University of Melbourne View Institution's Website 68 articles published in JoVE Biology Gas Chromatography-Mass Spectrometry-Based Targeted Metabolomics of Hard Coral Samples Jennifer L. Matthews1, Natasha Bartels1, Sheik Nadeem Elahee Doomun2, Simon K. Davy3, David P. De Souza2 1Climate Change Cluster (C3), University of Technology Sydney, 2Metabolomics Australia, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 3School of Biological Sciences, Victoria University of Wellington Here, we present the extraction and preparation of polar and semi-polar metabolites from a coral holobiont, as well as separated coral host tissue and Symbiodiniaceae cell fractions, for gas chromatography-mass spectrometry analysis. Medicine Erosion Identification in Metacarpophalangeal Joints in Rheumatoid Arthritis using High-Resolution Peripheral Quantitative Computed Tomography Yousif Al-Khoury1,2, Stephanie Finzel3, Camille Figueiredo4, Andrew J. Burghardt5, Kathryn S. Stok6, Lai-Shan Tam7, Isaac Cheng7, Justin J. Tse1, Sarah L. Manske1 1Department of Radiology, McCaig Institute for Bone and Joint Health, University of Calgary, 2Department of Biomedical Engineering, University of Calgary, 3University Medical Center Freiburg, 4Department of Rheumatology, University of São Paulo, 5Department of Radiology and Biomedical Imaging, University of California, San Francisco, 6Department of Biomedical Engineering, University of Melbourne, 7Department of Medicine and Therapeutics, The Chinese University of Hong Kong Bone erosions are an important pathological feature of rheumatoid arthritis. The purpose of this work is to introduce a training tool to provide users with guidance on identifying pathological cortical breaks on high resolution peripheral quantitative computed tomography images for erosion analysis. Medicine Using a Combination of Indirect Calorimetry, Infrared Thermography, and Blood Glucose Levels to Measure Brown Adipose Tissue Thermogenesis in Humans Lachlan Van Schaik1,3, Christine Kettle1, Rod A. Green1, Helen R. Irving1, Joseph A. Rathner1,2 1La Trobe Institute for Molecular Science, Department of Rural Clinical Sciences, La Trobe University, 2School of Biomedical Sciences, Department of Physiology, The University of Melbourne, 3Melbourne Medical School, Department of Rural Health, The University of Melbourne Here, we present a protocol to quantify the physiological significance of the impact of brown adipose tissue (BAT) activity on human metabolism. This is achieved by combining carbohydrate loading and indirect calorimetry with measurements of supraclavicular changes in temperature. This novel approach can help develop a pharmacological target for BAT thermogenesis in humans. Developmental Biology Methods for Studying Uterine Contributions to Pregnancy Establishment in an Ovariectomized Mouse Model Meaghan J. Griffiths*1,2, Jordan N. Higgins*1, Fiona L. Cousins3, Lauren R. Alesi1, Amy L. Winship*1, Karla J. Hutt*1 1Biomedicine Discovery Institute, Department of Anatomy and Developmental Biology, Monash University, 2Gynaecology Research Centre, Royal Women’s Hospital and Department of Obstetrics and Gynaecology, University of Melbourne, 3The Ritchie Centre, Hudson Institute for Medical Research and Department of Obstetrics and Gynaecology, Monash University Pregnancy establishment is a dynamic process involving complex embryo and uterine crosstalk. The precise contributions of the maternal uterine environment to these processes remain an active area of investigation. Here, detailed protocols are provided to aid in designing in vivo animal models to address these research questions. Bioengineering Experimental Quantification of Interactions Between Drug Delivery Systems and Cells In Vitro: A Guide for Preclinical Nanomedicine Evaluation Paula M. Cevaal1, Michael Roche2, Sharon R. Lewin2,3,4, Frank Caruso5, Matthew Faria6 1Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, 2Department of Infectious Diseases, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, 3Victorian Infectious Diseases Service, Royal Melbourne Hospital at the Peter Doherty Institute for Infection and Immunity, 4Department of Infectious Diseases, Alfred Hospital and Monash University, 5Department of Chemical Engineering, The University of Melbourne, 6Department of Biomedical Engineering, The University of Melbourne A workflow is demonstrated for the absolute quantification of drug carrier-cell interactions using flow cytometry to allow better rational evaluation of novel drug delivery systems. This workflow is applicable to drug carriers of any type. Immunology and Infection Rat Burn Model to Study Full-Thickness Cutaneous Thermal Burn and Infection Rajnikant Sharma1, Shekhar Yeshwante1, Quentin Vallé1, Maytham Hussein2, Varsha Thombare2, Sean Michael McCann1, Robert Maile3,4,5, Jian Li6, Tony Velkov2, Gauri Rao1 1UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, 2Department of Biochemistry & Pharmacology, School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, 3Department of Microbiology & Immunology, University of North Carolina School of Medicine, 4Department of Surgery, University of North Carolina at Chapel Hill, 5Curriculum in Toxicology and Environmental Medicine, University of North Carolina at Chapel Hill, 6Department of Microbiology, Monash Biomedicine Discovery Institute, Monash University A model mimicking the clinical scenario of burn injury and infection is necessary for furthering burn research. The present protocol demonstrates a simple and reproducible rat burn infection model comparable to that in humans. This facilitates the study of burn and infections following burn for developing new topical antibiotic treatments. Biology A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay Sebastian Bass-Stringer1,2, Colleen J. Thomas2,3, Clive N. May3, Paul Gregorevic4, Kate L. Weeks1,5,6, Julie R. McMullen1,2,5,6,7 1Baker Heart and Diabetes Institute, 2Department of Physiology, Anatomy and Microbiology, La Trobe University, 3Florey Institute of Neuroscience and Mental Health, University of Melbourne, 4Department of Physiology, Centre for Muscle Research (CMR), The University of Melbourne, 5Department of Diabetes, Central Clinical School, Monash University, 6Baker Department of Cardiometabolic Health, The University of Melbourne, 7Department of Physiology and Department of Medicine Alfred Hospital, Monash University A comprehensive laboratory protocol and analysis workflow are described for a rapid, cost-effective, and straightforward colorimetric cell-based assay to detect neutralizing elements against AAV6. Developmental Biology Spatiotemporal Subcellular Manipulation of the Microtubule Cytoskeleton in the Living Preimplantation Mouse Embryo using Photostatins Jessica Greaney1, Azelle Hawdon1, G. Gemma Stathatos1,2, Asma Aberkane1, Jennifer Zenker1 1Australian Regenerative Medicine Institute, Monash University, 2School of BioSciences, The University of Melbourne Typical microtubule inhibitors, used widely in basic and applied research, have far-reaching effects on cells. Recently, photostatins emerged as a class of photoswitchable microtubule inhibitors, capable of instantaneous, reversible, spatiotemporally precise manipulation of microtubules. This step-by-step protocol details the application of photostatins in a 3D live preimplantation mouse embryo. Biochemistry The Application of Open Searching-based Approaches for the Identification of Acinetobacter baumannii O-linked Glycopeptides Jessica M. Lewis*1, Pauline M. L. Coulon*1, Taylor A. McDaniels*1, Nichollas E. Scott1 1Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, The University of Melbourne Open searching enables the identification of glycopeptides decorated with previously unknown glycan compositions. Within this article, a streamlined approach for undertaking open searching and subsequent glycan-focused glycopeptide searches are presented for bacterial samples using Acinetobacter baumannii as a model. Medicine An In Vivo Mouse Model of Total Intravenous Anesthesia During Cancer Resection Surgery Julia A. Dubowitz1,2,3, Fabian Jost-Brinkmann1,4,5, Alexandra I. Ziegler1, Ryan D. Gillis1, Bernhard Riedel1,2,3,6, Erica K. Sloan1,2 1Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences, Monash University, 2Department of Anaesthesia, Division of Cancer Surgery, Peter MacCallum Cancer Centre, 3Department of Critical Care, Melbourne Medical School, University of Melbourne, 4Medical Department, Division of Hepatology and Gastroenterology, Charité - Universitätsmedizin Berlin, 5Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, 6Sir Peter MacCallum Department of Oncology, University of Melbourne This paper describes a method for modeling total intravenous anesthesia (TIVA) during cancer resection surgery in mice. The goal is to replicate key features of anesthesia delivery to patients with cancer. The method allows investigation of how anesthetic technique affects cancer recurrence after resection surgery. Neuroscience Dissection of Pelvic Autonomic Ganglia and Associated Nerves in Male and Female Rats Martin M. Bertrand1,2, Janet R. Keast1 1Department of Anatomy and Neuroscience, University of Melbourne, 2Department of Visceral Surgery, CHU de Nîmes The major pelvic ganglia contain parasympathetic and sympathetic neurons that innervate pelvic organs. Here we describe a dissection method and provide schematics for identification of these ganglia and their associated nerves. These methods can be applied to experimental manipulation of these ganglia in vivo or removal post-mortem for further study. Bioengineering Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior Francesco Carrara1, Douglas R. Brumley2, Andrew M. Hein3, Yutaka Yawata4,5, M. Mehdi Salek1, Kang Soo Lee1, Elzbieta Sliwerska1, Simon A. Levin6, Roman Stocker1 1Institute of Environmental Engineering, Department of Civil, Environmental and Geomatic Engineering, 2School of Mathematics and Statistics, University of Melbourne, 3Institute of Marine Sciences, University of California, Santa Cruz, 4Faculty of Life and Environmental Sciences, University of Tsukuba, 5Microbiology Research Center for Sustainability, University of Tsukuba, 6Department of Ecology and Evolutionary Biology, Princeton University A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level. Engineering Quantifying the Relative Thickness of Conductive Ferromagnetic Materials Using Detector Coil-Based Pulsed Eddy Current Sensors Nalika Ulapane1, Karthick Thiyagarajan2, David Hunt2, Jaime Valls Miro2 1Melbourne School of Engineering, University of Melbourne, 2Center for Autonomous Systems, University of Technology Sydney Here, we present a protocol to quantify the relative thickness (i.e., thickness as a percentage with respect to a reference) of conductive ferromagnetic materials using detector coil-based pulsed eddy current sensors, while overcoming the calibration requirement. Behavior An Instrumented Pull Test to Characterize Postural Responses Joy Tan1,2,4, Wesley Thevathasan2,3,4,5, Jennifer McGinley6, Peter Brown7, Thushara Perera1,4 1Department of Medical Bionics, The University of Melbourne, 2Department of Neurology, The Royal Melbourne Hospital, 3Department of Neurology, Austin Hospital, 4The Bionics Institute, 5Department of Medicine, The University of Melbourne, 6Department of Physiotherapy, The University of Melbourne, 7Medical Research Council Brain Network Dynamics Unit, University of Oxford Impairment of postural reflexes, termed postural instability, is difficult to quantify. Clinical assessments such as the pull test suffer issues with reliability and scaling. Here, we present an instrumented version of the pull test to objectively characterize postural responses. 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. Developmental Biology Electroretinogram Recording in Larval Zebrafish using A Novel Cone-Shaped Sponge-tip Electrode Jiaheng Xie1, Patricia R. Jusuf1, Patrick T. Goodbourn2, Bang V. Bui3 1School of Biosciences, University of Melbourne, 2Melbourne School of Psychological Sciences, University of Melbourne, 3Department of Optometry and Vision Sciences, University of Melbourne Here, we present a protocol that simplifies the measurement of light evoked electroretinogram responses from larval zebrafish. A novel cone-shaped sponge-tip electrode can help to make the study of visual development in larval zebrafish using the electroretinogram ERG easier to achieve with reliable outcomes and lower cost. Medicine An Improved and High Throughput Respiratory Syncytial Virus (RSV) Micro-neutralization Assay Lien Anh Ha Do1,2, Reuben Tse1, Jordan Nathanielsz1, Jeremy Anderson1, Darren Suryawijaya Ong1, Keith Chappell3, Kim Mulholland1,2,4, Paul V. Licciardi1,2 1 This study describes a high throughput, imaging-based micro-neutralization assay to determine the titer of neutralizing antibodies specific for respiratory syncytial virus (RSV). This assay format has been tested on different sample types. Immunology and Infection High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing Georges Khoury1, Damian F.J. Purcell1 1Department of Microbiology and Immunology, University of Melbourne A high throughput protocol for functional assessment of HIV efficient reactivation and clearance of latent proviruses is described and applied by testing the impact of interventions on HIV transcription and splicing. Representative results of the effect of latency reversing agents on LTR-driven transcription and splicing are provided. Chemistry Detection and Quantification of Plasmodium falciparum in Aqueous Red Blood Cells by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data Analysis Miguela Martin1, David Perez-Guaita1, Dean W. Andrew3, Jack S. Richards3,4, Bayden R. Wood1, Philip Heraud1,2 1Centre for Biospectroscopy, Monash University, 2Department of Microbiology, Faculty of Medicine, Nursing and Health Sciences, Monash University, 3Centre for Biomedical Research, Burnet Institute, 4Department of Medicine, University of Melbourne Here, we present a protocol for the detection and quantification of Plasmodium falciparum in infected aqueous red blood cells using an attenuated total reflection infrared spectrometer and multivariate data analysis. Medicine A Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice Zheng He1, Da Zhao1, Anna K. van Koeverden1, Christine T Nguyen1, Jeremiah K. H. Lim1, Vickie H. Y. Wong1, Algis J. Vingrys1, Bang V. Bui1 1Department of Optometry and Vision Sciences, University of Melbourne Chronic ocular hypertension is induced by applying a circumlimbal suture in rats and mice, leading to functional and structural deterioration of the retinal ganglion cells consistent with glaucoma. Neuroscience Operant Protocols for Assessing the Cost-benefit Analysis During Reinforced Decision Making by Rodents Mojtaba Kermani*1,3, Zahra Fatahi*2, Dechuan Sun3, Abbas Haghparast2, Chris French3,4 1Department of Optometry and Vision Science, The University of Melbourne, 2Neuroscience Research Center, Shahid Beheshti University of Medical Science, 3Department of Medicine, The University of Melbourne, 4Royal Melbourne Hospital A cost-benefit analysis is a weighing-scale approach that the brain performs during the course of decision making. Here, we propose a protocol to train rats on an operant-based decision-making paradigm where rats choose higher rewards at the expense of waiting for 15 s to receive them. Biology Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy Adam C. Parslow1,2, Andrew H.A. Clayton3, Peter Lock4, Andrew M. Scott1,2,5,6,7 1Tumour Targeting Laboratory, Olivia Newton-John Cancer Research Institute, 2School of Cancer Medicine, La Trobe University, 3Centre for Micro-Photonics, Faculty of Science, Engineering and Technology, Swinburne University of Technology, 4LIMS Bioimaging Facility, La Trobe Institute for Molecular Science, La Trobe University, 5Department of Medical Oncology, Olivia Newton-John Cancer and Wellnes Centre, Austin Health, 6Department of Medicine, University of Melbourne, 7Department of Molecular Imaging and Therapy, Austin Health Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface. Cancer Research Live Cell Imaging of the TGF- β/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System Hao Chen*1, Thomas M.B. Ware*1, Josephine Iaria1, Hong-Jian Zhu1 1Department of Surgery (RMH), University of Melbourne Here, we present a protocol for live cell imaging of TGF-β/Smad3 signaling activity using an adenovirus reporter system. This system tracks transcriptional activity in real-time and can be applied to both single cells in vitro and in live animalmodels. Bioengineering Force System with Vertical V-Bends: A 3D In Vitro Assessment of Elastic and Rigid Rectangular Archwires Madhur Upadhyay1, Raja Shah2, Sachin Agarwal3, Meenakshi Vishwanath4, Po-Jung Chen5, Takafumi Asaki6, Donald Peterson7 1Division of Orthodontics, University of Connecticut Health, 2Private Practice, 3Department of Orthodontics, University of Melbourne, 4Department of Orthodontics, University of Nebraska Medical Center, 5Department of Craniofacial Sciences, University of Connecticut Health, 6Biomedical Engineering, University of Hartford, 7Department of Mechanical Engineering, College of Engineering and Engineering Technology, Northern Illinois University The method presented here is designed to construct and validate an in vitro 3D model capable of measuring the force system generated by different archwires with V-bends placed between two brackets. Additional objectives are to compare this force system with different types of archwires and to previous models. Genetics Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry Sarah E. Ashley1,2, Braydon A. Meyer2,3, Justine A. Ellis2,3,4, David J. Martino2,3,5 1Molecular Genetics of Chronic Inflammation and Allergic Disease, Max-Delbrück Center for Molecular Medicine, 2 Identification of genetic variants contributing to complex human disease allows us to identify novel mechanisms. Here, we demonstrate a multiplex genotyping approach to candidate genes or gene pathway analysis that maximizes the coverage at low cost and is amenable to cohort-based studies. Bioengineering Creating a Structurally Realistic Finite Element Geometric Model of a Cardiomyocyte to Study the Role of Cellular Architecture in Cardiomyocyte Systems Biology Vijay Rajagopal1,2,3, Gregory Bass2,3, Shouryadipta Ghosh1,2,3, Hilary Hunt2,4, Cameron Walker5, Eric Hanssen6, Edmund Crampin2,3,4,7,8, Christian Soeller9 1Cell Structure and Mechanobiology Group, University of Melbourne, 2Systems Biology Laboratory, Melbourne School of Engineering, University of Melbourne, 3Department of Biomedical Engineering, University of Melbourne, 4School of Mathematics and Statistics, Faculty of Science, University of Melbourne, 5Department of Engineering Science, University of Auckland, 6Advanced Microscopy Facility, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, 7ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, University of Melbourne, 8School of Medicine, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, 9Living Systems Institute, University of Exeter This protocol outlines a novel method to create a spatially detailed finite element model of the intracellular architecture of cardiomyocytes from electron microscopy and confocal microscopy images. The power of this spatially detailed model is demonstrated using case studies in calcium signaling and bioenergetics. Neuroscience A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations Valerio Conti*1, Aurelie Carabalona*2,3,15, Emilie Pallesi-Pocachard2,3,4, Richard J. Leventer5,6,7, Fabienne Schaller2,3,8, Elena Parrini1, Agathe A. Deparis2,3, Françoise Watrin2,3, Emmanuelle Buhler2,3,8, Francesca Novara9, Stefano Lise10, Alistair T. Pagnamenta10, Usha Kini11, Jenny C. Taylor10, Orsetta Zuffardi9,12, Alfonso Represa2,3, David Antony Keays13, Renzo Guerrini1,14, Antonio Falace2,3, Carlos Cardoso2,3 1University of Florence, 2INSERM INMED, 3Aix-Marseille University, 4Plateforme Biologie Moléculaire et Cellulaire INMED, 5 Periventricular nodular heterotopia (PNH) is the most common form of malformation of cortical development (MCD) in adulthood but its genetic basis remains unknown in most sporadic cases. We have recently developed a strategy to identify novel candidate genes for MCDs and to directly confirm their causative role in vivo. Neuroscience A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments David G. Gonsalvez1, Jessica L. Fletcher1, Sang Won Yoo1, Rhiannon J. Wood1, Simon S. Murray1, Junhua Xiao1 1Department of Anatomy and Neuroscience, School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne This report outlines a simple approach to successfully induce experimental autoimmune neuritis (EAN) using the myelin protein zero (P0)180-199 peptide in combination with Freund's complete adjuvant and pertussis toxin. We present a sophisticated paradigm capable of accurately assessing the extent of functional deficits and neuropathology that occur in this EAN. Chemistry Measurements of Long-range Electronic Correlations During Femtosecond Diffraction Experiments Performed on Nanocrystals of Buckminsterfullerene Rebecca A. Ryan1, Sophie Williams1, Andrew V. Martin1, Ruben A. Dilanian1, Connie Darmanin2, Corey T. Putkunz1, David Wood3, Victor A. Streltsov4, Michael W.M. Jones5, Naylyn Gaffney6, Felix Hofmann7, Garth J. Williams8, Sebastien Boutet9, Marc Messerschmidt10, M. Marvin Seibert11, Evan K. Curwood11, Eugeniu Balaur2, Andrew G. Peele5, Keith A. Nugent2, Harry M. Quiney1, Brian Abbey2 1ARC Centre of Excellence in Advanced Molecular Imaging, School of Physics, University of Melbourne, 2Australian Research Council (ARC) Centre of Excellence in Advanced Molecular Imaging, Department of Chemistry and Physics, La Trobe Institute for Molecular Sciences, La Trobe University, 3Department of Physics, Imperial College London, 4Florey Institute of Neuroscience and Mental Health, 5Science and Engineering Faculty, Queensland University of Technology, 6Swinburne University of Technology, 7Department of Engineering Science, University of Oxford, 8Brookhaven National Laboratory, 9Linac Coherent Light Source, SLAC National Accelerator Laboratory, 10BioXFEL Science and Technology Center, 11Laboratory of Molecular Biophysics, Department of Cell and Molecular Biology, Uppsala University, 12Australian Synchrotron We describe an experiment designed to probe the electronic damage induced in nanocrystals of Buckminsterfullerene (C60) by intense, femtosecond pulses of X-rays. The experiment found that, surprisingly, rather than being stochastic, the X-ray induced electron dynamics in C60 are highly correlated, extending over hundreds of unit cells within the crystals1. Developmental Biology In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences Stefanie Dudczig1,2, Peter D. Currie2, Lucia Poggi3, Patricia R. Jusuf1,2 1School of Biosciences, The University of Melbourne, 2Australian Regenerative Medicine Institute (ARMI), Monash University, 3The David J Apple Center for Vision Research, Department of Ophthalmology, Heidelberg University Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose. Medicine Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS) Dominic J. Hare1,2, Kai Kysenius3, Bence Paul4, Beate Knauer5,6, Robert W. Hutchinson7, Ciaran O'Connor7, Fred Fryer8, Tom P. Hennessey8, Ashley I. Bush2, Peter J. Crouch3, Philip A. Doble1 1Elemental Bio-imaging Facility, University of Technology Sydney, 2Florey Institute of Neuroscience and Mental Health, The University of Melbourne, 3Department of Pathology, The University of Melbourne, 4School of Earth Sciences, The University of Melbourne, 5Research School, Ruhr University, 6Department of Physiology, Monash University, 7ESI Ltd., Bozeman, 8Agilent Technologies, Mulgrave Quantitatively mapping metals in tissue by laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) is a sensitive analytical technique that can provide new insight into how metals participate in normal function and disease processes. Here, we describe a protocol for quantitatively imaging metals in thin sections of mouse neurological tissue. Genetics The Use of Induced Somatic Sector Analysis (ISSA) for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development Antanas Spokevicius1, Lynette Taylor1, Emma Melder1, Kim Van Beveren1, Josquin Tibbits2, Nicky Creux3,4, Gerd Bossinger1 1School of Ecosystem and Forest Sciences, Faculty of Science, The University of Melbourne, 2Victorian AgriBiosciences Centre, La Trobe University R&D Park, 3College of Biological Sciences, Department of Plant Biology, University of California, Davis, 4Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria Here we present a protocol that facilitates the medium to high throughput functional characterization of gene and promoter constructs in tree secondary stem tissue within comparatively short time frames. It is efficient, easy to use and widely applicable to a range of tree species. Immunology and Infection A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood Clovis S. Palmer1,2,3, Joshua J. Anzinger4, Tiffany R. Butterfield4, Joseph M. McCune5, Suzanne M. Crowe1,2,6 1Centre for Biomedical Research, Macfarlane Burnet Institute for Medical Research and Public Health, 2Department of Infectious Diseases, Monash University, 3Department of Microbiology and Immunology, University of Melbourne, 4Department of Microbiology, The University of the West Indies, 5Division of Experimental Medicine, Department of Medicine, University of California, San Francisco, 6Department of Medicine, Monash University Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood. Immunology and Infection Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing Patrice Newton1, Eleanor A. Latomanski1, Hayley J. Newton1 1Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate. Neuroscience Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats Christine T. Nguyen1, Tina I. Tsai1, Zheng He1, Algis J. Vingrys1, Pei Y. Lee1, Bang V. Bui1 1Department of Optometry and Vision Sciences, University of Melbourne This protocol describes simultaneous measurement of electroretinogram and visual evoked potentials in anesthetized rats. Behavior Implantation and Recording of Wireless Electroretinogram and Visual Evoked Potential in Conscious Rats Jason Charng1, Zheng He1, Bang Bui1, Algis Vingrys1, Magnus Ivarsson1, Rebecca Fish2, Rachel Gurrell2, Christine Nguyen1 1Department of Optometry and Vision Sciences, University of Melbourne, 2Pfizer Neusentis We show surgical implantation and Recording procedures to measure visual electrophysiological signals from the eye (electroretinogram) and brain (visual evoked potential) in conscious rats, which is more analogous to the human condition where recordings are conducted without anesthesia confounds. Bioengineering Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber Weiqing Zhan*1, Diego Marre*1, Geraldine M. Mitchell1,2,3, Wayne A. Morrison1,2,3, Shiang Y. Lim1,2 1 This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling. Chemistry Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques Sunil Ratnayake1, Kristina Ford1, Antony Bacic1 1ARC Centre of Excellence in Plant Cell Walls, School of BioSciences, University of Melbourne This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR). Neuroscience Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice Mathusi Swaminathan*1, Elisa Hill-Yardin*1, Melina Ellis1, Matthew Zygorodimos2, Leigh A. Johnston3, Rachel M. Gwynne1, Joel C. Bornstein1 1Department of Physiology, The University of Melbourne, 2Melbourne School of Engineering, The University of Melbourne, 3Department of Electrical and Electronic Engineering, The University of Melbourne This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease. Medicine A Method of Trigonometric Modelling of Seasonal Variation Demonstrated with Multiple Sclerosis Relapse Data Tim Spelman1,2, Orla Gray3, Robyn Lucas4, Helmut Butzkueven1,2 1Department of Neurology, Royal Melbourne Hospital, 2Department of Medicine (RMH), The University of Melbourne, 3Department of Neurology, Ulster Hospital, 4National Centre for Epidemiology and Population Health, Australian National University Combining plot analysis with trigonometric regression is a robust method for exploring complex, cyclical phenomena such as relapse onset timing in multiple sclerosis (MS). This method enabled unbiased characterisation of seasonal trends in relapse onset permitting novel inferences around the influence of seasonal variation, ultraviolet radiation (UVR) and latitude. Developmental Biology Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes Sandy S.C. Hung1, Alice Pébay1, Raymond C.B. Wong1 1Centre for Eye Research Australia & Department of Ophthalmology, University of Melbourne This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors. Neuroscience Generation of Local CA1 γ Oscillations by Tetanic Stimulation Robert J. Hatch1, Christopher A. Reid1, Steven Petrou1 1The Florey Institute of Neuroscience and Mental Health, University of Melbourne Oscillations are fundamental network properties and are modulated by disease and drugs. Studying brain-slice oscillations allows characterization of isolated networks under controlled conditions. Protocols are provided for the preparation of acute brain slices for evoking CA1 γ oscillations. Medicine Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting Zerina Lokmic1,2, Elizabeth S. Ng1,3, Matthew Burton1, Edouard G. Stanley1,2,3, Anthony J. Penington1,2, Andrew G. Elefanty1,2,3 1 The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies. Medicine Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice Matthias Ernst1,2,3, Adele Preaudet1, Tracy Putoczki1,2 1The Walter and Eliza Hall Institute for Medical Research, 2The Department of Medical Biology, University of Melbourne, 3Olivia Newton-John Cancer Research Institute We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse. Medicine Techniques for Processing Eyes Implanted with a Retinal Prosthesis for Localized Histopathological Analysis: Part 2 Epiretinal Implants with Retinal Tacks David A.X. Nayagam1,2, Irfan Durmo3, Ceara McGowan1, Richard A. Williams2,4, Robert K. Shepherd1,5, Bionic Vision Australia Consortia, 1Bionics Institute, 2Department of Pathology, The University of Melbourne, 3Cochlear Limited, 4 Here we describe histological techniques for visualising ocular tissue directly adjacent to a metal epiretinal tack and retinal prosthesis. Neuroscience Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice Doris Tomas1, Augustinus H. Prijanto1, Emma L. Burrows1, Anthony J. Hannan1, Malcolm K. Horne1, Tim D. Aumann1 1Florey Institute of Neuroscience and Mental Health, The University of Melbourne This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice. Developmental Biology Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays Haley M. Peckham1, Anita H. Ferner1, Lauren Giuffrida1, Simon S. Murray1,2, Junhua Xiao1,2 1Department of Anatomy and Neuroscience, The University of Melbourne, 2The Florey Institute of Neuroscience and Mental Health Research, The University of Melbourne Here we present protocols that offer a flexible and strategic foundation for virally manipulating oligodendrocyte precursor cells to overexpress proteins of interest in order to specifically interrogate their role in oligodendrocytes via the in vitro model of central nervous system myelination. Immunology and Infection Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination Barbara D. Porter1, Belinda D. Ortika1, Catherine Satzke1,2 1Pneumococcal Research, Murdoch Childrens Research Institute, 2Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, The University of Melbourne Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping. Immunology and Infection High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays Danika L. Hill1,2, Emily M. Eriksson1,2, Louis Schofield1,2 1Division of Infection and Immunity, Walter and Eliza Hall Institute of Medical Research, 2Department of Medical Biology, University of Melbourne Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry. Environment Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry Colin Ruprecht1, Takayuki Tohge1, Alisdair Fernie1, Cara L. Mortimer2, Amanda Kozlo2, Paul D. Fraser2, Norma Funke1, Igor Cesarino3,4, Ruben Vanholme3,4, Wout Boerjan3,4, Kris Morreel3,4, Ingo Burgert5,6, Notburga Gierlinger5,6, Vincent Bulone7, Vera Schneider8, Andrea Stockero8, Juan Navarro-Aviñó9, Frank Pudel10, Bart Tambuyser11, James Hygate12, Jon Bumstead13, Louis Notley13, Staffan Persson1,14 1Max Planck Institute for Molecular Plant Physiology, 2School of Biological Sciences, Plant Molecular Science, Centre for Systems and Synthetic Biology, Royal Holloway, University of London, 3Department of Plant Systems Biology, VIB, 4Department of Plant Biotechnology and Bioinformatics, UGhent, 5Institute for Building Materials, ETH Zurich, 6Applied Wood Materials, EMPA, 7Division of Glycoscience, School of Biotechnology, AlbaNova University Center, Royal Institute of Technology (KTH), 8European Research and Project Office GmbH, 9ABBA Gaia S.L., 10Pflanzenöltechnologie, 11Capax Environmental Services, 12Green Fuels, 13Neutral Consulting Ltd, 14Plant Cell Biology Research Centre, School of Botany, University of Melbourne Plant biomass offers a renewable resource for multiple products, including fuel, feed, food, and a variety of materials. In this paper we investigate the properties of tobacco tree (Nicotiana glauca) and poplar as suitable sources for a biorefinery pipeline. Immunology and Infection Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay Eileen M. Dunne1, Zheng Q. Toh2, Mary John3, Jayne Manning1,4, Catherine Satzke*1,4, Paul Licciardi*2 1Pneumococcal Research, Murdoch Childrens Research Institute, 2Allergy & Immune Disorders, Murdoch Childrens Research Institute, 3Department of Otolaryngology, The University of Melbourne, 4Department of Microbiology & Immunology at the Peter Doherty Institute for Infection & Immunity, The University of Melbourne In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization. Immunology and Infection Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction Maha Habib1, Barbara D. Porter1, Catherine Satzke1,2 1Pneumococcal Research, Murdoch Childrens Research Institute, 2Department of Microbiology & Immunology, The University of Melbourne The Quellung reaction is the gold standard technique for serotyping Streptococcus pneumoniae. This technique utilizes a microscope and specific pneumococcal antisera and is commonly used in reference and research laboratories worldwide. Neuroscience Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons Nissa L. Carrodus*1, Kathleen Sue-Lyn Teng*1, Kathryn M. Munro1, Matthew J. Kennedy2, Jenny M. Gunnersen1,3 1Department of Anatomy & Neuroscience, The University of Melbourne, 2Department of Neurobiology, Duke University Medical Center, 3Florey Institute of Neuroscience & Mental Health, The University of Melbourne We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation. 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 Techniques for Processing Eyes Implanted With a Retinal Prosthesis for Localized Histopathological Analysis David A. X. Nayagam1,2,3, Ceara McGowan1, Joel Villalobos1, Richard A. Williams2,3, Cesar Salinas-LaRosa2,3, Penny McKelvie2,3, Irene Lo2,3, Meri Basa2,3, Justin Tan1, Chris E. Williams1,3,4 1Bionics Institute, 2 Techniques for visualizing retinal cytoarchitecture directly adjacent to individual electrodes within a retinal stimulator. Neuroscience Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation William G. A. Brown1, Karina Needham2, Bryony A. Nayagam2, Paul R. Stoddart1 1Biotactical Engineering, Faculty of Engineering and Industrial Science, Swinburne University of Technology, 2Department of Otolaryngology, The University of Melbourne Infrared nerve stimulation has been proposed as an alternative to electrical stimulation in a range of nerve types, including those associated with the auditory system. This protocol describes a patch clamp method for studying the mechanism of infrared nerve stimulation in a culture of primary auditory neurons. Chemistry Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies Pooja Sharma1,2, Mariam Kaywan-Lutfi1, Logesvaran Krshnan1,2, Eamon F. X. Byrne1,2, Melissa Joy Call1,2, Matthew Edwin Call1,2 1Structural Biology Division, The Walter and Eliza Hall Institute of Medical Research, 2The University of Melbourne Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications. Neuroscience Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster Christof Rickert1, Thomas Kunz1, Kerri-Lee Harris2, Paul Whitington2, Gerhard Technau1 1Institute of Genetics, University of Mainz, 2Department of Anatomy and Neuroscience, University of Melbourne We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy. Biology Glycan Profiling of Plant Cell Wall Polymers using Microarrays Isabel E. Moller1,2, Filomena A. Pettolino3, Charlie Hart1, Edwin R. Lampugnani1, William G.T. Willats4, Antony Bacic1,2 1Australian Centre of Excellence in Plant Cell Walls, School of Botany, University of Melbourne, 2Plant Cell Biology Research Centre, School of Botany, University of Melbourne, 3CSIRO Plant Industry, Black Mountain Laboratories, 4Department of Plant Biology and Biotechnology, University of Copenhagen A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts. Biology Extraction of Tissue Antigens for Functional Assays Andra Necula1, Rochna Chand1, Batool Albatat1, Stuart I. Mannering1,2 1Immunology and Diabetes Unit, St. Vincent's Institute of Medical Research, 2Department of Medicine, University of Melbourne A simple protocol for preparing extracts of human tissue to be used as a source of antigens in functional T-cell assays is described. This method allows T-cell responses to tissue-derived antigens to be measured in vitro. Biology ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells Sevgi Irtegun1, Yasmin M. Ramdzan1, Terrence D. Mulhern1, Danny M. Hatters1 1Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute The biarsenical dyes FlAsH and ReAsH bind specifically to tetracysteine motifs in proteins and can selectively label proteins in live cells. Recently this labeling strategy has been used to develop sensors for different protein conformations or oligomeric states. We describe the labeling approach and methods to quantitatively analyze binding. Immunology and Infection Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes Nancy Wang1, Richard Strugnell2, Odilia Wijburg2, Thomas Brodnicki1 1St Vincent’s Institute, Department of Medicine, The University of Melbourne, 2Department of Microbiology and Immunology, The University of Melbourne Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection. Bioengineering Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming Wah Chin Boon1, Karolina Petkovic-Duran2, Yonggang Zhu2, Richard Manasseh3, Malcolm K. Horne1, Tim D. Aumann1 1Florey Neuroscience Institutes and Centre for Neuroscience, University of Melbourne, 2Fluid Dynamics Group, CSIRO Materials Science and Engineering, 3Swinburne University of Technology, Faculty of Engineering and Industrial Sciences We describe a novel method for increasing cDNA yield from single-cell quantities of mRNA in otherwise standard laboratory reverse transcription reactions. The novelty resides in the use of a micromixer, which utilizes the phenomenon of acoustic microstreaming, to mix fluids at microliter scales more effectively than shaking, vortexing or trituration. Immunology and Infection Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice Kirsty R. Short1, Dimitri A. Diavatopoulos2, Patrick C. Reading1, Lorena E. Brown1, Kelly L. Rogers3, Richard A. Strugnell1, Odilia L.C. Wijburg1 1Department of Microbiology and Immunology, University of Melbourne, 2Laboratory of Pediatric Infectious Diseases, Radboud University Nijmegen Medical Centre, 3The Centre for Dynamic Imaging, The Walter and Eliza Hall Institute for Medical Research A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens. Biology Clonogenic Assay: Adherent Cells Haloom Rafehi1,2, Christian Orlowski1,2,3, George T. Georgiadis1, Katherine Ververis1,4, Assam El-Osta2,3, Tom C. Karagiannis1,2 1Epigenomic Medicine, BakerIDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 2Department of Pathology, The University of Melbourne, 3Epigenetics in Human Health and Disease, BakerIDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 4Department of Anatomy and Cellular Biology, The University of Melbourne The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells. Biology Evaluation of the Spatial Distribution of γH2AX following Ionizing Radiation Raja S. Vasireddy1,2,3, Michelle M. Tang1,2, Li-Jeen Mah2,3, George T. Georgiadis2, Assam El-Osta1,3, Tom C. Karagiannis2,3 1Epigenetics in Human Health and Disease, BakerIDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 2Epigenomic Medicine, BakerIDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 3Department of Pathology, University of Melbourne Microscopic analysis of γH2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced γH2AX formation within the nucleus. Biology Quantitation of γH2AX Foci in Tissue Samples Michelle M. Tang1,2, Li-Jeen Mah1,3, Raja S. Vasireddy1,2,3, George T. Georgiadis1, Assam El-Osta2,3, Simon G. Royce3,4,5, Tom C. Karagiannis1,3 1Epigenomic Medicine, Baker IDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 2Epigenetics in Human Health and Disease, Baker IDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 3Department of Pathology, The University of Melbourne, 4Department of Allergy and Immunology, Murdoch Children's Research Institute, Royal Children's Hospital, 5Department of Pediatrics, The University of Melbourne Quantitation of DNA double-strand breaks on the basis of γH2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of γH2AX foci in tissue samples. Biology Quantification of γH2AX Foci in Response to Ionising Radiation Li-Jeen Mah1,2, Raja S. Vasireddy1,2,3, Michelle M. Tang1,3, George T. Georgiadis1, Assam El-Osta2,3, Tom C. Karagiannis1,2 1Epigenomic Medicine, Baker IDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 2Department of Pathology, The University of Melbourne, 3Epigenetics in Human Health and Disease, Baker IDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct Quantification of DNA double-strand streaks using γH2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of γH2AX foci after exposure of cells to radiation.
