Royal Prince Alfred Hospital

5 ARTICLES PUBLISHED IN JoVE

Immunology and Infection

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

Jerry Zhou¹, Larissa Belov¹, Michael J. Solomon², Charles Chan³, Stephen J. Clarke⁴, Richard I. Christopherson¹

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.

Biology

Isolation of Small Noncoding RNAs from Human Serum

Samantha Khoury¹, Pamela Ajuyah¹, 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.

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 Ahmed³, 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.

Medicine

Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses

Chi Ching Goh^1,2, Jackson LiangYao Li¹, David Becker³, 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.

Biology

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury

Jully Gogoi-Tiwari¹, Julia Köhn-Gaone¹, Corey Giles², Dirk Schmidt-Arras³, Francis D. Gratte^1,4, Caryn L. Elsegood¹, 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.