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University of Newcastle

24 ARTICLES PUBLISHED IN JoVE

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Bioengineering

Polymer Microarrays for High Throughput Discovery of Biomaterials
Andrew L. Hook 1, Chien-Yi Chang 2, Jing Yang 1, David J. Scurr 1, Robert Langer 3, Daniel G. Anderson 3, Steve Atkinson 2, Paul Williams 2, Martyn C. Davies 1, Morgan R. Alexander 1
1Laboratory of Biophysics and Surface Analysis, University of Nottingham , 2School of Molecular Medical Sciences, University of Nottingham , 3David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

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Medicine

Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer
Lucy Murtha 1, Damian McLeod 1, Neil Spratt 1
1Biomedical 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.

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Neuroscience

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy
Victoria W. K. Tung 1, Stefano Di Marco 1, Rebecca Lim 2, Alan M. Brichta 2, Aaron J. Camp 1
1Discipline of Biomedical Science, School of Medical Sciences, Sydney Medical School, University of Sydney, 2School of Biomedical Sciences and Pharmacy, University of Newcastle

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

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Biology

Phosphopeptide Analysis of Rodent Epididymal Spermatozoa
Mark A. Baker 1, Louise Hetherington 1, Anita Weinberg 1, Tony Velkov 2
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.

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Biology

Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography
Rebecca Lim 1,2, Marcus J. Zavou 1, Phillipa-Louise Milton 3, Siow Teng Chan 1, Jean L. Tan 1, Hayley Dickinson 1,2, Sean V. Murphy 4, Graham Jenkin 1,2, Euan M. Wallace 1,2
1The Ritchie Centre, Monash Institute of Medical Research, 2Department of Obstetrics and Gynaecology, Monash Medical Centre, 3Animal Resource Centre, Perth, Australia, 4Wake Forest Institute for Regenerative Medicine

The assessment of respiratory physiology has traditionally relied upon techniques, which require restraint or sedation of the animal. Unrestrained whole-body plethysmography, however, provides precise, non-invasive, quantitative analysis of respiratory physiology in animal models. In addition, the technique allows repeated respiratory assessment of mice allowing for longitudinal studies.

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Medicine

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications
Sean V. Murphy 1, Amritha Kidyoor 1, Tanya Reid 1, Anthony Atala 1, Euan M. Wallace 2, Rebecca Lim 2
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.

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Medicine

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease
Sean W. Mateer 1, Jocelle Cardona 1, Ellen Marks 1, Bridie J. Goggin 1, Susan Hua 1, Simon Keely 1
1School 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.

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Behavior

The Emotional Stroop Task: Assessing Cognitive Performance under Exposure to Emotional Content
Moshe Shay Ben-Haim 1, Paul Williams 2, Zachary Howard 2, Yaniv Mama 3, Ami Eidels 2, Daniel Algom 1
1School of Psychological Sciences, Tel-Aviv University, 2School of Psychology, University of Newcastle, 3Department of Behavioral Sciences, Ariel University

The emotional Stroop effect (ESE) is the result of longer naming latencies to ink colors of emotion words than those of neutral words. This report refers to potential sources of confounding and includes a modal experiment that provides the means to control for them.

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Genetics

In Vitro Transcription Assays and Their Application in Drug Discovery
Xiao Yang 1, Cong Ma 1,2
1School 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.

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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
1Medical 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.

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Bioengineering

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations
Poonam Sharma 1, Julianne D. Twomey 1, Michelle Patkin 1, Adam H. Hsieh 1
1Fischell Department of Bioengineering, University of Maryland

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

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Biology

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System
Karla A. Mettrick 1, Nikki Lawrence 1, Claire Mason 1, Georgia M. Weaver 1, Tayla-Ann Corocher 1, Ian Grainge 1
1School 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.

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Developmental Biology

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts
Manish Kumar 1, Pradeep Tanwar 1
1Gynaecology 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.

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Neuroscience

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation
Jessica C. Stark *1, Euan Wallace 1,2, Rebecca Lim 1, 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.

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Developmental Biology

Analysis of Epididymal Protein Synthesis and Secretion
Wei Zhou 1,2, Petra Sipilä 3, Geoffry N. De Iuliis 1,2, Matthew D. Dun 2,4, Brett Nixon 1,2
1Priority 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.

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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
1Medical 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.

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Cancer Research

A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research
Zhuo Li *1, Xiao Yang *1, Yilu Zhang 1, Xiaolong Yang 1, Xinxin Cui 1, Yanjuan Zhang 1, Wenfeng Gong 1, Huichen Bai 1, Ning Liu 1, Zhiyu Tang 1, Mingming Guo 1, Kang Li 1, Tong Zhang 1, Lai Wang 1, Xiaomin Song 1
1BeiGene (Beijing) Co., Ltd.

We describe a human peripheral blood mononuclear cell (PBMC) — based humanized xenograft mouse model for translational immuno-oncology research. This protocol could serve as a general guideline for establishing and characterizing similar models for I-O therapy assessment.

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Bioengineering

Automated Counterflow Centrifugal System for Small-Scale Cell Processing
Anqi Li 1,2, Stephen Wilson 3, Ian Fitzpatrick 3, Mehri Barabadi 1,2, Siow Teng Chan 1, Mirja Krause 1,2, Gina Diamanta Kusuma 1,2, David James 3, Rebecca Lim 1,2,4
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.

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Engineering

Development of Efficient OLEDs from Solution Deposition
Manish Kumar 1,2, Luiz Pereira 1
1Department of Physics and i3N, Institute for Nanostructures, Nanomodulation and Nanofabrication, University of Aveiro, 2CeNTI, Centre for Nanotechnologies and Smart Materials

Presented here is a protocol for the fabrication of efficient, simple, solution-deposited organic light-emitting diodes with low roll-off.

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Biochemistry

Cell-Free Production of Proteoliposomes for Functional Analysis and Antibody Development Targeting Membrane Proteins
Wei Zhou 1, Hiroyuki Takeda 2
1Graduate School of Medicine, Ehime University, 2Proteo-Science Center, Ehime University

This protocol describes an efficient cell-free method for production of high-quality proteoliposome by bilayer-dialysis method using wheat cell-free system and liposomes. This method provides suitable means for functional analysis of membrane proteins, drug targets screening, and antibody development.

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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
1University of Newcastle, 2University of Sydney, 3Kolling Institute of Medical Research, Royal North Shore Hospital, 4University of Technology, Sydney

This protocol aims to fabricate 3D cardiac spheroids (CSs) by co-culturing cells in hanging drops. Collagen-embedded CSs are treated with doxorubicin (DOX, a cardiotoxic agent) at physiological concentrations to model heart failure. In vitro testing using DOX-treated CSs may be used to identify novel therapies for heart failure patients.

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Medicine

Short-Duration Hypothermia Induction in Rats using Models for Studies examining Clinical Relevance and Mechanisms
Daniel Omileke 1, Steven Bothwell 1, Daniel J. Beard 1, Nikolce Mackovski 1, Sara Azarpeykan 1, Kirsten Coupland 1, Adjanie Patabendige 1, Neil Spratt 1,2
1School 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. 

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Neuroscience

Recording Network Activity in Spinal Nociceptive Circuits Using Microelectrode Arrays
Jacqueline A Iredale 1, Jeremy G. Stoddard 2, Hannah R. Drury 1, Tyler J. Browne 1, Augustus Elton 2, Jessica F. Madden 1, Robert J. Callister 1, James S. Welsh 2, Brett A. Graham 1
1School 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.

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Medicine

Assessing Intracardiac Vortices with High Frame-Rate Echocardiography-Derived Blood Speckle Imaging in Newborns
Edward Crendal 1, Koert De Waal 2, Damien Vitiello 3
1John Hunter Hospital, Department of Cardiology, University of Newcastle, 2John Hunter Children’s Hospital, Department of Neonatology, University of Newcastle, 3Institute of Sport and Health Sciences of Paris (IS3P - URP 3625), Université Paris Cité

The present protocol uses echocardiography-derived blood speckle imaging technology to visualize intracardiac hemodynamics in newborns. The clinical utility of this technology is explored, the rotational body of fluid within the left ventricle (known as a vortex) is accessed, and its significance in understanding diastology is determined.

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