Monash University View Institution's Website 57 articles published in JoVE 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. 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. Neuroscience A Standardized Pipeline for Examining Human Cerebellar Grey Matter Morphometry using Structural Magnetic Resonance Imaging Rebecca Kerestes1, Shuo Han2, Srinivas Balachander3, Carlos Hernandez-Castillo4, Jerry L. Prince5,6, Jörn Diedrichsen7, Ian H. Harding1,8 1Department of Neuroscience, Central Clinical School, Monash University, 2Department of Biomedical Engineering, The Johns Hopkins University, 3Department of Psychiatry, National Institute of Mental Health & Neuro Sciences (NIMHANS), 4Faculty of Computer Science, Dalhousie University, 5Department of Electrical and Computer Engineering, The Johns Hopkins University, 6Department of Computer Science, The Johns Hopkins University, 7Brain and Mind Institute, Department for Statistical and Actuarial Sciences, Department for Computer Science, Western University, 8Monash Biomedical Imaging, Monash University A standardized pipeline is presented for examining cerebellum grey matter morphometry. The pipeline combines high-resolution, state-of-the-art approaches for optimized and automated cerebellum parcellation and voxel-based registration of the cerebellum for volumetric quantification. 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. Immunology and Infection Assessing Respiratory Immune Responses to Haemophilus Influenzae Lovisa Dousha1,2, Roleen Sharma1,2, Steven Lim2,3, James Ngui2,4, Ashley M. Buckle5, Paul T. King1,2 1Monash Lung and Sleep, Monash Medical Centre, 2Monash University Department of Medicine, Monash Medical Centre, 3Flow Cytometry Science Technology Platform, Francis Crick Institute, 4Clinical Immunology, Monash Medical Centre, 5Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University Haemophilus influenzae induces inflammation in the respiratory tract. This article will focus on the use of flow cytometry and confocal microscopy to define immune responses by phagocytes and lymphocytes in response to this bacterium. 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. Developmental Biology Examining Muscle Regeneration in Zebrafish Models of Muscle Disease Margo Montandon1, Peter D. Currie1, Avnika A. Ruparelia1 1Australian Regenerative Medicine Institute, Monash University Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease. Developmental Biology Accurate Follicle Enumeration in Adult Mouse Ovaries Amy L. Winship1, Urooza C. Sarma1, Lauren R. Alesi1, Karla J. Hutt1 1Development and Stem Cells Program, Monash Biomedicine Discovery Institute, and Department of Anatomy and Developmental Biology, Monash University Here, we describe, compare, and contrast two different techniques for accurate follicle counting of fixed mouse ovarian tissues. Developmental Biology Transillumination-Assisted Dissection of Specific Stages of the Mouse Seminiferous Epithelial Cycle for Downstream Immunostaining Analyses Juho-Antti Mäkelä1, Sheyla Cisneros-Montalvo1, Tiina Lehtiniemi1, Opeyemi Olotu1, Hue M. La3,4, Jorma Toppari1,2, Robin M. Hobbs3,4, Martti Parvinen1, Noora Kotaja1 1Institute of Biomedicine, Integrative Physiology and Pharmacology Unit, University of Turku, Turku, Finland, 2Department of Pediatrics, Turku University Hospital, Turku, Finland, 3Centre for Reproductive Health, Hudson Institute of Medical Research, Melbourne, VIC 3168, Australia, 4Australian Regenerative Medicine Institute, Monash University, Melbourne, VIC 3800, Australia This protocol describes transillumination-assisted microdissection of segments of adult mouse seminiferous tubules representing specific stages of seminiferous epithelial cycle, and cell types therein, and subsequent immunostaining of squash preparations and intact tubule segments. Chemistry Atomic Force Microscopy Combined with Infrared Spectroscopy as a Tool to Probe Single Bacterium Chemistry Kamila Kochan1, Anton Y. Peleg2,3, Philip Heraud1,2, Bayden R. Wood1 1Centre for Biospectroscopy and School of Chemistry, Monash University, 2Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Microbiology, Monash University, 3Department of Infectious Diseases, The Alfred Hospital and Central Clinical School, Monash University Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) provides a powerful platform for bacterial studies, enabling to achieve nanoscale resolution. Both, mapping of subcellular changes (e.g., upon cell division) as well as comparative studies of chemical composition (e.g., arising from drug resistance) can be conducted at a single cell level in bacteria. Immunology and Infection Supervised Machine Learning for Semi-Quantification of Extracellular DNA in Glomerulonephritis Kim Maree O'Sullivan1, Sarah Creed2, Poh-Yi Gan1, Stephen R. Holdsworth1,3 1Department of Medicine, Monash University, 2Monash Micro Imaging, Monash University, 3Immunology Department, Monash Health Extracellular DNA (ecDNA) released during cell death is proinflammatory and contributes to inflammation. Measurement of ecDNA at the site of injury can determine the efficacy of therapeutic treatment in the target organ. This protocol describes the use of a machine learning tool to automate measurement of ecDNA in kidney tissue. Neuroscience Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae Gonçalo M. Poças1,2, Pedro M. Domingos*2, Christen K. Mirth*1 1School of Biological Sciences, Monash University, 2Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA) Described here is a protocol that enables the colorimetric quantification of the amount of food eaten within a defined interval of time by Drosophila melanogaster larvae exposed to diets of different macronutrient quality. These assays are conducted in the context of a neuronal thermogenetic screen. Behavior Human Circadian Phenotyping and Diurnal Performance Testing in the Real World Elise R. Facer-Childs1,2,3, Benita Middleton1, Andrew P. Bagshaw2, Debra J. Skene1 1Chronobiology, Faculty of Health & Medical Sciences, University of Surrey, 2Centre for Human Brain Health, University of Birmingham, 3Turner Institute for Brain and Mental Health, School of Psychological Sciences, Monash University Here, we present a method to investigate diurnal rhythms in performance following accurate categorization of participants into circadian phenotype groups based on the Munich ChronoType Questionnaire, gold standard circadian phase biomarkers and actigraphic measures. Chemistry Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly Samuel C. Leguizamon1, Abdulla F. Alqubati1, Timothy F. Scott2,3 1Department of Chemical Engineering, University of Michigan, 2Department of Chemical Engineering, Monash University, 3Department of Materials Science and Engineering, Monash University A protocol is presented for the synthesis of information-encoded peptoid oligomers and for the sequence-directed self-assembly of these peptoids into molecular ladders using amines and aldehydes as dynamic covalent reactant pairs and Lewis acidic rare-earth metal triflates as multi-role reagents. Bioengineering Automated Counterflow Centrifugal System for Small-Scale Cell Processing Anqi Li1,2, Stephen Wilson3, Ian Fitzpatrick3, Mehri Barabadi1,2, Siow Teng Chan1, Mirja Krause1,2, Gina Diamanta Kusuma1,2, David James3, Rebecca Lim1,2,4 1The Ritchie Centre, Hudson Institute of Medical Research, 2Department of Obstetrics and Gynaecology, Monash University, 3Scinogy, 4Australian Regenerative Medicine Institute, Monash University Automation is key to upscaling and cost management in cell manufacturing. This manuscript describes the use of a counterflow centrifugal cell processing device for automating the buffer exchange and cell concentration steps for small-scale bioprocessing. Behavior Radiotracer Administration for High Temporal Resolution Positron Emission Tomography of the Human Brain: Application to FDG-fPET Sharna D. Jamadar1,2,3, Phillip G.D. Ward1,2,3, Alexandra Carey1,4, Richard McIntyre1,4, Linden Parkes1,3, Disha Sasan1, John Fallon1, Edwina Orchard1,2,3, Shenpeng Li1,5, Zhaolin Chen1,5, Gary F Egan1,2,3 1Monash Biomedical Imaging, Monash University, 2Australian Research Council Centre of Excellence for Integrative Brain Function, 3Turner Institute for Brain and Mental Health, Monash University, 4Department of Medical Imaging, Monash Health, 5Department of Electrical and Computer Systems Engineering, Monash University This manuscript describes two radiotracer administration protocols for FDG-PET (constant infusion and bolus plus infusion) and compares them to bolus administration. Temporal resolutions of 16 s are achievable using these protocols. Biology In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells Niels Alberts*1, Yasith Mathangasinghe*2, Nadinath B. Nillegoda3 1Department of Biomedical Sciences of Cells & Systems, University of Groningen, 2Department of Anatomy, Faculty of Medicine, University of Colombo, 3Australian Regenerative Medicine Institute (ARMI), Monash University Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells. Biology Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans Jean-Sébastien Teoh*1, Ming S. Soh*1, Joseph J. Byrne*1, Brent Neumann1 1Neuroscience Program, Monash Biomedicine Discovery Institute and Department of Anatomy and Developmental Biology, Monash University, Melbourne VIC 3800, Australia. This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in C. elegans to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations. Developmental Biology Culturing and Measuring Fetal and Newborn Murine Long Bones Veronica Uribe1, Alberto Rosello-Diez1 1Australian Regenerative Medicine Institute, Monash University Here, we present a method for ex vivo culture of long murine bones at both fetal and newborn stages, suitable for analyzing bone and cartilage development and homeostasis in controlled conditions while recapitulating the in vivo process. Bioengineering Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA Tatjana Michel1, Antonia Link1, Meike-Kristin Abraham1,2, Christian Schlensak1, Karlheinz Peter2,3, Hans-Peter Wendel1, Xiaowei Wang2,3, Stefanie Krajewski1 1Department of Thoracic and Cardiovascular Surgery, Clinical Research Laboratory, University Medical Center, 2Atherothrombosis and Vascular Biology, Baker Heart & Diabetes Institute, 3Department of Medicine, Monash University Here we describe a protocol for the generation of cationic nanoliposomes, which is based on the dry-film method and can be used for the safe and efficient delivery of in vitro transcribed messenger RNA. 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. Immunology and Infection Mouse Models Of Helicobacter Infection And Gastric Pathologies Kimberley D'Costa1, Michelle Chonwerawong1, Le Son Tran1, Richard L. Ferrero1,2 1Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, 2Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Microbiology, Monash University Mice represent an invaluable in vivo model to study infection and diseases caused by gastrointestinal microorganisms. Here, we describe the methods used to study bacterial colonization and histopathological changes in mouse models of Helicobacter pylori-related disease. Medicine Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease Louise Benning1, Samuel Robinson1,2, Marie Follo3, Lukas Andreas Heger1, Daniela Stallmann1, Daniel Duerschmied1, Christoph Bode1, Ingo Ahrens1,4, Marcus Hortmann1 1Department of Cardiology and Angiology I, Heart Center Freiburg University, Faculty of Medicine, University of Freiburg, 2Department of Medicine, Monash University, 3Department of Medicine I, Lighthouse Core Facility, Medical Center, University of Freiburg, 4Department of Cardiology, Augustinerinnen Hospital, Academic Teaching Hospital, University of Cologne Circulating microRNAs have shown promise as biomarkers for cardiovascular diseases and acute myocardial infarctions. In this study, we describe a protocol for miRNA extraction, reverse transcription, and digital PCR for the absolute quantification of miRNAs in the serum of patients with cardiovascular disease. Developmental Biology Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton Sandeep Gopal1, Roger Pocock1 1Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Department of Anatomy and Developmental Biology, Monash University We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure. Neuroscience Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation Jessica C. Stark*1, Euan Wallace1,2, Rebecca Lim1, Bryan Leaw*1 1The Ritchie Centre, Hudson Institute of Medical Research, 2Department of Obstetrics & Gynaecology, Monash University A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia. Biochemistry Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain Mayra A. Machuca1,2, Anna Roujeinikova1,2,3 1Infection and Immunity Program, Monash Biomedicine Discovery Institute, 2Department of Microbiology, Monash University, 3Department of Biochemistry and Molecular Biology, Monash University A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein. Immunology and Infection Visualizing Macrophage Extracellular Traps Using Confocal Microscopy Roleen Sharma1,2, Kim M. O'Sullivan2, Stephen R. Holdsworth2, Philip G. Bardin1,2, Paul T. King1,2 1Monash Lung and Sleep, Monash Medical Centre, 2Monash University Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples. Immunology and Infection Quantification of Monocyte Transmigration and Foam Cell Formation from Individuals with Chronic Inflammatory Conditions Thomas A. Angelovich1,2, Anna C. Hearps1,3, Anna Maisa1, Theodoros Kelesidis4, Anthony Jaworowski1,3 1Centre for Biomedical Research, Burnet Institute, 2School of Health and Biomedical Sciences, RMIT University, 3Department of Infectious Diseases, Monash University, 4University of California, Los Angeles We describe a protocol to measure transmigration by monocytes across human endothelial monolayers and their subsequent maturation into foam cells. This provides a versatile method to assess the atherogenic properties of monocytes isolated from people with different disease conditions and to evaluate factors in blood which may enhance this propensity. Medicine Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients Elena K. Schneider1, Felisa Reyes-Ortega1, Jian Li1,2, Tony Velkov1 1Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 2Monash Biomedicine Discovery Institute, Department of Microbiology, Monash University Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor. Immunology and Infection Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein Shubhendu Sen Roy1, Huy Cong Xuan Nguyen1, Thomas A. Angelovich2,3, Anna C. Hearps2, Diana Huynh1,4, Anthony Jaworowski2,4, Theodoros Kelesidis1 1University of California, Los Angeles, 2Centre for Biomedical Research, Burnet Institute, 3School of Health and Biomedical Sciences, RMIT University, 4Department of Infectious Diseases, Monash University We describe here a fluorometric cell-free biochemical assay for determination of HDL-lipid peroxidation. This rapid and reproducible assay can be used to determine HDL function in large scale studies and can contribute to our understanding of HDL function in human disease. Biochemistry Microcrystallography of Protein Crystals and In Cellulo Diffraction Marion Boudes*1, Damià Garriga*1, Fasséli Coulibaly1 1Infection and Immunity Program, Monash Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University A protocol is presented for X-ray crystallography using protein microcrystals. Two examples analyzing in vivo-grown microcrystals after purification or in cellulo are compared. Medicine Ovine Lumbar Intervertebral Disc Degeneration Model Utilizing a Lateral Retroperitoneal Drill Bit Injury Kai-Zheong Lim*1, Christopher D. Daly*1,2,3, Peter Ghosh4, Graham Jenkin3, David Oehme5, Justin Cooper-White6,7, Taryn Naidoo6, Tony Goldschlager1,8 1Department of Surgery, Monash University, 2Department of Neurosurgery, Monash University, 3The Ritchie Centre, Hudson Institute of Medical Research, 4Proteobioactives, Pty Ltd, 5 Intervertebral disc degeneration is a significant contributor to back pain and a leading cause of disability worldwide. Numerous animal models of intervertebral disc degeneration exist. We demonstrate an ovine model of intervertebral disc degeneration, utilizing a drill bit, which achieves a consistent disc injury and reproducible level of disc degeneration. Developmental Biology Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange Tim Pieters1,2,3,4, Lieven Haenebalcke1,2, Kenneth Bruneel1,2,4, Niels Vandamme1,2,4, Tino Hochepied1,2, Jolanda van Hengel5, Dagmar Wirth6, Geert Berx1,2,4, Jody J. Haigh7, Frans van Roy1,2,4, Steven Goossens1,2,3,4 1Department of Biomedical Molecular Biology, Ghent University, 2Inflammation Research Center, VIB, 3Center for Medical Genetics, Ghent University Hospital, 4Cancer Research Institute Ghent (CRIG), 5Department of Basic Medical Sciences, Faculty of Medicine and Health Sciences, Ghent University, 6Helmholtz Center for Infection Research, 7Mammalian Functional Genetics Laboratory, Division of Blood Cancers, Australian Centre for Blood Diseases, Department of Clinical Haematology, Monash University and Alfred Health Alfred Centre Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein. 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. Biology Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy Matthew McKenzie1,2, Sze C. Lim1, Michael R. Duchen3 1Centre for Genetic Diseases, Hudson Institute of Medical Research, 2The Department of Molecular and Translational Sciences, Monash University, 3Department of Physiology, University College London Mitochondria can utilize the electrochemical potential across their inner membrane (ΔΨm) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and ΔΨm in live cells using fluorescent dyes and confocal microscopy. 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. Developmental Biology Using Touch-evoked Response and Locomotion Assays to Assess Muscle Performance and Function in Zebrafish Tamar E. Sztal*1, Avnika A. Ruparelia*1, Caitlin Williams1, Robert J. Bryson-Richardson1 1School of Biological Sciences, Monash University Zebrafish are an excellent model to study muscle function and disease. During early embryogenesis zebrafish begin regular muscle contractions producing rhythmic swimming behavior, which is altered when the muscle is disrupted. Here we describe a touch-evoked response and locomotion assay to examine swimming performance as a measure of muscle function. 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. 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. Immunology and Infection The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport Natalie L. Trevaskis1, Luojuan Hu1, Suzanne M. Caliph1, Sifei Han1, Christopher J.H. Porter1 1Drug Delivery, Disposition, and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus) Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules. Biology Phosphopeptide Analysis of Rodent Epididymal Spermatozoa Mark A. Baker1, Louise Hetherington1, Anita Weinberg1, Tony Velkov2 1School of Environmental and Life Science, University of Newcastle, 2Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University Proteomic analysis of any cell type is highly dependent on both purity and pre-fractionation of the starting material in order to de-complexify the sample prior to liquid chromatography mass spectrometry (MS). By using back-flushing techniques, pure spermatozoa can be obtained from rodents. Following digestion, phosphopeptides can be enriched using TiO2. Medicine Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications Sean V. Murphy1, Amritha Kidyoor1, Tanya Reid1, Anthony Atala1, Euan M. Wallace2, Rebecca Lim2 1Wake Forest Institute for Regenerative Medicine, Wake Forest University Health Sciences, 2The Ritchie Centre, Monash Institute of Medical Research, Monash University We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines. Biology Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model Christian M. Nefzger*1,2, Sara Alaei*1,2, Anja S. Knaupp1,2, Melissa L. Holmes1,2, Jose M. Polo1,2 1Department of Anatomy and Developmental Biology, Monash University, 2Australian Regenerative Medicine Institute, Monash University Mouse embryonic fibroblast can be reprogrammed into induced pluripotent stem cells at low efficiency by the forced expression of transcription factors Oct-4, Sox-2, Klf-4, c-Myc. The rare intermediates of the reprogramming reaction are FACS isolated via labeling with antibodies against cell surface makers Thy-1.2, Ssea-1, and Epcam. Biology Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells Brigham J. Hartley1, Stewart A. Fabb1, Ben A.L. Finnin1, John M. Haynes1, Colin W. Pouton1 1Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University Reporter cell lines offer a means to visualize, track and isolate cells of interest from heterogeneous populations. However, gene-targeting using conventional homologous recombination in human embryonic stem cells is extremely inefficient. Herein, we describe targeting CNS midbrain specific transcription factor PITX3 locus with EGFP using zinc-finger nuclease enhanced homologous recombination. Medicine Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time Maree Bilandzic1, Kaye L. Stenvers1,2 1Reproductive Development and Cancer Laboratory, MIMR-PHI Institute of Medical Research, 2Department of Developmental Biology and Anatomy, Monash University Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process. Bioengineering Sample Drift Correction Following 4D Confocal Time-lapse Imaging Adam Parslow1, Albert Cardona2, Robert J. Bryson-Richardson1 1School of Biological Sciences, Monash University, 2Janelia Farm Research Campus, Howard Hughes Medical Institute Time-lapse microscopy allows the visualization of developmental processes. Growth or drift of samples during image acquisition reduces the ability to accurately follow and measure cell movements during development. We describe the use of open source image processing software to correct for three dimensional sample drift over time. 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. Medicine Intramyocardial Cell Delivery: Observations in Murine Hearts Tommaso Poggioli1,2, Padmini Sarathchandra1,2, Nadia Rosenthal2,3, Maria P. Santini1,2 1Magdi Yacoub Institute, Imperial College London, 2National Heart and Lung Institute, Imperial College London, 3Australian Regenerative Medicine Institute, Monash University Intramyocardial cell delivery in murine models of cardiovascular diseases, such as hypertension or myocardial infarction, is widely used to test the therapeutic potential of different cell types in regenerative studies. Therefore, a detailed description and a clear visualization of this surgical procedure will help to define the limits and advantages of cardiovascular cell therapeutic analyses in small rodents. Bioengineering Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier Be'eri Niego1, Robert L. Medcalf1 1Australian Centre for Blood Diseases, Monash University Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane. Medicine Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction Ryan Hodges1,2, Masayuki Endo1, Andre La Gerche3, Elisenda Eixarch4,5, Philip DeKoninck1, Vessilina Ferferieva3, Jan D'hooge3, Euan M. Wallace2, Jan Deprest1 1Division Woman and Child, Department Women, University Hospitals Leuven, 2The Ritchie Centre, Monash Institute of Medical Research, Department of Obstetrics and Gynaecology, Monash University, Victoria, Australia, 3Department of Cardiovascular Sciences, Katholieke Universiteit Leuven, 4 We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit. Medicine Bioluminescent Orthotopic Model of Pancreatic Cancer Progression Ming G. Chai1, Corina Kim-Fuchs1,2, Eliane Angst2, Erica K. Sloan1,3 1Monash Institute of Pharmaceutical Sciences, Monash University, 2Department of Visceral Surgery and Medicine, University of Bern, 3Cousins Center for Neuroimmunology, University of California Los Angeles Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging. Immunology and Infection Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens Erica L. Benard1, Astrid M. van der Sar2, Felix Ellett3, Graham J. Lieschke3, Herman P. Spaink1, Annemarie H. Meijer1 1Department of Molecular Cell Biology, Institute of Biology, Leiden University, 2Department of Medical Microbiology and Infection Control, VU University Medical Center, 3Australian Regenerative Medicine Institute, Monash University Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models. Immunology and Infection Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure Danielle L. Michell1, Karen L. Andrews1, Kevin J. Woollard1, Jaye P.F. Chin-Dusting1 1Vascular Pharmacology Laboratory, Baker IDI Heart and Diabetes Institute, Monash University This is a method to visualise leukocyte adhesion to the endothelium in harvested pressurised vessels. The technique enables studying vascular adhesion under shear flow with differing intraluminal pressures up to 200 mmHg thus mimic-ing the pathophysiological conditions of high blood pressure. Biology A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae Dalibor Mijaljica1, Mark Prescott1, Rodney J. Devenish1 1Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy. Biology Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis Tony Goldschlager1, Amany Abdelkader1, Jeffrey Kerr2, Ian Boundy2, Graham Jenkin1 1Monash Immunology and Stem Cell Laboratories, Monash University, 2Anatomy and Developmental Biology, Monash University Undecalcified bone histology provides important information for a variety of clinical and research applications. It is technically challenging, particularly with large size specimens. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them. Biology Anterior Cervical Discectomy and Fusion in the Ovine Model Tony Goldschlager1,2, Jeffrey V. Rosenfeld2, Ian R. Young1, Graham Jenkin1 1Monash Immunology and Stem Cell Laboratories (MISCL), Monash University, 2Department of Surgery, Monash University This video demonstrates the technique of anterior cervical discectomy and fusion in the ovine model.