Aix Marseille University View Institution's Website 21 articles published in JoVE Developmental Biology Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution Stephanie Ibrahim1, Catherine Robert2, Camille Humbert2, Clotilde Ferreira1, Gwenaëlle Collod2, Sonia Stefanovic1 1Center for Cardiovascular Disease & Nutrition, U1263, Aix-Marseille University, 2Marseille Medical Genetics, U1251, Aix-Marseille University Here, we present a protocol describing cell nuclei preparation. After microdissection and enzymatic dissociation of cardiac tissue into single cells, the progenitor cells were frozen, followed by isolation of pure viable cells, which were used for single-nucleus RNA sequencing and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing analyses. Biochemistry Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells Sebastien Mailfert*1, Karolina Wojtowicz*2, Sophie Brustlein*1, Ewa Blaszczak3, Nicolas Bertaux4, Marcin Łukaszewicz2, Didier Marguet1, Tomasz Trombik2 1CNRS, Inserm, Centre d'Immunologie de Marseille-Luminy, Aix Marseille Univ, 2Faculty of Biotechnology, University of Wroclaw, 3Faculty of Biological Sciences, University of Wroclaw, 4CNRS, Centrale Marseille, Institut Fresnel, Aix Marseille Univ This article aims to present a protocol on how to build a spot variation Fluorescence Correlation Spectroscopy (svFCS) microscope to measure molecular diffusion at the plasma membrane of living cells. Neuroscience Electrophoretic Delivery of γ-aminobutyric Acid (GABA) into Epileptic Focus Prevents Seizures in Mice Andrea Slezia*1,5, Christopher M. Proctor*2,3, Attila Kaszas1,4, George G. Malliaras2,3, Adam Williamson1,5 1Aix Marseille Université, Institut de Neurosciences des Systèmes (INS), 2Electrical Engineering Division, University of Cambridge, 3Department of Bioelectronics, Centre Microélectronique de Provence - Ecole Nationale Supérieure des Mines de Saint-Étienne (CMP-EMSE), 4Institut de Neurosciences de la Timone, Centre National de la Recherche Scientifique (CNRS) UMR 7289 & Aix- Marseille Université, 5Neuroengineering Research Group, Interdisciplinary Excellence Center, Department of Medical Microbiology and Immunobiology, University of Szeged The challenge of epilepsy research is to develop novel treatments for patients where classical therapy is inadequate. Using a new protocol—with the help of an implantable drug delivery system—we are able to control seizures in anesthetized mice by the electrophoretic delivery of GABA into the epileptic focus. Developmental Biology Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo Claire Chardès*1, Raphael Clement*1, Olivier Blanc1, Pierre-François Lenne1 1CNRS, Institut de Biologie du Développement de Marseille, Aix-Marseille Université We present a setup of optical tweezers coupled to a light sheet microscope, and its implementation to probe cell mechanics without beads in the Drosophila embryo. Genetics RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA Manon Torres1, Denis Becquet1, Séverine Guillen1, Bénédicte Boyer1, Mathias Moreno1, Marie-Pierre Blanchard2, Jean-Louis Franc1, Anne-Marie François-Bellan1 1CNRS, CRN2M-UMR7286, Faculté de Médecine Nord, Aix-Marseille Université, 2Plate-forme d'imagerie MRI, UMS3426, CNRS 141, Institut de Génétique Humaine This RNA pull-down method allows identifying the RNA targets of a long non-coding RNA (lncRNA). Based on the hybridization of home-made, designed anti-sense DNA oligonucleotide probes specific to this lncRNA in an appropriately fixed tissue or cell line, it efficiently allows the capture of all RNA targets of the lncRNA. Neuroscience A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis Masahiro Tsuji1, Jacques-Olivier Coq2, Yuko Ogawa1, Yumi Yamamoto1, Makiko Ohshima1 1Department of Regenerative Medicine and Tissue Engineering, National Cerebral and Cardiovascular Center, 2Institut de Neurosciences de la Timone, CNRS, Aix Marseille Université Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction. Neuroscience A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations Valerio Conti*1, Aurelie Carabalona*2,3,15, Emilie Pallesi-Pocachard2,3,4, Richard J. Leventer5,6,7, Fabienne Schaller2,3,8, Elena Parrini1, Agathe A. Deparis2,3, Françoise Watrin2,3, Emmanuelle Buhler2,3,8, Francesca Novara9, Stefano Lise10, Alistair T. Pagnamenta10, Usha Kini11, Jenny C. Taylor10, Orsetta Zuffardi9,12, Alfonso Represa2,3, David Antony Keays13, Renzo Guerrini1,14, Antonio Falace2,3, Carlos Cardoso2,3 1University of Florence, 2INSERM INMED, 3Aix-Marseille University, 4Plateforme Biologie Moléculaire et Cellulaire INMED, 5 Periventricular nodular heterotopia (PNH) is the most common form of malformation of cortical development (MCD) in adulthood but its genetic basis remains unknown in most sporadic cases. We have recently developed a strategy to identify novel candidate genes for MCDs and to directly confirm their causative role in vivo. Bioengineering An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur Martine Pithioux*1,2, Flavy Roseren*1,2, Christian Jalain4, Franck launay1,3, Philippe Charpiot5, Patrick chabrand1,2, Sandrine Roffino1, Edouard Lamy1,6 1CNRS, ISM, Inst Movement Sci, Aix Marseille Univ, 2Sainte-Marguerite Hospital, Institute for Locomotion, Department of Orthopaedics and Traumatology, APHM, 3Sainte-Marguerite Hospital, Institute for Locomotion, Department of Peadiatric Orthopaedics, APHM, 4Ecole centrale de Marseille, 5Faculté de Pharmacie, Laboratoire de Biochimie, 6Service Central de la Qualité et de l'Information Pharmaceutiques, APHM This study describes a reproducible and detailed protocol using a newly developed external fixator for distraction osteogenesis (DO) in a femoral rat model which permits physiological weight-bearing by the animal after removal of the external fixator. Biology An Easy Method for Plant Polysome Profiling Cécile Lecampion1,2,3, Maïna Floris1,2,3,4, Jean Raphaël Fantino5,6, Christophe Robaglia1,2,3, Christophe Laloi1,2,3 1Laboratoire de Génétique et Biophysique des Plantes, Aix-Marseille Université, 2UMR 7265 Biologie Végétale & Microbiologie Environnementales, CNRS, 3BIAM, CEA, 4Department of Biology, Biocenter, University of Copenhagen, 5Laboratoire de Chimie Bactérienne, 6CNRS, LCB UMR 7283, Aix Marseille Université This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs. Biology Implementation of a Coherent Anti-Stokes Raman Scattering (CARS) System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope Vasyl Mytskaniuk*1, Fabrice Bardin*2,3, Hassan Boukhaddaoui1,5, Herve Rigneault4, Nicolas Tricaud1 1INSERM U1051, Institut des Neurosciences de Montpellier (INM), Université de Montpellier, 2Université de Nîmes, 3CNRS, IES, UMR 5214, 4Aix-Marseille Université, CNRS, École Centrale Marseille, Institut Fresnel, UMR 7249, 5Montpellier RIO Imaging (MRI) Coherent anti-Stokes Raman scattering (CARS) microscopy based on inherent vibration of molecule bonds permits label-free chemically selective live cell imaging. This work presents the implementation of a complementary microscopy technique on a standard multiphoton laser scanning microscope based on a femtosecond Ti:sapphire laser and an OPO laser. Immunology and Infection A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis Jacques Yaacoub Bou Khalil1, Julien Andreani1, Didier Raoult1,2, Bernard La Scola1,2 1Faculty of Medicine and Pharmacy, Research Unit for Infectious and Tropical Emerging Diseases, Aix Marseille University, 2Pole of Infectious and Tropical Diseases, Clinical and Biological Sector, Federation of Bacteriology-Hygiene Virology, University Hospital Institute Mediterranean Infection We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba. Behavior Assessment of Social Cognition in Non-human Primates Using a Network of Computerized Automated Learning Device (ALDM) Test Systems Joël Fagot1, Yousri Marzouki1, Pascal Huguet1, Julie Gullstrand1, Nicolas Claidière1 1CNRS, Federation 3C, and Laboratory of Cognitive Psychology, Aix-Marseille University Fagot & Paleressompoulle1 have published an automated learning device (ALDM) aimed at testing individual cognitive abilities in semi-free ranging monkeys. The main goal of our protocol is to use a network of ALDM test units to study social cognition in non-human primates. Neuroscience Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts Norbert W. Lutz1, Evelyne Béraud2, Patrick J. Cozzone1 1Centre de Résonance Magnétique Biologique et Médicale, UMR 7339 CNRS, Faculté de Médecine, Aix-Marseille Université, 2Centre de Recherches en Oncologie Biologique et Oncopharmacologie, UMR 911 INSERM, Faculté de Médecine, Aix-Marseille Université The neurochemistry of mammalian brain is changed in many neurological and systemic diseases. Characteristic profiles of cerebral metabolites can be efficiently obtained based on crude extracts of brain tissue. To this end, high-resolution NMR spectroscopy is employed, enabling detailed quantitative analysis of metabolite concentrations (metabolomics). Biology High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli Natalie J. Saez1, Hervé Nozach2, Marilyne Blemont1, Renaud Vincentelli1 1Architecture et Fonction des Macromolécules Biologiques (AFMB), Aix-Marseille Université, 2 A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets. Medicine Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport Yves Molino1, Françoise Jabès1, Emmanuelle Lacassagne2, Nicolas Gaudin2, Michel Khrestchatisky2 1VECT-HORUS SAS, 2Aix-Marseille Université, CNRS, NICN UMR 7259 The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed. Medicine An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy Clément Ricard1,2, Fabio Stanchi1,3, Geneviève Rougon1,2, Franck Debarbieux1,2 1Developmental Biology Institute of Marseille, Aix Marseille University, 2European Research Center for Medical Imaging, Campus de la Timone, 3Vesalius Research Center, KU Leuven Campus Gasthuisberg We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy. Immunology and Infection Whole-cell MALDI-TOF Mass Spectrometry is an Accurate and Rapid Method to Analyze Different Modes of Macrophage Activation Richard Ouedraogo1, Aurélie Daumas1,2, Christian Capo1, Jean-Louis Mege1, Julien Textoris1 1Unité de Recherche sur les Maladies Infectieuses Tropicales et Emergentes (URMITE), CNRS UMR 7278, INSERM U1095, Aix Marseille Université, 2Service de médecine interne, post-urgence et thérapeutique, Hôpital de la Timone This protocol describes the use of whole-cell MALDI-TOF mass spectrometry on eukaryotic cells. Here, we illustrate the accuracy of this technique by analyzing the multiple activation states of macrophages in response to their microenvironment. Medicine Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis Keith K. Fenrich1, Pascal Weber1, Genevieve Rougon1,2, Franck Debarbieux1,2 1Developmental Biology Institute of Marseille-Luminy (IBDML), Aix Marseille University, 2Campus de la Timone, European Research Center for Medical Imaging (CERIMED) We describe a method for implanting and maintaining glass windows over the exposed spinal cords of adult mice for studying experimental autoimmune encephalomyelitis. These windows provide chronic optical access to the spinal cord for monitoring cell dynamics at the subcellular level in live animals using two-photon microscopy. Immunology and Infection Bone Marrow-derived Macrophage Production Virginie Trouplin1, Nicolas Boucherit1, Laurent Gorvel1, Filippo Conti1, Giovanna Mottola1,2, Eric Ghigo1 1CNRS UMR 7278, IRD198, INSERM U1095, Aix-Marseille Université, 2Department of Molecular Medicine and Medical Biotechnology, University of Naples "Federico II" Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow. Biology Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT) Vincent Rouger1,2,3, Nicolas Bertaux4,5,6, Tomasz Trombik1,2,3, Sébastien Mailfert1,2,3, Cyrille Billaudeau1,2,3, Didier Marguet1,2,3, Arnauld Sergé1,2,3 1Institut National de la Santé et de la Recherche Médicale, UMR 631, Parc scientifique de Luminy, 2Centre National de la Recherche Scientifique, UMR 6102, Parc scientifique de Luminy, 3Centre d'Immunologie de Marseille-Luminy, Aix-Marseille University, 4École Centrale Marseille, Technopôle de Château-Gombert, 5Institut Fresnel, Aix-Marseille University, 6Centre National de la Recherche Scientifique, UMR 6133, Aix-Marseille University Multiple-Target Tracing is a homemade algorithm developed for tracking individually labeled molecules within the plasma membrane of living cells. Efficiently detecting, estimating and tracing molecules over time at high-density provide a user-friendly, comprehensive tool to investigate nanoscale membrane dynamics. Neuroscience Isolating Nasal Olfactory Stem Cells from Rodents or Humans Stéphane D. Girard1,2, Arnaud Devéze3, Emmanuel Nivet1,2,4, Bruno Gepner5, François S. Roman2, François Féron1,6 1NICN, Aix Marseille University, 2LNPM, Aix Marseille University, 3ENT Department, Aix Marseille University, 4Gene expression Laboratory, The Salk Institute for Biological Studies, 5Laboratory of Speech and Language, Aix Marseille University, 6Centre d'Investigations Cliniques en Biothérapie, Aix Marseille University We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.