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University of Sydney

46 ARTICLES PUBLISHED IN JoVE

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Biology

Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)
Matthew V. N. O'Sullivan 1, Fei Zhou 1, Vitali Sintchenko 1, Fanrong Kong 1, Gwendolyn L. Gilbert 1
1Centre for Infectious Diseases and Microbiology, University of Sydney

An inexpensive, high throughput method for simultaneous detection of up to 43 molecular targets is described. Applications of mPCR/RLB include microbial typing and detection of multiple pathogens from clinical samples.

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Immunology and Infection

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing
Jerry Zhou 1, Larissa Belov 1, Michael J. Solomon 2, Charles Chan 3, Stephen J. Clarke 4, Richard I. Christopherson 1
1School of Molecular Bioscience, University of Sydney, 2Department of Surgery, Royal Prince Alfred Hospital, 3Department of Anatomical Pathology, Department of Anatomical Pathology, 4Department of Medicine, Concord Repatriation General Hospital

We described a procedure for the disaggregation of colorectal cancer (CRC) to produce viable single cells, which are then captured on customized antibody microarrays recognizing surface antigens (DotScan CRC microarray). Sub-populations of cells bound to the microarray can be profiled by fluorescence multiplexing using monoclonal antibodies tagged with fluorescent dyes.

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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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Medicine

Computerized Dynamic Posturography for Postural Control Assessment in Patients with Intermittent Claudication
Natalie Vanicek 1,2, Stephanie A. King 2, Risha Gohil 2,3, Ian C. Chetter 3, Patrick A Coughlin 3,4
1Discipline of Exercise and Sport Science, Faculty of Health Sciences, University of Sydney, 2Department of Sport, Health and Exercise Science, University of Hull, 3Academic Vascular Department, Hull Royal Infirmary, Hull and East Yorkshire Hospitals, 4Department of Vascular Surgery, Addenbrookes Hospital

Individuals with intermittent claudication exhibit poor balance compared to healthy controls. Computerized dynamic posturography is an objective method for measuring an individual's postural responses to balance disturbances. This provides an objective reflection of the person’s ability to respond to situations under which sensory stimuli are altered and unexpected perturbations occur.

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Immunology and Infection

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins
N. Bishara Marzook *1, Dean J. Procter *1, Helena Lynn 1, Yui Yamamoto 2, Jacquelyn Horsington 3, Timothy P. Newsome 1
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.

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Biology

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells
Darius J. R. Lane 1, Alfons Lawen 2
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.

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Medicine

A Modified Method for Heterotopic Mouse Heart Transplantion
Chuanmin Wang 1, Zane Wang 1, Richard Allen 1, G. Alex Bishop 1, Alexandra F. Sharland 1
1Collaborative Transplant Laboratory, University of Sydney

A technique is demonstrated for the microsurgical procedure for heterotopic transplantation of hearts in mice, including simplified methods for donor harvesting and recipient vessel anastomosis.

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Behavior

Behavioral Assessment of the Aging Mouse Vestibular System
Victoria W. K. Tung 1, Thomas J. Burton 2, Edward Dababneh 1, Stephanie L. Quail 1, Aaron J. Camp 1
1Discipline of Biomedical Science, University of Sydney, 2The Bosch Institute Animal Behavioural Facility, University of Sydney

Motor control and balance performance are known to deteriorate with age. This paper presents a number of standard noninvasive behavioral tests with the addition of a simple rotary stimulus to challenge the vestibular system and show changes in balance performance in a murine model of aging.

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Chemistry

Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy
Dexter D'Sa 1, Hak-Kim Chan 1, Hae-Won Kim 2, Wojciech Chrzanowski 1,2
1Faculty of Pharmacy, University of Sydney, 2Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University

Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems.

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Medicine

Remote Limb Ischemic Preconditioning: A Neuroprotective Technique in Rodents
Alice Brandli 1
1Discipline of Physiology and Bosch Institute, Sydney Medical School, University of Sydney

Remote ischemic preconditioning (RIP) is a method of conditioning tissues against damaging stress. We have established a method of remote ischemia at the hind limb, by inflating a sphygmomanometer cuff for 5-10 min. The neuroprotective capabilities of RIP have been demonstrated in a model of retinal degeneration in rodents.

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Neuroscience

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy
Nigel Tse 1, Marco Morsch 2, Nazanin Ghazanfari 1, Louise Cole 3, Archunan Visvanathan 1, Catherine Leamey 1, William D. Phillips 1
1Physiology and Bosch Institute, University of Sydney, 2Motor Neuron Disease Research Group, Australian School of Advanced Medicine, Macquarie University, 3Advanced Microscopy Facility, Bosch Institute, University of Sydney

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

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Behavior

The Use of the Puzzle Box as a Means of Assessing the Efficacy of Environmental Enrichment
Angela M. O'Connor 1, Thomas J. Burton 1,2, Catherine A. Leamey 1, Atomu Sawatari 1
1Discipline of Physiology, University of Sydney, 2Bosch Animal Behavioural Facility, University of Sydney

Environmental enrichment provides a potential protective effect against neurodegenerative disorders. Currently, however, there is no easy way of determining the efficacy of enrichment procedures. This protocol describes a simple “Puzzle Box” method for assessing an animal’s cognitive function, in order to reveal the effectiveness of environmental enrichment.

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Biology

Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium
Lucy Zhang 1, Victoria W. K. Tung 2, Miranda Mathews 2, Aaron J. Camp 2
1Discipline of Physiology, University of Sydney, 2Discipline of Biomedical Science, University of Sydney

Mitochondrial dysfunction is a hallmark of cellular senescence. This paper uses non-invasive near-infrared (NIr) treatment to improve mitochondrial function in the aging mouse vestibular sensory epithelium.

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Engineering

In Situ Neutron Powder Diffraction Using Custom-made Lithium-ion Batteries
William R. Brant 1, Siegbert Schmid 1, Guodong Du 2, Helen E. A. Brand 3, Wei Kong Pang 2,4,5, Vanessa K. Peterson 4, Zaiping Guo 2,5, Neeraj Sharma 6
1School of Chemistry, University of Sydney, 2Institute for Superconducting & Electronic Materials, University of Wollongong, 3Australian Synchrotron, 4Australian Nuclear Science and Technology Organisation, 5School of Mechanical, Materials, and Mechatronic Engineering, University of Wollongong, 6School of Chemistry, University of New South Wales

We describe the design and construction of an electrochemical cell for the examination of electrode materials using in situ neutron powder diffraction (NPD). We briefly comment on alternate in situ NPD cell designs and discuss methods for the analysis of the corresponding in situ NPD data produced using this cell.

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Neuroscience

Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning
Alice Brandli 1, Jonathan Stone 1
1Discipline of Physiology and Bosch Institute, Sydney Medical School, University of Sydney

The electroretinogram (ERG) is an electrical potential generated by the retina in response to light. This paper describes how to use the ERG to assess retinal function, in dark-adapted rats, and how it can be can be used to assess a neuroprotective intervention, in the present case remote ischemic preconditioning.

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Biology

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy
Victoria C Cogger *1,2,3, Jennifer N O'Reilly *1,2, Alessandra Warren 1,2,3, David G Le Couteur 1,2,3
1Centre for Education and Research on Ageing & ANZAC Research Institute, University of Sydney and Concord Hospital, 2Ageing and Alzheimers Institute, Concord Hospital, 3Charles Perkins Centre, University of Sydney

The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

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Engineering

Writing Bragg Gratings in Multicore Fibers
Emma Y. Lindley 1, Seong-sik Min 1,2, Sergio G. Leon-Saval 1,2, Nick Cvetojevic 1,2,3,4, Jon Lawrence 4, Simon C. Ellis 1,4, Joss Bland-Hawthorn 1,2
1Sydney Institute for Astronomy, School of Physics, University of Sydney, 2Institute of Photonics and Optical Science, School of Physics, University of Sydney, 3Center for Ultrahigh Bandwidth Devices for Optical Systems, School of Physics, University of Sydney, 4Australian Astronomical Observatory

We describe a technique for inscribing identical fiber Bragg gratings into each core of a multicore fiber. This is achieved by introducing an additional surface into the optical path to mitigate lensing by the curved surface of the fiber cladding.

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Medicine

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids
Kimberley A. Beaumont 1,2, Andrea Anfosso 1,2, Farzana Ahmed 3, Wolfgang Weninger *1,4,5, Nikolas K. Haass *1,3,5
1The Centenary Institute, 2Sydney Medical School, University of Sydney, 3The University of Queensland Diamantina Institute, Translational Research Institute, The University of Queensland, 4Department of Dermatology, Royal Prince Alfred Hospital, 5Discipline of Dermatology, University of Sydney

We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids.

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Developmental Biology

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol
Hananeh Fonoudi *1,2,3,8, Hassan Ansari *1,8, Saeed Abbasalizadeh 1, Gillian M Blue 6,7, Nasser Aghdami 1, David S Winlaw 6,7, Richard P Harvey 2,3,4, Alexis Bosman *2,3, Hossein Baharvand *1,5
1Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, 2Developmental and Stem Cell Biology Division, Victor Chang Cardiac Research Institute, 3St. Vincent´s Clinical School, Faculty of Medicine, University of New South Wales, 4School of Biotechnology and Biomolecular Sciences, University of New South Wales, 5Department of Developmental Biology, University of Science and Culture, 6Heart Centre for Children, The Children´s Hospital at Westmead, 7Sydney Medical School, University of Sydney, 8Department of Developmental Biology, University of Science and Culture, Tehran, Iran

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

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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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JoVE Journal

Palladium N-Heterocyclic Carbene Complexes: Synthesis from Benzimidazolium Salts and Catalytic Activity in Carbon-carbon Bond-forming Reactions
Ziya Sahin 1, Senem Akkoς 1,2, İlhan Özer İlhan 2, Veysel Kayser 1
1Faculty of Pharmacy, The University of Sydney, 2Department of Chemistry, Faculty of Sciences, Erciyes University

Detailed and generalized protocols are presented for the synthesis and subsequent purification of four palladium N-heterocyclic carbene complexes from benzimidazolium salts. The complexes were tested for catalytic activity in arylation and Suzuki-Miyaura reactions. For each reaction investigated, at least one of the four complexes successfully catalyzed the reaction.

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Developmental Biology

Isolation and Expansion of Adult Canine Hippocampal Neural Precursors
Thomas Duncan 1, Aileen Lowe 1, Marshall A. Dalton 2, Michael Valenzuela 1
1Regenerative Neuroscience Group, University of Sydney, 2Wellcome Trust Centre for Neuroimaging, Institute of Neurology, University College London

The canine brain is a valuable model in which to study adult neurogenesis. Presented here are protocols for isolating and expanding adult canine hippocampal neural precursor cells from primary brain tissue.

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Medicine

Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses
Chi Ching Goh 1,2, Jackson LiangYao Li 1, David Becker 3, Wolfgang Weninger 4,5,6, Veronique Angeli 2,7, Lai Guan Ng 1,2,5,8
1Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Biopolis, 2Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 3Lee Kong Chian School of Medicine, Nanyang Technological University, 4Centenary Institute for Cancer Medicine and Cell Biology, 5Discipline of Dermatology, University of Sydney, 6Department of Dermatology, Royal Prince Alfred Hospital, 7LSI Immunology Programme, National University of Singapore, 8School of Biological Sciences, Nanyang Technological University

This protocol describes the induction of an ischemia-reperfusion (IR) model on mouse ear skin using magnet clamping. Using a custom-built intravital imaging model, we study in vivo inflammatory responses post-reperfusion. The rationale behind the development of this technique is to extend the understanding of how leukocytes respond to skin IR injury.

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Biochemistry

Laboratory Scale Production and Purification of a Therapeutic Antibody
Zehra Elgundi 1, Vicki Sifniotis 1, Mouhamad Reslan 1, Esteban Cruz 1, Veysel Kayser 1
1Faculty of Pharmacy, University of Sydney

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

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Medicine

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT
Blake J. Cochran 1, William J. Ryder 2,3,4,5, Arvind Parmar 6, Kerstin Klaeser 4,5, Anthonin Reilhac 7, Georgios I. Angelis 4,5, Steven R. Meikle 4,5, Philip J. Barter 1,5, Kerry-Anne Rye 1,5
1School of Medical Sciences, Faculty of Medicine, UNSW Australia, 2Department of Nuclear Medicine, Concord Hospital, 3National Imaging Facility, University of Sydney, 4Brain and Mind Centre, University of Sydney, 5Faculty of Health Sciences, University of Sydney, 6Life Sciences, ANSTO, 7CERMEP

This study describes a protocol that uses 18F-FDG and positron emission tomography/computed tomography (PET/CT) imaging, together with kinetic modelling, to quantify the in vivo, real-time uptake of 18F-FDG into tissues.

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Medicine

Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model
Jaesung P. Choi 1,2, Xi Yang 3, Matthew Foley 4, Xian Wang 1, Xiangjian Zheng 1,2,3
1Lab of Cardiovascular Signaling, Centenary Institute, 2Faculty of Medicine, Sydney Medical School, University of Sydney, 3Department of Pharmacology, School of Basic Medical Sciences, Tianjin Medical University, 4Australian Centre for Microscopy & Microanalysis, University of Sydney

This protocol demonstrates the induction of cerebral cavernous malformation disease in a mouse model and uses contrast enhanced micro computed tomography to measure lesion burden. This method enhances the value of established mouse models to study the molecular basis and potential therapies for cerebral cavernous malformation and other cerebrovascular diseases.

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Medicine

Whole Genome Sequencing of Candida glabrata for Detection of Markers of Antifungal Drug Resistance
Chayanika Biswas *1, Sharon C-A. Chen *1,2,3, Catriona Halliday 1,2, Elena Martinez 1, Rebecca J. Rockett 1, Qinning Wang 1, Verlaine J. Timms 1, Rajat Dhakal 1, Rosemarie Sadsad 1, Karina J. Kennedy 4, Geoffrey Playford 4,5, Deborah J. Marriott 6, Monica A. Slavin 7, Tania C. Sorrell 1,3, Vitali Sintchenko 1,2,3
1Centre for Infectious Diseases and Microbiology-Public Health, Westmead Hospital, 2Centre for Infectious Diseases and Microbiology Laboratory Services, ICPMR, 3Marie Bashir Institute for Infectious Diseases and Biosecurity, The University of Sydney, 4Department of Microbiology and Infectious Diseases, Canberra Hospital and Health Services, Australian National University Medical School, 5Infection Management Services, Australian National University Medical School, 6Department of Microbiology and Infectious Diseases, St. Vincent's Hospital, 7Department of Infectious Diseases, Peter MacCallum Cancer Centre

This study implemented whole genome sequencing for analysis of mutations in genes conferring antifungal drug resistance in Candida glabrata. C. glabrata isolates resistant to echinocandins, azoles and 5-flucytosine, were sequenced to illustrate the methodology. Susceptibility profiles of the isolates correlated with presence or absence of specific mutation patterns in genes.

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Biochemistry

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows
Matthew B. O'Rourke 1,2, Matthew P. Padula 2, Caine Smith 3, Priscilla Youssef 4, Stuart Cordwell 1, Paul Witting 4, Greg Sutherland 3, Ben Crossett 1
1Mass Spectrometry Core Facility, University of Sydney, 2Proteomics Core Facility, University of Technology Sydney, 3Neuropathology Group, Discipline of Pathology, School of Medical Sciences, University of Sydney, 4Redox Biology Group, Discipline of Pathology, School of Medical Sciences, University of Sydney

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

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Medicine

'Boden Food Plate': Novel Interactive Web-based Method for the Assessment of Dietary Intake
Alice Meroni *1, Nyssa Jualim *1, Nicholas Fuller 1
1The Boden Institute of Obesity, Nutrition, Exercise & Eating Disorders, Charles Perkins Centre, University of Sydney

The 'Boden Food Plate' is an electronic food diary designed to be an interactive and fun method to collect dietary intake using visual depictions. The purpose of this study was to validate the web-based application against a traditional three-day estimated food diary method.

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Neuroscience

Simple Surgical Induction of Conductive Hearing Loss with Verification Using Otoscope Visualization and Behavioral Clap Startle Response in Rat
Francis A. M. Manno *1, Sinai H. C. Manno *2,3,4, Victor Ma 5, Fernando A. Barrios 1, William C Cho 5, Shuk Han Cheng 2,3,6, Condon Lau 4
1Instituto de Neurobiología, Universidad Nacional Autónoma de México, México, 2State Key Laboratory of Marine Pollution (SKLMP), City University of Hong Kong, Hong Kong, 3Department of Biomedical Sciences, College of Veterinary Medicine and Life Sciences, City University of Hong Kong, Hong Kong, 4Department of Physics, City University of Hong Kong, Hong Kong, 5Department of Clinical Oncology, Queen Elizabeth Hospital, Hong Kong, 6Department of Materials Science and Engineering, City University of Hong Kong, Hong Kong

Here, we present a protocol to establish a replicable conductive hearing loss induction via surgical tympanic membrane puncture and verification by otoscope visualization and behavioral assessment by clap startle.

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Genetics

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing
Bonnie Hiener 1, John-Sebastian Eden 1, Bethany A. Horsburgh 1, Sarah Palmer 1
1Centre for Virus Research, The Westmead Institute for Medical Research, University of Sydney

Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

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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. Leeson 1,2, Tailoi Chan-Ling 3,4, Michael D. Lovelace 3,5,6, Jeremy D. Brownlie 7, 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, 5Applied Neurosciences Program, Peter Duncan Neurosciences Research Unit, St. Vincent's Centre for Applied Medical Research, 6School of Medical Sciences, The University of New South Wales (UNSW) Medicine, Sydney, New South Wales, 7School of Environment and Science, Griffith University, Brisbane, Queensland, 8Florey Institute of Neuroscience and Mental Health, University of Melbourne

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.

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Environment

PARbars: Cheap, Easy to Build Ceptometers for Continuous Measurement of Light Interception in Plant Canopies
William T. Salter 1, Andrew M. Merchant 1, Matthew E. Gilbert 2, Thomas N. Buckley 2
1School of Life and Environmental Sciences, Sydney Institute of Agriculture, University of Sydney, 2Department of Plant Sciences, University of California, Davis

Here, we present detailed instructions on how to build and calibrate research quality ceptometers (light sensors that integrate light intensity across many sensors arrayed linearly along a horizontal bar).

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Behavior

Assessing the Autonomic and Behavioral Effects of Passive Motion in Rats using Elevator Vertical Motion and Ferris-Wheel Rotation
Francis A. M. Manno 1,2, Leilei Pan 3, Yuqi Mao 3, Yang Su 3, Sinai H. C. Manno 2,4,5, Shuk Han Cheng 4,5,6, Condon Lau 2, Yiling Cai 3
1School of Biomedical Engineering, Faculty of Engineering, University of Sydney, 2Department of Physics, City University of Hong Kong, 3Department of Nautical Injury Prevention, Faculty of Navy Medicine, Second Military Medical University, 4State Key Laboratory of Marine Pollution (SKLMP), City University of Hong Kong, 5Department of Biomedical Sciences, College of Veterinary Medicine and Life Sciences, City University of Hong Kong, 6Department of Materials Science and Engineering, City University of Hong Kong

Protocols are presented to assess the autonomic and behavioral effects of passive motion in rodents using elevator vertical motion and Ferris-wheel rotation.

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Neuroscience

Stochastic Noise Application for the Assessment of Medial Vestibular Nucleus Neuron Sensitivity In Vitro
Sebastian P. Stefani 1,2, Paul P. Breen 3, Jorge M. Serrador 4, Aaron J. Camp 1,2
1Discipline of Physiology, Sydney Medical School, University of Sydney, 2Bosch Institute, Sydney Medical School, University of Sydney, 3The MARCS Institute, Western Sydney University, 4Department of Pharmacology, Physiology & Neuroscience, Rutgers Biomedical and Health Sciences

Galvanic vestibular stimulation in humans exhibits improvements in vestibular function. However, it is unknown how these effects occur. Here, we describe how to apply sinusoidal and stochastic electrical noise and evaluate appropriate stimulus amplitudes in individual medial vestibular nucleus neurons in the C57BL/6 mouse.

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Immunology and Infection

In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages
Yunjia Zhang 1,2, Benjamin S. Rayner 1,2, Mathias Jensen 3, Clare L. Hawkins 1,2,3
1Heart Research Institute, 2Sydney Medical School, University of Sydney, 3Department of Biomedical Sciences, University of Copenhagen

Presented here is a protocol to detect macrophage extracellular trap (MET) production in live cell culture using microscopy and fluorescence staining. This protocol can be further extended to examine specific MET protein markers by immunofluorescence staining.

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JoVE Core

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model
Patrick Haubruck 1,2, Aimee C. Colbath 1,3, Yolanda Liu 1, Shihani Stoner 1, Cindy Shu 1, Christopher B. Little 1
1Raymond Purves Bone and Joint Research Laboratory, Institute of Bone and Joint Research, Kolling Institute, Royal North Shore Hospital, University of Sydney, 2HTRG - Heidelberg Trauma Research Group, Center for Orthopedics, Trauma Surgery and Spinal Cord Injury, Trauma and Reconstructive Surgery, Heidelberg University Hospital, 3Department of Large Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

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Bioengineering

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction
Christopher D. Roche 1,2,3,4, Carmine Gentile 1,2,3
1The University of Sydney, 2University of Technology Sydney (UTS), 3The Royal North Shore Hospital, 4University Hospital of Wales

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

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Cancer Research

An Orthotopic Resectional Mouse Model of Pancreatic Cancer
Tony C. Y. Pang 1,2,4,5, Zhihong Xu 1,2, Alpha Raj Mekapogu 1,2, Srinivasa Pothula 1,2, Therese M. Becker 3, David Goldstein 1, Romano C. Pirola 1,2, Jeremy S. Wilson 1,2, Minoti V. Apte 1,2
1Pancreatic Research Group, South Western Sydney Clinical School, University of New South Wales, 2Ingham Institute for Applied Medical Research, 3Centre for Circulating Tumour Cell Diagnostics and Research, Ingham Institute for Applied Medical Research, 4Surgical Innovations Unit, Westmead Hospital, 5Westmead Clinical School, University of Sydney

In the clinical context, patients with localized pancreatic cancer will undergo pancreatectomy followed by adjuvant treatment. This protocol reported here aims to establish a safe and effective method of modelling this clinical scenario in nude mice, through orthotopic implantation of pancreatic cancer followed by distal pancreatectomy and splenectomy.

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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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Neuroscience

Objectively Assessing Sports Concussion Utilizing Visual Evoked Potentials
Daryl H. Fong 1, Adrian J. Cohen 2, Dylan E. Mahony 2, Neil G. Simon 4, Joseph E. Herrera 3, Rebecca B. Baron 3, David Putrino 3
1School of Aerospace, Mechanical and Mechatronic Engineering, Faculty of Engineering and Information Technologies, University of Sydney, 2HeadsafeIP, 3Department of Rehabilitation Medicine, Icahn School of Medicine at Mount Sinai, 4St Vincent's Clinical School, Faculty of Medicine, University of New South Wales

A portable system capable of measuring steady-state visual-evoked potentials was developed and trialed on 65 amateur rugby players over 18 weeks to investigate SSVEP as a potential electrophysiological biomarker for concussion. Players' baselines were measured pre-season, with retesting for reliability, concussion, and recovery assessment being conducted within controlled time-periods, respectively.

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Neuroscience

Conventional and Threshold-Tracking Transcranial Magnetic Stimulation Tests for Single-handed Operation
Hatice Tankisi 1, James Howells 2, Bülent Cengiz 3, Gintaute Samusyte 4, Martin Koltzenburg 5, Hugh Bostock 6
1Department of Clinical Neurophysiology, Aarhus University Hospital, 2Central Clinical School, Faculty of Medicine and Health, University of Sydney, 3Department of Neurology, Gazi University Faculty of Medicine, 4Department of Neurology, Medical Academy, Lithuanian University of Health Sciences, 5Department of Clinical Neurophysiology, National Hospital for Neurology and Neurosurgery; Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, 6Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology

We present a suite of standardized single- and paired-pulse transcranial magnetic stimulation (TMS) recording protocols, with options for conventional amplitude measurements and threshold-tracking. This program can control three different types of magnetic stimulators and is designed to enable all tests to be performed conveniently by a single operator.

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Bioengineering

Ceramic Omnidirectional Bioprinting in Cell-Laden Suspensions for the Generation of Bone Analogs
Gagan Jalandhra 1, Sara Romanazzo 2, Stephanie Nemec 1, Iman Roohani 3, Kristopher A. Kilian 1,2
1School of Materials Science and Engineering, University of New South Wales, 2School of Chemistry, Australian Centre for NanoMedicine, University of New South Wales, 3School of Biomedical Engineering, University of Sydney

This protocol describes a 3D printing technique to fabricate bone-like structures by depositing a calcium phosphate ink in a gelatin-based granular support. Printed bone analogs are deposited in freeform, with flexibility for direct harvesting of the print or crosslinking within a living cell matrix for multiphasic constructs.

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Biology

Anesthesia and Intubation of Preadolescent Mouse Pups for Cardiothoracic Surgery
Jianxin Wu *1, Amy M. Nicks 1,2, Justin J. Skowno 3,4, Michael P. Feneley 1,2, Robert M. Graham 1,2, Siiri E. Iismaa *1,2
1Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, 2St Vincent’s Clinical School, University of New South Wales Sydney, 3Department of Anaesthesia, The Children’s Hospital at Westmead, 4School of Child and Adolescent Health, University of Sydney

Cardiothoracic surgical models in mice >7 days old require intubation, but this is challenging for preadolescent (8-14-day-old) mouse pups and there is little information on anesthetic regimens for intubation. Here, we present dosage regimens of ketamine/xylazine/atropine in 10-day-old C57BL/6J mouse pups that allow endotracheal intubation, while minimizing animal mortality.

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Cancer Research

Rapid Optical Clearing for Semi-High-Throughput Analysis of Tumor Spheroids
Gency Gunasingh 1, Alexander P. Browning 2, Nikolas K. Haass 1
1The University of Queensland Diamantina Institute, The University of Queensland, 2School of Mathematical Sciences, Queensland University of Technology

Tumor spheroids are becoming increasingly utilized to assess tumor cell-microenvironment interactions and therapy response. The present protocol describes a robust but simple method for semi-high-throughput imaging of 3D tumor spheroids using rapid optical clearing.

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Biology

High-Throughput Contractile Measurements of Hydrogel-Embedded Intact Mouse Muscle Fibers Using an Optics-Based System
Leander A. Vonk *1, Osman Esen *1, Michaela Yuen 1,4, Tyler J. Kirby 1,2,3
1Department of Physiology, Amsterdam UMC, 2Amsterdam Cardiovascular Sciences, Heart Failures and Arrhythmias, Amsterdam UMC, 3Amsterdam Movement Sciences, Tissue Function and Regeneration, Amsterdam UMC, 4Discipline of Child and Adolescent Health, Faculty of Health and Medicine, University of Sydney

Skeletal muscle function can be assessed by quantifying the contractility of isolated muscle fibers, traditionally using laborious, low-throughput approaches. Here, we describe an optics-based, high-throughput method to quantify the contractility of hydrogel-embedded muscle fibers. This approach has applications for drug screening and therapeutic development.

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