Hudson Institute of Medical Research 6 articles published in JoVE Developmental Biology Transillumination-Assisted Dissection of Specific Stages of the Mouse Seminiferous Epithelial Cycle for Downstream Immunostaining Analyses Juho-Antti Mäkelä1, Sheyla Cisneros-Montalvo1, Tiina Lehtiniemi1, Opeyemi Olotu1, Hue M. La3,4, Jorma Toppari1,2, Robin M. Hobbs3,4, Martti Parvinen1, Noora Kotaja1 1Institute of Biomedicine, Integrative Physiology and Pharmacology Unit, University of Turku, Turku, Finland, 2Department of Pediatrics, Turku University Hospital, Turku, Finland, 3Centre for Reproductive Health, Hudson Institute of Medical Research, Melbourne, VIC 3168, Australia, 4Australian Regenerative Medicine Institute, Monash University, Melbourne, VIC 3800, Australia This protocol describes transillumination-assisted microdissection of segments of adult mouse seminiferous tubules representing specific stages of seminiferous epithelial cycle, and cell types therein, and subsequent immunostaining of squash preparations and intact tubule segments. Bioengineering Automated Counterflow Centrifugal System for Small-Scale Cell Processing Anqi Li1,2, Stephen Wilson3, Ian Fitzpatrick3, Mehri Barabadi1,2, Siow Teng Chan1, Mirja Krause1,2, Gina Diamanta Kusuma1,2, David James3, Rebecca Lim1,2,4 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. Immunology and Infection Mouse Models Of Helicobacter Infection And Gastric Pathologies Kimberley D'Costa1, Michelle Chonwerawong1, Le Son Tran1, Richard L. Ferrero1,2 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. Neuroscience Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation Jessica C. Stark*1, Euan Wallace1,2, Rebecca Lim1, 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. 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 Ghosh4, Graham Jenkin3, David Oehme5, Justin Cooper-White6,7, Taryn Naidoo6, Tony Goldschlager1,8 1Department of Surgery, Monash University, 2Department of Neurosurgery, Monash University, 3The Ritchie Centre, Hudson Institute of Medical Research, 4Proteobioactives, Pty Ltd, 5 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. Biology Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy Matthew McKenzie1,2, Sze C. Lim1, Michael R. Duchen3 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.