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34 ARTICLES PUBLISHED IN JoVE

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Biology

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos
Félix Legendre *1,2, Neal Cody *1, Carole Iampietro 1, Julie Bergalet 1, Fabio Alexis Lefebvre 1,2, Gaël Moquin-Beaudry 1,2, Olivia Zhang 1, Xiaofeng Wang 1, Eric Lécuyer 1,2
1Institut de Recherches Cliniques de Montréal (IRCM), 2Department of Biochemistry, Université de Montréal

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

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Medicine

Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy
Marie-Yvonne Akoume 1, Anita Franco 1, Alain Moreau 1,2
1Viscogliosi Laboratory in Molecular Genetics of Musculoskeletal Diseases, Sainte-Justine University Hospital Research Center, 2Department of Stomatology, Faculty of Dentistry and Department of Biochemistry, Faculty of Medicine, Université de Montréal

A structured protocol is presented for a cell-based assay as a functional test to predict the prognosis of idiopathic scoliosis using cellular dielectric spectroscopy (CDS). The assay can be performed with fresh or frozen peripheral blood mononuclear cells (PBMCs) and the procedure is completed within 4 days.

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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. Bego 1, Édouard A. Côté 1, Éric A. Cohen 1,2
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.

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Immunology and Infection

Development of an IFN-γ ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation
Insaf Salem Fourati 1, Anne-Julie Grenier 1, Élyse Jolette 1, Natacha Merindol 1, Philippe Ovetchkine 2,3, Hugo Soudeyns 1,3
1Unité d'Immunopathologie Virale, Centre de Recherche du CHU Sainte-Justine, Department of Microbiology, Infectiology & Immunology, Faculty of Medicine, Université de Montréal, 2Infectious Diseases Service, CHU Sainte-Justine, Faculty of Medicine, Université de Montréal, 3Department of Paediatrics, Université de Montréal

Novel generations of functional assays such as gamma interferon (IFN-γ) ELISpot, which detect cytokine production at the single cell level and provide both quantitative and qualitative characterization of T cell responses can be used to assess cell-mediated immune responses directed against varicella zoster virus (VZV).

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Immunology and Infection

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay
Maxime Veillette 1, Mathieu Coutu *1, Jonathan Richard *1, Laurie-Anne Batraville *1, Anik Désormeaux 1, Michel Roger 1, Andrés Finzi 1
1Centre de recherche du CHUM, Department of Microbiology, Infectiology and Immunology, Université de Montréal

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

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Neuroscience

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain
Jimmy Lai 1, Marc-André Legault 1, Sébastien Thomas 1, Christian Casanova 1
1École d'optométrie, Université de Montréal

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

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Developmental Biology

Instrumentation of Near-term Fetal Sheep for Multivariate Chronic Non-anesthetized Recordings
Patrick Burns 1, Hai Lun Liu 2, Shikha Kuthiala 2, Gilles Fecteau 1, André Desrochers 1, Lucien Daniel Durosier 2, Mingju Cao 2, Martin G. Frasch 2,3,4
1Département de sciences cliniques, CHUV, Université de Montréal, St-Hyacinthe, QC, 2Département d'obstetriques et de gynécologie, CHU Ste-Justine Research Centre, Université de Montréal, 3Département de neurosciences, CHU Ste-Justine Centre de recherche, Université de Montréal, 4Centre de recherche en reproduction animale (CRRA), Université de Montréal, St-Hyacinthe, QC

The chronically instrumented non-anesthetized fetal sheep model is used to study human fetal development in health and disease, because it permits surgical placement and maintenance of catheters and electrodes, repetitive blood sampling, substance injection, recording of bioelectrical activity, and in vivo imaging. We describe the procedures required to establish this model.

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Immunology and Infection

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation
Antonio Layoun 1, Macha Samba 1, Manuela M. Santos 1,2
1Centre de recherche du Centre hospitalier de l'Université de Montréal, Institut du cancer de Montréal, 2Département de Médecine, Université de Montréal

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

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Biology

A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3
Alexa C. Robitaille *1,2,3, Mélissa K. Mariani *1,3, Audray Fortin 1, Nathalie Grandvaux 1,2,3
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.

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Medicine

A Magnetic Microbead Occlusion Model to Induce Ocular Hypertension-Dependent Glaucoma in Mice
Yoko A. Ito *1, Nicolas Belforte *1, Jorge L. Cueva Vargas 1, Adriana Di Polo 1
1Department of Neuroscience, CRCHUM and University of Montreal

Here, we present a protocol to induce ocular hypertension in the murine eye that results in the loss of retinal ganglion cells as observed in glaucoma. Magnetic microbeads are injected into the anterior chamber and attracted to the iridocorneal angle using a magnet to block the outflow of aqueous humour.

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Medicine

Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps
Ahmadali Sajadi 1,3, Chloé Provost 3, Brendon Pham 2, Jonathan Brouillette 1,3
1Department of Pharmacology, Université de Montréal, 2Department of Neuroscience, Université de Montréal, 3Hôpital du Sacré-coeur de Montréal Research Center, Université de Montréal

To study the effects of Aβo in vivo, we developed a model based on repeated hippocampal infusions of soluble Aβo coupled with continuous infusion of Aβo antibody (6E10) in the hippocampus using osmotic pumps to counteract the neurotoxic effect of Aβo.

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Immunology and Infection

Preparation and Applications of Organotypic Thymic Slice Cultures
Aditi Sood *1,2, Mengqi Dong *1,2, Heather J. Melichar 1,3
1Centre de Recherche, Hôpital Maisonneuve-Rosemont, 2Department of Microbiology, Infectiology and Immunology, Université de Montréal, 3Department of Medicine, Université de Montréal

We describe the preparation of thymic slices that, in combination with flow cytometry, can be used to model positive and negative selection of developing T cells. Thymic slices can also be adapted for the in situ analysis of thymocyte migration, localization, and signaling via immunofluorescence and two-photon microscopy.

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Immunology and Infection

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules
Ahmed Fouda 1, Mahasti Tahsini 2, Fatemeh Khodayarian 2, Fatimah Al-nafisah 2, Moutih Rafei 1,2,3
1Department of Microbiology, Infectiology and Immunology, Université de Montréal, 2Department of Pharmacology and Physiology, Université de Montréal, 3Molecular Biology Program, Université de Montréal

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

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Neuroscience

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy
Lara Eid 1,2, Martin Parent 1
1Centre de recherche de l'Institut universitaire en santé mentale de Québec, Department of Psychiatry and Neuroscience, Université Laval, 2Centre de recherche du CHU Sainte-Justine

Here, we provide an easy, low-cost, and time-efficient protocol to chemically fix primate brain tissue with acrolein fixative, allowing for long-term preservation that is compatible with pre-embedding immunohistochemistry for transmission electron microscopy.

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Biochemistry

Mouse Adipose Tissue Collection and Processing for RNA Analysis
Paul Tan 1,2, Émilie Pepin 1,4, Julie L. Lavoie 1,3,4
1Centre de Recherche du Centre Hospitalier, Université de Montréal, 2Department of Biochemistry and Molecular Medicine, Université de Montréal, 3Department of Kinesiology, Université de Montréal, 4Montreal Diabetes Research Center

The purpose of this paper is to present a step-by-step procedure to collect different white adipose tissues from mice, process the fat samples and extract RNA.

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Medicine

A Ligated Intestinal Loop Model in Anesthetized Specific Pathogen Free Chickens to Study Clostridium Perfringens Virulence
Eric Parent 1, Patrick Burns 2, André Desrochers 1, Martine Boulianne 1
1Department of Clinical Sciences, Faculty of Veterinary Medicine, Université de Montréal, 2University of Bristol

Here we present a protocol to surgically create 'intestinal ligated loops' in chicken small intestines. This procedure allows for the comparison of multiple Clostridium perfringens strains' virulence in situ in a single host. This method markedly decreases the number of chickens usually necessary for similar in vivo experiments.

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Neuroscience

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons
Lara Eid 1,2, Mathieu Lachance 1, Gilles Hickson 1,3, Elsa Rossignol 1,2,4
1Centre de recherche du CHU Sainte-Justine, 2Department of Neuroscience, Université de Montréal, 3Department of pathology and cellular biology, Université de Montréal, 4Department of Pediatrics, Université de Montréal

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

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Neuroscience

Analyzing the Size, Shape, and Directionality of Networks of Coupled Astrocytes
Steven Condamine 1, Dorly Verdier 1, Arlette Kolta 1,2
1Groupe de Recherche sur le Système Nerveux Central, and Département de Neurosciences, Université de Montréal, 2Faculté de Médecine Dentaire, Université de Montréal

Here we present a protocol to assess the organization of astrocytic networks. The described method minimizes bias to provide descriptive measures of these networks such as cell count, size, area, and position within a nucleus. Anisotropy is assessed with a vectorial analysis.

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Developmental Biology

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells
Amanda B.T. Hill 1,2, Jonathan E.B.T. Hill 1, Fabiana F. Bressan 3, Maria Angélica Miglino 4, Joaquim M. Garcia 1
1The School of Agricultural and Veterinary Studies (FCAV), Jaboticabal, São Paulo State University (UNESP), 2CRRF, Université de Montréal, 3FZEA, University of São Paulo, 4FMVZ, University of São Paulo

Herein, we describe a method for the isolation, expansion, and differentiation of mesenchymal stem cells from canine ovarian tissue.

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Immunology and Infection

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment
Syim Salahuddin *1,2, Elaine Thomson *1,2,3, Oussama Méziane 1,2, Omar Farnos 2, Amélie Pagliuzza 5, Nicolas Chomont 5,6, Ron Olivenstein 1, Cecilia Costiniuk 1,3,4, Mohammad-Ali Jenabian 2,3,6
1Research Institute McGill University Health Centre, 2Department of Biological Sciences, Université de Québec à Montréal, 3Department of Microbiology & Immunology, McGill University, 4Department of Medicine, Division of Infectious Diseases, McGill University, 5Centre de Recherche du Centre Hospitalier de l'Université de Montréal, 6Département de microbiologie, infectiologie et immunologie, Université de Montréal

We describe a method for processing bronchoalveolar lavage fluid and matched peripheral blood from chronically HIV-infected individuals on antiretroviral therapy to assess pulmonary HIV reservoirs. These methods result in the acquisition of highly pure CD4 T cells and alveolar macrophages that may subsequently be used for immunophenotyping and HIV DNA/RNA quantifications by ultrasensitive polymerase chain reaction.

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Neuroscience

A Novel and Translational Rat Model of Concussion Combining Force and Rotation with In Vivo Cerebral Microdialysis
Ian O. Massé 1, Luc Moquin 2, Chloé Provost 1, Samuel Guay 1, Alain Gratton 2, Louis De Beaumont 1
1Research Center, Hôpital du Sacré-Cœur de Montréal, 2Research Center, Douglas Institute

Neurotransmitter alteration is a mechanism of neural dysfunction that occurs after concussion and contributes to the sometimes-catastrophic long-term consequences. This rat model combines microdialysis, allowing in vivo neurotransmitter quantification, with a weight-drop technique exerting rapid acceleration and deceleration of the head and torso, an important factor of human craniocerebral trauma.

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Neuroscience

Thoracic Spinal Cord Hemisection Surgery and Open-Field Locomotor Assessment in the Rat
Andrew R. Brown 1,2, Marina Martinez 1,2,3
1Department of Neurosciences, Faculté de Médecine, Université de Montréal, 2Hôpital du Sacré-Cœur de Montréal, 3Groupe de Recherche sur le Système Nerveux Central (GRSNC), Université de Montréal

The rat thoracic spinal hemisection is a valuable and reproducible model of unilateral spinal cord injury to investigate the neural mechanisms of locomotor recovery and treatment efficacy. This article includes a detailed step-by-step guide to perform the hemisection procedure and to assess locomotor performance in an open-field arena.

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Medicine

Pre-clinical Model of Cardiac Donation after Circulatory Death
Henry Aceros *1, Leyla Joulali *2, Mélanie Borie 1, Roberto Vanin Pinto Ribeiro 3, Mitesh Vallabh Badiwala 3, Shant Der Sarkissian 1,4, Nicolas Noiseux 1,4
1Centre de recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), 2Deparment of pharmacology and physiology, Faculty of Medicine, Université de Montréal, 3Division of Cardiovascular Surgery, Toronto General Hospital, University Health Network, 4Department of surgery, Faculty of Medicine, Université de Montréal

This protocol shows a simple and flexible approach for the evaluation of new conditioning agents or strategies to increase the feasibility of cardiac donation after circulatory death.

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Behavior

Measuring the Switch Cost of Smartphone Use While Walking
Gabrielle Naïmé Mourra 1, David Brieugne 1, Emma Rucco 1, Élise Labonté-Lemoyne 1, François Courtemanche 1, Sylvain Sénécal 1, Marc Fredette 1, Ann-Frances Cameron 1, Jocelyn Faubert 2, Franco Lepore 2, François Bellavance 1, Pierre-Majorique Léger 1
1HEC Montréal, 2Université de Montréal

This study design measures the task-switching cost of using a smartphone while walking. Participants undergo two experimental conditions: a control condition (walking) and a multitasking condition (texting while walking). Participants switch between these tasks and a direction determining task. EEG data as well as behavioral measures are recorded.

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JoVE Journal

Efficient Transcriptionally Controlled Plasmid Expression System for Investigation of the Stability of mRNA Transcripts in Primary Alveolar Epithelial Cells
Francis Migneault 1,2,3, Frédéric Gagnon 2,3, Emmanuelle Brochiero 1,3, Yves Berthiaume 2,3, André Dagenais 2,3
1Centre de Recherche du Centre hospitalier de l'Université de Montréal (CRCHUM), 2Institut de Recherches Cliniques de Montréal (IRCM), 3Département de médecine, Faculté de médecine, Université de Montréal

Here, we present a tool that can be used to study the posttranscriptional modulation of a transcript in primary alveolar epithelial cells by using an inducible expression system coupled to a pipette electroporation technique.

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Immunology and Infection

Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment
Manuel Flores Molina 1,2, Thomas Fabre 1,2, Aurélie Cleret-Buhot 1, Geneviève Soucy 1,3, Liliane Meunier 1,4, Mohamed N. Abdelnabi 1,2, Nicolas Belforte 1,5, Simon Turcotte 1,4,6, Naglaa H. Shoukry 1,4,7
1Centre de Recherche du Centre hospitalier de l'Université de Montréal (CRCHUM), 2Département de Microbiologie, Infectiologie et Immunologie, Faculté de Médecine, Université de Montréal, 3Département de Pathologie et Biologie Cellulaire, Faculté de Médecine, Université de Montréal, 4Institut du Cancer de Montréal, 5Département de Neurosciences, Faculté de Médecine, Université de Montréal, 6Département de Chirurgie, Faculté de Médecine, Université de Montréal, 7Département de Médecine, Faculté de médecine, Université de Montréal

Here, we describe a simple and accessible strategy for visualizing, quantifying, and mapping immune cells in formalin-fixed paraffin-embedded tumor tissue sections. This methodology combines existing imaging and digital analysis techniques with the purpose of expanding the multiplexing capability and multiparameter analysis of imaging assays.

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Neuroscience

Electrocorticographic Recording of Cerebral Cortex Areas Manipulated Using an Adeno-Associated Virus Targeting Cofilin in Mice
Julien Dufort-Gervais 1, Robbert Havekes 2, Valérie Mongrain 1,3
1Center for Advanced Research in Sleep Medicine, Recherche CIUSSS-NIM, 2GELIFES, University of Groningen, 3Department of Neuroscience, Université de Montréal

This article describes a protocol for the manipulation of molecular targets in the cerebral cortex using adeno-associated viruses and for monitoring the effects of this manipulation during wakefulness and sleep using electrocorticographic recordings.

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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. Duquette 1, Frederic Saab 1, Etienne Caron 1,2
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.

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Neuroscience

Teasing Out the Interplay Between Natural Killer Cells and Nociceptor Neurons
Ali Ahmadi 1, Mohammad Balood 1, Katiane Roversi 1, Maryam Ahmadi 1, Moutih Rafei 1, Sebastien Talbot 1
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.

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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. Alvarez 4,5, Mitesh V. Badiwala 4,5, Shant Der Sarkissian 1,2, Nicolas Noiseux 1,2
1Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), 2Department of Surgery, Faculty of Medicine, Université de Montréal, 3Division of Cardiac Surgery, Dalhousie University, 4Division of Cardiovascular Surgery, Peter Munk Cardiac Centre, Toronto General Hospital, 5Department of Surgery, University of Toronto

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.

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Biology

Assessment of Gut Barrier Integrity in Mice Using Fluorescein-Isothiocyanate-Labeled Dextran
Claire Gerkins 1, Roy Hajjar 1,2, Manon Oliero 1, Manuela M. Santos 1,3
1Nutrition and Microbiome Laboratory, Institut du cancer de Montréal, Centre de recherche du Centre hospitalier de l’Université de Montréal (CRCHUM), 2Digestive Surgery Service, Centre hospitalier de l’Université de Montréal (CHUM), Faculty of Medicine, Université de Montréal, 3Department of Medicine, Faculty of Medicine, Université de Montréal

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.

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Neuroscience

A Human Cerebral Organoid Model of Neural Cell Transplantation
M. Iliana Ibanez-Rios 1, Melina Narlis 1, Colin A. Hammond 2, David J. H. F. Knapp 1,3
1Institut de Recherche en Immunologie et en Cancérologie, Université de Montréal, 2Terry Fox Laboratory, British Columbia Cancer Agency, 3Département de Pathologie et Biologie Cellulaire, Université de Montréal

Here, we describe a protocol for the transplantation and tracking of labeled neural cells into human cerebral organoids.

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Biology

Optimized Protocol for Generating Functional Pancreatic Insulin-secreting Cells from Human Pluripotent Stem Cells
Ines Cherkaoui 1, Qian Du 2, Dieter Egli 2, Shivani Misra 1,3, Guy A. Rutter 1,4,5
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.

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Medicine

Rat Model of Right-Sided Cardiac Remodeling and Arrhythmia Using Pulmonary Artery Banding
Charles-Alexandre LeBlanc 1, Marie-Ève Higgins 1, Martin G. Sirois 1,2, Jean-François Tanguay 1,3, Jean-Claude Tardif 1,3, Roddy Hiram 1,3
1Montreal Heart Institute, 2Department of Pharmacology and Physiology, Université de Montréal, 3Department of Medicine, Faculty of Medicine, Université de Montréal

Right heart failure (RHF) is characterized by right-sided cardiac dilation and hypertrophy, leading to ventricular and atrial malfunction. Cardiopulmonary conditions associated with RHF are accompanied by an increased risk for cardiac arrhythmias. This article describes a standardized model of pulmonary artery banding-induced RHF associated with enhanced ventricular and atrial arrhythmogenesis.

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