Universite de Montreal 7 articles published in JoVE Biology Optimized Protocol for Generating Functional Pancreatic Insulin-secreting Cells from Human Pluripotent Stem Cells Ines Cherkaoui1, Qian Du2, Dieter Egli2, Shivani Misra1,3, Guy A. Rutter1,4,5 1Department of Metabolism, Digestion and Reproduction, Faculty of Medicine, Cell Biology and Functional Genomics, 2Departments of Pediatrics, Naomi Berrie Diabetes Center, Obstetrics and Gynecology, Columbia Stem Cell Initiative, Columbia University Irving Medical Center, Columbia University, 3Department of Diabetes, Imperial College Healthcare NHS Trust, 4Faculté de Médicine, Université de Montréal, 5Lee Kong Chian Imperial Medical School, Nanyang Technological University This article presents a protocol for directed differentiation and functional analysis of β-cell like cells. We describe optimal culture conditions and passages for human pluripotent stem cells before generating insulin-producing pancreatic cells. The six-stage differentiation progresses from definitive endoderm formation to functional β-cell like cells secreting insulin in response to glucose. Biology Assessment of Gut Barrier Integrity in Mice Using Fluorescein-Isothiocyanate-Labeled Dextran Claire Gerkins1, Roy Hajjar1,2, Manon Oliero1, Manuela M. Santos1,3 1 In the present study, fluorescein-isothiocyanate-labeled (FITC) dextran is administered to mice via oral gavage to evaluate intestinal permeability both in vivo and in plasma and fecal samples. As gut barrier function is affected in many disease processes, this direct and quantitative assay can be used in diverse research areas. Neuroscience Teasing Out the Interplay Between Natural Killer Cells and Nociceptor Neurons Ali Ahmadi1, Mohammad Balood1, Katiane Roversi1, Maryam Ahmadi1, Moutih Rafei1, Sebastien Talbot1 1Department of Pharmacology and Physiology, Université de Montréal Nociceptor neurons and NK cells actively interact in an inflammatory context. A co-culture approach enables studying this interplay. Medicine Large-Animal Model of Donation after Circulatory Death and Normothermic Regional Perfusion for Cardiac Assessment Khalil Khalil*1,2, Roberto V. P. Ribeiro*3, Julgans S. Alvarez4,5, Mitesh V. Badiwala4,5, Shant Der Sarkissian1,2, Nicolas Noiseux1,2 1 The protocol describes a large-animal (porcine) model of donation after circulatory death, followed by thoracoabdominal normothermic regional perfusion that closely simulates the clinical scenario in heart transplantation, and has the potential to facilitate therapeutic studies and strategies. Biochemistry Immunopeptidomics: Isolation of Mouse and Human MHC Class I- and II-Associated Peptides for Mass Spectrometry Analysis Isabelle Sirois*1, Maxim Isabelle*1, Jérôme D. Duquette1, Frederic Saab1, Etienne Caron1,2 1CHU Sainte-Justine Research Center, 2Department of Pathology and Cellular Biology, Faculty of Medicine, Université de Montréal Here, we present a protocol for the purification of MHC class I and class II peptide complexes from mouse and human cell lines providing high-quality immunopeptidomics data. The protocol focuses on sample preparation using commercially available antibodies. Biology A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3 Alexa C. Robitaille*1,2,3, Mélissa K. Mariani*1,3, Audray Fortin1, Nathalie Grandvaux1,2,3 1CRCHUM - Research center, Centre Hospitalier de l'Université de Montréal, Université de Montréal, 2Department of Biochemistry and Molecular Medicine, Université de Montréal, 3Faculty of Medicine, Université de Montréal Here we describe a procedure allowing a detailed analysis of the phosphorylation-dependent activation of the IRF3 transcription factor. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple phosphospecific antibodies. Immunology and Infection Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System. Mariana G. Bego1, Édouard A. Côté1, Éric A. Cohen1,2 1Laboratory of Human Retrovirology, Institut de Recherches Cliniques de Montréal (IRCM), 2Department of Microbiology, Infectiology, and Immunology, Université de Montréal Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.
