Aix Marseille University

20 articles published in JoVE

• 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 Trombik^{2 1}CNRS, 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 Kaszas^1,4, George G. Malliaras^2,3, Adam Williamson^{1,5 1}Aix 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 Lenne^{1 1}CNRS, 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-Bellan^{1 1}CNRS, 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 Ohshima^{1 1}Department 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-Pocachard^2,3,4, Richard J. Leventer^5,6,7, Fabienne Schaller^2,3,8, Elena Parrini¹, Agathe A. Deparis^2,3, Françoise Watrin^2,3, Emmanuelle Buhler^2,3,8, Francesca Novara⁹, Stefano Lise¹⁰, Alistair T. Pagnamenta¹⁰, Usha Kini¹¹, Jenny C. Taylor¹⁰, Orsetta Zuffardi^9,12, Alfonso Represa^2,3, David Antony Keays¹³, Renzo Guerrini^1,14, Antonio Falace^2,3, Carlos Cardoso^{2,3 1}University 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 launay^1,3, Philippe Charpiot⁵, Patrick chabrand^1,2, Sandrine Roffino¹, Edouard Lamy^{1,6 1}CNRS, 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 Lecampion^1,2,3, Maïna Floris^1,2,3,4, Jean Raphaël Fantino^5,6, Christophe Robaglia^1,2,3, Christophe Laloi^{1,2,3 1}Laboratoire 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 Boukhaddaoui^1,5, Herve Rigneault⁴, Nicolas Tricaud^{1 1}INSERM 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 Raoult^1,2, Bernard La Scola^{1,2 1}Faculty 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 Claidière^{1 1}CNRS, 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. Cozzone^{1 1}Centre 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 Vincentelli^{1 1}Architecture 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 Khrestchatisky^{2 1}VECT-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 Ricard^1,2, Fabio Stanchi^1,3, Geneviève Rougon^1,2, Franck Debarbieux^{1,2 1}Developmental 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 Daumas^1,2, Christian Capo¹, Jean-Louis Mege¹, Julien Textoris^{1 1}Unité 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 Rougon^1,2, Franck Debarbieux^{1,2 1}Developmental 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 Mottola^1,2, Eric Ghigo^{1 1}CNRS 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 Rouger^1,2,3, Nicolas Bertaux^4,5,6, Tomasz Trombik^1,2,3, Sébastien Mailfert^1,2,3, Cyrille Billaudeau^1,2,3, Didier Marguet^1,2,3, Arnauld Sergé^{1,2,3 1}Institut 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. Girard^1,2, Arnaud Devéze³, Emmanuel Nivet^1,2,4, Bruno Gepner⁵, François S. Roman², François Féron^{1,6 1}NICN, 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.