Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico 10 articles published in JoVE Genetics Dual Immunofluorescence of γH2AX and 53BP1 in Human Peripheral Lymphocytes Aurora Falaschi1, Anna Chiaramonte1,2, Serena Testi1, Roberto Scarpato1 1Unità di Genetica, Dipartimento di Biologia, University of Pisa, 2 This protocol presents a method to assess the formation and repair of DNA double-strand breaks through the simultaneous detection of γH2AX and 53BP1 foci in interphase nuclei of bleomycin-treated human peripheral lymphocytes. Biology Microvascular and Macrovascular Endothelial Cell Isolation and Purification from Lung-Derived Samples Roberto Plebani1,2, Alessandra D'Alessandro2, Paola Lanuti2,3, Pasquale Simeone2,3, Massimiliano Cinalli1, Ilaria Righi4, Alessandro Palleschi4,5, Matteo Mucci1,2, Marco Marchisio2,3, Francesco Cappabianca1, Marina Camera6,7, Felice Mucilli1, Mario Romano1,2 1Department of Medical, Oral and Biotechnological Sciences, “G. d’Annunzio” University of Chieti-Pescara, 2Center on Advanced Studies and Technology (CAST), “G. d’Annunzio” University of Chieti-Pescara, 3Department of Medicine and Aging Sciences, University “G. d’Annunzio” of Chieti-Pescara, 4 Although challenging, the isolation of pulmonary endothelial cells is essential for studies on lung inflammation. The present protocol describes a procedure for the high-yield, high-purity isolation of macrovascular and microvascular endothelial cells. 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. Medicine Laparoscopic Oocyte Retrieval and Cryopreservation during Vaginoplasty for Treatment of Mayer-Rokitansky-Kuster-Hauser Syndrome Valeria Stella Vanni*1,2, Alessandra Alteri*1,2, Lucia De Santis1,2, Greta Chiara Cermisoni1, Elisa Rabellotti1, Diana Delprato1, Marta Parma1, Enrico Papaleo1, Luigi Fedele3, Massimo Candiani1,2 1Obstetrics and Gynaecology Unit, IRCCS San Raffaele Scientific Institute, 2Vita-Salute San Raffaele University, 3 A dedicated team can offer Mayer-Rokitansky-Kuster-Hauser patients the option to perform controlled ovarian stimulation and oocyte cryopreservation at the time of laparoscopic vaginoplasty. 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. 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 Using Robotic Systems to Process and Embed Colonic Murine Samples for Histological Analyses Fulvia Milena Cribiù*1, Claudia Burrello*2, Roberta Tacchi1, Francesca Boggio1, Dario Ricca1, Flavio Caprioli3,4, Stefano Ferrero1, Federica Facciotti2 1Pathology Unit, Fondazione IRCCS Cà Granda, Ospedale Maggiore Policlinico, 2Department of Experimental Oncology, European Institute of Oncology, 3 Lack of standardization for murine tissue processing reduces the quality of murine histopathological analysis as compared to human specimens. Here, we present a protocol to perform histopathological examination of murine inflamed and uninflamed colonic tissues to show the feasibility of robotic systems routinely used for processing and embedding human samples. 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 Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids Viviana Meraviglia*1, Alessandra Zanon*1, Alexandros A. Lavdas1, Christine Schwienbacher1, Rosamaria Silipigni2, Marina Di Segni2, Huei-Sheng Vincent Chen3, Peter P. Pramstaller1, Andrew A. Hicks1, Alessandra Rossini1 1Center for Biomedicine, European Academy Bozen/Bolzano (EURAC), 2 Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids. Biology Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts Deborah Mattinzoli1, Piergiorgio Messa2, Alessandro Corbelli1, Masami Ikehata1, Anna Mondini1, Cristina Zennaro3, Silvia Armelloni1, Min Li1, Laura Giardino1, Maria Pia Rastaldi1 1 Treatment of primary mouse osteoblasts with retinoic acid produces a homogeneous population of ramified cells bearing morphological and molecular features of osteocytes. The method overcomes the difficulty of obtaining and maintaining primary osteocytes in culture, and can be advantageous to study cells derived from transgenic models.