JoVE Logo

Sign In

Monash University

65 ARTICLES PUBLISHED IN JoVE

image

Biology

Anterior Cervical Discectomy and Fusion in the Ovine Model
Tony Goldschlager 1,2, Jeffrey V. Rosenfeld 2, Ian R. Young 1, Graham Jenkin 1
1Monash Immunology and Stem Cell Laboratories (MISCL), Monash University, 2Department of Surgery, Monash University

This video demonstrates the technique of anterior cervical discectomy and fusion in the ovine model.

image

Biology

Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis
Tony Goldschlager 1, Amany Abdelkader 1, Jeffrey Kerr 2, Ian Boundy 2, Graham Jenkin 1
1Monash Immunology and Stem Cell Laboratories, Monash University, 2Anatomy and Developmental Biology, Monash University

Undecalcified bone histology provides important information for a variety of clinical and research applications. It is technically challenging, particularly with large size specimens. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them.

image

Immunology and Infection

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites
Sittiporn Pattaradilokrat 1, Jian Li 1,2, Xin-zhuan Su 1
1National Institute of Allergy and Infectious Diseases, National Institutes of Health, 2School of Life Science, Xiamen University

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

image

Biology

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae
Dalibor Mijaljica 1, Mark Prescott 1, Rodney J. Devenish 1
1Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

image

Immunology and Infection

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure
Danielle L. Michell 1, Karen L. Andrews 1, Kevin J. Woollard 1, Jaye P.F. Chin-Dusting 1
1Vascular Pharmacology Laboratory, Baker IDI Heart and Diabetes Institute, Monash University

This is a method to visualise leukocyte adhesion to the endothelium in harvested pressurised vessels. The technique enables studying vascular adhesion under shear flow with differing intraluminal pressures up to 200 mmHg thus mimic-ing the pathophysiological conditions of high blood pressure.

image

Bioengineering

Simultaneous Synthesis of Single-walled Carbon Nanotubes and Graphene in a Magnetically-enhanced Arc Plasma
Jian Li 1, Alexey Shashurin 1, Madhusudhan Kundrapu 1, Michael Keidar 1
1Department of Mechanical and Aerospace Engineering, The George Washington University

Anodic arc discharge is one of the most practical and efficient methods to synthesize various carbon nanostructures. To increase the arc controllability and flexibility, a non-uniform magnetic field was introduced to process the one-step synthesis of large-scale graphene flakes and high-purity single-walled carbon nanotubes.

image

Immunology and Infection

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens
Erica L. Benard 1, Astrid M. van der Sar 2, Felix Ellett 3, Graham J. Lieschke 3, Herman P. Spaink 1, Annemarie H. Meijer 1
1Department of Molecular Cell Biology, Institute of Biology, Leiden University, 2Department of Medical Microbiology and Infection Control, VU University Medical Center, 3Australian Regenerative Medicine Institute, Monash University

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

image

Medicine

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction
Ryan Hodges 1,2, Masayuki Endo 1, Andre La Gerche 3, Elisenda Eixarch 4,5, Philip DeKoninck 1, Vessilina Ferferieva 3, Jan D'hooge 3, Euan M. Wallace 2, Jan Deprest 1
1Division Woman and Child, Department Women, University Hospitals Leuven, 2The Ritchie Centre, Monash Institute of Medical Research, Department of Obstetrics and Gynaecology, Monash University, Victoria, Australia, 3Department of Cardiovascular Sciences, Katholieke Universiteit Leuven, 4Fetal and Perinatal Medicine Research Group, Institut d'Investigacions Biomediques August Pi i Sunyer (IDIBAPS), 5Maternal-Fetal Medicine Department, ICGON, Hospital Clínic, Universitat de Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER)

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

image

Medicine

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression
Ming G. Chai 1, Corina Kim-Fuchs 1,2, Eliane Angst 2, Erica K. Sloan 1,3
1Monash Institute of Pharmaceutical Sciences, Monash University, 2Department of Visceral Surgery and Medicine, University of Bern, 3Cousins Center for Neuroimmunology, University of California Los Angeles

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

image

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.

image

Bioengineering

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier
Be'eri Niego 1, Robert L. Medcalf 1
1Australian Centre for Blood Diseases, Monash University

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

image

Medicine

Intramyocardial Cell Delivery: Observations in Murine Hearts
Tommaso Poggioli 1,2, Padmini Sarathchandra 1,2, Nadia Rosenthal 2,3, Maria P. Santini 1,2
1Magdi Yacoub Institute, Imperial College London, 2National Heart and Lung Institute, Imperial College London, 3Australian Regenerative Medicine Institute, Monash University

Intramyocardial cell delivery in murine models of cardiovascular diseases, such as hypertension or myocardial infarction, is widely used to test the therapeutic potential of different cell types in regenerative studies. Therefore, a detailed description and a clear visualization of this surgical procedure will help to define the limits and advantages of cardiovascular cell therapeutic analyses in small rodents.

image

Bioengineering

Sample Drift Correction Following 4D Confocal Time-lapse Imaging
Adam Parslow 1, Albert Cardona 2, Robert J. Bryson-Richardson 1
1School of Biological Sciences, Monash University, 2Janelia Farm Research Campus, Howard Hughes Medical Institute

Time-lapse microscopy allows the visualization of developmental processes. Growth or drift of samples during image acquisition reduces the ability to accurately follow and measure cell movements during development. We describe the use of open source image processing software to correct for three dimensional sample drift over time.

image

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.

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

Medicine

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time
Maree Bilandzic 1, Kaye L. Stenvers 1,2
1Reproductive Development and Cancer Laboratory, MIMR-PHI Institute of Medical Research, 2Department of Developmental Biology and Anatomy, Monash University

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

image

Biology

Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model
Christian M. Nefzger *1,2, Sara Alaei *1,2, Anja S. Knaupp 1,2, Melissa L. Holmes 1,2, Jose M. Polo 1,2
1Department of Anatomy and Developmental Biology, Monash University, 2Australian Regenerative Medicine Institute, Monash University

Mouse embryonic fibroblast can be reprogrammed into induced pluripotent stem cells at low efficiency by the forced expression of transcription factors Oct-4, Sox-2, Klf-4, c-Myc. The rare intermediates of the reprogramming reaction are FACS isolated via labeling with antibodies against cell surface makers Thy-1.2, Ssea-1, and Epcam.

image

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.

image

Biology

Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells
Brigham J. Hartley 1, Stewart A. Fabb 1, Ben A.L. Finnin 1, John M. Haynes 1, Colin W. Pouton 1
1Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University

Reporter cell lines offer a means to visualize, track and isolate cells of interest from heterogeneous populations. However, gene-targeting using conventional homologous recombination in human embryonic stem cells is extremely inefficient. Herein, we describe targeting CNS midbrain specific transcription factor PITX3 locus with EGFP using zinc-finger nuclease enhanced homologous recombination.

image

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.

image

JoVE Journal

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood
Clovis S. Palmer 1,2,3, Joshua J. Anzinger 4, Tiffany R. Butterfield 4, Joseph M. McCune 5, Suzanne M. Crowe 1,2,6
1Centre for Biomedical Research, Macfarlane Burnet Institute for Medical Research and Public Health, 2Department of Infectious Diseases, Monash University, 3Department of Microbiology and Immunology, University of Melbourne, 4Department of Microbiology, The University of the West Indies, 5Division of Experimental Medicine, Department of Medicine, University of California, San Francisco, 6Department of Medicine, Monash University

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

image

Developmental Biology

Using Touch-evoked Response and Locomotion Assays to Assess Muscle Performance and Function in Zebrafish
Tamar E. Sztal *1, Avnika A. Ruparelia *1, Caitlin Williams 1, Robert J. Bryson-Richardson 1
1School of Biological Sciences, Monash University

Zebrafish are an excellent model to study muscle function and disease. During early embryogenesis zebrafish begin regular muscle contractions producing rhythmic swimming behavior, which is altered when the muscle is disrupted. Here we describe a touch-evoked response and locomotion assay to examine swimming performance as a measure of muscle function.

image

Biochemistry

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification
Danielle L. Michell *1, Ryan M. Allen *1, Stuart R. Landstreet 1, Shilin Zhao 2, Cynthia L. Toth 1, Quanhu Sheng 3, Kasey C. Vickers 1,2
1Department of Medicine, Vanderbilt University School of Medicine, 2Center for Quantitative Sciences, Vanderbilt University School of Medicine, 3Department of Cancer Biology, Vanderbilt University School of Medicine

This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

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

Biology

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy
Matthew McKenzie 1,2, Sze C. Lim 1, Michael R. Duchen 3
1Centre for Genetic Diseases, Hudson Institute of Medical Research, 2The Department of Molecular and Translational Sciences, Monash University, 3Department of Physiology, University College London

Mitochondria can utilize the electrochemical potential across their inner membrane (ΔΨm) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and ΔΨm in live cells using fluorescent dyes and confocal microscopy.

image

Bioengineering

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair
Jian Li 1, Holger Jahr 2,3, Wei Zheng 4, Pei-Gen Ren 1
1Center for Translational Medicine Research and Development, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, 2Department of Orthopedic Surgery, Maastricht UMC+, 3Department of Orthopaedic Surgery, University Hospital RWTH, 4Research Laboratory for Biomedical Optics and Molecular Imaging, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

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

Ovine Lumbar Intervertebral Disc Degeneration Model Utilizing a Lateral Retroperitoneal Drill Bit Injury
Kai-Zheong Lim *1, Christopher D. Daly *1,2,3, Peter Ghosh 4, Graham Jenkin 3, David Oehme 5, Justin Cooper-White 6,7, Taryn Naidoo 6, Tony Goldschlager 1,8
1Department of Surgery, Monash University, 2Department of Neurosurgery, Monash University, 3The Ritchie Centre, Hudson Institute of Medical Research, 4Proteobioactives, Pty Ltd, 5Department of Neurosurgery, St Vincent's Hospital, 6Australian Institute for Bioengineering and Nanotechnology, University of Queensland, 7School of Chemical Engineering, University of Queensland, 8Department of Neurosurgery, Monash Health

Intervertebral disc degeneration is a significant contributor to back pain and a leading cause of disability worldwide. Numerous animal models of intervertebral disc degeneration exist. We demonstrate an ovine model of intervertebral disc degeneration, utilizing a drill bit, which achieves a consistent disc injury and reproducible level of disc degeneration.

image

Biochemistry

Microcrystallography of Protein Crystals and In Cellulo Diffraction
Marion Boudes *1, Damià Garriga *1, Fasséli Coulibaly 1
1Infection and Immunity Program, Monash Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University

A protocol is presented for X-ray crystallography using protein microcrystals. Two examples analyzing in vivo-grown microcrystals after purification or in cellulo are compared.

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

Immunology and Infection

Quantification of Monocyte Transmigration and Foam Cell Formation from Individuals with Chronic Inflammatory Conditions
Thomas A. Angelovich 1,2, Anna C. Hearps 1,3, Anna Maisa 1, Theodoros Kelesidis 4, Anthony Jaworowski 1,3
1Centre for Biomedical Research, Burnet Institute, 2School of Health and Biomedical Sciences, RMIT University, 3Department of Infectious Diseases, Monash University, 4University of California, Los Angeles

We describe a protocol to measure transmigration by monocytes across human endothelial monolayers and their subsequent maturation into foam cells. This provides a versatile method to assess the atherogenic properties of monocytes isolated from people with different disease conditions and to evaluate factors in blood which may enhance this propensity.

image

JoVE Core

Preparation of Aligned Steel Fiber Reinforced Cementitious Composite and Its Flexural Behavior
Ru Mu 1, Luansu Wei 1, Xiaowei Wang 1, Hui Li 1, Longbang Qing 1, Jian Zhou 1, Quanming Zhao 2
1School of Civil and Transportation Engineering, Hebei University of Technology, 2School of Information Engineering, Hebei University of Technology

This protocol describes an approach for manufacturing aligned steel fiber reinforced cementitious composite by applying a uniform electromagnetic field. Aligned steel fiber reinforced cementitious composite exhibits superior mechanical properties to ordinary fiber reinforced concrete.

image

Immunology and Infection

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein
Shubhendu Sen Roy 1, Huy Cong Xuan Nguyen 1, Thomas A. Angelovich 2,3, Anna C. Hearps 2, Diana Huynh 1,4, Anthony Jaworowski 2,4, Theodoros Kelesidis 1
1University of California, Los Angeles, 2Centre for Biomedical Research, Burnet Institute, 3School of Health and Biomedical Sciences, RMIT University, 4Department of Infectious Diseases, Monash University

We describe here a fluorometric cell-free biochemical assay for determination of HDL-lipid peroxidation. This rapid and reproducible assay can be used to determine HDL function in large scale studies and can contribute to our understanding of HDL function in human disease.

image

Immunology and Infection

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy
Roleen Sharma 1,2, Kim M. O'Sullivan 2, Stephen R. Holdsworth 2, Philip G. Bardin 1,2, Paul T. King 1,2
1Monash Lung and Sleep, Monash Medical Centre, 2Monash University

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

image

Developmental Biology

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae
Felix Ellett 1, Daniel Irimia 1
1BioMEMS Resource Center, Department of Surgery, Massachusetts General Hospital–Harvard Medical School–Shriners Burns Hospital

Microinjection of zebrafish embryos and larvae is a crucial but challenging technique used in many zebrafish models. Here, we present a range of microscale tools to aid in the stabilization and orientation of zebrafish for both microinjection and imaging.

image

Chemistry

Detection and Quantification of Plasmodium falciparum in Aqueous Red Blood Cells by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data Analysis
Miguela Martin 1, David Perez-Guaita 1, Dean W. Andrew 3, Jack S. Richards 3,4, Bayden R. Wood 1, Philip Heraud 1,2
1Centre for Biospectroscopy, Monash University, 2Department of Microbiology, Faculty of Medicine, Nursing and Health Sciences, Monash University, 3Centre for Biomedical Research, Burnet Institute, 4Department of Medicine, University of Melbourne

Here, we present a protocol for the detection and quantification of Plasmodium falciparum in infected aqueous red blood cells using an attenuated total reflection infrared spectrometer and multivariate data analysis.

image

Immunology and Infection

Mouse Models Of Helicobacter Infection And Gastric Pathologies
Kimberley D'Costa 1, Michelle Chonwerawong 1, Le Son Tran 1, Richard L. Ferrero 1,2
1Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, 2Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Microbiology, Monash University

Mice represent an invaluable in vivo model to study infection and diseases caused by gastrointestinal microorganisms. Here, we describe the methods used to study bacterial colonization and histopathological changes in mouse models of Helicobacter pylori-related disease.

image

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.

image

Biochemistry

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain
Mayra A. Machuca 1,2, Anna Roujeinikova 1,2,3
1Infection and Immunity Program, Monash Biomedicine Discovery Institute, 2Department of Microbiology, Monash University, 3Department of Biochemistry and Molecular Biology, Monash University

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

image

Developmental Biology

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton
Sandeep Gopal 1, Roger Pocock 1
1Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Department of Anatomy and Developmental Biology, Monash University

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

image

Medicine

Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease
Louise Benning 1, Samuel Robinson 1,2, Marie Follo 3, Lukas Andreas Heger 1, Daniela Stallmann 1, Daniel Duerschmied 1, Christoph Bode 1, Ingo Ahrens 1,4, Marcus Hortmann 1
1Department of Cardiology and Angiology I, Heart Center Freiburg University, Faculty of Medicine, University of Freiburg, 2Department of Medicine, Monash University, 3Department of Medicine I, Lighthouse Core Facility, Medical Center, University of Freiburg, 4Department of Cardiology, Augustinerinnen Hospital, Academic Teaching Hospital, University of Cologne

Circulating microRNAs have shown promise as biomarkers for cardiovascular diseases and acute myocardial infarctions. In this study, we describe a protocol for miRNA extraction, reverse transcription, and digital PCR for the absolute quantification of miRNAs in the serum of patients with cardiovascular disease.

image

Bioengineering

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA
Tatjana Michel 1, Antonia Link 1, Meike-Kristin Abraham 1,2, Christian Schlensak 1, Karlheinz Peter 2,3, Hans-Peter Wendel 1, Xiaowei Wang 2,3, Stefanie Krajewski 1
1Department of Thoracic and Cardiovascular Surgery, Clinical Research Laboratory, University Medical Center, 2Atherothrombosis and Vascular Biology, Baker Heart & Diabetes Institute, 3Department of Medicine, Monash University

Here we describe a protocol for the generation of cationic nanoliposomes, which is based on the dry-film method and can be used for the safe and efficient delivery of in vitro transcribed messenger RNA.

image

JoVE Journal

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction
Dan Zhou *1,2, Lei Tan *1, Jian Li *3, Tanbin Liu 1, Yi Hu 1, Yalan Li 1, Sachiyo Kawamoto 4, Chengyu Liu 5, Shiyin Guo 3, Aibing Wang 1
1Lab of Animal Models and Functional Genomics (LAMFG), The Key Laboratory of Animal Vaccine & Protein Engineering, College of Veterinary Medicine, Hunan Agricultural University (HUNAU), 2Department of Pathology, Georgetown University Medical School, 3College of Food Science and Technology, Hunan Agricultural University (HUNAU), 4Lab of Molecular Cardiology (LMC), National Heart, Lung, and Blood Institute (NHLBI)/National Institutes of Health (NIH), 5Transgenic Core, National Heart, Lung, and Blood Institute (NHLBI)/National Institutes of Health (NIH)

Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology.

image

Developmental Biology

Culturing and Measuring Fetal and Newborn Murine Long Bones
Veronica Uribe 1, Alberto Rosello-Diez 1
1Australian Regenerative Medicine Institute, Monash University

Here, we present a method for ex vivo culture of long murine bones at both fetal and newborn stages, suitable for analyzing bone and cartilage development and homeostasis in controlled conditions while recapitulating the in vivo process.

image

JoVE Core

Home-Based Prescribed Pulmonary Exercise in Patients with Stable Chronic Obstructive Pulmonary Disease
Xiaodan Liu 1,2, Peijun Li 3, Jian Li 3, Lu Xiao 1, Ning Li 3, Yufan Lu 3, Zhengrong Wang 3, Jianqing Su 3, Zhenwei Wang 4, Chunlei Shan 1,2, Weibing Wu 3
1School of Rehabilitation Science, Shanghai University of Traditional Chinese Medicine, 2Institute of Rehabilitation Medicine, Shanghai Academy of Traditional Chinese Medicine, 3Department of Sports Medicine, Shanghai University of Sport, 4Department of Respiratory Medicine, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine

Presented here is a protocol to investigate the effects of home-based prescribed pulmonary exercise in stable chronic obstructive pulmonary disease (COPD) patients, which is modified based on traditional Chinese exercises according to dyspnea and limited exercise capacity observed in COPD patients.

image

Neuroscience

In Vivo Imaging of Cerebrospinal Fluid Transport through the Intact Mouse Skull using Fluorescence Macroscopy
Amanda M Sweeney *1, Virginia Plá *1, Ting Du 1, Guojun Liu 1, Qian Sun 1, Sisi Peng 1, Benjamin A. Plog 1, Benjamin T. Kress 1, Xiaowei Wang 1, Humberto Mestre 1, Maiken Nedergaard 1,2
1Center for Translational Neuromedicine, University of Rochester Medical Center, 2Center for Translational Neuromedicine, University of Copenhagen

Transcranial optical imaging allows wide-field imaging of cerebrospinal fluid transport in the cortex of live mice through an intact skull.

image

Biology

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells
Niels Alberts *1, Yasith Mathangasinghe *2, Nadinath B. Nillegoda 3
1Department of Biomedical Sciences of Cells & Systems, University of Groningen, 2Department of Anatomy, Faculty of Medicine, University of Colombo, 3Australian Regenerative Medicine Institute (ARMI), Monash University

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

image

Behavior

Radiotracer Administration for High Temporal Resolution Positron Emission Tomography of the Human Brain: Application to FDG-fPET
Sharna D. Jamadar 1,2,3, Phillip G.D. Ward 1,2,3, Alexandra Carey 1,4, Richard McIntyre 1,4, Linden Parkes 1,3, Disha Sasan 1, John Fallon 1, Edwina Orchard 1,2,3, Shenpeng Li 1,5, Zhaolin Chen 1,5, Gary F Egan 1,2,3
1Monash Biomedical Imaging, Monash University, 2Australian Research Council Centre of Excellence for Integrative Brain Function, 3Turner Institute for Brain and Mental Health, Monash University, 4Department of Medical Imaging, Monash Health, 5Department of Electrical and Computer Systems Engineering, Monash University

This manuscript describes two radiotracer administration protocols for FDG-PET (constant infusion and bolus plus infusion) and compares them to bolus administration. Temporal resolutions of 16 s are achievable using these protocols.

image

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.

image

JoVE Journal

Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly
Samuel C. Leguizamon 1, Abdulla F. Alqubati 1, Timothy F. Scott 2,3
1Department of Chemical Engineering, University of Michigan, 2Department of Chemical Engineering, Monash University, 3Department of Materials Science and Engineering, Monash University

A protocol is presented for the synthesis of information-encoded peptoid oligomers and for the sequence-directed self-assembly of these peptoids into molecular ladders using amines and aldehydes as dynamic covalent reactant pairs and Lewis acidic rare-earth metal triflates as multi-role reagents.

image

Behavior

Human Circadian Phenotyping and Diurnal Performance Testing in the Real World
Elise R. Facer-Childs 1,2,3, Benita Middleton 1, Andrew P. Bagshaw 2, Debra J. Skene 1
1Chronobiology, Faculty of Health & Medical Sciences, University of Surrey, 2Centre for Human Brain Health, University of Birmingham, 3Turner Institute for Brain and Mental Health, School of Psychological Sciences, Monash University

Here, we present a method to investigate diurnal rhythms in performance following accurate categorization of participants into circadian phenotype groups based on the Munich ChronoType Questionnaire, gold standard circadian phase biomarkers and actigraphic measures.

image

Immunology and Infection

Supervised Machine Learning for Semi-Quantification of Extracellular DNA in Glomerulonephritis
Kim Maree O'Sullivan 1, Sarah Creed 2, Poh-Yi Gan 1, Stephen R. Holdsworth 1,3
1Department of Medicine, Monash University, 2Monash Micro Imaging, Monash University, 3Immunology Department, Monash Health

Extracellular DNA (ecDNA) released during cell death is proinflammatory and contributes to inflammation. Measurement of ecDNA at the site of injury can determine the efficacy of therapeutic treatment in the target organ. This protocol describes the use of a machine learning tool to automate measurement of ecDNA in kidney tissue.

image

Neuroscience

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae
Gonçalo M. Poças 1,2, Pedro M. Domingos *2, Christen K. Mirth *1
1School of Biological Sciences, Monash University, 2Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA)

Described here is a protocol that enables the colorimetric quantification of the amount of food eaten within a defined interval of time by Drosophila melanogaster larvae exposed to diets of different macronutrient quality. These assays are conducted in the context of a neuronal thermogenetic screen.

image

Chemistry

Atomic Force Microscopy Combined with Infrared Spectroscopy as a Tool to Probe Single Bacterium Chemistry
Kamila Kochan 1, Anton Y. Peleg 2,3, Philip Heraud 1,2, Bayden R. Wood 1
1Centre for Biospectroscopy and School of Chemistry, Monash University, 2Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Microbiology, Monash University, 3Department of Infectious Diseases, The Alfred Hospital and Central Clinical School, Monash University

Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) provides a powerful platform for bacterial studies, enabling to achieve nanoscale resolution. Both, mapping of subcellular changes (e.g., upon cell division) as well as comparative studies of chemical composition (e.g., arising from drug resistance) can be conducted at a single cell level in bacteria.

image

Developmental Biology

Accurate Follicle Enumeration in Adult Mouse Ovaries
Amy L. Winship 1, Urooza C. Sarma 1, Lauren R. Alesi 1, Karla J. Hutt 1
1Development and Stem Cells Program, Monash Biomedicine Discovery Institute, and Department of Anatomy and Developmental Biology, Monash University

Here, we describe, compare, and contrast two different techniques for accurate follicle counting of fixed mouse ovarian tissues.

image

Developmental Biology

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease
Margo Montandon 1, Peter D. Currie 1, Avnika A. Ruparelia 1
1Australian Regenerative Medicine Institute, Monash University

Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease.

image

Immunology and Infection

Assessing Respiratory Immune Responses to Haemophilus Influenzae
Lovisa Dousha 1,2, Roleen Sharma 1,2, Steven Lim 2,3, James Ngui 2,4, Ashley M. Buckle 5, Paul T. King 1,2
1Monash Lung and Sleep, Monash Medical Centre, 2Monash University Department of Medicine, Monash Medical Centre, 3Flow Cytometry Science Technology Platform, Francis Crick Institute, 4Clinical Immunology, Monash Medical Centre, 5Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University

Haemophilus influenzae induces inflammation in the respiratory tract. This article will focus on the use of flow cytometry and confocal microscopy to define immune responses by phagocytes and lymphocytes in response to this bacterium.

image

Medicine

An In Vivo Mouse Model of Total Intravenous Anesthesia During Cancer Resection Surgery
Julia A. Dubowitz 1,2,3, Fabian Jost-Brinkmann 1,4,5, Alexandra I. Ziegler 1, Ryan D. Gillis 1, Bernhard Riedel 1,2,3,6, Erica K. Sloan 1,2
1Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences, Monash University, 2Department of Anaesthesia, Division of Cancer Surgery, Peter MacCallum Cancer Centre, 3Department of Critical Care, Melbourne Medical School, University of Melbourne, 4Medical Department, Division of Hepatology and Gastroenterology, Charité - Universitätsmedizin Berlin, 5Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, 6Sir Peter MacCallum Department of Oncology, University of Melbourne

This paper describes a method for modeling total intravenous anesthesia (TIVA) during cancer resection surgery in mice. The goal is to replicate key features of anesthesia delivery to patients with cancer. The method allows investigation of how anesthetic technique affects cancer recurrence after resection surgery.

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

Neuroscience

A Standardized Pipeline for Examining Human Cerebellar Grey Matter Morphometry using Structural Magnetic Resonance Imaging
Rebecca Kerestes 1, Shuo Han 2, Srinivas Balachander 3, Carlos Hernandez-Castillo 4, Jerry L. Prince 5,6, Jörn Diedrichsen 7, Ian H. Harding 1,8
1Department of Neuroscience, Central Clinical School, Monash University, 2Department of Biomedical Engineering, The Johns Hopkins University, 3Department of Psychiatry, National Institute of Mental Health & Neuro Sciences (NIMHANS), 4Faculty of Computer Science, Dalhousie University, 5Department of Electrical and Computer Engineering, The Johns Hopkins University, 6Department of Computer Science, The Johns Hopkins University, 7Brain and Mind Institute, Department for Statistical and Actuarial Sciences, Department for Computer Science, Western University, 8Monash Biomedical Imaging, Monash University

A standardized pipeline is presented for examining cerebellum grey matter morphometry. The pipeline combines high-resolution, state-of-the-art approaches for optimized and automated cerebellum parcellation and voxel-based registration of the cerebellum for volumetric quantification.

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

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

Biology

Visualizing Mitophagy with Fluorescent Dyes for Mitochondria and Lysosome
Bilin Liu 1,2, Anqi Li 1, Yuan Qin 3, Lei Chen 1, Meng Gao 1, Guohua Gong 1,2
1Institute for Regenerative Medicine, Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, 2Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, 3Department of Pharmacy, Shanghai East Hospital, Tongji University

Mitophagy is the primary mechanism of mitochondrial quality control. However, the evaluation of mitophagy in vivo is hindered by the lack of reliable quantitative assays. Presented here is a protocol for the observation of mitophagy in living cells using a cell-permeant green-fluorescent mitochondria dye and a red-fluorescent lysosome dye.

image

Developmental Biology

Methods for Studying Uterine Contributions to Pregnancy Establishment in an Ovariectomized Mouse Model
Meaghan J. Griffiths *1,2, Jordan N. Higgins *1, Fiona L. Cousins 3, Lauren R. Alesi 1, Amy L. Winship *1, Karla J. Hutt *1
1Biomedicine Discovery Institute, Department of Anatomy and Developmental Biology, Monash University, 2Gynaecology Research Centre, Royal Women’s Hospital and Department of Obstetrics and Gynaecology, University of Melbourne, 3The Ritchie Centre, Hudson Institute for Medical Research and Department of Obstetrics and Gynaecology, Monash University

Pregnancy establishment is a dynamic process involving complex embryo and uterine crosstalk. The precise contributions of the maternal uterine environment to these processes remain an active area of investigation. Here, detailed protocols are provided to aid in designing in vivo animal models to address these research questions.

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved