PSL Research University

12 articles published in JoVE

• Biology

  
  In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing Georgios Galaris¹, Jérémy H. Thalgott¹, Eliott Teston¹, Franck P.G. Lebrin^{1,2,3 1}Einthoven Laboratory for Experimental Vascular Medicine, Department of Internal Medicine (Nephrology), Leiden University Medical Center, ²Institute Physics for Medicine Paris, INSERM U1273, ESPCI Paris, CNRS FRE 2031, ³MEMOLIFE Laboratory of Excellence and PSL Research University This assay utilizes mouse embryonic stem cells differentiated into embryoid bodies cultured in 3D-collagen gel to analyze the biological processes that control sprouting angiogenesis in vitro. The technique can be applied for testing drugs, modeling diseases, and for studying specific genes in the context of deletions that are embryonically lethal.

• Neuroscience

  
  Whole-Brain 3D Activation and Functional Connectivity Mapping in Mice using Transcranial Functional Ultrasound Imaging Adrien Bertolo*¹, Mohamed Nouhoum*¹, Silvia Cazzanelli*¹, Jeremy Ferrier*³, Jean-Charles Mariani², Andrea Kliewer^4,2, Benoit Belliard¹, Bruno-Félix Osmanski³, Thomas Deffieux*¹, Sophie Pezet*¹, Zsolt Lenkei*², Mickael Tanter*^{1 1}Physics for Medicine Paris, ESPCI Paris, INSERM, CNRS, PSL Research University, ²Institute of Psychiatry and Neurosciences of Paris, INSERM U1266, Université de Paris, ³Iconeus, ⁴Department of Pharmacology and Toxicology, Jena University Hospital - Friedrich Schiller University Jena This protocol describes the quantification of volumetric cerebral hemodynamic variations in the mouse brain using functional ultrasound (fUS). Procedures for 3D functional activation map following sensory stimulation as well as resting-state functional connectivity are provided as illustrative examples, in anesthetized and awake mice.

• Medicine

  
  Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System Sandra Johnen¹, Nina Harmening^2,3, Corinne Marie^4,5, Daniel Scherman⁴, Zsuzsanna Izsvák⁶, Zoltán Ivics⁷, Peter Walter¹, Gabriele Thumann^{2,3 1}Department of Ophthalmology, University Hospital RWTH Aachen, ²Experimental Ophthalmology, University of Geneva, ³Department of Ophthalmology, University Hospitals of Geneva, ⁴Université de Paris, CNRS, INSERM, UTCBS, Unité des technologies Chimiques et Biologiques pour la Santé, ⁵Chimie ParisTech, PSL Research University, ⁶Max Delbrück Center for Molecular Medicine in the Helmholtz Association, ⁷Division of Medical Biotechnology, Paul-Ehrlich-Institute We have developed a protocol to transfect primary human pigment epithelial cells by electroporation with the gene encoding pigment epithelium-derived factor (PEDF) using the Sleeping Beauty (SB) transposon system. Successful transfection was demonstrated by quantitative polymerase chain reaction (qPCR), immunoblotting, and enzyme-linked immunosorbent assay (ELISA).

• Biology

  
  Quantifying Spatiotemporal Parameters of Cellular Exocytosis in Micropatterned Cells Hugo Lachuer^1,2,3, Pallavi Mathur^1,2,3, Kevin Bleakley⁴, Kristine Schauer^{1,2,3 1}Unité Mixte de Recherche 144 CNRS, Molecular Mechanisms of Intracellular Transport group, Institut Curie, 75005 Paris, France, ²PSL Research University, Paris, France, ³Sorbonne Université, Paris, France, ⁴INRIA, Université Paris-Sud, PSL Live imaging of lysosomal exocytosis on micropatterned cells allows a spatial quantification of this process. Morphology normalization using micropatterns is an outstanding tool to uncover general rules about the spatial distribution of cellular processes.

• Environment

  
  Data Collection on Marine Litter Ingestion in Sea Turtles and Thresholds for Good Environmental Status Marco Matiddi¹, Giuseppe A. deLucia², Cecilia Silvestri¹, Gaëlle Darmon³, Jesús Tomás⁴, Christopher K. Pham⁵, Andrea Camedda², Frederic Vandeperre^5,6, Françoise Claro⁷, Yakup Kaska⁸, Helen Kaberi⁹, Ohiana Revuelta⁴, Raffaella Piermarini¹, Roberto Daffina¹, Marco Pisapia¹, Daniela Genta¹, Doğan Sözbilen⁸, Mohamed N. Bradai¹⁰, Yasmina Rodríguez⁵, Delphine Gambaiani³, Catherine Tsangaris⁹, Olfa Chaieb¹⁰, Judicaëlle Moussier⁷, Ana L. Loza¹¹, Claude Miaud³, INDICIT consortium, ¹Italian National Institute for Environmental Protection and Research (ISPRA), ²Institute for Coastal Marine Environment-National Research Council (IAMC-CNR), ³EPHE, PSL Research University, UMR 5175 CE3FE, CNRS, UM, Univ P. Valery, SupAgro, IRD, INRA, Biogéographie et Écologie des Vertébrés, ⁴Cavanilles Institute of Biodiversity and Evolutionary Biology, University of Valencia, ⁵Departamento de Oceanografia e Pescas, Instituto do Mar/Okeanos, Universidade dos Açores, ⁶MARE - Marine and Environmental Sciences Centre, Universidade dos Açores, ⁷ The protocol focuses on the collection of sea turtle samples, describing all the steps from the animal recovery and necropsy to the classification and quantification of ingested marine litter. Moreover, the representative results show how to use the collected data to elaborate the possible thresholds for Good Environmental Status.

• Neuroscience

  
  Dissection of Local Ca²⁺ Signals in Cultured Cells by Membrane-targeted Ca²⁺ Indicators Hiroko Bannai^1,2, Matsumi Hirose², Fumihiro Niwa^2,3, Katsuhiko Mikoshiba^{2 1}Japan Science and Technology Agency, PRESTO, ²Laboratory for Developmental Neurobiology, RIKEN Center for Brain Science, ³École Normale Supèrieure, Institut de Biologie de l'ENS (IBENS), Institut national de la santè et de la recherche mèdicale (INSERM), Centre national de la recherche scientifique (CNRS), École Normale Supèrieure, PSL Research University Here we present a protocol for Ca²⁺ imaging in neurons and glial cells, which enables the dissection of Ca²⁺ signals at subcellular resolution. This process is applicable to all cell types that allow the expression of genetically encoded Ca²⁺ indicators.

• Genetics

  
  Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions Charles Decraene^1,2, Amanda Bortolini Silveira¹, Marc Michel¹, François-Clément Bidard^1,3,4, Jean-Yves Pierga^1,3,5, Marc-Henri Stern^1,6, Charlotte Proudhon^{1 1}Circulating 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.

• Neuroscience

  
  High-resolution Volume Imaging of Neurons by the Use of Fluorescence eXclusion Method and Dedicated Microfluidic Devices Céline Braïni¹, Angelo Mottolese¹, Ivan Ferrante¹, Sylvain Monnier², Catherine Villard^{1 1}Laboratoire Physico-Chimie Curie, Institut Curie, Institut Pierre-Gilles de Gennes pour la microfluidique, Université PSL, CNRS, ²UMR 144 Institut Curie, Université PSL, CNRS Volume is an important parameter regarding physiological and pathological characteristics of cells. We describe a fluorescent exclusion method allowing full-field measurement of in vitro neuronal volume with sub-micrometric axial resolution required for the analysis of neurites and dynamic structures implied in neuronal growth.

• Biology

  
  Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture Cedric Allier¹, Romaric Vincent¹, Fabrice Navarro², Mathilde Menneteau², Lamya Ghenim^3,4, Xavier Gidrol³, Thomas Bordy¹, Lionel Hervé¹, Olivier Cioni¹, Sabine Bardin⁵, Michel Bornens⁵, Yves Usson^4,6, Sophie Morales^{1 1}CEA, 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.

• Biology

  
  Pulling Membrane Nanotubes from Giant Unilamellar Vesicles Coline Prévost*^1,2,3, Feng-Ching Tsai*^1,4, Patricia Bassereau^1,4, Mijo Simunovic^{1,5 1}Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University, CNRS UMR168, ²Department of Genetics and Complex Diseases, T. H. Chan School of Public Health, Harvard Medical School, ³Department of Cell Biology, Harvard Medical School, ⁴Sorbonne Universités, UPMC University Paris 06, ⁵Center for Studies in Physics and Biology, The Rockefeller University Many proteins in the cell sense and induce membrane curvature. We describe a method to pull membrane nanotubes from lipid vesicles to study the interaction of proteins or any curvature-active molecule with curved membranes in vitro.

• Developmental Biology

  
  Transcriptional Analysis by Nascent RNA FISH of In Vivo Trophoblast Giant Cells or In Vitro Short-term Cultures of Ectoplacental Cone Explants Catherine Corbel¹, Edith Heard^{1 1}Unité de Génétique et Biologie du Développement, Institut Curie, PSL Research University, CNRS UMR 3215, INSERM U934 Trophoblast giant cells (TGCs) play a key role in the placenta to ensure a healthy pregnancy. We present a protocol for assessing the transcriptional status of genes in TGCs by nascent fluorescent in situ hybridization on cryostat sections of post-implantation embryos or short-term cultures of embryonic day 7 ectoplacental cones.

• Developmental Biology

  
  Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo Beatrice C. Durand^{1,2,3,4 1}Institut Curie, ²UMR 3387, CNRS, ³PSL Research University, ⁴Université Paris-Sud In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.