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Istituto Italiano di Tecnologia

15 ARTICLES PUBLISHED IN JoVE

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Bioengineering

Measurement of Tension Release During Laser Induced Axon Lesion to Evaluate Axonal Adhesion to the Substrate at Piconewton and Millisecond Resolution
Massimo Vassalli 1, Michele Basso 2, Francesco Difato 3
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.

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JoVE Journal

Fabrication, Operation and Flow Visualization in Surface-acoustic-wave-driven Acoustic-counterflow Microfluidics
Marco Travagliati 1,2, Richie Shilton 1, Fabio Beltram 2, Marco Cecchini 2
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.

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Immunology and Infection

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry
Olimpia Gamucci 1, Alice Bertero 1,2, Maria Ada Malvindi 3, Stefania Sabella 3, Pier Paolo Pompa 3, Barbara Mazzolai 1, Giuseppe Bardi 1
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.

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Biology

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster
Pietro Laneve 1,2, Angela Giangrande 1
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.

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Neuroscience

Closed-loop Neuro-robotic Experiments to Test Computational Properties of Neuronal Networks
Jacopo Tessadori 1, Michela Chiappalone 1
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.

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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 Cohen 3, Miri Goldin 4, Marieteresa Tedesco 5, Yael Hanein 3, Eshel Ben-Jacob 4, Ari Barzilai 1, Michela Chiappalone 2, Paolo Bonifazi 1,4
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.

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Bioengineering

Optical Control of Living Cells Electrical Activity by Conjugated Polymers
Nicola Martino 1, Caterina Bossio 1, Susana Vaquero Morata 1, Guglielmo Lanzani 1,2, Maria Rosa Antognazza 1
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.

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Behavior

An Operant Intra-/Extra-dimensional Set-shift Task for Mice
Diego Scheggia 1, Francesco Papaleo 1
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.

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Biochemistry

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase
Elisa Romeo 1, Silvia Pontis 1, Stefano Ponzano 1, Fabiola Bonezzi 1, Marco Migliore 1, Simona Di Martino 1, Maria Summa 1, Daniele Piomelli 1,2
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.

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Education

Measuring Neuromuscular Junction Functionality
Emanuele Rizzuto 1, Simona Pisu 2, Carmine Nicoletti 2, Zaccaria Del Prete 1,3, Antonio Musarò 2,3
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.

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Neuroscience

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits
Agnes Thalhammer 1, Fanny Jaudon 1, Lorenzo A. Cingolani 1
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.

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Neuroscience

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays
Gabriella Panuccio 1, Ilaria Colombi 1, Michela Chiappalone 1,2
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.

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Medicine

Inducing and Characterizing Vesicular Steatosis in Differentiated HepaRG Cells
Silvia Di Cocco 1,2, Laura Belloni 3, Abigail D.G. Nunn 3, Debora Salerno 3, Silvia Piconese 2,4, Massimo Levrero 2,3,5,6, Natalia Pediconi 1,3
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. 

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Genetics

Engineering Intracellular Protein Sensors in Mammalian Cells
Marina Duhkinova 1, Claudia Crina 1, Ron Weiss 2, Velia Siciliano 1
1Istituto Italiano di Tecnologia, 2Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology

Here, we present a protocol for engineering genetically-encoded intracellular protein sensor-actuator(s). The device specifically detects target proteins through intracellular antibodies (intrabodies) and responds by switching on gene transcriptional output. A general framework is built to rapidly replace intrabodies, enabling rapid detection of any desired protein, without altering the general architecture.

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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 Peruzzi 3, Alfredo Nicosia 4, Antonella Folgori 5, Stefania Capone 5, Angela Santoni 2,6, Francesca Di Rosa 1
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.

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