University of Geneva View Institution's Website 33 articles published in JoVE Medicine Dynamic Light Scattering Analysis for the Determination of the Particle Size of Iron-Carbohydrate Complexes Michael Burgert1, Cintia Baptista Marques2, Gerrit Borchard2, Erik Philipp1, Maria Wilhelm1, Amy Alston1, Reinaldo Digigow1 1Vifor Pharma Group, Vifor Pharma Management Ltd, 2Section of Pharmaceutical Sciences, School of Pharmaceutical Sciences of Western Switzerland (ISPSO), University of Geneva Dynamic light scattering (DLS) has emerged as a suitable assay for evaluating the particle size and distribution of intravenously administered iron-carbohydrate complexes. However, the protocols lack standardization and need to be modified for each iron-carbohydrate complex analyzed. The present protocol describes the application and special considerations for the analysis of iron sucrose. Biology Bioinformatics Resources for the Study of Glycan-Mediated Protein Interactions Frédérique Lisacek1,2,3 1Proteome Informatics Group, SIB Swiss Institute of Bioinformatics, 2Computer Science Department, University of Geneva, 3Section of Biology, University of Geneva This protocol illustrates how to explore, compare, and interpret human protein glycomes with online resources. Neuroscience Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue (FACS-RTT) to Determine the Cellular Origin of Radioactive Signal Quentin Amossé1,2, Kelly Ceyzériat1,3,4, Stergios Tsartsalis1,5, Benjamin B. Tournier1,2, Philippe Millet1,2 1Division of Adult Psychiatry, Department of Psychiatry, University Hospitals of Geneva, 2Department of Psychiatry, University of Geneva, 3Division of Nuclear medicine and Molecular Imaging, University Hospitals of Geneva, 4Division of Radiation Oncology, Department of Oncology, University Hospitals of Geneva, 5Department of Brain Sciences, Faculty of Medicine, Imperial College London Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue (FACS-RTT) is a powerful tool to study the role of the 18 kDa translocator protein or Serotonin 5HT2A-receptor expression in Alzheimer's Disease at a cellular scale. This protocol describes the ex-vivo application of FACS-RTT in the TgF344-AD rat model. Biology Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy Thais Bascuas*1,2, Martina Kropp*1,2, Nina Harmening1,2, Brittany M. Wong1, Sandra Johnen3, Zsuzsanna Izsvák4, Gabriele Thumann1,2 1Experimental Ophthalmology, University of Geneva, 2Department of Ophthalmology, University Hospitals of Geneva, 3Department of Ophthalmology, University Hospital RWTH Aachen, 4Max Delbrück Center for Molecular Medicine Here, a protocol to isolate and transfect primary iris and retinal pigment epithelial cells from various mammals (mice, rat, rabbit, pig, and bovine) is presented. The method is ideally suited to study ocular gene therapy approaches in various set-ups for ex vivo analyses and in vivo studies transferable to humans. Bioengineering Production of Autologous Platelet-Rich Plasma for Boosting In Vitro Human Fibroblast Expansion Sarah Berndt1,2, Antoine Turzi2, Ali Modarressi1 1Department of Plastic, Reconstructive and Aesthetic Surgery, Geneva University Hospitals, Faculty of Medicine, Geneva University, 2Regen Lab SA This protocol presents a device that produces PRP to boost the in vitro expansion of cells in a 100% autologous fibroblast culture system. 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). Medicine Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells Thais Bascuas1,2, Martina Kropp1,2, Nina Harmening1,2, Mohammed Asrih1, Zsuzsanna Izsvák3, Gabriele Thumann1,2 1Experimental Ophthalmology, University of Geneva, 2Department of Ophthalmology, University Hospitals of Geneva, 3Max Delbrück Center for Molecular Medicine We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration. Biochemistry Spectrophotometric Screening for Potential Inhibitors of Cytosolic Glutathione S-Transferases Shannon K. D. Robin1,2, Marc Ansari*1,3, Chakradhara Rao S. Uppugunduri*1,3 1Research platform of Pediatric Onco-Hematology, Department of Paediatrics, Gynaecology and Obstetrics, Faculty of Medicine, University of Geneva, 2Section of Biology, Faculty of Science, University of Geneva, 3Onco-Hematology Unit, Department of Women, Children-Adolescents, University Hospitals of Geneva Glutathione S-transferases (GSTs) are detoxification enzymes involved in the metabolism of numerous chemotherapeutic drugs. Overexpression of GSTs is correlated with cancer chemotherapy resistance. One way to counter this phenotype is to use inhibitors. This protocol describes a method using a spectrophotometric assay to screen for potential GST inhibitors. Genetics A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands Clément Immarigeon1, François Karch1, Robert K. Maeda1 1Department of Genetics and Evolution, University of Geneva Here, we present a protocol to dissociate and sort a specific cell population from the Drosophila male accessory glands (secondary cells) for RNA sequencing and RT-qPCR. Cell isolation is accomplished through FACS purification of GFP-expressing secondary cells after a multistep-dissociation process requiring dissection, proteases digestion and mechanical dispersion. Bioengineering Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases Erika Cosset*1, Manon Locatelli*2, Antoine Marteyn2, Pierre Lescuyer3, Florence Dall Antonia3, Flavio Maurizio Mor4, Olivier Preynat-Seauve5, Luc Stoppini4, Vannary Tieng2 1Laboratory of Tumor Immunology, Translational Research Center in Onco-Hematology, Department of Internal Medicine Specialties, Faculty of Medicine, University of Geneva, 2Department of Pathology and Immunology, University Medical Center, University of Geneva, 3Laboratory of Toxicology and Therapeutic Drug Monitoring, Geneva University Hospitals, 4Tissue Engineering Laboratory, Hepia/HES-SO, University of Applied Sciences Western Switzerland, 5Laboratory of Experimental cell therapy, Department of Diagnostics, Geneva University Hospitals This study introduces and describes protocols to derive two specific human neural organoids as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid. Neuroscience Dynamic Inter-subject Functional Connectivity Reveals Moment-to-Moment Brain Network Configurations Driven by Continuous or Communication Paradigms Thomas A.W. Bolton1,2, Delphine Jochaut3, Anne-Lise Giraud3, Dimitri Van De Ville1,2 1Institute of Bioengineering, École Polytechnique Fédérale de Lausanne (EPFL), 2Department of Radiology and Medical Informatics, University of Geneva, 3Department of Neuroscience, University of Geneva The goal of the described approach is to determine at what moments of the paradigm (temporal perspective), and between which regions (spatial perspective), significant reconfigurations in functional connectivity occur on functional magnetic resonance imaging recordings during which a time-locked stimulus is played. Genetics qKAT: Quantitative Semi-automated Typing of Killer-cell Immunoglobulin-like Receptor Genes Jyothi Jayaraman1,2,3,4, Vitalina Kirgizova1, Da Di1,5, Christopher Johnson1,6, Wei Jiang1,7, James A. Traherne1 1Department of Pathology, University of Cambridge, 2Department of Physiology, Development and Neuroscience, University of Cambridge, 3Department of Obstetrics and Gynaecology, University of Cambridge School of Medicine, NIHR Cambridge Biomedical Research Centre, 4Centre for Trophoblast Research, University of Cambridge, 5Department of Genetics & Evolution, University of Geneva, 6Royal Papworth Hospital, 7Department of Plant Sciences, University of Cambridge Quantitative killer cell immunoglobulin-like receptor (KIR) semi-automated typing (qKAT) is a simple, high-throughput, and cost-effective method to copy number type KIR genes for their application in population and disease association studies. Environment Laboratory and Field Protocol for Estimating Sheet Erosion Rates from Dendrogeomorphology Jose Maria Bodoque1, Juan Antonio Ballesteros-Cánovas2,3, Juan Manuel Rubiales4,5, Markus Stoffel2,3 1University of Castilla-La Mancha (UCLM), 2Department of Earth Sciences, University of Geneva, 3Institute for Environmental Sciences, University of Geneva, 4Departamento de Sistemas y Recursos Naturales, Universidad Politécnica de Madrid, 5Departamento de Biodiversidad, Ecología y Evolución, Universidad Complutense de Madrid Characterizing erosion from dendrogeomorphology has usually focused on accurately finding the starting time of root exposure, by examining macroscopic or cell level changes caused by exposure. Here, we offer a detailed description of different novel techniques to obtain more precise erosion rates from highly accurate microtopographic data. Immunology and Infection Simultaneous Study of the Recruitment of Monocyte Subpopulations Under Flow In Vitro Patricia Ropraz1, Beat A. Imhof2, Thomas Matthes1, Bernhard Wehrle-Haller3, Adama Sidibé3 1Hematology service, Centre Médical Universitaire (CMU), Medical faculty, University of Geneva, 2Department of Pathology and Immunology, Centre Médical Universitaire (CMU), Medical faculty, University of Geneva, 3Department of Cell Physiology and Metabolism, Centre Médical Universitaire (CMU), Medical faculty, University of Geneva Here, we present an integrated protocol that measures monocyte subpopulation trafficking under flow in vitro by use of specific surface markers and confocal fluorescence microscopy. This protocol can be used to explore sequential recruitment steps as well as to profile other leukocyte subtypes using other specific surface markers. Biology Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles Nikolai Klena*1, Davide Gambarotto*1, Maeva Le Guennec1, Susanne Borgers1, Paul Guichard1, Virginie Hamel1 1Department of Cell Biology, Sciences III, University of Geneva We have developed a strategy to purify and image a large number of centrioles in different orientations amenable for super-resolution microscopy and single-particle averaging. Cancer Research Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma Archana Vijaya Kumar1,2, Carmen Donate2, Beat A. Imhof1, Thomas Matthes2 1Department of Pathology and Immunology, University of Geneva, 2Hematology Service, University Hospital, Geneva Mantle cell lymphoma (MCL) is a difficult to treat B cell disorder and it is equally difficult to establish a xenograft mouse model of primary MCL to study and develop therapeutics. Here, we describe the successful establishment of MCL xenografts in mice to help understand its underlying biology. Neuroscience Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture Virginie Sabado1, Emi Nagoshi1 1Department of Genetics and Evolution, University of Geneva The goal of this protocol is to establish ex vivo Drosophila larval brain culture optimized to monitor circadian molecular rhythms with long-term fluorescence time-lapse imaging. The application of this method to pharmacological assays is also discussed. Medicine Surgical Training for the Implantation of Neocortical Microelectrode Arrays Using a Formaldehyde-fixed Human Cadaver Model Pierre Mégevand1,2, Alain Woodtli1, Aude Yulzari1, G. Rees Cosgrove3, Shahan Momjian4, Bojan V. Stimec5, Marco V. Corniola4, Jean H. D. Fasel5 1Wyss Center for Bio and Neuroengineering, Geneva, 2Division of Neurology, Department of Clinical Neuroscience, Geneva University Hospitals, 3 We designed a procedure in which a formaldehyde-fixed human cadaver is used to assist neurosurgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain. Biochemistry In Vitro Polymerization of F-actin on Early Endosomes Olivia Muriel1,2, Cameron Christopher Scott1, Jorge Larios1, Vicent Mercier1, Jean Gruenberg1 1Department of Biochemistry, University of Geneva, 2Department of Fundamental Microbiology, University of Lausanne Early endosome functions depend on F-actin polymerization. Here, we describe a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to biochemical and genetic manipulations. Chemistry Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS Claudia A. Simões-Pires*1, Vincent Zwick*1, Sylvian Cretton1, Muriel Cuendet1 1Section des Sciences Pharmaceutiques, Universités des Geneva–Lausanne (EPGL) The present method serves to identify isoform-specific inhibitors of histone deacetylases (HDAC) in HeLa cells by the UHPLC-MS analysis of multiple substrates. This is an antibody-free method developed to reflect HDAC1 and HDAC6 activity in the living cell environment, in contrast to single-isoform cell-free assays. Biology Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes Stephanie Chanon1, Christine Durand1, Aurelie Vieille-Marchiset1, Maud Robert2, Charna Dibner3, Chantal Simon1, Etienne Lefai1 1CarMeN Laboratory, INSERM U1060, INRA 1397, University of Lyon, 2Department of digestive and bariatric surgery, Obesity Integrated Center, University Hospital of Edouard Herriot, Hospices Civils de Lyon, Lyon 1 University, 3Division of Endocrinology, Diabetes, Hypertension and Nutrition, Department of Clinical Medicine, Faculty of Medicine, University of Geneva In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose. Medicine Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation Filippo Molica1, Séverine Nolli2, Pierre Fontana2, Brenda Renata Kwak1 1Department of Pathology and Immunology, University of Geneva, 2Division of Angiology and Haemostasis & Geneva Platelet Group, Geneva University Hospitals We describe a straightforward method for the isolation of washed platelets from human blood followed by agonist-induced platelet aggregation measurements by turbidimetry. As an example we apply this method for studying the aggregation response of human platelets to collagen after a pre-incubation with the Pannexin1 channel inhibitor Brilliant Blue FCF. Immunology and Infection Measuring Phagosome pH by Ratiometric Fluorescence Microscopy Paula Nunes1, Daniele Guido1, Nicolas Demaurex1 1Department of Cellular Physiology and Metabolism, University of Geneva Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy. Immunology and Infection Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time Yalin Emre1, Stephane Jemelin1, Beat A. Imhof1 1Department of Pathology and Immunology, University of Geneva We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice. Biology A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells Farokh Dotiwala1, Isabelle Fellay2, Luis Filgueira2, Denis Martinvalet3, Judy Lieberman1, Michael Walch2 1 We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease. Behavior Methods to Test Visual Attention Online Amanda Yung1, Pedro Cardoso-Leite2, Gillian Dale3, Daphne Bavelier2,4, C. Shawn Green3 1Center for Visual Science, University of Rochester, 2Faculty of Psychology and Educational Sciences, University of Geneva, 3Department of Psychology, University of Wisconsin-Madison, 4Department of Brain and Cognitive Sciences, University of Rochester To replicate laboratory settings, online data collection methods for visual tasks require tight control over stimulus presentation. We outline methods for the use of a web application to collect performance data on two tests of visual attention. Bioengineering Harmonic Nanoparticles for Regenerative Research Flavio Ronzoni1, Thibaud Magouroux2, Remi Vernet1, Jérôme Extermann3, Darragh Crotty4, Adriele Prina-Mello5, Daniel Ciepielewski6, Yuri Volkov4, Luigi Bonacina2, Jean-Pierre Wolf2, Marisa Jaconi1 1Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, 2Physics Department, GAP-Biophotonics, University of Geneva, 3Laboratoire d'Optique Biomédicale (LOB), Faculté des Sciences et Techniques de l'Ingénieur, École Polytechnique Fédérale de Lausanne, 4Department of Clinical Medicine, School of Medicine, Trinity College Dublin, 5School of Medicine and CRANN, Trinity College Dublin, 6Nikon AG Instruments Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented. Biology Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System Xuezhi Zhang1, Thierry Soldati1 1Department of Biochemistry, University of Geneva We adapted a set of protocols for the measurement of reactive oxygen species (ROS) that can be applied in various amoeba and mammalian cellular models for qualitative and quantitative studies. Bioengineering Manufacturing Devices and Instruments for Easier Rat Liver Transplantation Graziano Oldani1,2, Stephanie Lacotte3, Lorenzo Orci1, Philippe Morel4, Gilles Mentha1, Christian Toso1 1Transplantation Division, Department of Surgery, University of Geneva Hospitals, 2Department of Surgery, University of Pavia, 3Department of Surgery, University of Geneva, 4Division of Abdominal Surgery, Department of Surgery, University of Geneva Hospitals We describe the design of the “quick-linker” device for easier orthotopic rat liver transplantation. Medicine Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens Dorothée Sturm1,2, Lorella Marselli3, Florian Ehehalt1,2, Daniela Richter1, Marius Distler2, Stephan Kersting1,2, Robert Grützmann2, Krister Bokvist4, Philippe Froguel5, Robin Liechti6, Anne Jörns7, Paolo Meda8, Gustavo Bruno Baretton9, Hans-Detlev Saeger2, Anke M. Schulte10, Piero Marchetti3, Michele Solimena1 1Molecular Diabetology, Paul Langerhans Institute Dresden, 2Department of GI-, Thoracic- and Vascular Surgery, University Hospital Carl Gustav Carus, University of Technology Dresden, 3Department of Endocrinology and Metabolism, Metabolic Unit University of Pisa, 4Labs DC0522, Lilly Corporate Center, 5Genomics, Faculty of Medicine Imperial College London, 6Vital-IT, SIB Swiss Institute of Bioinformatics, 7Clinical Biochemistry, Hannover Medical School, 8Cell Physiology and Metabolism, Medical School, University of Geneva, 9Department of Pathology, University Hospital Carl Gustav Carus, University of Technology Dresden, 10R&D DIAB Division / Translational Medicine, Sanofi-Aventis Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection. Medicine Orthotopic Liver Transplantation in Rats Graziano Oldani1,2, Stephanie Lacotte3, Philippe Morel4, Gilles Mentha1, Christian Toso1 1Transplantation Division, Department of Surgery, University of Geneva Hospitals, 2Department of Surgery, University of Pavia, 3Department of Surgery, University of Geneva, 4Division of Abdominal Surgery, Department of Surgery, University of Geneva Hospitals We present an easy-to-establish revision of the classical two-cuff technique for orthotopic liver transplantation in rat. Medicine How to Measure Cortical Folding from MR Images: a Step-by-Step Tutorial to Compute Local Gyrification Index Marie Schaer1, Meritxell Bach Cuadra2,3, Nick Schmansky4, Bruce Fischl4, Jean-Philippe Thiran2, Stephan Eliez1 1Department of Psychiatry, University of Geneva School of Medicine, 2Signal Processing Laboratory, École Polytechnique Fédérale de Lausanne, 3Department of Radiology, University Hospital Center and University of Lausanne, 4Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital Measuring gyrification (cortical folding) at any age represents a window into early brain development. Hence, we previously developed an algorithm to measure local gyrification at thousands of points over the hemisphere1. In this paper, we detail the computation of this local gyrification index. Neuroscience Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation Julien Brechbühl1, Gaëlle Luyet1, Fabian Moine1, Ivan Rodriguez2, Marie-Christine Broillet1 1Department of Pharmacology and Toxicology, University of Lausanne, 2Department of Genetics and Evolution, University of Geneva In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.