Universite Grenoble Alpes 13 articles published in JoVE Genetics Development of Knock-Out Muscle Cell Lines using Lentivirus-Mediated CRISPR/Cas9 Gene Editing Mathilde Beaufils1, Amandine Tourel1, Anne Petiot1, Nicole B. Halmai2, Dave J. Segal2, John Rendu1, Isabelle Marty1 1University Grenoble Alpes, INSERM, U1216, CHU Grenoble Alpes, Grenoble Institut Neurosciences, 2Genome Center, University of California, Davis The protocol describes how to generate knock-out myoblasts using CRISPR/Cas9, starting from the design of guide-RNAs to the cellular cloning and characterization of the knock-out clones. Chemistry Biological Samples Preparation for Speciation at Cryogenic Temperature using High-Resolution X-Ray Absorption Spectroscopy Caroline Bissardon1, Marie-Pierre Isaure2, Emmanuel Lesuisse3, Mauro Rovezzi4,5, Eric Lahera4,5, Olivier Proux4,5, Sylvain Bohic1,6 1University Grenoble Alpes, INSERM UA7, Synchrotron Radiation for Biomedicine (STROBE), 2 This protocol presents a detailed procedure to prepare biological cryosamples for synchrotron-based X-ray absorption spectroscopy experiments. We describe all the steps required to optimize sample preparation and cryopreservation with examples of the protocol with cancer and phytoplankton cells. This method provides a universal standard of sample cryo-preparation. Biochemistry High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water Kevin Pounot1,2, Markus Appel2, Christian Beck1,2, Martin Weik3, Giorgio Schirò3, Yann Fichou4, Tilo Seydel2, Frank Schreiber1 1Institut für Angewandte Physik, Universität Tübingen, 2Institut Max von Laue - Paul Langevin (ILL), 3Institut de Biologie Structurale, Université Grenoble Alpes, 4Institut Européen de Chimie et Biologie, Bordeaux INP, Chimie et Biologie des Membranes et des Nanoobjets (CBMN), Université de Bordeaux Neutron backscattering spectroscopy offers a nondestructive and label-free access to the ps-ns dynamics of proteins and their hydration water. The workflow is presented with two studies on amyloid proteins: on the time-resolved dynamics of lysozyme during aggregation and on the hydration water dynamics of tau upon fiber formation. Chemistry Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies Sofia Jaho1, Niels Junius1,3, Franck Borel1, Yoann Sallaz-Damaz1, Jean-Baptiste Salmon2, Monika Budayova-Spano1 1Université Grenoble Alpes, CEA, CNRS, IBS, 2CNRS, Solvay LOF, UMR 5258, Université Bordeaux, 3ELVESYS SAS This paper details the fabrication protocol of microfluidic chips developed for on-chip protein crystallization with the dialysis method and in situ X-ray diffraction experiments. The microfabrication process makes it possible to integrate a semipermeable regenerated cellulose dialysis membrane with any molecular weight cut-off, between two layers of the chip. Biology Optimization of Crystal Growth for Neutron Macromolecular Crystallography Elham Vahdatahar1, Niels Junius1,2, Monika Budayova - Spano1 1CEA, CNRS, IBS, Université Grenoble Alpes, 2ELVESYS SAS Structural studies of biomacromolecules by crystallography require high-quality crystals. Here we demonstrate a protocol that can be used by OptiCrys (a fully automated instrument developed in our lab) and/or microdialysis buttons for growing large high-quality crystals based on knowledge of the crystallization phase diagram. Engineering Characterization of SiN Integrated Optical Phased Arrays on a Wafer-Scale Test Station Nicola A. Tyler1, Sylvain Guerber1, Daivid Fowler1, Stephane Malhouitre1, Stephanie Garcia1, Philippe Grosse1, Bertrand Szelag1 1University Grenoble Alpes and CEA, LETI, Minatec Campus Here, we describe the operation of a SiN integrated photonic circuit containing optical phased arrays. The circuits are used to emit low divergence laser beams in the near infrared and steer them in two dimensions. Biochemistry Cell Culture on Silicon Nitride Membranes and Cryopreparation for Synchrotron X-ray Fluorescence Nano-analysis Caroline Bissardon1, Solveig Reymond1, Murielle Salomé2, Lionel André2, Sam Bayat1, Peter Cloetens2, Sylvain Bohic1,2 1Inserm, UA7, Synchrotron Radiation for Biomedicine (STROBE), Université Grenoble Alpes, 2ID16A Beamline, ESRF, The European Synchrotron Presented here is a protocol for cell culture on silicon nitride membranes and plunge-freezing prior to X-ray fluorescence imaging with a synchrotron cryogenic X-ray nanoprobe. When only room temperature nano-analysis is provided, the frozen samples can be further freeze-dried. These are critical steps to obtain information on the intracellular elemental composition. Biochemistry Structure Solution of the Fluorescent Protein Cerulean Using MeshAndCollect Stephanie Hutin*1, Gianluca Santoni*1, Ulrich Zander2, Nicolas Foos1, Sylvain Aumonier1, Guillaume Gotthard1, Antoine Royant1,3, Christoph Mueller-Dieckmann1, Gordon Leonard1 1European Synchrotron Radiation Facility, Structural Biology Group, 2European Molecular Biology Laboratory, 3Univ. Grenoble Alpes, CNRS, CEA, IBS (Institut de Biologie Structurale) We present the use of the MeshAndCollect protocol to obtain a complete diffraction data set, for use in subsequent structure determination, composed of partial diffraction data sets collected from many small crystals of the fluorescent protein Cerulean. Biochemistry Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment Magali Court1,2, Marie Malier1,2, Arnaud Millet1,2,3 1Team Mechanobiology, Immunity and Cancer, Institute for Advanced Biosciences, INSERM U1209, CNRS UMR5309, 2Université Grenoble Alpes, 3Pôle Recherche, Centre Hospitalier Universitaire des Alpes We present a protocol to obtain proteomic signatures of human macrophages and apply this to determination of the impact of a low oxygen environment on macrophage polarization. Biochemistry Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics Imen Bouchnak1, Lucas Moyet1, Daniel Salvi1, Marcel Kuntz1, Norbert Rolland1 1Université Grenoble Alpes, INRA, CNRS, CEA Here, we describe a method to purify intact chloroplasts from Arabidopsis leaves and their three main sub-compartments (envelope, stroma, and thylakoids), using a combination of differential centrifugations, continuous Percoll gradients, and discontinuous sucrose gradients. The method is valuable for subplastidial and subcellular localization of proteins by immunoblotting and proteomics. Biology Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture Cedric Allier1, Romaric Vincent1, Fabrice Navarro2, Mathilde Menneteau2, Lamya Ghenim3,4, Xavier Gidrol3, Thomas Bordy1, Lionel Hervé1, Olivier Cioni1, Sabine Bardin5, Michel Bornens5, Yves Usson4,6, Sophie Morales1 1CEA, LETI, DTBS, LISA, Université Grenoble Alpes, 2CEA, LETI, DTBS, LBAM, Université Grenoble Alpes, 3CEA, INSERM, BIG, Université Grenoble Alpes, 4CNRS, FR CNRS 3425, 5CNRS, UMR 144, Molecular Mechanisms of Intracellular Transport, PSL Research University, Institut Curie, 6TIMC-IMAG Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks. Environment Dispersion of Nanomaterials in Aqueous Media: Towards Protocol Optimization Inder Kaur1, Laura-Jayne Ellis1, Isabella Romer1, Ratna Tantra2, Marie Carriere3,4, Soline Allard5, Martine Mayne-L'Hermite5, Caterina Minelli6, Wolfgang Unger7, Annegret Potthoff8, Steffi Rades7, Eugenia Valsami-Jones1 1School of Geography, Earth and Environmental Sciences, University of Birmingham, 2Analytical Science, National Physical Laboratory, 3INAC-LCIB, Université Grenoble Alpes, 4CEA, INAC-SyMMES, 5NIMBE, CEA, CNRS, Université Paris-Saclay, CEA Saclay, 6Chemical, Medical and Environmental Science, National Physical Laboratory, 7BAM Division 6.1 'Surface Analysis and Interfacial Chemistry', BAM Federal Institute for Materials Research and Testing, 8Fraunhofer Institute for Ceramic Technologies and Systems Here, we present a step-wise protocol for the dispersion of nanomaterials in aqueous media with real-time characterization to identify the optimal sonication conditions, intensity, and duration for improved stability and uniformity of nanoparticle dispersions without impacting the sample integrity. Neuroscience Somatosensory Event-related Potentials from Orofacial Skin Stretch Stimulation Takayuki Ito1,2,3, David J. Ostry1,4, Vincent L. Gracco1,5 1Haskins Laboratories, 2Speech and Cognition Department, Gipsa-lab, CNRS, 3Univ. Grenoble-Alpes, 4Department of Psychology, McGill University, 5School of Communication Science and Disorders, McGill University This paper introduces a method for obtaining somatosensory event-related potentials following orofacial skin stretch stimulation. The current method can be used to evaluate the contribution of somatosensory afferents to both speech production and speech perception.
