Istituto Italiano di Tecnologia View Institution's Website 18 articles published in JoVE 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. 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. Neuroscience Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays Gabriella Panuccio1, Ilaria Colombi1, Michela Chiappalone1,2 1Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia, 2Rehab Technologies, Istituto Italiano di Tecnologia We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface. Neuroscience Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits Agnes Thalhammer1, Fanny Jaudon1, Lorenzo A. Cingolani1 1Center for Synaptic Neuroscience and Technology, Istituto Italiano di Tecnologia This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits. 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. Biochemistry Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase Elisa Romeo1, Silvia Pontis1, Stefano Ponzano1, Fabiola Bonezzi1, Marco Migliore1, Simona Di Martino1, Maria Summa1, Daniele Piomelli1,2 1Drug Discovery and Development, Istituto Italiano di Tecnologia, 2Departments of Anatomy and Neurobiology, Pharmacology, and Biological Chemistry, University of California, Irvine School of Medicine Here, we describe the preparation and use of an activity-based probe (ARN14686, undec-10-ynyl-N-[(3S)-2-oxoazetidin-3-yl]carbamate) that allows for the detection and quantification of the active form of the proinflammatory enzyme N-acylethanolamine acid amidase (NAAA), both in vitro and ex vivo. Bioengineering Optical Control of Living Cells Electrical Activity by Conjugated Polymers Nicola Martino1, Caterina Bossio1, Susana Vaquero Morata1, Guglielmo Lanzani1,2, Maria Rosa Antognazza1 1Center for Nano Science and Technology @PoliMi, Istituto Italiano di Tecnologia, 2Dipartimento di Fisica, Politecnico di Milano A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described. Behavior An Operant Intra-/Extra-dimensional Set-shift Task for Mice Diego Scheggia1, Francesco Papaleo1 1Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia Attentional set-shifting cognitive abnormalities are a core feature of many psychiatric diseases. Here, we present the development of a novel automatic system for the effective study of attentional set-shifting abilities, executive functions and other cognitive abilities in mice with high translational validity to human studies. Neuroscience Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits Sivan Kanner*1, Marta Bisio*2, Gilad Cohen3, Miri Goldin4, Marieteresa Tedesco5, Yael Hanein3, Eshel Ben-Jacob4, Ari Barzilai1, Michela Chiappalone2, Paolo Bonifazi1,4 1Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel-Aviv University, 2Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia, 3School of Electrical Engineering, Tel-Aviv University, 4School of Physics and Astronomy, Tel-Aviv University, 5Department of Informatics, Bioengineering, Robotics and System Engineering, University of Genova This manuscript describes a protocol to grow in vitro modular networks consisting of spatially confined, functionally inter-connected neuronal circuits. A polymeric mask is used to pattern a protein layer to promote cellular adhesion over the culturing substrate. Plated neurons grow on coated areas establishing spontaneous connections and exhibiting electrophysiological activity. Neuroscience Closed-loop Neuro-robotic Experiments to Test Computational Properties of Neuronal Networks Jacopo Tessadori1, Michela Chiappalone1 1Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia In this paper, an experimental framework to perform closed-loop experiments is presented, in which information processing (i.e., coding and decoding) and learning of neuronal assemblies are studied during the continuous interaction with a robotic body. Bioengineering From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope Carmine Di Rienzo1,2, Enrico Gratton3, Fabio Beltram1, Francesco Cardarelli2 1NEST Laboratory, Scuola Normale Superiore, 2Center for Nanotechnology Innovation, Instituto Italiano di Tecnologia, 3Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups. Biology Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster Pietro Laneve1,2, Angela Giangrande1 1Département de Génomique Fonctionnelle et Cancer, Institut de Génétique et de Biologie Moléculaire et Cellulaire, 2Center for Life NanoScience, Istituto Italiano di Tecnologia The aim of this publication is to visualize and discuss the operative steps of an Enhanced Northern Blot protocol on RNA extracted from Drosophila melanogaster embryos, cells, and tissues. This protocol is particularly useful for the efficient detection of small RNA species. Immunology and Infection Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry Olimpia Gamucci1, Alice Bertero1,2, Maria Ada Malvindi3, Stefania Sabella3, Pier Paolo Pompa3, Barbara Mazzolai1, Giuseppe Bardi1 1Center for Micro-BioRobotics @SSSA, Istituto Italiano di Tecnologia, 2Department of Biology, University of Pisa, 3Center for Biomolecular Nanotechnologies @UniLe, Istituto Italiano di Tecnologia Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry. Engineering Fabrication, Operation and Flow Visualization in Surface-acoustic-wave-driven Acoustic-counterflow Microfluidics Marco Travagliati1,2, Richie Shilton1, Fabio Beltram2, Marco Cecchini2 1NEST Center for Nanotechnology Innovation, Istituto Italiano di Tecnologia, 2NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR In this video we first describe fabrication and operation procedures of a surface acoustic wave (SAW) acoustic counterflow device. We then demonstrate an experimental setup that allows for both qualitative flow visualization and quantitative analysis of complex flows within the SAW pumping device. Bioengineering Measurement of Tension Release During Laser Induced Axon Lesion to Evaluate Axonal Adhesion to the Substrate at Piconewton and Millisecond Resolution Massimo Vassalli1, Michele Basso2, Francesco Difato3 1Institute of Biophysics, National Research Council of Italy, 2Dipartimento di Sistemi e Informatica, Università di Firenze, 3Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia We measured the tension release in an axon that was partially lesioned with a laser dissector by simultaneous force spectroscopy measurement performed on an optically-trapped probe adhered to the membrane of the axon. The developed experimental protocol evaluates the axon adhesion to the culture substrate. Engineering Fabrication of Nano-engineered Transparent Conducting Oxides by Pulsed Laser Deposition Paolo Gondoni1, Matteo Ghidelli1, Fabio Di Fonzo1,2, Andrea Li Bassi1,2, Carlo S. Casari1 1Department of Energy and NEMAS - Center for NanoEngineered Materials and Surfaces, Politecnico di Milano, 2Center for Nano Science and Technology, Instituto Italiano di Tecnologia We describe the experimental method to deposit nanostructured oxide thin films by nanosecond Pulsed Laser Deposition (PLD) in the presence of a background gas. By using this method Al-doped ZnO (AZO) films, from compact to hierarchically structured as nano-tree forests, can be deposited. Neuroscience Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory Neurons Anna Boccaccio1,2, Claudia Sagheddu1, Anna Menini1,3 1SISSA, International School for Advanced Studies, 2Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 3SISSA Unit, Italian Institute of Technology Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.