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Royal Prince Alfred Hospital

6 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

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

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

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Biology

Isolation of Small Noncoding RNAs from Human Serum
Samantha Khoury 1, Pamela Ajuyah 1, Nham Tran 2,3
1School of Medical and Molecular Biosciences, Faculty of Science, University of Technology, Sydney, 2Centre for Health Technologies, Faculty of Engineering and Information Technology, University of Technology, Sydney, 3The Sydney Head and Neck Cancer Institute, Sydney Cancer Centre, Royal Prince Alfred Hospital

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

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Medicine

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

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

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Medicine

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

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

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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

Ultrasound Assessment of Aortic and Carotid Artery Intima-media Thickness in Children and Adolescents
Reeja Nasir 1, Michael Skilton 1, Adrienne Gordon 1,2
1The Boden Initiative, Charles Perkins Centre, University of Sydney, 2Department of Obstetrics, Gynaecology and Neonatology, Royal Prince Alfred Hospital

The present article describes the methodological considerations for the noninvasive assessment of abdominal aortic and carotid intima-media thickness using B-mode ultrasonography. This technique is commonly used in the developmental origins of health and disease research as a surrogate for early arterial changes.

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