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. Lebrin1,2,3 1Einthoven 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 Kliewer4,2, Benoit Belliard1, Bruno-Félix Osmanski3, Thomas Deffieux*1, Sophie Pezet*1, Zsolt Lenkei*2, Mickael Tanter*1 1Physics 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 Harmening2,3, Corinne Marie4,5, Daniel Scherman4, Zsuzsanna Izsvák6, Zoltán Ivics7, Peter Walter1, Gabriele Thumann2,3 1Department 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 Lachuer1,2,3, Pallavi Mathur1,2,3, Kevin Bleakley4, Kristine Schauer1,2,3 1Unité 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 Vandeperre5,6, 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 Bannai1,2, Matsumi Hirose2, Fumihiro Niwa2,3, Katsuhiko Mikoshiba2 1Japan 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 Decraene1,2, Amanda Bortolini Silveira1, Marc Michel1, François-Clément Bidard1,3,4, Jean-Yves Pierga1,3,5, Marc-Henri Stern1,6, Charlotte Proudhon1 1Circulating 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 Villard1 1Laboratoire 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 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. Biology Pulling Membrane Nanotubes from Giant Unilamellar Vesicles Coline Prévost*1,2,3, Feng-Ching Tsai*1,4, Patricia Bassereau1,4, Mijo Simunovic1,5 1Laboratoire 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 Heard1 1Unité 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. Durand1,2,3,4 1Institut 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.