San Raffaele Scientific Institute 8 articles published in JoVE Medicine Fertility Sparing Procedure using Carbon Dioxide Fiber Laser Vaporization of Ovarian Endometrioma Massimo Candiani1, Jessica Ottolina1, Iacopo Tandoi1, Ludovica Bartiromo1, Matteo Schimberni1, Roberta Villanacci1, Stefano Ferrari1 1Gynecological Surgery and Endometriosis Departmental Unit, San Raffaele Scientific Institute In this protocol, CO2 fiber laser technique is demonstrated for the surgical treatment of ovarian endometriosis, which represents a viable alternative in terms of fertility preservation with the major advantage of not being dependent on the surgeon's skills and personal experience. Immunology and Infection Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies Gloria Delfanti1, Alessandra Perini*1,2, Elisa Zappa*1,2, Maya Fedeli1,2 1Experimental Immunology Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, 2Vita-Salute San Raffaele University We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo studies. Biology Exploiting Live Imaging to Track Nuclei During Myoblast Differentiation and Fusion Giorgia Careccia*1,2, Federica Colombo*1,3, Mario Tirone1, Alessandra Agresti1, Marco E. Bianchi1,2, Samuel Zambrano*1,2, Emilie Vénéreau*1 1Chromatin Dynamics Unit, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) San Raffaele Scientific Institute, 2Vita-Salute San Raffaele University, 3Department of Electronics, Information and Bioengineering, Politecnico di Milano Skeletal muscle differentiation is a highly dynamic process, which particularly relies on nuclear positioning. Here, we describe a method to track nuclei movements by live cell imaging during myoblast differentiation and myotube formation and to perform a quantitative characterization of nuclei dynamics by extracting information from automatic tracking. Cancer Research Immunoglobulin Gene Sequence Analysis In Chronic Lymphocytic Leukemia: From Patient Material To Sequence Interpretation Andreas Agathangelidis*1, Lesley Ann Sutton*2,3, Anastasia Hadzidimitriou1, Cristina Tresoldi4, Anton W. Langerak5, Chrysoula Belessi6, Frederic Davi7, Richard Rosenquist2,3, Kostas Stamatopoulos1,2, Paolo Ghia8 1Institute of Applied Biosciences, Centre for Research and Technology Hellas, 2Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, 3Department of Molecular Medicine and Surgery, Karolinska Institutet, 4Division of Immunology, Transplantation and Infectious, IRCCS San Raffaele Scientific Institute, 5Department of Immunology, Laboratory for Medical Immunology, Erasmus University Medical Center, 6Hematology Department, Nikea General Hospital, 7Assistance publique - Hôpitaux de Paris (AP-HP), Hopital Pitié-Salpêtrière, Department of Hematology, and UPMC University Paris 06, UMRS 1138, 8Division of Experimental Oncology, IRCCS Istituto Scientifico San Raffaele and Università Vita-Salute San Raffaele Herein, we present a protocol that details the technical aspects and essential requirements to ensure robust IG gene sequence analysis in patients with chronic lymphocytic leukemia (CLL), based on the accumulated experience of the European Research initiative on CLL (ERIC). Developmental Biology Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection Ombretta Guardiola1, Gennaro Andolfi1, Mario Tirone2,3, Francescopaolo Iavarone1, Silvia Brunelli2, Gabriella Minchiotti1 1Stem Cell Fate Laboratory, Institute of Genetics and Biophysics "A. Buzzati Traverso", CNR, 2School of Medicine and Surgery, University of Milano-Bicocca, 3Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis. Neuroscience TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis Federica Daniele1, Eliana S. Di Cairano1, Stefania Moretti1, Giovanni Piccoli2, Carla Perego1,3 1Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, 2San Raffaele Scientific Institute and Vita-Salute University, 3CEND Center of Excellence in Neurodegenerative Diseases, Università degli Studi di Milano This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported. Medicine From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia Benedetta Apollonio1,2, Maria Teresa Sabrina Bertilaccio1, Umberto Restuccia3, Pamela Ranghetti1, Federica Barbaglio1, Paolo Ghia1,4, Federico Caligaris-Cappio1,4, Cristina Scielzo1 1Division of Molecular Oncology, IRCCS, San Raffaele Scientific Institute, 2 Here we describe a protocol that couples two proteomic techniques, namely 2-dimensional Electrophoresis (2DE) and Mass Spectrometry (MS), to identify differentially expressed/post-translational modified proteins among two or more groups of primary samples. This approach, together with functional experiments, allows the identification and characterization of prognostic markers/therapeutic targets. Immunology and Infection Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice Marcella Facchini1,2, Ida De Fino1,2, Camilla Riva1,2, Alessandra Bragonzi1 1Infections and Cystic Fibrosis Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, 2Italian Cystic Fibrosis Research Foundation We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.
