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University of Geneva

31 ARTICLES PUBLISHED IN JoVE

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Neuroscience

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation
Julien Brechbühl 1, Gaëlle Luyet 1, Fabian Moine 1, Ivan Rodriguez 2, Marie-Christine Broillet 1
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.

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Medicine

Orthotopic Liver Transplantation in Rats
Graziano Oldani 1,2, Stephanie Lacotte 3, Philippe Morel 4, Gilles Mentha 1, Christian Toso 1
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.

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Medicine

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens
Dorothée Sturm 1,2, Lorella Marselli 3, Florian Ehehalt 1,2, Daniela Richter 1, Marius Distler 2, Stephan Kersting 1,2, Robert Grützmann 2, Krister Bokvist 4, Philippe Froguel 5, Robin Liechti 6, Anne Jörns 7, Paolo Meda 8, Gustavo Bruno Baretton 9, Hans-Detlev Saeger 2, Anke M. Schulte 10, Piero Marchetti 3, Michele Solimena 1
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.

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Bioengineering

Manufacturing Devices and Instruments for Easier Rat Liver Transplantation
Graziano Oldani 1,2, Stephanie Lacotte 3, Lorenzo Orci 1, Philippe Morel 4, Gilles Mentha 1, Christian Toso 1
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.

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Biology

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System
Xuezhi Zhang 1, Thierry Soldati 1
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.

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Bioengineering

Harmonic Nanoparticles for Regenerative Research
Flavio Ronzoni 1, Thibaud Magouroux 2, Remi Vernet 1, Jérôme Extermann 3, Darragh Crotty 4, Adriele Prina-Mello 5, Daniel Ciepielewski 6, Yuri Volkov 4, Luigi Bonacina 2, Jean-Pierre Wolf 2, Marisa Jaconi 1
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.

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Behavior

Methods to Test Visual Attention Online
Amanda Yung 1, Pedro Cardoso-Leite 2, Gillian Dale 3, Daphne Bavelier 2,4, C. Shawn Green 3
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.

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Biology

A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells
Farokh Dotiwala 1, Isabelle Fellay 2, Luis Filgueira 2, Denis Martinvalet 3, Judy Lieberman 1, Michael Walch 2
1Cellular and Molecular Medicine Program, Boston Children’s Hospital and Harvard Medical School, 2Department of Medicine, University of Fribourg, 3Centre Médical Universitaire, University of Geneva

We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease.

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Immunology and Infection

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time
Yalin Emre 1, Stephane Jemelin 1, Beat A. Imhof 1
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.

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Immunology and Infection

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy
Paula Nunes 1, Daniele Guido 1, Nicolas Demaurex 1
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.

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Medicine

Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation
Filippo Molica 1, Séverine Nolli 2, Pierre Fontana 2, Brenda Renata Kwak 1
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.

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JoVE Journal

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes
Stephanie Chanon 1, Christine Durand 1, Aurelie Vieille-Marchiset 1, Maud Robert 2, Charna Dibner 3, Chantal Simon 1, Etienne Lefai 1
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.

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JoVE Journal

In Vitro Polymerization of F-actin on Early Endosomes
Olivia Muriel 1,2, Cameron Christopher Scott 1, Jorge Larios 1, Vicent Mercier 1, Jean Gruenberg 1
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.

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Cancer Research

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma
Archana Vijaya Kumar 1,2, Carmen Donate 2, Beat A. Imhof 1, Thomas Matthes 2
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.

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Medicine

Surgical Training for the Implantation of Neocortical Microelectrode Arrays Using a Formaldehyde-fixed Human Cadaver Model
Pierre Mégevand 1,2, Alain Woodtli 1, Aude Yulzari 1, G. Rees Cosgrove 3, Shahan Momjian 4, Bojan V. Stimec 5, Marco V. Corniola 4, Jean H. D. Fasel 5
1Wyss Center for Bio and Neuroengineering, Geneva, 2Division of Neurology, Department of Clinical Neuroscience, Geneva University Hospitals, 3Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, 4Division of Neurosurgery, Department of Clinical Neuroscience, Geneva University Hospitals, 5Clinical Anatomy Research Group, Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva

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.

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Neuroscience

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture
Virginie Sabado 1, Emi Nagoshi 1
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.

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Environment

Laboratory and Field Protocol for Estimating Sheet Erosion Rates from Dendrogeomorphology
Jose Maria Bodoque 1, Juan Antonio Ballesteros-Cánovas 2,3, Juan Manuel Rubiales 4,5, Markus Stoffel 2,3
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.

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Biology

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles
Nikolai Klena *1, Davide Gambarotto *1, Maeva Le Guennec 1, Susanne Borgers 1, Paul Guichard 1, Virginie Hamel 1
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.

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Immunology and Infection

Simultaneous Study of the Recruitment of Monocyte Subpopulations Under Flow In Vitro
Patricia Ropraz 1, Beat A. Imhof 2, Thomas Matthes 1, Bernhard Wehrle-Haller 3, Adama Sidibé 3
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.

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Genetics

qKAT: Quantitative Semi-automated Typing of Killer-cell Immunoglobulin-like Receptor Genes
Jyothi Jayaraman 1,2,3,4, Vitalina Kirgizova 1, Da Di 1,5, Christopher Johnson 1,6, Wei Jiang 1,7, James A. Traherne 1
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.

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Neuroscience

Dynamic Inter-subject Functional Connectivity Reveals Moment-to-Moment Brain Network Configurations Driven by Continuous or Communication Paradigms
Thomas A.W. Bolton 1,2, Delphine Jochaut 3, Anne-Lise Giraud 3, Dimitri Van De Ville 1,2
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.

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Bioengineering

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases
Erika Cosset *1, Manon Locatelli *2, Antoine Marteyn 2, Pierre Lescuyer 3, Florence Dall Antonia 3, Flavio Maurizio Mor 4, Olivier Preynat-Seauve 5, Luc Stoppini 4, Vannary Tieng 2
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.

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Genetics

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands
Clément Immarigeon 1, François Karch 1, Robert K. Maeda 1
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.

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Biochemistry

Spectrophotometric Screening for Potential Inhibitors of Cytosolic Glutathione S-Transferases
Shannon K. D. Robin 1,2, 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.

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Medicine

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells
Thais Bascuas 1,2, Martina Kropp 1,2, Nina Harmening 1,2, Mohammed Asrih 1, Zsuzsanna Izsvák 3, Gabriele Thumann 1,2
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.

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Medicine

Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System
Sandra Johnen 1, Nina Harmening 2,3, Corinne Marie 4,5, Daniel Scherman 4, Zsuzsanna Izsvák 6, Zoltán Ivics 7, Peter Walter 1, Gabriele Thumann 2,3
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).

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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 Harmening 1,2, Brittany M. Wong 1, Sandra Johnen 3, Zsuzsanna Izsvák 4, Gabriele Thumann 1,2
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.

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Neuroscience

Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue (FACS-RTT) to Determine the Cellular Origin of Radioactive Signal
Quentin Amossé 1,2, Kelly Ceyzériat 1,3,4, Stergios Tsartsalis 1,5, Benjamin B. Tournier 1,2, Philippe Millet 1,2
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.

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Biology

Bioinformatics Resources for the Study of Glycan-Mediated Protein Interactions
Frédérique Lisacek 1,2,3
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.

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Medicine

Dynamic Light Scattering Analysis for the Determination of the Particle Size of Iron-Carbohydrate Complexes
Michael Burgert 1, Cintia Baptista Marques 2, Gerrit Borchard 2, Erik Philipp 1, Maria Wilhelm 1, Amy Alston 1, Reinaldo Digigow 1
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.

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Aquatic Animal Models for Studies in Regenerative Medicine
Cacialli Pietro 1
1Department of Pathology and Immunology, School of Medicine, University of Geneva

Aquatic Animal Models for Studies in Regenerative Medicine

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