Hospital for Sick Children View Institution's Website 10 articles published in JoVE Biology A Fluorescence-Based Assay of Membrane Potential for High-Throughput Functional Study of Two Endogenous Ion Channels in Two Epithelial Cell Lines Sunny Xia1,3, Michelle Di Paola3, Nicola L. Jones2,4,5, Christine E. Bear1,3,5 1Molecular Medicine, Hospital for Sick Children, 2Cell Biology, Hospital for Sick Children, 3Department of Physiology, University of Toronto, 4Department of Paediatrics, University of Toronto, 5Department of Biochemistry, University of Toronto This protocol describes a method for the study of electrogenic membrane proteins by measuring changes in membrane potential. This assay provides a platform for the functional readout of multiple ion channels endogenously expressed in epithelial cell lines. Immunology and Infection Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software Amanda J. Morris1, Alvin Li1, Lindsay Jackson1, Yvonne C. W. Yau1,2, Valerie Waters1,3 1Translational Medicine, Hospital for Sick Children, 2Microbiology, Department of Pediatric Laboratory Medicine, Hospital for Sick Children, 3Infectious Diseases, Department of Pediatrics, Hospital for Sick Children Antimicrobial-induced changes to Pseudomonas aeruginosa biofilm architecture differ among clinical isolates cultured from patients with cystic fibrosis and chronic pulmonary infection. Following confocal microscopy, COMSTAT software can be utilized to quantify variations in biofilm architecture (e.g., surface area, thickness, biomass) for individual isolates to assess the efficacy of anti-infective agents. Immunology and Infection Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients Lindsay Jackson1, William DePas2, Amanda J. Morris1, Kevin Guttman1, Yvonne C W Yau1,3, Valerie Waters1,4 1Translational Medicine, Hospital for Sick Children, 2Center for Microbial Pathogenesis, Department of Pediatrics, University of Pittsburgh, 3Microbiology, Department of Pediatric Laboratory Medicine, Hospital for Sick Children, 4Infectious Diseases, Department of Pediatrics, Hospital for Sick Children This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients. Medicine Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents Martin Sidler1,2,3, Karen J. Aitken1, Jia Xin Jiang4, Darius J. Bägli1,2,3 1Developmental and Stem Cell Biology, Research Institute, The Hospital for Sick Children, 2Institute of Medical Science, University of Toronto, 3Pediatric Urology, The Hospital for Sick Children, 4Molecular Structure and Function, Research Institute, The Hospital for Sick Children Traditional modeling of partial bladder outlet obstruction in rodents is fraught with animal mortality. A denervation injury from dissection around the proximal urethra and bladder neck is also of major concern. We developed and evaluated a safe and reliable mid-urethral obstruction model, avoiding the shortcomings of the traditional model. Immunology and Infection Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model Trevor Beaudoin1, Sarah Kennedy2, Yvonne Yau3, Valerie Waters4 1Physiology and Experimental Medicine, Hospital for Sick Children, 2Department of Clinical Microbiology, Royal College of Surgeons in Ireland, 3Department of Laboratory Medicine and Pathobiology, University of Toronto, 4Department of Pediatrics, University of Toronto This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs. Bioengineering Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds Sharareh Shojaie1, Joyce Lee1, Jinxia Wang1, Cameron Ackerley1, Martin Post1 1Department of Physiology and Experimental Medicine, Peter Gilgan Centre for Research and Learning, Hospital for Sick Children This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting. Biology Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein Paul D. W. Eckford1, Canhui Li1, Christine E. Bear1,2,3 1Programme in Molecular Structure and Function, Hospital for Sick Children, 2Department of Biochemistry, University of Toronto, 3Department of Physiology, University of Toronto Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules. Developmental Biology Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus Steven J. Deimling1, Rami R. Halabi2,3, Stephanie A. Grover4, Jean H. Wang2,5, Thomas A. Drysdale2,4,5 1Developmental and Stem Cell Biology, Hospital for Sick Children, 2Children's Health Research Institute, University of Western Ontario, 3Department of Physiology and Pharmacology, University of Western Ontario, 4Neurosciences and Mental Health, Hospital for Sick Children, 5Department of Paediatrics, University of Western Ontario The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system. Medicine A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics Kelly Burrell1, Sameer Agnihotri1, Michael Leung2, Ralph DaCosta2, Richard Hill2, Gelareh Zadeh3 1Brain Tumor Research Centre, Hospital for Sick Children, Toronto Medical Discovery Tower, 2Ontario Cancer Institute, Princess Margaret Hospital, 3Neurosurgery, Toronto Western Hospital We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution. Neuroscience Multiple-mouse Neuroanatomical Magnetic Resonance Imaging Jun Dazai*1, Shoshana Spring*1, Lindsay S. Cahill1, R. Mark Henkelman1,2 1Mouse Imaging Centre, Hospital for Sick Children, 2Department of Medical Biophysics and Medical Imaging, University of Toronto Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.
