Universite-Pierre-et-Marie-Curie View Institution's Website 9 articles published in JoVE Cancer Research Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy Elisa Peranzoni*1, Houcine Bougherara*1, Sarah Barrin*1, Audrey Mansuet-Lupo2,3, Marco Alifano4, Diane Damotte2,3, Emmanuel Donnadieu1 1Institut Cochin, Inserm U1016-CNRS UMR8104, Université Paris Descartes, 2Department of Pathology, Paris Centre University Hospitals, Université Paris Descartes, 3INSERM U1138, Cancer and Immune Escape, Cordeliers Research Center, University Pierre and Marie Curie, 4Department of Thoracic Surgery, Paris Centre University Hospitals, University Paris Descartes This protocol describes a method to image resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy. Developmental Biology Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry Lorea Iturri1,2, Javier Saenz Coronilla1, Yvan Lallemand1, Elisa Gomez Perdiguero1 1Department of Developmental and Stem Cell Biology, CNRS UMR3738, Department of Immunology, Institut Pasteur, 2Cellule Pasteur UPMC, University Pierre et Marie Curie While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors. Biology Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry Sandrine Schmutz1, Mariana Valente2,3,4,5, Ana Cumano2, Sophie Novault1 1Flow Cytometry Core Facility, Center for Translational Research-Technical Core, Institut Pasteur, 2Unit for Lymphopoiesis, Immunology Department, INSERM U1223, University Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Institut Pasteur, 3Stem-Cell Microenvironments in Repair/Regeneration Team, Instituto de Investigação e Inovação em Saúde (i3s), INEB - Instituto de Engenharia Biomédica, 4ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 5Stem Cells and Regenerative Medicine Team, UMRS 1166, ICAN - Institute of Cardiometabolism And Nutrition, UPMC - Université Pierre et Marie Curie - Paris 6, INSERM This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs. Developmental Biology Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres Miguel A. Lopez-Ramirez*1,2, Charles-Félix Calvo*3, Emma Ristori1, Jean-Léon Thomas1,3, Stefania Nicoli1 1Yale Cardiovascular Research Center, Internal Medicine, Yale University, 2Department of Medicine, University of California, San Diego, 3APHP Groupe Hospitalier Pitié-Salpètrière, Université Pierre and Marie Curie Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres. Developmental Biology Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays Cyrille Ramond1,3, Antonio Bandeira2, Claire Berthault3, Pablo Pereira3, Ana Cumano3, Odile Burlen-Defranoux3 1Research Center Growth and Signaling, INSERM U845, Institut Cochin, 2Unit for Biology of Lymphocyte Populations, Immunology Department, Institut Pasteur and CIMI, Unity of Treg Biology and Therapy, University of Pierre & Marie Curie, 3Unit for Lymphopoiesis, Immunology Department, INSERM U668, University Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Institut Pasteur This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny. Biology Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies Matthias Garten1, Sophie Aimon2, Patricia Bassereau1, Gilman E. S. Toombes3 1Institut Curie, Centre de Recherche, CNRS, UMR 168, PhysicoChimie Curie, Université Pierre et Marie Curie, 2Kavli Institute for Brain and Mind, University of California, San Diego, 3Molecular Physiology and Biophysics Section, National Institute for Neurological Disorders and Stroke, National Institute of Health The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods — electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology. Medicine Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue Elena Dossi1, Thomas Blauwblomme2,3, Rima Nabbout2,4, Gilles Huberfeld2,5, Nathalie Rouach1 1Neuroglial Interactions in Cerebral Physiopathology, Center for Interdisciplinary Research in Biology, CNRS UMR 7241, INSERM U1050, Collège de France, 2Infantile Epilepsies & Brain Plasticity, INSERM U1129, PRES, Paris Descartes University, Sorbonne Paris Cité, CEA, 3Neurosurgery Department, Necker Hospital, AP-HP, Paris Descartes University, 4Rare Epilepsies Reference Center, Necker Hospital, AP-HP, Paris Descartes University, 5Neurophysiology Department, La Pitié-Salpêtrière Hospital, AP-HP, Sorbonne and Pierre and Marie Curie University We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail. Medicine Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl Marie-Noëlle Delyfer1,2,3,4, Najate Aït-Ali1,2,3, Hawa Camara1,2,3, Emmanuelle Clérin1,2,3, Jean-François Korobelnik4, José-Alain Sahel1,2,3, Thierry Léveillard1,2,3 1U968, Institut National de la Santé et de la Recherche Médicale, 2UMR S 968, Université Pierre et Marie Curie, 3UMR 7210, Centre National de la Recherche Scientifique, 4Départment d'Ophtalmologie, Centre Hospitalier Universitaire de Bordeaux We used retinal samples from retinectomy for a transcriptomic analysis of retinal detachment. We developed a procedure that allows RNA conservation between the surgical blocks and the laboratory. We standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for microarray analysis. Biology Genomic Transformation of the Picoeukaryote Ostreococcus tauri Gerben van Ooijen1, Kirsten Knox1, Katalin Kis1, François-Yves Bouget2,3, Andrew J. Millar1 1SynthSys, University of Edinburgh, 2Centre National de la Recherche Scientifique, Université Pierre et Marie Curie, Paris 06, 3UMR 7621, Laboratoire d'Océanographie Microbienne, Observatoire Océanologique, Banyuls-sur-Mer, Université Pierre et Marie Curie, Paris 06 This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.