Institut Pasteur View Institution's Website 33 articles published in JoVE Biology Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses Amaia Dominguez-Belloso1, Sandrine Schmutz2, Sophie Novault2, Laetitia Travier*1, Aleksandra Deczkowska*1 1Brain-Immune Communication Lab, Institut Pasteur, 2Cytometry platform, CB UTechS, Institut Pasteur This study uses flow cytometry and two different gating strategies on isolated perfused mice brain choroid plexuses; this protocol identifies the main immune cell subsets that populate this brain structure. Biology Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe Aline Stedman1,3, Antonin Levy1,4, Philippe J. Sansonetti1,2,5, Giulia Nigro1,6 1Molecular Microbial Pathogenesis Unit, Institut Pasteur, 2Chaire de Microbiologie et Maladies Infectieuses, Collège de France, 3Institut de Biologie Paris Seine (IBPS) - Developmental Biology Unit, Sorbonne Université, CNRS UMR7622, INSERM U1156, 4Molecular Radiotherapy, INSERM U1030, Gustave Roussy, Université Paris-Saclay, 5The Center for Microbes, Development and Health, Institut Pasteur Shanghai and Chinese Academy of Sciences, 6Microenvironment and Immunity Unit, Institut Pasteur The present protocol describes a method to detect reactive oxygen species (ROS) in the intestinal murine organoids using qualitative imaging and quantitative cytometry assays. This work can be potentially extended to other fluorescent probes to test the effect of selected compounds on ROS. Immunology and Infection Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis Thomas Labadie1, Quentin Grassin2, Christophe Batéjat2, Jean-Claude Manuguerra2, India Leclercq2,3 1Department of Infection Biology, London School of Hygiene and Tropical Medicine, 2Institut Pasteur, Unité Environnement et Risques Infectieux, Cellule d'Intervention Biologique d'Urgence (CIBU), 3Cellule Pasteur, Université de Paris Reported here is a protocol for the quantification of infectious viral particles using real-time monitoring of electrical impedance of infected cells. A practical application of this method is presented by quantifying influenza A virus decay under different physicochemical parameters mimicking environmental conditions. Bioengineering Substructure Analyzer: A User-Friendly Workflow for Rapid Exploration and Accurate Analysis of Cellular Bodies in Fluorescence Microscopy Images Géraud Heckler1, Christelle Aigueperse1, Liza Hettal1, Quentin Thuillier1, Fabrice de Chaumont2, Stéphane Dallongeville2, Isabelle Behm-Ansmant1 1Université de Lorraine, CNRS, IMoPA F54000 Nancy France, 2Institut Pasteur, BioImage Analysis Unit, CNRS UMR 3691, Paris, France. We present a freely available workflow built for rapid exploration and accurate analysis of cellular bodies in specific cell compartments in fluorescence microscopy images. This user-friendly workflow is designed on the open-source software Icy and also uses ImageJ functionalities. The pipeline is affordable without knowledge in image analysis. Immunology and Infection Field Postmortem Rabies Rapid Immunochromatographic Diagnostic Test for Resource-Limited Settings with Further Molecular Applications Stephanie Mauti1, Monique Léchenne2, Service Naïssengar3, Abdallah Traoré4, Vessaly Kallo5,6, Casimir Kouakou7, Emmanuel Couacy-Hymann7, Morgane Gourlaouen8, Céline Mbilo9,10, Pati Patient Pyana11, Enos Madaye3, Ibrahima Dicko4, Pascal Cozette1, Paola De Benedictis8, Hervé Bourhy1, Jakob Zinsstag9,10, Laurent Dacheux1 1Unit Lyssavirus Epidemiology and Neuropathology, National Reference Center for Rabies and WHO Collaborating Center for Reference and Research on Rabies, Institut Pasteur, 2Environment and Sustainability Institute, University of Exeter, Penryn Campus, 3Institut de Recherche en Elevage pour le Développement, 4Laboratoire Central Vétérinaire, 5 We present a complete protocol for postmortem diagnosis of animal rabies under field conditions using a rapid immunochromatographic diagnostic test (RIDT), from brain biopsy sampling to final interpretation. We also describe further applications using the device for molecular analysis and viral genotyping. Biochemistry Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening Karine Nozeret1, Aurélia Pernin1, Nienke Buddelmeijer1 1Institut Pasteur, Unité Biologie et génétique de la paroi bactérienne, Paris, France ; CNRS, UMR 2001 « Microbiologie intégrative et Moléculaire », Paris, France ; INSERM, Équipe Avenir, Paris, France Presented here is a sensitive fluorescence assay to monitor apolipoprotein N-acyltransferase activity using diacylglyceryl peptide and alkyne-phospholipids as substrates with click-chemistry. Immunology and Infection In Vitro ELISA Test to Evaluate Rabies Vaccine Potency Corinne Jallet1, Noël Tordo1,2 1Unit Antiviral Strategies, OIE Reference Laboratories for Rift Valley Fever Virus & Crimean Congo Hemorrhagic Fever Virus, Institut Pasteur, 2Institut Pasteur de Guinée, Conakry Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production. Neuroscience A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain Anaelle da Costa*1, Christophe Prehaud*1, Florian Bakoa1, Philippe Afonso2, Pierre-Emmanuel Ceccaldi2, Pierre Lafaye3, Monique Lafon1 1Institut Pasteur, CNRS UMR 3569, Unité de Neuroimmunologie Virale, 2Institut Pasteur, Unité d’Epidémiologie et de Pathophysiologie des Virus Oncogènes, Université Sorbonne Paris Cité/Paris Diderot, 3Institut Pasteur, PFIA, DTPS Here we present a protocol describing the setting of an in cellulo BBB (Blood brain barrier)-Minibrain polyester porous membrane culture insert system in order to evaluate the transport of biomolecules or infectious agents across a human BBB and their physiological impact on the neighboring brain cells. Biology The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events Mark C. Anderson1,2, Louise Injarabian1,2,3, Antonin Andre1,2,3, Regis Tournebize1,2,4, Benoit S. Marteyn1,2 1Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, 2INSERM Unité 1202, Institut Pasteur, 3Institut de Biochimie et Génétique Cellulaires, Université Bordeaux Segalen, 4Unit of Technology and Service Photonic BioImaging, Institut Pasteur Here, we present a protocol to detect the presence of neutrophils in fixed/permeabilized histology sections and assess the activation state of live purified neutrophils. In particular, the MUB40 peptide binds lactoferrin present in neutrophil-specific and tertiary granules. Exposure of the granule contents through either permeabilization or neutrophil activation allows for the marking of neutrophils. Immunology and Infection Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-α Alba Llibre*1,2, Vincent Bondet*1,2, Mathieu P. Rodero3, David Hunt4, Yanick J. Crow3,5, Darragh Duffy1,2 1Immunobiology of Dendritic Cells, Institut Pasteur, 2INSERM U1223, 3Laboratory of Neurogenetics and Neuroinflammation, INSERM UMR1163, Institut Imagine, 4MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, 5Manchester Centre for Genomic Medicine, University of Manchester Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-α subtypes in human samples. Biology Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy Cécile Leduc1, Audrey Salles2, Spencer L. Shorte2, Sandrine Etienne-Manneville1 1Cell Polarity, Migration and Cancer Unit, UMR 3691, CNRS, Institut Pasteur, 2UTechS Photonic BioImaging (Imagopole) Citech, Institut Pasteur The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells Immunology and Infection Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice Anna Zychlinsky Scharff1, Matthew L. Albert1,2, Molly A. Ingersoll1 1Unité d’Immunobiologie des Cellules Dendritiques, Department of Immunology, Institut Pasteur, INSERM U818, 2Genentech The established model of transurethral catheterization of mice allows the study of bladder pathologies, including urinary tract infection, but can only be performed in females. A new model of male transurethral instillation, presented here, will enable research in an area marked by strong clinical and epidemiological differences between the sexes. Developmental Biology Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle Coralie Cazin1, Aurelie Chiche1, Han Li1 1Cellular Plasticity & Disease Modelling, Department of Developmental & Stem Cell Biology, CNRS UMR 3738, Institut Pasteur Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo. Developmental Biology Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry Lorea Iturri1,2, Javier Saenz Coronilla1, Yvan Lallemand1, Elisa Gomez Perdiguero1 1Department of Developmental and Stem Cell Biology, CNRS UMR3738, Department of Immunology, Institut Pasteur, 2Cellule Pasteur UPMC, University Pierre et Marie Curie While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors. 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. Biology Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry Sandrine Schmutz1, Mariana Valente2,3,4,5, Ana Cumano2, Sophie Novault1 1Flow Cytometry Core Facility, Center for Translational Research-Technical Core, Institut Pasteur, 2Unit for Lymphopoiesis, Immunology Department, INSERM U1223, University Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Institut Pasteur, 3Stem-Cell Microenvironments in Repair/Regeneration Team, Instituto de Investigação e Inovação em Saúde (i3s), INEB - Instituto de Engenharia Biomédica, 4ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 5Stem Cells and Regenerative Medicine Team, UMRS 1166, ICAN - Institute of Cardiometabolism And Nutrition, UPMC - Université Pierre et Marie Curie - Paris 6, INSERM This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs. Immunology and Infection Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus Fabio Cafini*1,2, Nguyen Thi Le Thuy*3, Federico Román4, José Prieto5, Sarah Dubrac6,7, Tarek Msadek6,7, Kazuya Morikawa1 1Division of Biomedical Science, Faculty of Medicine, University of Tsukuba, 2Department of Basic Biomedical Science, Universidad Europea de Madrid, 3Human Biology Program, School of Integrative and Global Majors, University of Tsukuba, 4Laboratory of Nosocomial Infections, Department of Bacteriology, Centro Nacional de MicrobiologÍa, Instituto de Salud Carlos III, 5Division of Microbiology, Department of Medicine, School of Medicine, Universidad Complutense, 6Biology of Gram-Positive Pathogens, Department of Microbiology, Institut Pasteur, Paris, France, 7ERL3526, CNRS, Paris, France We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus. Developmental Biology A Versatile Mounting Method for Long Term Imaging of Zebrafish Development Estelle Hirsinger1,3, Ben Steventon1,2 1Department of Developmental and Stem Cell Biology, Institut Pasteur, 2Department of Genetics, University of Cambridge, 3IBPS- Laboratoire de Biologie du Developpement (LBD), CNRS, UPMC, UMR 7622, INSERM ERL U1156 Here, we present a versatile mounting method that allows for the long-term time-lapse imaging of the posterior body development of live zebrafish embryos without perturbing normal development. Genetics Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations Thibaut Perchet1, Sylvestre Chea1, Milena Hasan2, Ana Cumano1, Rachel Golub1 1Unit for Lymphopoieseis, Immunology Department, INSERM U1223, University Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Institut Pasteur, 2Center for Translational Science, Institut Pasteur, INSERM UMS20 This protocol describes how to assess the expression of a large array of genes at the clonal level. Single-cell RT-qPCR produces highly reliable results with a strong sensitivity for hundreds of samples and genes. Immunology and Infection Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites Laura Mac-Daniel1, Matthew R. Buckwalter2, Pascale Gueirard1, Robert Ménard1 1Unité de Biologie et Génétique du Paludisme, Institut Pasteur, 2Unité dImmunobiologie des Cellules Dendritiques, Institut Pasteur We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite. Developmental Biology Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells Laura Gouder1,2,3, Jean-Yves Tinevez4, Hany Goubran-Botros1,2,3, Alexandra Benchoua5, Thomas Bourgeron1,2,3, Isabelle Cloëz-Tayarani1,2,3 1Human Genetics and Cognitive Functions, Institut Pasteur, 2CNRS URA 2182 'Genes, synapses and cognition', Institut Pasteur, 3Human Genetics and Cognitive Functions, Université Paris Diderot, Sorbonne Paris Cité, 4Plateforme d' Imagerie Dynamique, Imagopole, CiTech, Institut Pasteur, 5Neuroplasticity and Therapeutics, CECS, I-STEM, AFM, Evry Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced pluripotent stem cells. Developmental Biology Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays Cyrille Ramond1,3, Antonio Bandeira2, Claire Berthault3, Pablo Pereira3, Ana Cumano3, Odile Burlen-Defranoux3 1Research Center Growth and Signaling, INSERM U845, Institut Cochin, 2Unit for Biology of Lymphocyte Populations, Immunology Department, Institut Pasteur and CIMI, Unity of Treg Biology and Therapy, University of Pierre & Marie Curie, 3Unit for Lymphopoiesis, Immunology Department, INSERM U668, University Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Institut Pasteur This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny. Developmental Biology Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes Estelle Hirsinger1, João Emanuel Carvalho2, Christine Chevalier1,3, Georges Lutfalla4, Jean-François Nicolas1, Nadine Peyriéras5, Michael Schubert2 1Département de Biologie du Développement et Cellules Souches, Institut Pasteur, 2Laboratoire de Biologie du Développement de Villefranche-sur-Mer (UMR7009 CNRS/UPMC Univ Paris 06), Sorbonne Universités, 3Equipe Epigenetic Control of Normal and Pathological Hematopoiesis, Centre de Recherche en Cancérologie de Marseille, 4Unité de Dynamique des Interactions Membranaires Normales et Pathologiques, CNRS UMR5235/DAA/cc107/Université Montpellier II, 5Plateforme BioEmergences IBiSA FBI, CNRS-NED, Institut de Neurobiologie Alfred Fessard We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations. Immunology and Infection Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions Maria J. Mazon Moya1, Emma Colucci-Guyon2, Serge Mostowy1 1Section of Microbiology, MRC Centre for Molecular Bacteriology and Infection, Imperial College London, 2Département de Biologie du Développement et des Cellules Souches, Institut Pasteur, Unité Macrophages et Développement de l'Immunité To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo. Neuroscience Using Insect Electroantennogram Sensors on Autonomous Robots for Olfactory Searches Dominique Martinez1, Lotfi Arhidi1, Elodie Demondion2, Jean-Baptiste Masson3, Philippe Lucas2 1UMR 7503, Laboratoire Lorrain de Recherche en Informatique et ses Applications (LORIA), Centre National de la Recherche Scientifique (CNRS), 2UMR 1392 iEES-Paris, Institut d'Ecologie et des Sciences de l'Environnement de Paris, 3Physics of Biological Systems, Institut Pasteur We describe a protocol for using insect antennae in the form of electroantennograms (EAGs) on autonomous robots. Our experimental design allows stable recordings within a day and resolves individual odor patches up to 10 Hz. The efficiency of EAG sensors for olfactory searches is demonstrated in driving a robot toward an odor source. Bioengineering A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence (FUEL) Joseph Dragavon1, Carolyn Sinow2, Alexandra D. Holland1, Abdessalem Rekiki1, Ioanna Theodorou3, Chelsea Samson4, Samantha Blazquez1, Kelly L. Rogers5, Régis Tournebize1,6,7, Spencer L. Shorte1 1Plate-Forme d'Imagerie Dynamique, Imagopole, Institut Pasteur, 2Department of Radiation Oncology, Stanford School of Medicine, 3 Expanding the foundation and applicability of Fluorescence by Unbound Excitation from Luminescence (FUEL) by surveying the relevant principles and demonstrating its compatibility with a multitude of fluorophores and antibody-targeted conditions. Immunology and Infection High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses Marianne Lucas-Hourani1, Hélène Munier-Lehmann2, Olivier Helynck2, Anastassia Komarova1, Philippe Desprès3, Frédéric Tangy1, Pierre-Olivier Vidalain1 1Unité de Génomique Virale et Vaccination, Virology Department, Institut Pasteur, CNRS UMR3569, 2Unité de Chimie et Biocatalyse, Biochemistry and Structural Biology Department, Institut Pasteur, CNRS UMR3523, 3Unité des Interactions Moléculaires Flavivirus-Hôtes, Virology Department, Institut Pasteur In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries. Bioengineering Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties Olivier Galy1, Kais Zrelli1, Patricia Latour-Lambert2, Lyndsey Kirwan3, Nelly Henry1,3 1Physicochime Curie, CNRS UMR 168, Institut Curie, Sorbonne Universités, UPMC, 2Unité de Génétique des Biofilms, Institut Pasteur, 3Laboratoire Jean Perrin, CNRS UMR 8237, Sorbonne Universités, UPMC This paper shows an original methodology based on the remote actuation of magnetic particles seeded in a bacterial biofilm and the development of dedicated magnetic tweezers to measure in situ the local mechanical properties of the complex living material built by micro-organisms at interfaces. Immunology and Infection A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions Mandy Muller*1,2, Patricia Cassonnet*1, Michel Favre1, Yves Jacob1,3, Caroline Demeret1 1Unité de Génétique, Papillomavirus et Cancer Humain (GPCH), Institut Pasteur, 2Cellule Pasteur, Université Sorbonne Paris Cité, 3Center for Cancer Systems Biology (CCSB), Harvard Medical School, Department of Cancer Biology, Dana Farber Cancer Institute This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA. Immunology and Infection Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens Charlotte Keller*1, Nora Mellouk*1, Anne Danckaert2, Roxane Simeone3, Roland Brosch3, Jost Enninga1, Alexandre Bobard1 1Dynamique des Interactions Hôte Pathogène, Institut Pasteur, Paris, France, 2Imagopole, Institut Pasteur, Paris, France, 3Pathogenomique Mycobacterienne Integrée, Institut Pasteur, Paris, France We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface. Neuroscience Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation Gabriel Lepousez1, Mariana Alonso1, Sebastian Wagner1, Benjamin W. Gallarda1, Pierre-Marie Lledo1 1Laboratory for Perception and Memory, Institut Pasteur and Centre National de la Recherche Scientifique (CNRS) Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED. Neuroscience Using Affordable LED Arrays for Photo-Stimulation of Neurons Matthew Valley1, Sebastian Wagner1, Benjamin W. Gallarda1, Pierre-Marie Lledo1 1Laboratory for Perception and Memory, Institut Pasteur and Centre National de la Recherche Scientifique (CNRS) Adult-born neurons expressing ChR2 can be manipulated in slice electrophysiological preparations in order to examine their contribution towards the function of olfactory neural circuits. Immunology and Infection Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency Stéphanie Beaucourt1, Antonio V. Bordería1, Lark L. Coffey1, Nina F. Gnädig1, Marta Sanz-Ramos1, Yasnee Beeharry1, Marco Vignuzzi1 1Viral Populations and Pathogenesis lab and CNRS 3015, Institut Pasteur The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.