Universidad Autonoma de Madrid 11 articles published in JoVE Biochemistry Cryo-EM and Single-Particle Analysis with Scipion A. Jiménez-Moreno1, L. del Caño1, M. Martínez1, E. Ramírez-Aportela1, A. Cuervo1, R. Melero1, R. Sánchez-García1, D. Strelak1,2,3, E. Fernández-Giménez1, F.P. de Isidro-Gómez1, D. Herreros1, P. Conesa1, Y. Fonseca1, D. Maluenda1, J. Jiménez de la Morena1, J.R. Macías1, P. Losana1, R. Marabini1, J.M. Carazo1, C.O.S. Sorzano1,4 1Centro Nacional de Biotecnología, Campus Universidad Autónoma de Madrid, 2Faculty of Informatics, Masaryk University, 3Institute of Computer Science, Masaryk University, 4Campus Urbanización Montepríncipe, Universidad San Pablo CEU Single-particle analysis in cryo-electron microscopy is one of the main techniques used to determine the structure of biological ensembles at high resolution. Scipion provides the tools to create the whole pipeline to process the information acquired by the microscope and achieve a 3D reconstruction of the biological specimen. Neuroscience Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration María Portela-Lomba1,3, Diana Simón1, Cristina Russo2, Javier Sierra1, María Teresa Moreno-Flores3 1Facultad de CC Experimentales, Universidad Francisco de Vitoria, 2Dept. Biomedical and Biotechnological Sciences, Section of Physiology, University of Catania, 3Dept. Anatomía, Histología y Neurociencia, Facultad de Medicina, Universidad Autónoma de Madrid We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers. Biology Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In Situ Jesús Espada1,2, Elisa Carrasco3, María I. Calvo-Sánchez1,4, Sandra Fernández-Martos1, Juan José Montoya5 1Ramón y Cajal Institute for Health Research (IRYCIS), Ramón y Cajal University Hospital, 2 The aim of this protocol is to induce transient in vivo production of nonlethal levels of reactive oxygen species (ROS) in mouse skin, further promoting physiological responses in the tissue. Immunology and Infection Imaging the Human Immunological Synapse Ana Bello-Gamboa*1,2, Juan Manuel Izquierdo1,2, Marta Velasco1,2, Solange Moreno1,2, Alejandro Garrido1,2, Laura Meyers1,2, Juan Carlos Palomino3, Víctor Calvo1,2, Manuel Izquierdo*1,2 1Instituto de Investigaciones Biomédicas Alberto Sols, CSIC-Universidad Autónoma de Madrid, 2Departamento de Bioquímica, Instituto de Investigaciones Biomédicas Alberto Sols CSIC-UAM, Facultad de Medicina, Universidad Autónoma de Madrid, 3Unidad de Audiovisuales, Facultad de Medicina, Universidad Autónoma de Madrid This protocol images both the immunological synapse formation and the subsequent polarized secretory traffic towards the immunological synapse. Cellular conjugates were formed between a superantigen-pulsed Raji cell (acting as an antigen-presenting cell) and a Jurkat clone (acting as an effector helper T lymphocyte). Immunology and Infection Rescue of Recombinant Zika Virus from a Bacterial Artificial Chromosome cDNA Clone Ginés Ávila-Pérez1, Jun-Gyu Park1, Aitor Nogales1, Fernando Almazán2, Luis Martínez-Sobrido1 1Department of Microbiology and Immunology, University of Rochester Medical Center, 2Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid The recent epidemic of Zika virus highlights the importance of establishing reverse genetic approaches to develop vaccines and/or therapeutic strategies. Here, we describe the protocol to rescue an infectious recombinant Zika virus from a full-length cDNA clone assembled in a bacterial artificial chromosome under the control of the human cytomegalovirus immediate-early promoter. Immunology and Infection Image Processing Protocol for the Analysis of the Diffusion and Cluster Size of Membrane Receptors by Fluorescence Microscopy Carlos Oscar S. Sorzano*1,2, Laura Martínez-Muñoz*3,4, Graciela Cascio3,5, Eva M. García-Cuesta3, J. Vargas1,6, Mario Mellado3, Jose Miguel Rodriguez Frade3 1Department of Macromolecular Structures, Centro Nacional de Biotecnología, Campus Univ. Autónoma de Madrid, 2Campus Urb. Montepríncipe s/n, Univ. San Pablo CEU, 3Department of Immunology and Oncology, Centro Nacional de Biotecnología, Campus Univ. Autónoma de Madrid, 4Department of Cell Signaling, Centro Andaluz de Biología Molecular y Medicina Regenerativa (CSIC), 5Meyer Cancer Center, 6Department of Anatomy and Cell Biology, McGill Univ. Here, we present a protocol for single particle tracking image analysis that allows quantitative evaluation of diffusion coefficients, types of motion and cluster sizes of single particles detected by fluorescence microscopy. Environment BtM, a Low-cost Open-source Datalogger to Estimate the Water Content of Nonvascular Cryptogams María Leo*1, Angel Lareo*2, Carlos Garcia-Saura2, Joaquín Hortal3,4, Nagore G. Medina5 1Real Jardín Botánico (CSIC-RJB), 2Grupo de Neurocomputación Biológica, Dpto. de Ingeniería Informática, Escuela Politécnica Superior, Universidad Autónoma de Madrid, 3Department of Biogeography and Global Change, Museo Nacional de Ciencias Naturales (MNCN-CSIC), 4Centre for Ecology, Evolution and Environmental Changes (cE3c), Faculdade de Ciências da Universidade de Lisboa, 5Department of Botany, Faculty of Science, University of South Bohemia We present a simple and cost-effective method to build an open-source datalogger that measures the conductance of nonvascular cryptogams together with the environmental temperature and humidity. We describe the hardware design of the datalogger and provide step-by-step assembly instructions, the list of required open-source logging software, the code to run the datalogger, and a calibration protocol. Chemistry Microfluidic-based Synthesis of Covalent Organic Frameworks (COFs): A Tool for Continuous Production of COF Fibers and Direct Printing on a Surface Afshin Abrishamkar1, David Rodríguez-San-Miguel2, Jorge Andrés Rodríguez Navarro3, Romen Rodriguez-Trujillo4, David B. Amabilino5, Ruben Mas-Ballesté2, Félix Zamora2,6,7, Andrew J. deMello1, Josep Puigmarti-Luis1 1Institute of Chemical and Bioengineering, Department of Chemistry and Applied Bioscience, ETH Zurich, 2Departamento de Química Inorgánica, Universidad Autónoma de Madrid, 3Departamento de Química Inorgánica, Universidad de Granada, 4Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), 5School of Chemistry, University of Nottingham, 6Condensed Matter Physics Center (IFMAC), Universidad Autónoma de Madrid, 7Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA Nanociencia) We present a novel microfluidic-based method for synthesis of covalent organic frameworks (COFs). We demonstrate how this approach can be used to produce continuous COF fibers, and also 2D or 3D COF structures on surfaces. Neuroscience Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology Anna Castells-Nobau*1, Bonnie Nijhof*1, Ilse Eidhof1, Louis Wolf2, Jolanda M. Scheffer-de Gooyert1, Ignacio Monedero3,4, Laura Torroja3, Jeroen A.W.M. van der Laak2,5, Annette Schenck*1 1Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, 2Microscopical Imaging Centre (MIC), Radboud University Medical Center, 3Department of Biology, Universidad Autónoma de Madrid, 4Department of Clinical and Experimental Medicine, Linköping University, 5Department of Pathology, Radboud University Medical Center Two image analysis algorithms, "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics" were created, to automatically quantify nine morphological features of the Drosophila neuromuscular junction (NMJ). Immunology and Infection Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry Alfonso Rubio-Navarro1, Melania Guerrero-Hue1, Beatriz Martín-Fernandez2, Isabel Cortegano3, Elena Olivares-Alvaro2, Natalia de las Heras2, Mario Alía3, Belén de Andrés3, María Luisa Gaspar3, Jesús Egido1, Juan Antonio Moreno1 1Renal, Vascular and Diabetes Research Lab, IIS-Fundaciòn Jiménez Dìaz, Autonoma University, 2Department of Physiology, Faculty of Medicine, Complutense University, 3Department of Immunology, Centro Nacional de Microbiologìa, Instituto de Salud Carlos III (ISCIII) This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model. Chemistry Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography Samantha M. Desmarais1, Felipe Cava2, Miguel A. de Pedro3, Kerwyn Casey Huang1,4 1Department of Bioengineering, Stanford University, 2Department of Molecular Biology and Laboratory for Molecular Infection Medicine Sweden, Umeå Centre for Microbial Research, Umeå University, 3Campus de Cantoblanco, Universidad Autonoma de Madrid, 4Department of Microbiology and Immunology, Stanford University School of Medicine The bacterial cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by peptides. Ultra Performance Liquid Chromatography provides high resolution and throughput for novel discoveries of peptidoglycan composition. We present a procedure for the isolation of cell walls (sacculi) and their subsequent preparation for analysis via UPLC.
