University of Milan View Institution's Website 22 articles published in JoVE Developmental Biology AMEBaS: Automatic Midline Extraction and Background Subtraction of Ratiometric Fluorescence Time-Lapses of Polarized Single Cells Rafael Badain1, Daniel S. C. Damineli1,2, Maria Teresa Portes3, José Feijó4, Stefano Buratti5, Giorgia Tortora6, Hugo Neves de Oliveira1, Roberto M. Cesar Jr1 1Institute of Mathematics and Statistics, University of São Paulo, 2Center for Mathematics, Computing and Cognition, Federal University of ABC, 3Department of Botany, Institute of Biosciences, University of São Paulo, 4Cell Biology and Molecular Genetics Department, University of Maryland, 5Department of Biosciences, University of Milan, 6Department of Physics, Politecnico di Milano Current methods for analyzing the intracellular dynamics of polarized single cells are often manual and lack standardization. This manuscript introduces a novel image analysis pipeline for automating midline extraction of single polarized cells and quantifying spatiotemporal behavior from time lapses in a user-friendly online interface. Cancer Research Biobank for Translational Medicine: Standard Operating Procedures for Optimal Sample Management Giuseppina Bonizzi*1, Maria Capra*1, Cristina Cassi1, Giulio Taliento1, Oriana Pala2, Elham Sajjadi2,3, Konstantinos Venetis2,3, Mariia Ivanova2, Massimo Monturano4, Giuseppe Renne2, Lorenzo Zattoni1,3, Elena Guerini-Rocco2,3, Giuseppe Viale2,3, Roberto Orecchia5, Nicola Fusco1,2,3 1Biobank for Translational and Digital Medicine Unit, IEO, European Institute of Oncology IRCCS, 2Division of Pathology, IEO, European Institute of Oncology IRCCS, 3Department of Oncology and Hemato-Oncology, University of Milan, 4Patient Safety & Risk Management Service, IEO, European Institute of Oncology IRCCS, 5Scientific Directorate, IEO, European Institute of Oncology IRCCS Biobanks are crucial resources for biomedical research and the Biobank for Translational and Digital Medicine Unit at the European Institute of Oncology is a model in this field. Here, we provide a detailed description of biobanks' standard operating procedures for the management of different types of human biological samples. Medicine Investigation of the Electrophysiological and Thermographic Safety Parameters of Surgical Energy Devices During Thyroid and Parathyroid Surgery in a Porcine Model Hsin-Yi Tseng1, Tzu-Yen Huang1, Jia Joanna Wang1,2, Yi-Chu Lin1, I-Cheng Lu3, Feng-Yu Chiang4, Gianlorenzo Dionigi5,6, Gregory W. Randolph7, Che-Wei Wu1,8 1Department of Otorhinolaryngology-Head and Neck Surgery, International Thyroid Surgery Center, Kaohsiung Medical University Hospital, Kaohsiung Medical University, 2Department of Otolaryngology-Head and Neck Surgery, Kaohsiung Municipal Siaogang Hospital, Kaohsiung Medical University, 3Department of Anesthesiology, Kaohsiung Municipal Siaogang Hospital, Kaohsiung Medical University, 4Department of Otolaryngology-Head and Neck Surgery, E-Da Hospital, I-Shou University, 5Division of General Surgery, Endocrine Surgery Section, Istituto Auxologico Italiano (IRCCS), 6Department of Pathophysiology and Transplantation, Faculty of Medicine and Surgery, University of Milan, 7Division of Thyroid and Parathyroid Endocrine Surgery, Department of Otolaryngology-Head and Neck Surgery, Massachusetts Eye and Ear Infirmary, Harvard Medical School, 8Center for Liquid Biopsy and Cohort Research, and Faculty of Medicine, College of Medicine, Kaohsiung Medical University The safe application of newly developed surgical energy devices in thyroid/parathyroid surgery attracts the attention of surgeons. Animal experimental models can avoid unnecessary trials and errors in human surgery. This report aims to demonstrate electrophysiological and thermographic methods to evaluate the safety parameters of SEDs in thyroid/parathyroid surgery. Medicine Transthoracic Echocardiography to Assess Post-Resuscitation Left Ventricular Dysfunction After Acute Myocardial Infarction and Cardiac Arrest in Pigs Daria De Giorgio1, Davide Olivari1, Francesca Fumagalli1, Lidia Staszewsky1, Giuseppe Ristagno2,3 1Istituto di Ricerche Farmacologiche Mario Negri IRCCS, 2University of Milan, 3 Transthoracic echocardiography is the first-line diagnostic test for post-resuscitation left ventricular dysfunction and structural changes in a pig model of cardiac arrest. Behavior Assessment of Spatial Lingual Tactile Sensitivity using a Gratings Orientation Test Noemi Sofia Rabitti1, Marta Appiani1, Camilla Cattaneo1, Rebecca Ford2, Monica Laureati1 1Department of Food, Environmental and Nutritional Sciences (DeFENS), University of Milan, 2Division of Food, Nutrition, and Dietetics, School of Biosciences, University of Nottingham This work illustrates a standard procedure and threshold determination by the R-index to assess spatial lingual tactile sensitivity using a gratings orientation test. Cancer Research Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma Greta Donisi*1, Marialuisa Barbagallo*2, Giovanni Capretti1,3, Gennaro Nappo1,3, Panteleimon G. Takis4,5,6, Alessandro Zerbi1,3, Federica Marchesi2,7, Nina Cortese2 1Section of Pancreatic Surgery, Humanitas Clinical and Research Center-IRCCS, 2Department of Immunology and Inflammation, Humanitas Clinical and Research Center-IRCCS, 3Department of Biomedical Sciences, Humanitas University, 4Giotto Biotech S.R.L., 5Section of Bioanalytical Chemistry, Division of Systems Medicine, Department of Metabolism, Digestion and Reproduction, Imperial College London, 6National Phenome Centre, Department of Metabolism, Digestion and Reproduction, Faculty of Medicine, Imperial College London, 7Department of Medical Biotechnology and Translational Medicine, University of Milan Pancreatic juice is a precious source of biomarkers for human pancreatic cancer. We describe here a method for intraoperative collection procedure. To overcome the challenge of adopting this procedure in murine models, we suggest an alternative sample, tumor interstitial fluid, and describe here two protocols for its isolation. Neuroscience Brain Morphology of Cannabis Users With or Without Psychosis: A Pilot MRI Study Giuseppe Delvecchio*1, Lucio Oldani*2, Gian Mario Mandolini2, Alessandro Pigoni3, Valentina Ciappolino2, Giandomenico Schiena2, Matteo Lazzaretti2, Elisabetta Caletti2, Viviana Barbieri1, Claudia Cinnante4, Fabio Triulzi1,4, Paolo Brambilla1,2 1Department of Pathophysiology and Transplantation, University of Milan, 2 This is a 3T magnetic resonance imaging study aiming to investigate grey matter volume differences between cannabis-induced psychosis patients and non-psychotic chronic cannabis users. Developmental Biology In Vitro Culture Strategy for Oocytes from Early Antral Follicle in Cattle Rodrigo G. Barros1, Valentina Lodde1, Federica Franciosi1, Alberto M. Luciano1 1Reproductive and Developmental Biology Laboratory, Department of Health, Animal Science and Food Safety, University of Milan We describe the procedures for isolation of growing oocytes from ovarian follicles at early stages of development, as well as the setup of an in vitro culture system which can support the growth and differentiation up to the fully-grown stage. Medicine Integrating Augmented Reality Tools in Breast Cancer Related Lymphedema Prognostication and Diagnosis Marco Invernizzi1, Letterio Runza2, Alessandro De Sire1, Lorenzo Lippi1, Concetta Blundo3, Donatella Gambini4, Renzo Boldorini5,6, Stefano Ferrero2,7, Nicola Fusco2,7 1Physical and Rehabilitative Medicine, Department of Health Sciences, University of Eastern Piedmont "A. Avogadro", 2 Breast cancer related lymphedema is frequent in breast cancer survivors, but there are not widely employed guidelines for its diagnosis and quantification. Here, we introduce a reliable and cost-effective protocol to define, quantify, and compare upper limb volume in breast cancer patients. Immunology and Infection Isolation, Transfection, and Culture of Primary Human Monocytes Karlie Plaisance-Bonstaff1,2, Celeste Faia2, Dorota Wyczechowska1, Duane Jeansonne1, Cecilia Vittori1,3, Francesca Peruzzi1,2 1Department of Medicine, Louisiana State University Health Sciences Center, 2Department of Microbiology, Immunology, and Parasitology, Louisiana State University Health Sciences Center, 3Department of Biomedical and Clinical Sciences L. Sacco, University of Milan Presented here is an optimized protocol for isolating, culturing, transfecting, and differentiating human primary monocytes from HIV-infected individuals and healthy controls. Developmental Biology Quantification of Self-renewal in Murine Mammosphere Cultures Thalia Vlachou1, Xieraili Aobuli1, Errico D'Elia1, Angela Santoro1, Maria Cristina Moroni1, Pier Giuseppe Pelicci1,2 1Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, 2Department of Oncology and Hemato-Oncology, University of Milan Here, we describe the implementation and interpretation of the results of an in vitro mammosphere self-renewal quantitative assay. Behavior Treatment of Ankle Osteoarthritis with Total Ankle Replacement Through a Lateral Transfibular Approach Federico G. Usuelli1, Riccardo D'Ambrosi1,2, Luigi Manzi1, Camilla Maccario1,2, Cristian Indino1 1Galeazzi Orthopedic Institute for Research and Care, 2Dipartimento di Scienze Biomediche per la Salute, Università degli Studi di Milano We present our preoperative, operative, and postoperative protocols for the treatment of osteoarthritis of the ankle with total ankle replacement via lateral transfibular approach. Medicine Autologous Microfractured and Purified Adipose Tissue for Arthroscopic Management of Osteochondral Lesions of the Talus Riccardo D'Ambrosi1,2, Cristian Indino1, Camilla Maccario1, Luigi Manzi1, Federico Giuseppe Usuelli1 1IRCCS Istituto Ortopedico Galeazzi - C.A.S.C.O. Piede e Caviglia, 2Dipartimento di Scienze Biomediche per la Salute, Università degli Studi di Milano The aim of this study is to report a protocol for the arthroscopic treatment of osteochondral lesions of the talus using microfractured and purified adipose-derived stem cells. Immunology and Infection Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers Giulia Ercoli1, Gianluca Lopez1,2, Camilla Ciapponi1,3, Chiara Corti1,2, Luca Despini4, Donatella Gambini5, Letterio Runza1, Concetta Blundo4, Amedeo Sciarra1, Nicola Fusco1,6 1 Heterogeneous distribution of HER2-positive cells can be observed in a subset of breast cancers and generates clinical dilemmas. Here, we introduce a reliable and cost-effective protocol to define, quantify, and compare HER2 intra-tumor genetic heterogeneity in a large series of heterogeneously processed breast cancers. Developmental Biology Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos Lorena Benedetti1,2,3, Anna Ghilardi4, Laura Prosperi4, Maura Francolini*1, Luca Del Giacco*4 1Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, 2Department of Neuroscience; Department of Cell Biology, Howard Hughes Medical Institute, Yale University School of Medicine, 3Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, 4Department of BioSciences, Università degli Studi di Milano Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest. Biochemistry Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve Cristina Banfi1, Anna Guarino2, Maura Brioschi1, Stefania Ghilardi1, Valeria Mastrullo1, Elena Tremoli1, Gianluca Polvani2,3,4 1Centro Cardiologico Monzino IRCCS, 2Cardiovascular Tissue Bank of Milan, Centro Cardiologico Monzino IRCCS, 3Department of Clinical Sciences and Community Health, Cardiovascular Section, University of Milan, 4Department of Cardiovascular Disease, Development and Innovation Cardiac Surgery Unit, Centro Cardiologico Monzino IRCCS The protein composition of the human mitral valve is still partially unknown, because its analysis is complicated by low cellularity and therefore by low protein biosynthesis. This work provides a protocol to efficiently extract protein for the analysis of the mitral valve proteome. Developmental Biology Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes Federica Franciosi1, Irene Tessaro1,2, Rozenn Dalbies-Tran3, Cecile Douet3, Fabrice Reigner4, Stefan Deleuze5, Pascal Papillier3, Ileana Miclea6, Valentina Lodde1, Alberto M. Luciano1, Ghylene Goudet3 1Department of Health, Animal Science and Food Safety, University of Milan, 2IRCCS. Istituto Ortopedico Galeazzi, 3PRC, CNRS, IFCE, Université de Tours, INRA, 4PAO, INRA, 5Clinique des Animaux de Compagnie et des Équidés, Université de Liège, 6University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania This manuscript describes an experimental approach to morphologically and biochemically characterize horse oocytes. Specifically, the present work illustrates how to collect immature and mature horse oocytes by ultrasound-guided ovum pick-up (OPU) and how to investigate chromosome segregation, spindle morphology, global histone acetylation, and mRNA expression. Medicine A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status Wendy E. Heywood*1, Anna Baud*1, Emily Bliss1, Ernestas Sirka1, Jonathan M. Schott2, Henrik Zetterberg3, Daniela Galimberti4, Neil J. Sebire5, Kevin Mills1 1Centre for Translational Omics, Genetics and Genomic Medicine Deptartment, Great Ormond Street Institute of Child Health, University College London, 2Dementia Research Centre, Institute of Neurology, University College London, 3Clinical Neurochemistry Laboratory, Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry, The Sahlgrenska Academy, University of Gothenburg, 4Neurology Unit, Department of Pathophysiology and Transplantation, University of Milan, 5Great Ormond Street Hospital for Children, University College London We describe a high-throughput, multiplex, and targeted proteomic cerebrospinal fluid (CSF) assay developed with potential for clinical translation. The test can quantitate potential markers and risk factors for neurodegeneration, such as the apolipoprotein E variants (E2, E3 and E4), and measure their allelic expression. Medicine Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury Stephana Carelli*1, Toniella Giallongo*1, Claudio Gerace1, Anthea De Angelis1, Michele D. Basso2, Anna Maria Di Giulio1, Alfredo Gorio1 1Laboratory of Pharmacology, Department of Health Sciences, University of Milan, 2Department of Neuroscience, College of Medicine, The Ohio State University Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells. Biology Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells Matteo Fossati1,2,3, Nica Borgese2,3,4, Sara Francesca Colombo2,3, Maura Francolini1,2 1Fondazione Filarete, 2Department of Biotechnology and Translational Medicine, University of Milan, 3Neuroscience Institute, National Research Council (CNR), 4Department of Health Science, "Magna Graecia" University of Catanzaro We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment. Medicine Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR Marco Pacifici1, Serena Delbue2, Ferdous Kadri1, Francesca Peruzzi1 1Medical School and Stanley S. Scott Cancer Center, LSU Health Sciences Center, 2Department of Biomedical, Surgery and Dental Sciences, University of Milan We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens. Biology Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors Michela Frascoli1, Michele Proietti1, Fabio Grassi1,2 1Institute for Research in Biomedicine, Bellinzona (Switzerland), 2Dipartimento di Biologia e Genetica per le Scienze Mediche, Universitá degli Studi di Milano A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.