Universita degli Studi di Milano 11 articles published in JoVE 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. Chemistry Applying Hyperspectral Reflectance Imaging to Investigate the Palettes and the Techniques of Painters Michele Caccia1, Simone Caglio1, Anna Galli1,2, Matteo Interlenghi2, Isabella Castiglioni2,3, Marco Martini1 1Dipartimento di Scienza dei Materiali, Università degli Studi di Milano-Bicocca, 2CNR-IBFM, 3Dipartimento di Fisica, Università degli Studi di Milano-Bicocca Hyperspectral Reflectance Imaging hypercubes include remarkable information into a large amount of data. Therefore, the request for automated protocols to manage and study the datasets is widely justified. The combination of Spectral Angle Mapper, data manipulation, and a user-adjustable analysis method constitutes a key-turn for exploring the experimental results. Developmental Biology Fluorescent Immunolocalization of Arabinogalactan Proteins and Pectins in the Cell Wall of Plant Tissues Mário Costa1,2, Ana Marta Pereira3, Sílvia Coimbra1,2 1Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, 2Laboratório Associado para a Química Verde, 3Dipartimento di Bioscienze, Universitá degli Studi di Milano This protocol describes in detail how the plant material for immunolocalization of Arabinogalactan proteins and pectins is fixed, embedded in a hydrophilic acrylic resin, sectioned and mounted on glass slides. We show cell wall related epitopes will be detected with specific antibodies. Biology A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells Georgia Pennarossa1, Sergio Ledda2, Sharon Arcuri1, Fulvio Gandolfi3, Tiziana A. L. Brevini1 1Laboratory of Biomedical Embryology, Department of Health, Animal Science and Food Safety and Center for Stem Cell Research, Università degli Studi di Milano, 2Department of Veterinary Medicine, University of Sassari, 3Department of Agricultural and Environmental Sciences - Production, Landscape, Agroenergy and Center for Stem Cell Research, Università degli Studi di Milano We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology. Developmental Biology Minimum Volume Vitrification of Immature Feline Oocytes Martina Colombo1, Gaia C. Luvoni1 1Dipartimento di Scienze Veterinarie per la Salute, la Produzione Animale e la Sicurezza Alimentare 'Carlo Cantoni', Università degli Studi di Milano This manuscript describes a protocol for the minimum volume vitrification of immature cat oocytes with laboratory-made media on commercial supports. It covers every step from oocyte isolation from ex vivo gonads to vitrification and warming. Immunology and Infection Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos Marco Cafora1,2, Francesca Forti3, Federica Briani3, Daniela Ghisotti3, Anna Pistocchi2 1Dipartimento di Scienze Cliniche e Comunità, Università degli Studi di Milano, 2Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, LITA, 3Dipartimento di Bioscienze, Università degli Studi di Milano Presented here is a protocol for Pseudomonas aeruginosa infection and phage therapy application in cystic fibrosis (CF) zebrafish embryos. 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. Developmental Biology Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients Chiara Assunta Pilato*1,2, Ilaria Stadiotti*1, Angela Serena Maione1, Valentina Saverio1, Valentina Catto3, Fabrizio Tundo3, Antonio Dello Russo3, Claudio Tondo2,3, Giulio Pompilio1,2, Michela Casella*3, Elena Sommariva*1 1Vascular Biology and Regenerative Medicine Unit, Centro Cardiologico Monzino-IRCCS, 2Department of Clinical Sciences and Community Health, Università degli Studi di Milano, 3Heart Rhythm Center, Centro Cardiologico Monzino-IRCCS In this article, a method to isolate cardiac mesenchymal stromal cells from endomyocardial bioptic samples of arrhythmogenic cardiomyopathy patients is provided. Their characterization and the protocol to boost their adipogenic differentiation are described. Bioengineering In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules Luisa Fiandra1, Serena Mazzucchelli1, Marta Truffi1, Michela Bellini2, Luca Sorrentino1, Fabio Corsi1 1Dipartimento di Scienze Biomediche e Cliniche Luigi Sacco, Università di Milano, 2Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca A method to establish an in vitro model of blood-brain barrier based on a co-culture of rat brain microvascular endothelial cells and astrocytes is described and validated. This system proved to be a valid tool to study the effect of nanoformulation on the trans-barrier permeation of fluorescent molecules. Developmental Biology Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts Tiziana A.L. Brevini1, Georgia Pennarossa1, Sara Maffei1, Alessandro Zenobi1, Fulvio Gandolfi1 1Laboratory of Biomedical Embryology, Unistem, Centre for Stem Cell Research, Università degli Studi di Milano Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications. 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.