Universite-Paris-Descartes View Institution's Website 20 articles published in JoVE Bioengineering A Reliable Porcine Fascio-Cutaneous Flap Model for Vascularized Composite Allografts Bioengineering Studies Victor Pozzo1,2,4, Golda Romano1,2,4, Marion Goutard1,2,4, Elise Lupon1,2,4, Pierre Tawa1,2,4, Aylin Acun3,4,5, Alec R. Andrews2, Corentin B. Taveau1,2,4,6, Basak E. Uygun1,2,3,4, Mark A. Randolph1,2,4, Curtis L. Cetrulo1,2,4, Alexandre G. Lellouch1,2,4,6 1Division of Plastic and Reconstructive Surgery, Massachusetts General Hospital, Harvard Medical School, 2Vascularized Composite Allotransplantation Laboratory, Center for Transplantation Sciences, Massachusetts General Hospital, Harvard Medical School, 3Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, Harvard Medical School, 4Shriners Hospital for Children, 5Department of Biomedical Engineering, Widener University, 6Service de Chirurgie Plastique, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris (APHP), Université Paris Descartes The present protocol describes the porcine fascio-cutaneous flap model and its potential use in vascularized composite tissue research. Immunology and Infection Partial Heterotopic Hindlimb Transplantation Model in Rats Marion Goutard1,2,3, Mark A. Randolph1,2,3, Corentin B. Taveau1,2,3,4, Elise Lupon1,2,3, Laurent Lantieri4, Korkut Uygun1,2,3, Curtis L. Cetrulo Jr.*1,2,3, Alexandre G. Lellouch*1,2,3,4 1Division of Plastic and Reconstructive Surgery, Massachusetts General Hospital, Harvard Medical School, 2Vascularized Composite Allotransplantation Laboratory, Center for Transplantation Sciences, Massachusetts General Hospital, Harvard Medical School, 3Shriners Hospital for Children, 4Service de Chirurgie Plastique, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris (APHP), Université de Paris This paper presents a partial heterotopic osteomyocutaneous flap transplantation protocol in rats and its potential outcomes in the mid-term follow-up. Immunology and Infection Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue Nikaïa Smith1,2,3,4, Nassima Bekaddour2,3,4, Nicolas Leboulanger4,5, Yolande Richard*6, Jean-Philippe Herbeuval*2,3,4 1Institute of Molecular Virology, Ulm University Medical Center, 2CNRS UMR-8601, Centre Interdisciplinaire Chimie Biologie, 3Team Chemistry & Biology, Modeling & Immunology for Therapy, Université Paris Descartes, 4Université Paris Descartes, Sorbonne Paris Cité, 5Pediatric Otolaryngology Department, Hôpital Necker-Enfants Malades, Assistance Publique Hôpitaux de Paris, 6Université de Paris, Institut Cochin In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Genetics Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis Sophie Outh-Gauer1, Jérémy Augustin1, Marion Mandavit2, Ophélie Grard2, Thomas Denize1, Marine Nervo1, Charles Lépine1, Marc Rassy2, Eric Tartour2,3, Cécile Badoual1,2 1Department of Pathology, Georges Pompidou European Hospital, Assistance publique - Hôpitaux de Paris (APHP), Paris Descartes University, 2Paris Cardiovascular Research Center (PARCC), Institut national de la santé et de la recherche médicale (Inserm) U970, 3Laboratory of Immunology, Georges Pompidou European Hospital, APHP, Paris Descartes University Human papillomavirus (HPV) RNA chromogenic in situ hybridization is considered to be one of the gold standards for active human papillomavirus infection detection within tumors. It allows the visualization of HPV E6-E7 mRNA expression with localization and semiquantitative evaluation of its signal. Neuroscience Long-term Sensory Conflict in Freely Behaving Mice Filipa França de Barros1,2, Julie Carcaud2, Mathieu Beraneck1,2 1Integrative Neuroscience and Cognition Center, UMR8002, CNRS, 2Sorbonne Paris Cité, Université Paris Descartes The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Genetics Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions Charles Decraene1,2, Amanda Bortolini Silveira1, Marc Michel1, François-Clément Bidard1,3,4, Jean-Yves Pierga1,3,5, Marc-Henri Stern1,6, Charlotte Proudhon1 1Circulating Tumor Biomarkers Laboratory, SiRIC, Translational Research Department, Institut Curie, PSL Research University, 2CNRS UMR144, Institut Curie, PSL Research University, 3Department of Medical Oncology, Institut Curie, PSL Research University, 4University Versailles Saint-Quentin-en-Yvelines, 5University Paris Descartes, 6Inserm U830, Institut Curie, PSL Research University Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes. Cancer Research Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells Clémence Granier1, Emeline Vinatier2,3,4, Elia Colin2,3, Marion Mandavit2,3, Charles Dariane2,3,5, Virginie Verkarre6, Lucie Biard7, Rami El Zein2, Corinne Lesaffre2, Isabelle Galy-Fauroux2, Hélène Roussel2,3,6, Eléonore De Guillebon8, Charlotte Blanc2,3, Antonin Saldmann4, Cécile Badoual2,6, Alain Gey*4, Éric Tartour*2,4 1PARCC-INSERM U970, Université Paris Descartes, 2INSERM U970, Université Paris Descartes, 3Equipe Labellisée Ligue Contre le Cancer, 4Department of Immunology, Hopital Européen Georges Pompidou, 5Department of Medical Urology, Hopital Européen Georges Pompidou, 6Department of Pathology, Hopital Européen Georges Pompidou, 7Université Paris Diderot Paris 7, 8Department of medical oncology, Hopital Européen Georges Pompidou Studying tumor microenvironment may identify prognostic or predictive biomarkers of clinical response to immunotherapy. Presented here, is an innovative method based on in situ fluorescence multispectral imaging to analyze and count automatically various subpopulations of CD8+ T cells. This reproducible and reliable technique is suitable for large cohort analyses. Cancer Research Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy Elisa Peranzoni*1, Houcine Bougherara*1, Sarah Barrin*1, Audrey Mansuet-Lupo2,3, Marco Alifano4, Diane Damotte2,3, Emmanuel Donnadieu1 1Institut Cochin, Inserm U1016-CNRS UMR8104, Université Paris Descartes, 2Department of Pathology, Paris Centre University Hospitals, Université Paris Descartes, 3INSERM U1138, Cancer and Immune Escape, Cordeliers Research Center, University Pierre and Marie Curie, 4Department of Thoracic Surgery, Paris Centre University Hospitals, University Paris Descartes This protocol describes a method to image resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy. Immunology and Infection Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells Pascale Vonaesch1, Philippe J. Sansonetti1,2, Pamela Schnupf3 1Unité de Pathogénie Microbienne Moléculaire, INSERM U786, Institut Pasteur, 2Microbiologie et Maladies Infectieuses, Collège de France, 3Laboratory of Intestinal Immunity, Institut Imagine-INSERM UMR 1163, Université Paris Descartes-Sorbonne Paris Cité We describe a method for the qualitative and quantitative analysis of stress granule formation in mammalian cells after the cells are challenged with bacteria and a number of different stresses. This protocol can be applied to investigate the cellular stress granule response in a wide range of host-bacterial interactions. Genetics Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions Oriana Romano1,2, Ingrid Cifola3, Valentina Poletti4, Marco Severgnini3, Clelia Peano3, Gianluca De Bellis3, Fulvio Mavilio1,4, Annarita Miccio1,2,4,5 1Center for Genome Research, Department of Life Sciences, University of Modena and Reggio Emilia, 2Laboratory of Chromatin and Gene Regulation During Development, Imagine Institute, 3Institute for Biomedical Technologies, CNR, 4Généthon, 5Sorbonne Paris Cité - Université Paris Descartes Here, we describe a protocol for genome-wide mapping of the integration sites of Moloney murine leukemia virus-based retroviral vectors in human cells. Medicine Continuous Manual Exchange Transfusion for Patients with Sickle Cell Disease: An Efficient Method to Avoid Iron Overload Bérengère Koehl1, Florence Missud1, Laurent Holvoet1, Ghislaine Ithier1, Oliver Sakalian-Black2, Zinedine Haouari1, Emmanuelle Lesprit3, André Baruchel4, Malika Benkerrou1 1Reference Centre of Sickle Cell Disease, Hematology Unit, Robert Debré Hospital, AP-HP, 2School of Pharmacy, Université Paris Descartes, 3Etablissement Français du Sang, Robert Debré Hospital, AP-HP, 4Hematology Unit,, Robert Debré Hospital, AP-HP, Univsité Paris Diderot We have outlined a method of continuous manual exchange transfusion for the treatment of sickle cell disease in patients. This safe protocol was designed to effectively limit iron overload in patients in need of chronic transfusions and can be used extensively without any special equipment. Medicine ADSC-sheet Transplantation to Prevent Stricture after Extended Esophageal Endoscopic Submucosal Dissection Guillaume Perrod1,2,3, Laetitia Pidial3, Sophie Camilleri1,4, Alexandre Bellucci1,3,5, Amaury Casanova3, Thomas Viel3, Bertrand Tavitian1,3,5, Chirstophe Cellier1,2, Olivier Clément1,3,5, Gabriel Rahmi1,2,3 1Assistance Publique-Hôpitaux de Paris, Université Paris Descartes Sorbonne Paris Cité, 2Department of Gastroenterology, Hôpital Européen Georges Pompidou, 3UMR-S970, Université Paris Descartes Sorbonne Paris Cité, 4Department of Pathology, Hôpital Européen Georges Pompidou, 5Department of Radiology, Hôpital Européen Georges Pompidou This study reports a successful method of endoscopical adipose tissue-derived stromal cell (ADSC)-sheet transplantation for esophageal stricture prevention after an extended endoscopic submucosal dissection (ESD) in a swine model. Biology The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress Luke A. White1, Emily V. Stevenson1, J. Winny Yun1, Randa Eshaq1, Norman R. Harris1, David K. Mills2, Alireza Minagar3, Pierre-Olivier Couraud4, J. Steven Alexander1 1Molecular and Cellular Physiology, Louisiana State University Health Sciences Center in Shreveport, 2Biological Sciences, Louisiana Tech University, 3Neurology, Louisiana State University Health Sciences Center in Shreveport, 4Institut Cochin, Inserm U1016, Cnrs Umr8104, Université Paris Descartes Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields. Neuroscience The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation Félix Leroy1, Boris Lamotte d'Incamps1 1Centre National de la Recherche Scientifique (UMR 8119), Centre de Neurophysique, Physiologie et Pathologie, Université Paris Descartes We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots. Immunology and Infection "Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM) Florence Marie-Anaïs1, Julie Mazzolini1, Pierre Bourdoncle1, Florence Niedergang1 1Inserm U1016, Institut Cochin, CNRS UMR 8104, Université Paris Descartes, Sorbonne Paris Cité We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission. Chemistry Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR Riccardo Balzan1, Laetitia Fernandes1, Arnaud Comment2, Laetitia Pidial3, Bertrand Tavitian3, Paul R. Vasos1 1LCBPT - UMR8601, Institut de Chimie, Université Paris Descartes, 2Institute of Physics of Biological Systems, EPFL, 3PARCC - INSERM U970, Université Paris Descartes The sensitivity enhancement provided by dissolution dynamic nuclear polarization (DNP) enables following metabolic processes in real time by NMR and MRI. The characteristics and performances of a dedicated dissolution DNP setup designed for study enzymatic reactions are discussed. Medicine Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue Elena Dossi1, Thomas Blauwblomme2,3, Rima Nabbout2,4, Gilles Huberfeld2,5, Nathalie Rouach1 1Neuroglial Interactions in Cerebral Physiopathology, Center for Interdisciplinary Research in Biology, CNRS UMR 7241, INSERM U1050, Collège de France, 2Infantile Epilepsies & Brain Plasticity, INSERM U1129, PRES, Paris Descartes University, Sorbonne Paris Cité, CEA, 3Neurosurgery Department, Necker Hospital, AP-HP, Paris Descartes University, 4Rare Epilepsies Reference Center, Necker Hospital, AP-HP, Paris Descartes University, 5Neurophysiology Department, La Pitié-Salpêtrière Hospital, AP-HP, Sorbonne and Pierre and Marie Curie University We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail. Immunology and Infection Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature Keira Melican1,2, Flore Aubey1,2, Guillaume Duménil1,2 1INSERM U970, Paris Cardiovascular Research Centre, 2Faculté de Médecine Paris Descartes, Université Paris Descartes Neisseria meningitidis is a human specific pathogen that infects blood vessels. In this protocol human microvessels are introduced into a mouse by grafting human skin onto immunocompromised mice. Bacteria adhere extensively to the human vessels, leading to vascular damage and development of the purpuric rash typically observed in human cases. Immunology and Infection Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes Hélène Salmon1,2, Ana Rivas-Caicedo1,2, François Asperti-Boursin1,2, Camille Lebugle1,2, Pierre Bourdoncle1,2, Emmanuel Donnadieu1,2 1Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), 2Inserm, U1016, Paris, France This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes. Immunology and Infection Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides Nazzy Pakpour1, Kong Wai Cheung1, Lattha Souvannaseng1, Jean-Paul Concordet2, Shirley Luckhart1 1Department of Medical Microbiology and Immunology, University of California, Davis, 2Département Génétique et Développement, Institut Cochin, Université Paris Descartes Oligonucleotides can be used to site specifically substitute a single nucleotide of transfected target genes in both Anopheles gambiae and Anopheles stephensi cells.