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25 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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Biology

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins
Andrius Masedunskas 1,2, Natalie Porat-Shliom 1, Muhibullah Tora 1, Oleg Milberg 1,3, Roberto Weigert 1
1Intracellular Membrane Trafficking Unit, Oral and Pharyngeal Cancer Branch National Institute of Dental and Craniofacial Research, National Institutes of Health, 2Department of Biology, University of North Carolina at Chapel Hill , 3Department of Chemical & Biochemical Engineering and Department of Biomedical Engineering, Rutgers University

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

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Medicine

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum
Mazen Amatoury 1, Vera Merheb 1, Jessica Langer 1, Xin Maggie Wang 2, Russell Clive Dale 1, Fabienne Brilot 1
1Institute for Neuroscience and Muscle Research, The Kids Research Institute at the Children's Hospital at Westmead, The University of Sydney, 2Flow Cytometry Centre, Westmead Millennium Institute for Medical Research

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

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Chemistry

Amide Coupling Reaction for the Synthesis of Bispyridine-based Ligands and Their Complexation to Platinum as Dinuclear Anticancer Agents
Michael G. Apps 1, Ben W. Johnson 2, Oliver B. Sutcliffe 3, Sarah D. Brown 4, Nial J. Wheate 1
1Faculty of Pharmacy, The University of Sydney, 2School of Science and Health, University of Western Sydney, 3Division of Chemistry and Environmental Science, School of Science and the Environment, Manchester Metropolitan University, 4Nature Publishing Group

This protocol describes the use of amide coupling reactions of isonicotinic acid and diaminoalkanes to form bridging ligands suitable for use in the synthesis of multinuclear platinum complexes, which combine aspects of the anticancer drugs BBR3464 and picoplatin.

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Engineering

Integrating a Triplet-triplet Annihilation Up-conversion System to Enhance Dye-sensitized Solar Cell Response to Sub-bandgap Light
Andrew Nattestad 1, Yuen Yap Cheng 2, Rowan W. MacQueen 2, Gordon G. Wallace 1, Timothy W. Schmidt 3
1ARC Centre of Excellence for Electromaterials Science (ACES), Intelligent Polymer Research Institute (IPRI), The University of Wollongong, 2School of Chemistry, The University of Sydney, 3School of Chemistry, The University of New South Wales

An integrated device, incorporating a dye-sensitized solar cell and triplet-triplet annihilation up-conversion unit was produced, affording enhanced light harvesting, from a wider section of the solar spectrum. Under modest irradiation levels a significantly enhanced response to low energy photons was demonstrated, yielding a record figure of merit for dye-sensitized solar cells.

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Biology

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs
Wilson Wong 1, Ryan Farr 1, Mugdha Joglekar 1, Andrzej Januszewski 2, Anandwardhan Hardikar 1
1Diabetes and Islet Biology Group, NHMRC Clinical Trials Centre, Faculty of Medicine, The University of Sydney, 2Biomarkers Laboratory, NHMRC Clinical Trials Centre, Faculty of Medicine, The University of Sydney

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

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Neuroscience

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination
Yuyi You 1,2, Vivek K. Gupta 1, Nitin Chitranshi 1, Brittany Reedman 1, Alexander Klistorner 1,2, Stuart L. Graham 1,2
1Department of Ophthalmology, Australian School of Advanced Medicine, Macquarie University, 2Save Sight Institute, The University of Sydney

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

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Bioengineering

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell
Yunfeng Chen *1, Baoyu Liu *2, Lining Ju *3, Jinsung Hong *1, Qinghua Ji 4,5, Wei Chen 6, Cheng Zhu 1
1Woodruff School of Mechanical Engineering, Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, 2Coulter Department of Biomedical Engineering, Georgia Institute of Technology, 3Charles Perkins Centre, The University of Sydney, 4Institute of Biophysics, Laboratory of RNA Biology, Chinese Academy of Sciences, 5University of Chinese Academy of Sciences, 6School of Medicine and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

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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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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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Neuroscience

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging
Morven A. Cameron 1, Orsolya Kekesi 1,2, John W. Morley 1,2, Alba Bellot-Saez 1,2, Sindy Kueh 2, Paul Breen 1, André van Schaik 1, Jonathan Tapson 1, Yossi Buskila 1,2
1The MARCS Institute, Western Sydney University, 2School of Medicine, Western Sydney University

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for >24 h.

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Engineering

Characterization of Ultra-fine Grained and Nanocrystalline Materials Using Transmission Kikuchi Diffraction
Gwénaëlle Proust 1, Patrick Trimby 2, Sandra Piazolo 3, Delphine Retraint 4
1School of Civil Engineering, The University of Sydney, 2Australian Centre for Microscopy and Microanalysis, The University of Sydney, 3Department of Earth and Planetary Sciences, Macquarie University, 4Charles Delaunay Institute, LASMIS, UMR STMR CNRS 6281, University of Technology of Troyes

This paper provides a detailed method to characterize the microstructure of ultra-fine grained and nanocrystalline materials using a scanning electron microscope equipped with a standard electron backscatter diffraction system. Metal alloys and minerals presenting refined microstructures are analyzed using this technique, showing the diversity of its possible applications.

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Biology

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury
Jully Gogoi-Tiwari 1, Julia Köhn-Gaone 1, Corey Giles 2, Dirk Schmidt-Arras 3, Francis D. Gratte 1,4, Caryn L. Elsegood 1, Geoffrey W. McCaughan 5,6,7, Grant A. Ramm 8,9, John K. Olynyk 10,11, Janina E.E. Tirnitz-Parker 1,12
1School of Biomedical Sciences & Curtin Health Innovation Research Institute, Curtin University, 2School of Public Health & Curtin Health Innovation Research Institute, Curtin University, 3Institute of Biochemistry, Christian-Albrechts-University, 4School of Veterinary and Life Sciences, Murdoch University, 5Centenary Institute of Cancer Medicine and Cell Biology, The University of Sydney, 6Royal Prince Alfred Hospital, 7A.W. Morrow Gastroenterology and Liver Centre, 8QIMR Berghofer Medical Research Institute, 9Faculty of Medicine and Biomedical Sciences, The University of Queensland, 10Fiona Stanley and Fremantle Hospitals, 11School of Medical and Health Sciences, Edith Cowan University, 12School of Medicine and Pharmacology, University of Western Australia

Here we describe a common method to induce chronic liver injury in mice by feeding of a choline-deficient and ethionine-supplemented (CDE) diet. We demonstrate health monitoring, liver perfusion, isolation, and preservation. A time course of six weeks can inform about liver injury, pathohistology, fibrosis, inflammatory, and liver progenitor cell responses.

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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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Immunology and Infection

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis
Rekha Marimuthu 1,2, Habib Francis 1,2, Suat Dervish 3, Stephen C.H. Li 4, Heather Medbury *1,2, Helen Williams *1,2
1Department of Surgery, Vascular Biology Research Centre, Westmead Hospital, 2Westmead Clinical School, Department of Surgery, The University of Sydney, 3Westmead Research Hub, Westmead Institute for Medical Research, 4Institute for Clinical Pathology and Medical Research, Westmead Hospital

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

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Chemistry

Employing Pressurized Hot Water Extraction (PHWE) to Explore Natural Products Chemistry in the Undergraduate Laboratory
Curtis C. Ho 1, Bianca J. Deans *1, Jeremy Just *1, Gregory G. Warr 2, Shane Wilkinson 2, Jason A. Smith 1, Alex C. Bissember 1
1School of Natural Sciences - Chemistry, University of Tasmania, 2School of Chemistry, The University of Sydney

Here, we employ a pressurized hot water extraction (PHWE) method, which utilizes an unmodified household espresso machine to introduce undergraduates to natural products chemistry in the laboratory. Two experiments are presented: PHWE of eugenol and acetyleugenol from cloves and PHWE of seselin and (+)-epoxysuberosin from the Australian plant Correa reflexa.

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Developmental Biology

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development
Yong Wang *1, Ding Zhang *2, Fang Zhou 2, Meijun Zhou 2, Qiujie Li 1, Jingyu Chen 3, Jun Yang 1,2
1Department of Physiology and Department of Cardiology of the Second Affiliated Hospital, Zhejiang University School of Medicine, 2Department of Cell Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, 3Wuxi Lung Transplant Center, Wuxi People's Hospital affiliated to Nanjing Medical University

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

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Medicine

Intratracheal Administration of Dry Powder Formulation in Mice
Yingshan Qiu 1, Qiuying Liao 1, Michael Y.T. Chow 1,2, Jenny K.W. Lam 1
1Department of Pharmacology and Pharmacy, LKS Faculty of Medicine, The University of Hong Kong, 2Advanced Drug Delivery Group, Sydney Pharmacy School, Faculty of Medicine and Health, The University of Sydney

Dry powder formulations for inhalation have great potential in treating respiratory diseases. Before entering human studies, it is necessary to evaluate the efficacy of the dry powder formulation in preclinical studies. A simple and noninvasive method of the administration of dry powder in mice through the intratracheal route is presented.

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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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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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Bioengineering

Molecular Spring Constant Analysis by Biomembrane Force Probe Spectroscopy
Peyman Obeidy 1, Haoqing Wang 1,2,3, Mingqin Du 1, Huiqian Hu 1,4, Fang Zhou 1, Haoruo Zhou 5, Hao Huang 5, Yunduo Charles Zhao 1,2, Lining Arnold Ju 1,2,3
1School of Biomedical Engineering, Faculty of Engineering, The University of Sydney, 2Charles Perkins Centre, The University of Sydney, 3Heart Research Institute, 4Department of Chemistry, The Hong Kong University of Science and Technology, 5School of Aerospace, Mechanical and Mechatronic Engineering, Faculty of Engineering, The University of Sydney

A biomembrane force probe (BFP) is an in situ dynamic force spectroscopy (DFS) technique. BFP can be used to measure the spring constant of molecular interactions on living cells. This protocol presents spring constant analysis for molecular bonds detected by BFP.

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Biology

Intravital Subcellular Microscopy of the Mammary Gland
Yeap Ng 1, Andrius Masedunskas 1,2, Marco Heydecker 1, Seham Ebrahim 1,3, Roberto Weigert 1, Ian H. Mather 1
1Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 2Charles Perkins Centre, Central Clinical School, Faculty of Medicine and Health, The University of Sydney, 3Department of Molecular Physiology and Biological Physics, School of Medicine, University of Virginia

The present protocol describes a facile technique for the intravital imaging of the lactating mouse mammary gland by laser scanning confocal and multiphoton microscopy.

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Neuroscience

Computational Modeling of Retinal Neurons for Visual Prosthesis Research - Fundamental Approaches
Ariastity Pratiwi 1, Orsolya Kekesi 1, Gregg Suaning 1
1The University of Sydney

We summarize a workflow to computationally model a retinal neuron's behaviors in response to electrical stimulation. The computational model is versatile and includes automation steps that are useful in simulating a range of physiological scenarios and anticipating the outcomes of future in vivo/in vitro studies.

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Medicine

Delivery of Cardioactive Therapeutics in a Porcine Myocardial Infarction Model
Dinesh Selvakumar *1,2, Emma Wilkie *1, Tejas Deshmukh 1,2, Dhanya Ravindran 1, Yasuhito Kotake 2, Juntang Lu 2, Tony Barry 2, Vu Tran 2, Hugh Paterson 3, Alfred Hing 4, Timothy Campbell 2, Saurabh Kumar 2, Eddy Kizana 1,2, James J. H. Chong 1,2
1Centre for Heart Research, The Westmead Institute for Medical Research, The University of Sydney, 2Department of Cardiology, Westmead Hospital, 3Sydney Imaging, Core Research Facility, The University of Sydney, 4Department of Cardiothoracic Surgery, Liverpool Hospital

The present protocol describes three methods of administering cardioactive therapeutic agents in a porcine model. Female landrace swine received treatment through either: (1) thoracotomy and transepicardial injection, (2) catheter-based transendocardial injection, or (3) intravenous infusion via jugular vein osmotic minipump.

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Biology

Fluorescence Micropipette Aspiration Assay to Investigate Red Blood Cell Mechanosensing
Jasmine Jin 1, Haoqing Jerry Wang 1,2,3, Yiyao Catherine Chen 1, Blake Russell 1, Allan Sun 1,2,3,4, Yao Wang 1, Lining Arnold Ju 1,2,3,4
1School of Biomedical Engineering, The University of Sydney, 2Charles Perkins Centre, The University of Sydney, 3Heart Research Institute, 4The University of Sydney Nano Institute (Sydney Nano), The University of Sydney

The exploration of cellular behavior under mechanical stress is pivotal for advances in cellular mechanics and mechanobiology. We introduce the Fluorescence Micropipette Aspiration (fMPA) technique, a novel method combining controlled mechanical stimulation with comprehensive analysis of intracellular signaling in single cells. This technique investigates new in-depth studies of live-cell mechanobiology.

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