5,6, Jeremy D. Brownlie7, Michael W. Weible II*1,4,7, Ben J. Gu*8 1Griffith Institute for Drug Discovery, Griffith University, 2Australian Institute for Bioengineering and Nanotechnology, University of Queensland, 3Discipline of Anatomy and Histology, School of Medical Science, University of Sydney, 4Bosch Institute, University of Sydney, 5 Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads. Chemistry Employing Pressurized Hot Water Extraction (PHWE) to Explore Natural Products Chemistry in the Undergraduate Laboratory Curtis C. Ho1, Bianca J. Deans*1, Jeremy Just*1, Gregory G. Warr2, Shane Wilkinson2, Jason A. Smith1, Alex C. Bissember1 1School of Natural Sciences - Chemistry, University of Tasmania, 2School of Chemistry, The University of Sydney Here, we employ a pressurized hot water extraction (PHWE) method, which utilizes an unmodified household espresso machine to introduce undergraduates to natural products chemistry in the laboratory. Two experiments are presented: PHWE of eugenol and acetyleugenol from cloves and PHWE of seselin and (+)-epoxysuberosin from the Australian plant Correa reflexa. Immunology and Infection Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis Rekha Marimuthu1,2, Habib Francis1,2, Suat Dervish3, Stephen C.H. Li4, Heather Medbury*1,2, Helen Williams*1,2 1Department of Surgery, Vascular Biology Research Centre, Westmead Hospital, 2Westmead Clinical School, Department of Surgery, The University of Sydney, 3Westmead Research Hub, Westmead Institute for Medical Research, 4Institute for Clinical Pathology and Medical Research, Westmead Hospital Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory). Genetics Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing Bonnie Hiener1, John-Sebastian Eden1, Bethany A. Horsburgh1, Sarah Palmer1 1Centre for Virus Research, The Westmead Institute for Medical Research, University of Sydney Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir. Medicine 'Boden Food Plate': Novel Interactive Web-based Method for the Assessment of Dietary Intake Alice Meroni*1, Nyssa Jualim*1, Nicholas Fuller1 1The Boden Institute of Obesity, Nutrition, Exercise & Eating Disorders, Charles Perkins Centre, University of Sydney The 'Boden Food Plate' is an electronic food diary designed to be an interactive and fun method to collect dietary intake using visual depictions. The purpose of this study was to validate the web-based application against a traditional three-day estimated food diary method. Biochemistry Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows Matthew B. O'Rourke1,2, Matthew P. Padula2, Caine Smith3, Priscilla Youssef4, Stuart Cordwell1, Paul Witting4, Greg Sutherland3, Ben Crossett1 1Mass Spectrometry Core Facility, University of Sydney, 2Proteomics Core Facility, University of Technology Sydney, 3Neuropathology Group, Discipline of Pathology, School of Medical Sciences, University of Sydney, 4Redox Biology Group, Discipline of Pathology, School of Medical Sciences, University of Sydney This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry. Medicine Whole Genome Sequencing of Candida glabrata for Detection of Markers of Antifungal Drug Resistance Chayanika Biswas*1, Sharon C-A. Chen*1,2,3, Catriona Halliday1,2, Elena Martinez1, Rebecca J. Rockett1, Qinning Wang1, Verlaine J. Timms1, Rajat Dhakal1, Rosemarie Sadsad1, Karina J. Kennedy4, Geoffrey Playford4,5, Deborah J. Marriott6, Monica A. Slavin7, Tania C. Sorrell1,3, Vitali Sintchenko1,2,3 1Centre for Infectious Diseases and Microbiology-Public Health, Westmead Hospital, 2Centre for Infectious Diseases and Microbiology Laboratory Services, ICPMR, 3Marie Bashir Institute for Infectious Diseases and Biosecurity, The University of Sydney, 4Department of Microbiology and Infectious Diseases, Canberra Hospital and Health Services, Australian National University Medical School, 5Infection Management Services, Australian National University Medical School, 6 This study implemented whole genome sequencing for analysis of mutations in genes conferring antifungal drug resistance in Candida glabrata. C. glabrata isolates resistant to echinocandins, azoles and 5-flucytosine, were sequenced to illustrate the methodology. Susceptibility profiles of the isolates correlated with presence or absence of specific mutation patterns in genes. Biology The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury Jully Gogoi-Tiwari1, Julia Köhn-Gaone1, Corey Giles2, Dirk Schmidt-Arras3, Francis D. Gratte1,4, Caryn L. Elsegood1, Geoffrey W. McCaughan5,6,7, Grant A. Ramm8,9, John K. Olynyk10,11, Janina E.E. Tirnitz-Parker1,12 1School of Biomedical Sciences & Curtin Health Innovation Research Institute, Curtin University, 2School of Public Health & Curtin Health Innovation Research Institute, Curtin University, 3Institute of Biochemistry, Christian-Albrechts-University, 4School of Veterinary and Life Sciences, Murdoch University, 5Centenary Institute of Cancer Medicine and Cell Biology, The University of Sydney, 6Royal Prince Alfred Hospital, 7A.W. Morrow Gastroenterology and Liver Centre, 8QIMR Berghofer Medical Research Institute, 9Faculty of Medicine and Biomedical Sciences, The University of Queensland, 10Fiona Stanley and Fremantle Hospitals, 11School of Medical and Health Sciences, Edith Cowan University, 12School of Medicine and Pharmacology, University of Western Australia Here we describe a common method to induce chronic liver injury in mice by feeding of a choline-deficient and ethionine-supplemented (CDE) diet. We demonstrate health monitoring, liver perfusion, isolation, and preservation. A time course of six weeks can inform about liver injury, pathohistology, fibrosis, inflammatory, and liver progenitor cell responses. Medicine Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model Jaesung P. Choi1,2, Xi Yang3, Matthew Foley4, Xian Wang1, Xiangjian Zheng1,2,3 1Lab of Cardiovascular Signaling, Centenary Institute, 2Faculty of Medicine, Sydney Medical School, University of Sydney, 3Department of Pharmacology, School of Basic Medical Sciences, Tianjin Medical University, 4Australian Centre for Microscopy & Microanalysis, University of Sydney This protocol demonstrates the induction of cerebral cavernous malformation disease in a mouse model and uses contrast enhanced micro computed tomography to measure lesion burden. This method enhances the value of established mouse models to study the molecular basis and potential therapies for cerebral cavernous malformation and other cerebrovascular diseases. Chemistry Palladium N-Heterocyclic Carbene Complexes: Synthesis from Benzimidazolium Salts and Catalytic Activity in Carbon-carbon Bond-forming Reactions Ziya Sahin1, Senem Akkoς1,2, İlhan Özer İlhan2, Veysel Kayser1 1Faculty of Pharmacy, The University of Sydney, 2Department of Chemistry, Faculty of Sciences, Erciyes University Detailed and generalized protocols are presented for the synthesis and subsequent purification of four palladium N-heterocyclic carbene complexes from benzimidazolium salts. The complexes were tested for catalytic activity in arylation and Suzuki-Miyaura reactions. For each reaction investigated, at least one of the four complexes successfully catalyzed the reaction. Medicine Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT Blake J. Cochran1, William J. Ryder2,3,4,5, Arvind Parmar6, Kerstin Klaeser4,5, Anthonin Reilhac7, Georgios I. Angelis4,5, Steven R. Meikle4,5, Philip J. Barter1,5, Kerry-Anne Rye1,5 1School of Medical Sciences, Faculty of Medicine, UNSW Australia, 2Department of Nuclear Medicine, Concord Hospital, 3National Imaging Facility, University of Sydney, 4Brain and Mind Centre, University of Sydney, 5Faculty of Health Sciences, University of Sydney, 6Life Sciences, ANSTO, 7CERMEP This study describes a protocol that uses 18F-FDG and positron emission tomography/computed tomography (PET/CT) imaging, together with kinetic modelling, to quantify the in vivo, real-time uptake of 18F-FDG into tissues. Engineering Characterization of Ultra-fine Grained and Nanocrystalline Materials Using Transmission Kikuchi Diffraction Gwénaëlle Proust1, Patrick Trimby2, Sandra Piazolo3, Delphine Retraint4 1School of Civil Engineering, The University of Sydney, 2Australian Centre for Microscopy and Microanalysis, The University of Sydney, 3Department of Earth and Planetary Sciences, Macquarie University, 4Charles Delaunay Institute, LASMIS, UMR STMR CNRS 6281, University of Technology of Troyes This paper provides a detailed method to characterize the microstructure of ultra-fine grained and nanocrystalline materials using a scanning electron microscope equipped with a standard electron backscatter diffraction system. Metal alloys and minerals presenting refined microstructures are analyzed using this technique, showing the diversity of its possible applications. Biochemistry Laboratory Scale Production and Purification of a Therapeutic Antibody Zehra Elgundi1, Vicki Sifniotis1, Mouhamad Reslan1, Esteban Cruz1, Veysel Kayser1 1Faculty of Pharmacy, University of Sydney This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography. Medicine Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses Chi Ching Goh1,2, Jackson LiangYao Li1, David Becker3, Wolfgang Weninger4,5,6, Veronique Angeli2,7, Lai Guan Ng1,2,5,8 1Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Biopolis, 2Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 3Lee Kong Chian School of Medicine, Nanyang Technological University, 4Centenary Institute for Cancer Medicine and Cell Biology, 5Discipline of Dermatology, University of Sydney, 6Department of Dermatology, Royal Prince Alfred Hospital, 7LSI Immunology Programme, National University of Singapore, 8School of Biological Sciences, Nanyang Technological University This protocol describes the induction of an ischemia-reperfusion (IR) model on mouse ear skin using magnet clamping. Using a custom-built intravital imaging model, we study in vivo inflammatory responses post-reperfusion. The rationale behind the development of this technique is to extend the understanding of how leukocytes respond to skin IR injury. Developmental Biology Isolation and Expansion of Adult Canine Hippocampal Neural Precursors Thomas Duncan1, Aileen Lowe1, Marshall A. Dalton2, Michael Valenzuela1 1Regenerative Neuroscience Group, University of Sydney, 2Wellcome Trust Centre for Neuroimaging, Institute of Neurology, University College London The canine brain is a valuable model in which to study adult neurogenesis. Presented here are protocols for isolating and expanding adult canine hippocampal neural precursor cells from primary brain tissue. Developmental Biology Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol Hananeh Fonoudi*1,2,3,8, Hassan Ansari*1,8, Saeed Abbasalizadeh1, Gillian M Blue6,7, Nasser Aghdami1, David S Winlaw6,7, Richard P Harvey2,3,4, Alexis Bosman*2,3, Hossein Baharvand*1,5 1Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, 2Developmental and Stem Cell Biology Division, Victor Chang Cardiac Research Institute, 3St. Vincent´s Clinical School, Faculty of Medicine, University of New South Wales, 4School of Biotechnology and Biomolecular Sciences, University of New South Wales, 5Department of Developmental Biology, University of Science and Culture, 6Heart Centre for Children, The Children´s Hospital at Westmead, 7Sydney Medical School, University of Sydney, 8Department of Developmental Biology, University of Science and Culture, Tehran, Iran Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications. Engineering Writing Bragg Gratings in Multicore Fibers Emma Y. Lindley1, Seong-sik Min1,2, Sergio G. Leon-Saval1,2, Nick Cvetojevic1,2,3,4, Jon Lawrence4, Simon C. Ellis1,4, Joss Bland-Hawthorn1,2 1Sydney Institute for Astronomy, School of Physics, University of Sydney, 2Institute of Photonics and Optical Science, School of Physics, University of Sydney, 3Center for Ultrahigh Bandwidth Devices for Optical Systems, School of Physics, University of Sydney, 4Australian Astronomical Observatory We describe a technique for inscribing identical fiber Bragg gratings into each core of a multicore fiber. This is achieved by introducing an additional surface into the optical path to mitigate lensing by the curved surface of the fiber cladding. Medicine Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids Kimberley A. Beaumont1,2, Andrea Anfosso1,2, Farzana Ahmed3, Wolfgang Weninger*1,4,5, Nikolas K. Haass*1,3,5 1The Centenary Institute, 2Sydney Medical School, University of Sydney, 3The University of Queensland Diamantina Institute, Translational Research Institute, The University of Queensland, 4Department of Dermatology, Royal Prince Alfred Hospital, 5Discipline of Dermatology, University of Sydney We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids. Biology Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization Robert Welschinger1, Linda J. Bendall1 1Centre for Cancer Research, Westmead Millennium Institute for Medical Research and University of Sydney This protocol describes the use of bromodeoxyuridine (BrdU) uptake to permit the temporal tracking of cells that were in S phase at a specific point in time. Addition of DNA dyes and antibody labeling facilitates detailed analysis of the fate of the S phase cells at later times. Bioengineering Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell Yunfeng Chen*1, Baoyu Liu*2, Lining Ju*3, Jinsung Hong*1, Qinghua Ji4,5, Wei Chen6, Cheng Zhu1 1Woodruff School of Mechanical Engineering, Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, 2Coulter Department of Biomedical Engineering, Georgia Institute of Technology, 3Charles Perkins Centre, The University of Sydney, 4Institute of Biophysics, Laboratory of RNA Biology, Chinese Academy of Sciences, 5University of Chinese Academy of Sciences, 6School of Medicine and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte. Neuroscience Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination Yuyi You1,2, Vivek K. Gupta1, Nitin Chitranshi1, Brittany Reedman1, Alexander Klistorner1,2, Stuart L. Graham1,2 1Department of Ophthalmology, Australian School of Advanced Medicine, Macquarie University, 2Save Sight Institute, The University of Sydney Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection. Neuroscience Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning Alice Brandli1, Jonathan Stone1 1Discipline of Physiology and Bosch Institute, Sydney Medical School, University of Sydney The electroretinogram (ERG) is an electrical potential generated by the retina in response to light. This paper describes how to use the ERG to assess retinal function, in dark-adapted rats, and how it can be can be used to assess a neuroprotective intervention, in the present case remote ischemic preconditioning. Medicine Remote Limb Ischemic Preconditioning: A Neuroprotective Technique in Rodents Alice Brandli1 1Discipline of Physiology and Bosch Institute, Sydney Medical School, University of Sydney Remote ischemic preconditioning (RIP) is a method of conditioning tissues against damaging stress. We have established a method of remote ischemia at the hind limb, by inflating a sphygmomanometer cuff for 5-10 min. The neuroprotective capabilities of RIP have been demonstrated in a model of retinal degeneration in rodents. Biology A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy Victoria C Cogger*1,2,3, Jennifer N O'Reilly*1,2, Alessandra Warren1,2,3, David G Le Couteur1,2,3 1Centre for Education and Research on Ageing & ANZAC Research Institute, University of Sydney and Concord Hospital, 2Ageing and Alzheimers Institute, Concord Hospital, 3Charles Perkins Centre, University of Sydney The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells. Biology Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs Wilson Wong1, Ryan Farr1, Mugdha Joglekar1, Andrzej Januszewski2, Anandwardhan Hardikar1 1Diabetes and Islet Biology Group, NHMRC Clinical Trials Centre, Faculty of Medicine, The University of Sydney, 2Biomarkers Laboratory, NHMRC Clinical Trials Centre, Faculty of Medicine, The University of Sydney Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs. Biology Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium Lucy Zhang1, Victoria W. K. Tung2, Miranda Mathews2, Aaron J. Camp2 1Discipline of Physiology, University of Sydney, 2Discipline of Biomedical Science, University of Sydney Mitochondrial dysfunction is a hallmark of cellular senescence. This paper uses non-invasive near-infrared (NIr) treatment to improve mitochondrial function in the aging mouse vestibular sensory epithelium. Behavior The Use of the Puzzle Box as a Means of Assessing the Efficacy of Environmental Enrichment Angela M. O'Connor1, Thomas J. Burton1,2, Catherine A. Leamey1, Atomu Sawatari1 1Discipline of Physiology, University of Sydney, 2Bosch Animal Behavioural Facility, University of Sydney Environmental enrichment provides a potential protective effect against neurodegenerative disorders. Currently, however, there is no easy way of determining the efficacy of enrichment procedures. This protocol describes a simple “Puzzle Box” method for assessing an animal’s cognitive function, in order to reveal the effectiveness of environmental enrichment. Neuroscience The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy Nigel Tse1, Marco Morsch2, Nazanin Ghazanfari1, Louise Cole3, Archunan Visvanathan1, Catherine Leamey1, William D. Phillips1 1Physiology and Bosch Institute, University of Sydney, 2Motor Neuron Disease Research Group, Australian School of Advanced Medicine, Macquarie University, 3Advanced Microscopy Facility, Bosch Institute, University of Sydney The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes. Engineering In Situ Neutron Powder Diffraction Using Custom-made Lithium-ion Batteries William R. Brant1, Siegbert Schmid1, Guodong Du2, Helen E. A. Brand3, Wei Kong Pang2,4,5, Vanessa K. Peterson4, Zaiping Guo2,5, Neeraj Sharma6 1School of Chemistry, University of Sydney, 2Institute for Superconducting & Electronic Materials, University of Wollongong, 3Australian Synchrotron, 4Australian Nuclear Science and Technology Organisation, 5School of Mechanical, Materials, and Mechatronic Engineering, University of Wollongong, 6School of Chemistry, University of New South Wales We describe the design and construction of an electrochemical cell for the examination of electrode materials using in situ neutron powder diffraction (NPD). We briefly comment on alternate in situ NPD cell designs and discuss methods for the analysis of the corresponding in situ NPD data produced using this cell. Engineering Integrating a Triplet-triplet Annihilation Up-conversion System to Enhance Dye-sensitized Solar Cell Response to Sub-bandgap Light Andrew Nattestad1, Yuen Yap Cheng2, Rowan W. MacQueen2, Gordon G. Wallace1, Timothy W. Schmidt3 1ARC Centre of Excellence for Electromaterials Science (ACES), Intelligent Polymer Research Institute (IPRI), The University of Wollongong, 2School of Chemistry, The University of Sydney, 3School of Chemistry, The University of New South Wales An integrated device, incorporating a dye-sensitized solar cell and triplet-triplet annihilation up-conversion unit was produced, affording enhanced light harvesting, from a wider section of the solar spectrum. Under modest irradiation levels a significantly enhanced response to low energy photons was demonstrated, yielding a record figure of merit for dye-sensitized solar cells. Chemistry Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy Dexter D'Sa1, Hak-Kim Chan1, Hae-Won Kim2, Wojciech Chrzanowski1,2 1Faculty of Pharmacy, University of Sydney, 2Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems. Behavior Behavioral Assessment of the Aging Mouse Vestibular System Victoria W. K. Tung1, Thomas J. Burton2, Edward Dababneh1, Stephanie L. Quail1, Aaron J. Camp1 1Discipline of Biomedical Science, University of Sydney, 2The Bosch Institute Animal Behavioural Facility, University of Sydney Motor control and balance performance are known to deteriorate with age. This paper presents a number of standard noninvasive behavioral tests with the addition of a simple rotary stimulus to challenge the vestibular system and show changes in balance performance in a murine model of aging. Medicine A Modified Method for Heterotopic Mouse Heart Transplantion Chuanmin Wang1, Zane Wang1, Richard Allen1, G. Alex Bishop1, Alexandra F. Sharland1 1Collaborative Transplant Laboratory, University of Sydney A technique is demonstrated for the microsurgical procedure for heterotopic transplantation of hearts in mice, including simplified methods for donor harvesting and recipient vessel anastomosis. Chemistry Amide Coupling Reaction for the Synthesis of Bispyridine-based Ligands and Their Complexation to Platinum as Dinuclear Anticancer Agents Michael G. Apps1, Ben W. Johnson2, Oliver B. Sutcliffe3, Sarah D. Brown4, Nial J. Wheate1 1Faculty of Pharmacy, The University of Sydney, 2School of Science and Health, University of Western Sydney, 3Division of Chemistry and Environmental Science, School of Science and the Environment, Manchester Metropolitan University, 4Nature Publishing Group This protocol describes the use of amide coupling reactions of isonicotinic acid and diaminoalkanes to form bridging ligands suitable for use in the synthesis of multinuclear platinum complexes, which combine aspects of the anticancer drugs BBR3464 and picoplatin. Biology A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells Darius J. R. Lane1, Alfons Lawen2 1Molecular Pharmacology and Pathology Program, Department of Pathology & Bosch Institute, University of Sydney, 2Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University Ascorbate plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. Here we describe a medium-throughput, specific and inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture. Immunology and Infection Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins N. Bishara Marzook*1, Dean J. Procter*1, Helena Lynn1, Yui Yamamoto2, Jacquelyn Horsington3, Timothy P. Newsome1 1School of Molecular Bioscience, University of Sydney, 2Department of Medicine, Center for Vascular Research, 3Asia Pacific Centre for Animal Health, Faculty of Veterinary Science, University of Melbourne A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently tagged proteins simultaneously using the method of transient dominant selection is described here. Medicine Computerized Dynamic Posturography for Postural Control Assessment in Patients with Intermittent Claudication Natalie Vanicek1,2, Stephanie A. King2, Risha Gohil2,3, Ian C. Chetter3, Patrick A Coughlin3,4 1Discipline of Exercise and Sport Science, Faculty of Health Sciences, University of Sydney, 2Department of Sport, Health and Exercise Science, University of Hull, 3Academic Vascular Department, Hull Royal Infirmary, Hull and East Yorkshire Hospitals, 4 Individuals with intermittent claudication exhibit poor balance compared to healthy controls. Computerized dynamic posturography is an objective method for measuring an individual's postural responses to balance disturbances. This provides an objective reflection of the person’s ability to respond to situations under which sensory stimuli are altered and unexpected perturbations occur. Medicine High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum Mazen Amatoury1, Vera Merheb1, Jessica Langer1, Xin Maggie Wang2, Russell Clive Dale1, Fabienne Brilot1 1Institute for Neuroscience and Muscle Research, The Kids Research Institute at the Children's Hospital at Westmead, The University of Sydney, 2Flow Cytometry Centre, Westmead Millennium Institute for Medical Research Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders. Neuroscience An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy Victoria W. K. Tung1, Stefano Di Marco1, Rebecca Lim2, Alan M. Brichta2, Aaron J. Camp1 1Discipline of Biomedical Science, School of Medical Sciences, Sydney Medical School, University of Sydney, 2School of Biomedical Sciences and Pharmacy, University of Newcastle Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies. Medicine Murine Model of Wound Healing Louise Dunn1,2, Hamish C. G Prosser1,2, Joanne T. M. Tan1,2, Laura Z. Vanags1,2, Martin K. C. Ng1,2,3, Christina A. Bursill1,2 1The Heart Research Institute, 2Sydney Medical School, University of Sydney, 3Cardiology Department, Royal Prince Alfred Hospital A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings. Neuroscience Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells Jin Y. Huang1, Klaus M. Stiefel2, Dario A. Protti3 1Discipline of Biomedical Science, School of Medical Sciences, Sydney Medical School and Bosch Institute, University of Sydney, 2The MARCS Institute, University of Western Sydney, 3Discipline of Physiology, School of Medical Sciences, Sydney Medical School and Bosch Institute, University of Sydney This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking. Engineering Fabricating Metamaterials Using the Fiber Drawing Method Alessandro Tuniz1, Richard Lwin1, Alexander Argyros1, Simon C. Fleming1, Boris T. Kuhlmey1 1Institute of Photonics and Optical Sciences (IPOS), School of Physics, University of Sydney Metamaterials at terahertz frequencies offer unique opportunities, but are challenging to fabricate in bulk. We adapt the fabrication procedure for microstructured polymer optical fibers to inexpensively fabricate metamaterials potentially on an industrial scale. We produce polymethylmethacrylate fibers containing ~10 μm diameter indium wires separated by ~100 μm, which exhibit a terahertz plasmonic response. Immunology and Infection Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing Jerry Zhou1, Larissa Belov1, Michael J. Solomon2, Charles Chan3, Stephen J. Clarke4, Richard I. Christopherson1 1School of Molecular Bioscience, University of Sydney, 2Department of Surgery, Royal Prince Alfred Hospital, 3Department of Anatomical Pathology, Department of Anatomical Pathology, 4Department of Medicine, Concord Repatriation General Hospital We described a procedure for the disaggregation of colorectal cancer (CRC) to produce viable single cells, which are then captured on customized antibody microarrays recognizing surface antigens (DotScan CRC microarray). Sub-populations of cells bound to the microarray can be profiled by fluorescence multiplexing using monoclonal antibodies tagged with fluorescent dyes. Biology Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB) Matthew V. N. O'Sullivan1, Fei Zhou1, Vitali Sintchenko1, Fanrong Kong1, Gwendolyn L. Gilbert1 1Centre for Infectious Diseases and Microbiology, University of Sydney An inexpensive, high throughput method for simultaneous detection of up to 43 molecular targets is described. Applications of mPCR/RLB include microbial typing and detection of multiple pathogens from clinical samples.
