University of Newcastle

15 articles published in JoVE

• Neuroscience

  
  Recording Network Activity in Spinal Nociceptive Circuits Using Microelectrode Arrays Jacqueline A Iredale¹, Jeremy G. Stoddard², Hannah R. Drury¹, Tyler J. Browne¹, Augustus Elton², Jessica F. Madden¹, Robert J. Callister¹, James S. Welsh², Brett A. Graham^{1 1}School of Biomedical Sciences and Pharmacy, University of Newcastle, ²School of Electrical Engineering, University of Newcastle The combined use of microelectrode array technology and 4-aminopyridine-induced chemical stimulation for investigating network-level nociceptive activity in the spinal cord dorsal horn is outlined.

• Medicine

  
  Short-Duration Hypothermia Induction in Rats using Models for Studies examining Clinical Relevance and Mechanisms Daniel Omileke¹, Steven Bothwell¹, Daniel J. Beard¹, Nikolce Mackovski¹, Sara Azarpeykan¹, Kirsten Coupland¹, Adjanie Patabendige¹, Neil Spratt^{1,2 1}School of Biomedical Sciences and Pharmacy, University of Newcastle, ²Department of Neurology, John Hunter Hospital, Hunter New England Local Health District This article describes two methods of whole-body short-duration hypothermia induction in rats. The first, rapid induction method, employs active cooling using fans and ethanol spray for a rapid decrease in temperature. The second method is a gradual cooling method. This is achieved using the combination of isoflurane anesthesia and the reduction of temperature settings on the homeothermic heat mat. This results in a gradual decrease in core body temperature without the use of any external cooling devices. 

• Bioengineering

  
  Cardiac Spheroids as in vitro Bioengineered Heart Tissues to Study Human Heart Pathophysiology Poonam Sharma^1,2,3,4, Carmine Gentile^{2,3,4 1}University of Newcastle, ²University of Sydney, ³Kolling Institute of Medical Research, Royal North Shore Hospital, ⁴University of Technology, Sydney This protocol aims to fabricate 3D cardiac spheroids (CSs) by co-culturing cells in hanging drops. Collagen-embedded CSs are treated with doxorubicin (DOX, a cardiotoxic agent) at physiological concentrations to model heart failure. In vitro testing using DOX-treated CSs may be used to identify novel therapies for heart failure patients.

• Cancer Research

  
  A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager Xiajie Zhang^1,2, Brianna C. Morten^1,2, Rodney J. Scott^1,2,3, Kelly A. Avery-Kiejda^{1,2 1}Medical Genetics, Hunter Medical Research Institute, ²Priority Research Centre for Cancer Research, Innovation and Translation, School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, University of Newcastle, ³Pathology North, John Hunter Hospital The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

• Developmental Biology

  
  Analysis of Epididymal Protein Synthesis and Secretion Wei Zhou^1,2, Petra Sipilä³, Geoffry N. De Iuliis^1,2, Matthew D. Dun^2,4, Brett Nixon^{1,2 1}Priority Research Centre for Reproductive Science, School of Environmental and Life Sciences, University of Newcastle, ²Hunter Medical Research Institute, ³Department of Physiology, Turku Center for Disease Modeling, Institute of Biomedicine, University of Turku, ⁴School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, University of Newcastle Here, we report the immunofluorescence localization of dynamin to illustrate the protocols for the detection of proteins in paraffin-embedded mouse epididymal sections and those of an immortalized epididymal cell line (mECap18). We also describe the protocols for the isolation of secretory proteins from both epididymal fluid and conditioned cell media.

• Developmental Biology

  
  Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts Manish Kumar¹, Pradeep Tanwar^{1 1}Gynaecology Oncology Group, School of Biomedical Sciences and Pharmacy, University of Newcastle We present a method for isolation and culture of the mouse Wolffian duct (WD). We also demonstrate a detailed procedure for whole mount immunostaining of cultured/freshly isolated WDs with fluorescently tagged antibodies. Together, these techniques enable the study of WD development, coiling, and differentiation.

• Genetics

  
  In Vitro Transcription Assays and Their Application in Drug Discovery Xiao Yang¹, Cong Ma^{1,2 1}School of Environmental and Life Sciences, University of Newcastle, ²Department of Applied Biology and Chemical Technology, The State Key Laboratory of Chirosciences, The Hong Kong Polytechnic University In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

• Biology

  
  Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines Brianna C. Morten^1,2, Rodney J. Scott^1,2,3, Kelly A. Avery-Kiejda^{1,2 1}Medical Genetics, Hunter Medical Research Institute, ²Priority Research Centre for Cancer, School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, University of Newcastle, ³Pathology North, John Hunter Hospital This protocol describes the use of three different methods for analyzing cell proliferation in breast cancer cell lines. This includes the use of conventional cell counting, luminescence-based cell viability, and cell counting through the use of a cell imager. Each offers advantages for the reproducible measurement of cell proliferation.

• Biology

  
  Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System Karla A. Mettrick¹, Nikki Lawrence¹, Claire Mason¹, Georgia M. Weaver¹, Tayla-Ann Corocher¹, Ian Grainge^{1 1}School of Environmental and Life Sciences, University of Newcastle We describe here a system utilizing a site-specific, reversible in vivo protein block to stall and collapse replication forks in Escherichia coli. The establishment of the replication block is evaluated by fluorescence microscopy and neutral-neutral 2-dimensional agarose gel electrophoresis is used to visualize replication intermediates.

• Medicine

  
  Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease Sean W. Mateer¹, Jocelle Cardona¹, Ellen Marks¹, Bridie J. Goggin¹, Susan Hua¹, Simon Keely^{1 1}School of Biomedical Science and Pharmacy, University of Newcastle This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

• Developmental Biology

  
  Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula Sergey Kurdyukov¹, Youhong Song¹, Terence W-Y Tiew¹, Xin-Ding Wang¹, Kim E. Nolan¹, Ray J. Rose^{1 1}School of Environmental and Life Sciences, University of Newcastle, Callaghan, New South Wales, Australia The goal is to illustrate that the model legume Medicago truncatula can be readily utilized to investigate the regulation of early plant embryogenesis to complement the non-legume Arabidopsis model. Pod morphology is linked to zygotic embryogenesis stages and a protocol to collect embryos using tissue culture is also provided.

• Biology

  
  Phosphopeptide Analysis of Rodent Epididymal Spermatozoa Mark A. Baker¹, Louise Hetherington¹, Anita Weinberg¹, Tony Velkov^{2 1}School 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

  
  Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke Kathryn A. Skelding¹, Jacinta M. Arellano², David A. Powis³, John A. Rostas^{1 1}School of Biomedical Sciences and Pharmacy, Hunter Medical Research Institute, The University of Newcastle, ²School of Health and Human Sciences, Southern Cross University, ³School of Medicine and Public Health, The University of Newcastle We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury. This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

• Neuroscience

  
  An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy Victoria W. K. Tung¹, Stefano Di Marco¹, Rebecca Lim², Alan M. Brichta², Aaron J. Camp^{1 1}Discipline of Biomedical Science, School of Medical Sciences, Sydney Medical School, University of Sydney, ²School of Biomedical Sciences and Pharmacy, University of Newcastle Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

• Medicine

  
  Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer Lucy Murtha¹, Damian McLeod¹, Neil Spratt^{1 1}Biomedical Sciences and Pharmacy, The University of Newcastle A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.