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The Centenary Institute

3 ARTICLES PUBLISHED IN JoVE

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.

Cancer Research

Rapid Optical Clearing for Semi-High-Throughput Analysis of Tumor Spheroids

Gency Gunasingh¹, Alexander P. Browning², Nikolas K. Haass¹

¹The University of Queensland Diamantina Institute, The University of Queensland, ²School of Mathematical Sciences, Queensland University of Technology

Tumor spheroids are becoming increasingly utilized to assess tumor cell-microenvironment interactions and therapy response. The present protocol describes a robust but simple method for semi-high-throughput imaging of 3D tumor spheroids using rapid optical clearing.