JoVE Logo

Sign In

The University of Melbourne

36 ARTICLES PUBLISHED IN JoVE

image

Biology

Quantification of γH2AX Foci in Response to Ionising Radiation
Li-Jeen Mah 1,2, Raja S. Vasireddy 1,2,3, Michelle M. Tang 1,3, George T. Georgiadis 1, Assam El-Osta 2,3, Tom C. Karagiannis 1,2
1Epigenomic Medicine, Baker IDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 2Department of Pathology, The University of Melbourne, 3Epigenetics in Human Health and Disease, Baker IDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct

Quantification of DNA double-strand streaks using γH2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of γH2AX foci after exposure of cells to radiation.

image

Biology

Quantitation of γH2AX Foci in Tissue Samples
Michelle M. Tang 1,2, Li-Jeen Mah 1,3, Raja S. Vasireddy 1,2,3, George T. Georgiadis 1, Assam El-Osta 2,3, Simon G. Royce 3,4,5, Tom C. Karagiannis 1,3
1Epigenomic Medicine, Baker IDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 2Epigenetics in Human Health and Disease, Baker IDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 3Department of Pathology, The University of Melbourne, 4Department of Allergy and Immunology, Murdoch Children's Research Institute, Royal Children's Hospital, 5Department of Pediatrics, The University of Melbourne

Quantitation of DNA double-strand breaks on the basis of γH2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of γH2AX foci in tissue samples.

image

Biology

Evaluation of the Spatial Distribution of γH2AX following Ionizing Radiation
Raja S. Vasireddy 1,2,3, Michelle M. Tang 1,2, Li-Jeen Mah 2,3, George T. Georgiadis 2, Assam El-Osta 1,3, Tom C. Karagiannis 2,3
1Epigenetics in Human Health and Disease, BakerIDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 2Epigenomic Medicine, BakerIDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 3Department of Pathology, University of Melbourne

Microscopic analysis of γH2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced γH2AX formation within the nucleus.

image

Biology

Clonogenic Assay: Adherent Cells
Haloom Rafehi 1,2, Christian Orlowski 1,2,3, George T. Georgiadis 1, Katherine Ververis 1,4, Assam El-Osta 2,3, Tom C. Karagiannis 1,2
1Epigenomic Medicine, BakerIDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 2Department of Pathology, The University of Melbourne, 3Epigenetics in Human Health and Disease, BakerIDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 4Department of Anatomy and Cellular Biology, The University of Melbourne

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

image

Immunology and Infection

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes
Nancy Wang 1, Richard Strugnell 2, Odilia Wijburg 2, Thomas Brodnicki 1
1St Vincent’s Institute, Department of Medicine, The University of Melbourne, 2Department of Microbiology and Immunology, The University of Melbourne

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

image

Bioengineering

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming
Wah Chin Boon 1, Karolina Petkovic-Duran 2, Yonggang Zhu 2, Richard Manasseh 3, Malcolm K. Horne 1, Tim D. Aumann 1
1Florey Neuroscience Institutes and Centre for Neuroscience, University of Melbourne, 2Fluid Dynamics Group, CSIRO Materials Science and Engineering, 3Swinburne University of Technology, Faculty of Engineering and Industrial Sciences

We describe a novel method for increasing cDNA yield from single-cell quantities of mRNA in otherwise standard laboratory reverse transcription reactions. The novelty resides in the use of a micromixer, which utilizes the phenomenon of acoustic microstreaming, to mix fluids at microliter scales more effectively than shaking, vortexing or trituration.

image

Chemistry

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies
Pooja Sharma 1,2, Mariam Kaywan-Lutfi 1, Logesvaran Krshnan 1,2, Eamon F. X. Byrne 1,2, Melissa Joy Call 1,2, Matthew Edwin Call 1,2
1Structural Biology Division, The Walter and Eliza Hall Institute of Medical Research, 2The University of Melbourne

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

image

Medicine

Techniques for Processing Eyes Implanted With a Retinal Prosthesis for Localized Histopathological Analysis
David A. X. Nayagam 1,2,3, Ceara McGowan 1, Joel Villalobos 1, Richard A. Williams 2,3, Cesar Salinas-LaRosa 2,3, Penny McKelvie 2,3, Irene Lo 2,3, Meri Basa 2,3, Justin Tan 1, Chris E. Williams 1,3,4
1Bionics Institute, 2Department of Anatomical Pathology, St Vincent's Hospital Melbourne, 3Department of Pathology, University of Melbourne, 4Medical Bionics Department, University of Melbourne

Techniques for visualizing retinal cytoarchitecture directly adjacent to individual electrodes within a retinal stimulator.

image

Neuroscience

Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation
William G. A. Brown 1, Karina Needham 2, Bryony A. Nayagam 2, Paul R. Stoddart 1
1Biotactical Engineering, Faculty of Engineering and Industrial Science, Swinburne University of Technology, 2Department of Otolaryngology, The University of Melbourne

Infrared nerve stimulation has been proposed as an alternative to electrical stimulation in a range of nerve types, including those associated with the auditory system. This protocol describes a patch clamp method for studying the mechanism of infrared nerve stimulation in a culture of primary auditory neurons.

image

Immunology and Infection

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay
Eileen M. Dunne 1, Zheng Q. Toh 2, Mary John 3, Jayne Manning 1,4, Catherine Satzke *1,4, Paul Licciardi *2
1Pneumococcal Research, Murdoch Childrens Research Institute, 2Allergy & Immune Disorders, Murdoch Childrens Research Institute, 3Department of Otolaryngology, The University of Melbourne, 4Department of Microbiology & Immunology at the Peter Doherty Institute for Infection & Immunity, The University of Melbourne

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

image

Neuroscience

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons
Nissa L. Carrodus *1, Kathleen Sue-Lyn Teng *1, Kathryn M. Munro 1, Matthew J. Kennedy 2, Jenny M. Gunnersen 1,3
1Department of Anatomy & Neuroscience, The University of Melbourne, 2Department of Neurobiology, Duke University Medical Center, 3Florey Institute of Neuroscience & Mental Health, The University of Melbourne

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

image

Immunology and Infection

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction
Maha Habib 1, Barbara D. Porter 1, Catherine Satzke 1,2
1Pneumococcal Research, Murdoch Childrens Research Institute, 2Department of Microbiology & Immunology, The University of Melbourne

The Quellung reaction is the gold standard technique for serotyping Streptococcus pneumoniae. This technique utilizes a microscope and specific pneumococcal antisera and is commonly used in reference and research laboratories worldwide.

image

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.

image

Immunology and Infection

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination
Barbara D. Porter 1, Belinda D. Ortika 1, Catherine Satzke 1,2
1Pneumococcal Research, Murdoch Childrens Research Institute, 2Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, The University of Melbourne

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

image

Developmental Biology

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays
Haley M. Peckham 1, Anita H. Ferner 1, Lauren Giuffrida 1, Simon S. Murray 1,2, Junhua Xiao 1,2
1Department of Anatomy and Neuroscience, The University of Melbourne, 2The Florey Institute of Neuroscience and Mental Health Research, The University of Melbourne

Here we present protocols that offer a flexible and strategic foundation for virally manipulating oligodendrocyte precursor cells to overexpress proteins of interest in order to specifically interrogate their role in oligodendrocytes via the in vitro model of central nervous system myelination.

image

Neuroscience

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice
Doris Tomas 1, Augustinus H. Prijanto 1, Emma L. Burrows 1, Anthony J. Hannan 1, Malcolm K. Horne 1, Tim D. Aumann 1
1Florey Institute of Neuroscience and Mental Health, The University of Melbourne

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

image

Medicine

Techniques for Processing Eyes Implanted with a Retinal Prosthesis for Localized Histopathological Analysis: Part 2 Epiretinal Implants with Retinal Tacks
David A.X. Nayagam 1,2, Irfan Durmo 3, Ceara McGowan 1, Richard A. Williams 2,4, Robert K. Shepherd 1,5,
1Bionics Institute, 2Department of Pathology, The University of Melbourne, 3Cochlear Limited, 4Department of Anatomical Pathology, St Vincent's Hospital Melbourne, 5Medical Bionics Department, The University of Melbourne

Here we describe histological techniques for visualising ocular tissue directly adjacent to a metal epiretinal tack and retinal prosthesis.

image

Medicine

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting
Zerina Lokmic 1,2, Elizabeth S. Ng 1,3, Matthew Burton 1, Edouard G. Stanley 1,2,3, Anthony J. Penington 1,2, Andrew G. Elefanty 1,2,3
1Murdoch Childrens Research Institute, The Royal Children’s Hospital, 2Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, 3Department of Anatomy and Developmental Biology, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton

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.

image

Medicine

A Method of Trigonometric Modelling of Seasonal Variation Demonstrated with Multiple Sclerosis Relapse Data
Tim Spelman 1,2, Orla Gray 3, Robyn Lucas 4, Helmut Butzkueven 1,2
1Department of Neurology, Royal Melbourne Hospital, 2Department of Medicine (RMH), The University of Melbourne, 3Department of Neurology, Ulster Hospital, 4National Centre for Epidemiology and Population Health, Australian National University

Combining plot analysis with trigonometric regression is a robust method for exploring complex, cyclical phenomena such as relapse onset timing in multiple sclerosis (MS). This method enabled unbiased characterisation of seasonal trends in relapse onset permitting novel inferences around the influence of seasonal variation, ultraviolet radiation (UVR) and latitude.

image

Neuroscience

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice
Mathusi Swaminathan *1, Elisa Hill-Yardin *1, Melina Ellis 1, Matthew Zygorodimos 2, Leigh A. Johnston 3, Rachel M. Gwynne 1, Joel C. Bornstein 1
1Department of Physiology, The University of Melbourne, 2Melbourne School of Engineering, The University of Melbourne, 3Department of Electrical and Electronic Engineering, The University of Melbourne

This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

image

Genetics

The Use of Induced Somatic Sector Analysis (ISSA) for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development
Antanas Spokevicius 1, Lynette Taylor 1, Emma Melder 1, Kim Van Beveren 1, Josquin Tibbits 2, Nicky Creux 3,4, Gerd Bossinger 1
1School of Ecosystem and Forest Sciences, Faculty of Science, The University of Melbourne, 2Victorian AgriBiosciences Centre, La Trobe University R&D Park, 3College of Biological Sciences, Department of Plant Biology, University of California, Davis, 4Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria

Here we present a protocol that facilitates the medium to high throughput functional characterization of gene and promoter constructs in tree secondary stem tissue within comparatively short time frames. It is efficient, easy to use and widely applicable to a range of tree species.

image

Medicine

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS)
Dominic J. Hare 1,2, Kai Kysenius 3, Bence Paul 4, Beate Knauer 5,6, Robert W. Hutchinson 7, Ciaran O'Connor 7, Fred Fryer 8, Tom P. Hennessey 8, Ashley I. Bush 2, Peter J. Crouch 3, Philip A. Doble 1
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.

image

Developmental Biology

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences
Stefanie Dudczig 1,2, Peter D. Currie 2, Lucia Poggi 3, Patricia R. Jusuf 1,2
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.

image

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. Schneider 1, Felisa Reyes-Ortega 1, Jian Li 1,2, Tony Velkov 1
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.

image

Neuroscience

A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments
David G. Gonsalvez 1, Jessica L. Fletcher 1, Sang Won Yoo 1, Rhiannon J. Wood 1, Simon S. Murray 1, Junhua Xiao 1
1Department of Anatomy and Neuroscience, School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne

This report outlines a simple approach to successfully induce experimental autoimmune neuritis (EAN) using the myelin protein zero (P0)180-199 peptide in combination with Freund's complete adjuvant and pertussis toxin. We present a sophisticated paradigm capable of accurately assessing the extent of functional deficits and neuropathology that occur in this EAN.

image

Neuroscience

Operant Protocols for Assessing the Cost-benefit Analysis During Reinforced Decision Making by Rodents
Mojtaba Kermani *1,3, Zahra Fatahi *2, Dechuan Sun 3, Abbas Haghparast 2, Chris French 3,4
1Department of Optometry and Vision Science, The University of Melbourne, 2Neuroscience Research Center, Shahid Beheshti University of Medical Science, 3Department of Medicine, The University of Melbourne, 4Royal Melbourne Hospital

A cost-benefit analysis is a weighing-scale approach that the brain performs during the course of decision making. Here, we propose a protocol to train rats on an operant-based decision-making paradigm where rats choose higher rewards at the expense of waiting for 15 s to receive them.

image

JoVE Core

An Instrumented Pull Test to Characterize Postural Responses
Joy Tan 1,2,4, Wesley Thevathasan 2,3,4,5, Jennifer McGinley 6, Peter Brown 7, Thushara Perera 1,4
1Department of Medical Bionics, The University of Melbourne, 2Department of Neurology, The Royal Melbourne Hospital, 3Department of Neurology, Austin Hospital, 4The Bionics Institute, 5Department of Medicine, The University of Melbourne, 6Department of Physiotherapy, The University of Melbourne, 7Medical Research Council Brain Network Dynamics Unit, University of Oxford

Impairment of postural reflexes, termed postural instability, is difficult to quantify. Clinical assessments such as the pull test suffer issues with reliability and scaling. Here, we present an instrumented version of the pull test to objectively characterize postural responses.

image

Biochemistry

The Application of Open Searching-based Approaches for the Identification of Acinetobacter baumannii O-linked Glycopeptides
Jessica M. Lewis *1, Pauline M. L. Coulon *1, Taylor A. McDaniels *1, Nichollas E. Scott 1
1Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, The University of Melbourne

Open searching enables the identification of glycopeptides decorated with previously unknown glycan compositions. Within this article, a streamlined approach for undertaking open searching and subsequent glycan-focused glycopeptide searches are presented for bacterial samples using Acinetobacter baumannii as a model.

image

Developmental Biology

Spatiotemporal Subcellular Manipulation of the Microtubule Cytoskeleton in the Living Preimplantation Mouse Embryo using Photostatins
Jessica Greaney 1, Azelle Hawdon 1, G. Gemma Stathatos 1,2, Asma Aberkane 1, Jennifer Zenker 1
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.

image

Biology

A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay
Sebastian Bass-Stringer 1,2, Colleen J. Thomas 2,3, Clive N. May 3, Paul Gregorevic 4, Kate L. Weeks 1,5,6, Julie R. McMullen 1,2,5,6,7
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.

image

Neuroscience

Intraventricular Drug Delivery and Sampling for Pharmacokinetics and Pharmacodynamics Study
Sara Oberrauch 1, Jing Lu 1, Linda Cornthwaite-Duncan 1, Maytham Hussein 1, Jian Li 2, Gauri Rao 3, Tony Velkov 1
1Department of Biochemistry & Pharmacology, School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, 2Department of Microbiology, Biomedicine Discovery Institute, Monash University, 3UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill

Delivery of therapeutics directly into the central nervous system is one way of circumventing the blood-brain barrier. The present protocol demonstrates intracerebroventricular injection for subsequent collection of cerebrospinal fluid and bodily organs. This facilitates the investigation of drug pharmacokinetics and pharmacodynamics in animal models for developing new treatments.

image

Bioengineering

Experimental Quantification of Interactions Between Drug Delivery Systems and Cells In Vitro: A Guide for Preclinical Nanomedicine Evaluation
Paula M. Cevaal 1, Michael Roche 2, Sharon R. Lewin 2,3,4, Frank Caruso 5, Matthew Faria 6
1Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, 2Department of Infectious Diseases, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, 3Victorian Infectious Diseases Service, Royal Melbourne Hospital at the Peter Doherty Institute for Infection and Immunity, 4Department of Infectious Diseases, Alfred Hospital and Monash University, 5Department of Chemical Engineering, The University of Melbourne, 6Department of Biomedical Engineering, The University of Melbourne

A workflow is demonstrated for the absolute quantification of drug carrier-cell interactions using flow cytometry to allow better rational evaluation of novel drug delivery systems. This workflow is applicable to drug carriers of any type.

image

Immunology and Infection

Rat Burn Model to Study Full-Thickness Cutaneous Thermal Burn and Infection
Rajnikant Sharma 1, Shekhar Yeshwante 1, Quentin Vallé 1, Maytham Hussein 2, Varsha Thombare 2, Sean Michael McCann 1, Robert Maile 3,4,5, Jian Li 6, Tony Velkov 2, Gauri Rao 1
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.

image

Medicine

Using a Combination of Indirect Calorimetry, Infrared Thermography, and Blood Glucose Levels to Measure Brown Adipose Tissue Thermogenesis in Humans
Lachlan Van Schaik 1,3, Christine Kettle 1, Rod A. Green 1, Helen R. Irving 1, Joseph A. Rathner 1,2
1La Trobe Institute for Molecular Science, Department of Rural Clinical Sciences, La Trobe University, 2School of Biomedical Sciences, Department of Physiology, The University of Melbourne, 3Melbourne Medical School, Department of Rural Health, The University of Melbourne

Here, we present a protocol to quantify the physiological significance of the impact of brown adipose tissue (BAT) activity on human metabolism. This is achieved by combining carbohydrate loading and indirect calorimetry with measurements of supraclavicular changes in temperature. This novel approach can help develop a pharmacological target for BAT thermogenesis in humans.

image

Biology

Gas Chromatography-Mass Spectrometry-Based Targeted Metabolomics of Hard Coral Samples
Jennifer L. Matthews 1, Natasha Bartels 1, Sheik Nadeem Elahee Doomun 2, Simon K. Davy 3, David P. De Souza 2
1Climate Change Cluster (C3), University of Technology Sydney, 2Metabolomics Australia, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 3School of Biological Sciences, Victoria University of Wellington

Here, we present the extraction and preparation of polar and semi-polar metabolites from a coral holobiont, as well as separated coral host tissue and Symbiodiniaceae cell fractions, for gas chromatography-mass spectrometry analysis.

image

Neuroscience

Dual Extracellular Recordings in the Mouse Hippocampus and Prefrontal Cortex
Dechuan Sun 1,2, Mona Amiri 1, Luke Weston 1,2, Chris French 1
1Neural Dynamics Laboratory, Department of Medicine, The University of Melbourne, 2Department of Electrical and Electronic Engineering, The University of Melbourne

This protocol outlines the use of a custom-designed recording device and electrodes to record local field potentials and investigate information flow in the mouse hippocampus and prefrontal cortex.

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved