Sapienza University of Rome 11 articles published in JoVE Biochemistry An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA Núria Crua Asensio1, Stefano Rizzieri2, Alessandro Cuomo2, Gian Gaetano Tartaglia1,3, Elsa Zacco1 1Center for Human Technology, Italian Institute of Technology (IIT), 2Department of Experimental Oncology, European Institute of Oncology (IEO), 3Department of Biology and Biotechnologies ‘Charles Darwin’, Sapienza University of Rome Here, we present an optimized in vitro method to uncover, quantify, and validate protein interactors of specific RNA sequences, using total protein extract from human cells, streptavidin beads coated with biotinylated RNA, and mass spectrometry analysis. Immunology and Infection A DNA/Ki67-Based Flow Cytometry Assay for Cell Cycle Analysis of Antigen-Specific CD8 T Cells in Vaccinated Mice Sonia Simonetti*1,2, Ambra Natalini*1,2, Giovanna Peruzzi3, Alfredo Nicosia4, Antonella Folgori5, Stefania Capone5, Angela Santoni2,6, Francesca Di Rosa1 1Institute of Molecular Biology and Pathology, National Research Council of Italy (CNR), 2Department of Molecular Medicine, University of Rome “Sapienza”, 3Center for Life Nano Science, Istituto Italiano di Tecnologia, 4Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, 5Reithera Srl, 6IRCCS, Neuromed Clonal expansion is a key feature of antigen-specific T cell response. However, the cell cycle of antigen-responding T cells has been poorly investigated, partly because of technical limitations. We describe a flow cytometric method to analyze clonally expanding antigen-specific CD8 T cells in spleen and lymph nodes of vaccinated mice. Developmental Biology Human Blastocyst Biopsy and Vitrification Roberta Maggiulli1, Adriano Giancani1,2, Danilo Cimadomo1, Filippo M. Ubaldi1, Laura Rienzi1 1G.EN.E.R.A. Centers for Reproductive Medicine, 2DAHFMO, Unit of Histology and Medical Embryology, Sapienza University of Rome Blastocyst biopsy and vitrification are required to efficiently conduct preimplantation genetic testing. An approach entailing the sequential opening of the zona pellucida and retrieval of 7-8 trophectoderm cells in day 5-7 post-insemination limits both the number of manipulations required and the exposure of the embryo to sub-optimal environmental conditions. Medicine Inducing and Characterizing Vesicular Steatosis in Differentiated HepaRG Cells Silvia Di Cocco1,2, Laura Belloni3, Abigail D.G. Nunn3, Debora Salerno3, Silvia Piconese2,4, Massimo Levrero2,3,5,6, Natalia Pediconi1,3 1Department of Molecular Medicine, Sapienza University, 2Department of Internal Medicine - DMISM, Sapienza University, 3Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, 4Pasteur Institute Italy-Fondazione Cenci Bolognetti, 5INSERM U1052-Cancer Research Center of Lyon, 6Department of Hepatology, Croix Rousse Hospital, Hospices Civils de Lyon In this study, we describe a detailed protocol for inducing liver vesicular steatosis in differentiated HepaRG cells with the fatty acid salt sodium oleate and employ methods for detection and quantification of lipid accumulation, including coherent anti-Stokes Raman scattering (CARS) microscopy, cytofluorimetric analysis, Oil red O staining, and qPCR. Neuroscience Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors Maria G. Garone1, Valeria de Turris2, Alessandro Soloperto2, Carlo Brighi2, Riccardo De Santis3, Francesca Pagani2,4, Silvia Di Angelantonio2,4, Alessandro Rosa1,2 1Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Italy, 2Center for Life Nano Science, Istituto Italiano di Tecnologia, Italy, 3Laboratory of Stem Cell biology and Molecular Embryology, The Rockefeller University, USA, 4Department of Physiology and Pharmacology, Sapienza University of Rome, Italy This protocol allows rapid and efficient conversion of induced pluripotent stem cells into motor neurons with a spinal or cranial identity, by ectopic expression of transcription factors from inducible piggyBac vectors. Developmental Biology Isolation, Propagation, and Prion Protein Expression During Neuronal Differentiation of Human Dental Pulp Stem Cells Stefano Martellucci1,2, Costantino Santacroce1, Valeria Manganelli2, Francesca Santilli1,2, Luca Piccoli3, Michele Cassetta3, Roberta Misasi2, Maurizio Sorice2, Vincenzo Mattei1,2 1Laboratory of Experimental Medicine and Environmental Pathology, Sabina Universitas - Rieti University Hub, 2Department of Experimental Medicine, Sapienza University of Rome, 3Department of Science Dentistry and Maxillofacial, Sapienza University of Rome Here we present a protocol for human Dental Pulp Stem Cells isolation and propagation in order to evaluate the prion protein expression during neuronal differentiation process. Immunology and Infection A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration Floriana Cappiello1, Bruno Casciaro1, Maria Luisa Mangoni1 1Department of Biochemical Sciences, Sapienza University of Rome Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure. Medicine Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report Paolo Giuseppe Limoli1, Enzo Maria Vingolo2, Celeste Limoli1, Sergio Zaccaria Scalinci3, Marcella Nebbioso4 1Low Vision Research Centre of Milan, 2Department of Ophthalmology, A. Fiorini Hospital, Sapienza University of Rome, 3Glaucoma and Low Vision Study Center, Department of General Surgery and Organ Transplants, University of Bologna, 4Department of Sense Organs, Faculty of Medicine and Odontology, Sapienza University of Rome The goal of this study is to assess whether the suprachoroidal graft of adipose-derived stem cells included in the stromal vascular fraction and platelets derived from platelet-rich plasma by the Limoli Retinal Restoration Technique can improve visual acuity and retinal sensitivity responses in eyes affected by dry age-related macular degeneration. Neuroscience Electroencephalographic, Heart Rate, and Galvanic Skin Response Assessment for an Advertising Perception Study: Application to Antismoking Public Service Announcements Giulia Cartocci1, Myriam Caratù2, Enrica Modica3, Anton Giulio Maglione1, Dario Rossi3, Patrizia Cherubino4, Fabio Babiloni1 1Department of Molecular Medicine, Sapienza University of Rome, 2Department of Communication and Social Research, Sapienza University of Rome, 3Department of Anatomical, Histological, Forensic, and Orthopedic Sciences, Sapienza University of Rome, 4BrainSigns SRL The following protocol describes a series of operational and computational steps required to properly estimate the emotional and cerebral reaction of a group of subjects towards a selected number of Public Service Announcements (PSAs) against smoking, aired in the USA and Europe during the period between 1998 and 2015. Neuroscience Measuring Neuromuscular Junction Functionality Emanuele Rizzuto1, Simona Pisu2, Carmine Nicoletti2, Zaccaria Del Prete1,3, Antonio Musarò2,3 1Department of Mechanical and Aerospace Engineering, Sapienza University of Rome, 2Institute Pasteur Cenci-Bolognetti, DAHFMO-Unit of Histology and Medical Embryology, Sapienza University of Rome, 3Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic. Biology Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes Evan Lee Graham1, Cristina Balla1,2, Hannabeth Franchino1, Yonathan Melman1, Federica del Monte*1, Saumya Das*1 1Cardiovascular Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, 2Division of Cardiology, Sapienza University Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.
