Ludwig-Maximilians-Universitat-Munchen View Institution's Website 26 articles published in JoVE Biochemistry Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics Frederik Steiert1,2, Tamara Heermann1, Nikolas Hundt3, Petra Schwille1 1Department of Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, 2Department of Physics, Technical University Munich, 3Department of Cellular Physiology, Biomedical Center (BMC), Ludwig-Maximilians-Universität München This protocol describes an iSCAT-based image processing and single-particle tracking approach that enables the simultaneous investigation of the molecular mass and the diffusive behavior of macromolecules interacting with lipid membranes. Step-by-step instructions for sample preparation, mass-to-contrast conversion, movie acquisition, and post-processing are provided alongside directions to prevent potential pitfalls. Medicine Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model Manuel Lasch1,2,3, Mykhailo Vladymyrov4, Dominic van den Heuvel1,5, Philipp Götz1,3, Elisabeth Deindl1,3, Hellen Ishikawa-Ankerhold1,5 1Walter-Brendel-Centre of Experimental Medicine, University Hospital, Ludwig-Maximilians-Universität München, 2Department of Otorhinolaryngology, Head & Neck Surgery, University Hospital, Ludwig-Maximilians-Universität München, 3Biomedical Center, Institute of Cardiovascular Physiology and Pathophysiology, Faculty of Medicine, Ludwig-Maximilians-Universität München, 4TKI, University of Bern, 5Department of Internal Medicine I and Cardiology, University Hospital, Ludwig-Maximilians-Universität München The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis. Biology Live-cell Imaging of Single-Cell Arrays (LISCA) - a Versatile Technique to Quantify Cellular Kinetics Anita Reiser1, Daniel Woschée1, Simon Maximilian Kempe1, Joachim Oskar Rädler1 1Faculty of Physics and Center for NanoScience (CeNS), Ludwig-Maximilians-Universität München, Geschwister-Scholl-Platz 1, 80539 München, Germany We present a method for the acquisition of fluorescence reporter time courses from single cells using micropatterned arrays. The protocol describes the preparation of single-cell arrays, the setup and operation of live-cell scanning time-lapse microscopy and an open-source image analysis tool for automated preselection, visual control and tracking of cell-integrated fluorescence time courses per adhesion site. Medicine Implantation of Combined Telemetric ECG and Blood Pressure Transmitters to Determine Spontaneous Baroreflex Sensitivity in Conscious Mice René D. Rötzer1, Verena F. Brox1, Konstantin Hennis1, Stefan B. Thalhammer1, Martin Biel1,2, Christian Wahl-Schott3, Stefanie Fenske1,2 1Center for Integrated Protein Science (CIPS-M) and Center for Drug Research, Department of Pharmacy, Ludwig-Maximilians-Universität München, 2German Center for Cardiovascular Research (DZHK), Partner Site Munich Heart Alliance, 3Hannover Medical School, Institute for Neurophysiology The baroreflex is a heart-rate regulation mechanism by the autonomic nervous system in response to blood-pressure changes. We describe a surgical technique to implant telemetry transmitters for continuous and simultaneous measurement of electrocardiogram and blood pressure in mice. This can determine spontaneous baroreflex sensitivity, an important prognostic marker for cardiovascular disease. Immunology and Infection Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow Hilger Jagau1,2, Ina-Kristin Behrens1,3, Michael Steinert1,4, Simone Bergmann1 1Institut für Mikrobiologie, Technische Universität Braunschweig, 2Devision of Infection Medicine, Department of Clinical Science, Lund University, 3Medical Microbiology and Hospital Epidemiology, Max von Pettenkofer Institute, Ludwig Maximilians University, 4Department of Molecular Infection Biology, Helmholtz Center for Infection Research This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures. Genetics Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches Shao-Yen Kao*1, Elena Nikonova*1, Keshika Ravichandran*1, Maria L. Spletter1,2 1Biomedical Center, Department of Physiological Chemistry, Ludwig-Maximilians-University Munich, 2Center for Integrated Protein Science Munich (CIPSM), Department of Chemistry, Ludwig-Maximilians-University Munich Drosophila flight muscle is a powerful model to study transcriptional regulation, alternative splicing, metabolism, and mechanobiology. We present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched samples ideal for proteomics and deep-sequencing. These samples can offer important mechanistic insights into diverse aspects of muscle development. Behavior Longitudinal Two-Photon Imaging of Dorsal Hippocampal CA1 in Live Mice Alessandro F. Ulivi1, Tim P. Castello-Waldow1, Ghabiba Weston1,2, Long Yan3, Ryohei Yasuda3, Alon Chen1,4, Alessio Attardo1 1Dept. of Stress Neurobiology and Neurogenetics, Max Planck Institute of Psychiatry, 2Graduate School of Systemic Neurosciences, Ludwig Maximilians University, 3Max Planck Florida Institute for Neuroscience, 4Dept. of Neurobiology, Weizmann Institute of Science This method describes a chronic preparation that allows optical access to the hippocampus of living mice. This preparation can be used to perform longitudinal optical imaging of neuronal structural plasticity and activity-evoked cellular plasticity over a period of several weeks. Engineering Preparing an Isotopically Pure 229Th Ion Beam for Studies of 229mTh Lars von der Wense1, Benedict Seiferle1, Ines Amersdorffer1, Peter G. Thirolf1 1Faculty of Physics, Ludwig-Maximilians-Universität München We present a protocol for the generation of an isotopically purified low-energy 229Th ion beam from a 233U source. This ion beam is used for the direct detection of the 229mTh ground-state decay via the internal-conversion decay channel. We also measure the internal conversion lifetime of 229mTh as well. Behavior Investigating the Deployment of Visual Attention Before Accurate and Averaging Saccades via Eye Tracking and Assessment of Visual Sensitivity Luca Wollenberg1,2, Heiner Deubel1, Martin Szinte1,3 1Allgemeine und Experimentelle Psychologie, Ludwig-Maximilians-Universität München, 2Graduate School of Systemic Neurosciences, Ludwig-Maximilians-Universität München, 3Department of Cognitive Psychology, Vrije Universiteit Amsterdam This experimental protocol combined eye tracking and the assessment of presaccadic visual sensitivity in a dual task paradigm, consisting of a free choice saccade task and a visual discrimination task, to investigate the deployment of visual spatial attention before both, accurate and averaging saccades. Medicine Generation of Human 3D Lung Tissue Cultures (3D-LTCs) for Disease Modeling Michael Gerckens1,2,3, Hani N. Alsafadi4,5,6, Darcy E. Wagner4,5,6, Michael Lindner2,7, Gerald Burgstaller*1,2,3, Melanie Königshoff*1,2,3,8 1Comprehensive Pneumology Center, Ludwig-Maximilians-Universität and Helmholtz Zentrum Munich, 2German Center of Lung Research (DZL), 3Translational Lung Research and CPC-M bioArchive, Helmholtz Zentrum München, Comprehensive Pneumology Center Munich DZL/CPC-M, 4Department of Experimental Medical Science, Lung Bioengineering and Regeneration, Lund University, 5Wallenberg Center for Molecular Medicine, Lund University, 6Stem Cell Centre, Lund University, 7Asklepios Fachkliniken Munich-Gauting, 8Department of Medicine, Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Here, we present a protocol for the preparation of agarose-filled human precision-cut lung slices from resected patient tissue that are suitable for generating 3D lung tissue cultures to model human lung diseases in biological and biomedical studies. Genetics Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins Isabell-Christin Fiedler1, Arne Weiberg2, Karina van der Linde1 1Department of Biology, Regensburg University, 2Department of Biology, Ludwig-Maximilians University of Munich This work describes the cloning of an Ustilago maydis Trojan horse strain for the in situ delivery of secreted maize proteins into three different types of maize tissues. Biochemistry High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy Christophe Jung1, Max Schnepf1, Peter Bandilla1, Ulrich Unnerstall1, Ulrike Gaul1 1Gene Center and Department of Biochemistry, Center for Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system. Biochemistry Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments Louisa Jutta Dietz1, Anuroop Venkateswaran Venkatasubramani1, Annika Müller-Eigner2, Martin Hrabe de Angelis3,4,5, Axel Imhof1, Lore Becker3, Shahaf Peleg2,6,7 1Munich Center of Integrated Protein Science and Biomedical Center, Ludwig-Maximilians University of Munich, 2Laboratory for Metabolism and Epigenetics in Aging, Leibniz Institute for Farm Animal Biology (FBN), 3German Mouse Clinic, Helmholtz Zentrum Munich, German Research Center for Environment and Health (GmbH), 4German Center for Diabetes Research (DZD), 5Chair of Experimental Genetics, School of Life Science Weihenstephan, Technische Universität München, 6Laboratory for Metabolism and Epigenetics in Brain Aging, Institute of Neuroregeneration & Neurorehabilitation of Qingdao University, 7Molecular Biology Division, Biomedical Center, Faculty of Medicine, Ludwig-Maximilians University of Munich Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity. Biochemistry Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy Tanja D. Becke1,2,3, Stefan Ness2, Stefanie Sudhop1,3, Hermann E. Gaub3, Markus Hilleringmann2, Arndt F. Schilling*4, Hauke Clausen-Schaumann*1,3 1Center for Applied Tissue Engineering and Regenerative Medicine, Munich University of Applied Sciences, 2FG Protein Biochemistry & Cellular Microbiology, Munich University of Applied Sciences, 3Center for Nano Science, Ludwig-Maximilians-Universität München, 4Klinik für Unfallchirurgie, Orthopädie und Plastische Chirurgie, University Medical Center Göttingen This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin. Genetics Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation Kathrin Davari*1, Johannes Lichti*1, Caroline C. Friedel2, Elke Glasmacher1,3 1Institute for Diabetes and Obesity (IDO), German Center for Diabetes Research (DZD), Helmholtz Zentrum München, 2Institute for Informatics, Ludwig-Maximilians-Universität München, 3Roche Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Penzberg This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells. Biology Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities Bettina Bölter1, Franka Seiler1, Jürgen Soll1 1Department Biologie I-Botanik, Ludwig-Maximilians-Universität Here, we present a protocol for studying plant growth behavior and especially phenotypes in a reproducible manner. We show how to provide variable and at the same time stable light conditions. Proper analyses depend on sufficient sample numbers and valid statistical evaluations. Neuroscience Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations Rosa Gómez-Villafuertes*1,2,3, Lucía Paniagua-Herranz*1,2,3, Sergio Gascon*4,5, David de Agustín-Durán1,2,3, María de la O Ferreras1,2,3, Juan Carlos Gil-Redondo1,2,3, María José Queipo1,2,3, Aida Menendez-Mendez1,2,3, Ráquel Pérez-Sen1,2,3, Esmerilda G. Delicado1,2,3, Javier Gualix1,2,3, Marcos R. Costa6, Timm Schroeder7, María Teresa Miras-Portugal1,2,3, Felipe Ortega1,2,3 1Biochemistry and Molecular Biology Department, Faculty of Veterinary medicine, Complutense University, 2University Institute for Neurochemistry Research (IUIN), 3Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), 4Institute of Stem Cell Research, Helmholtz Center Munich, Neuherberg/Munich, Germany Physiological Genomics, Biomedical Center, Ludwig-Maximilians University Munich, 5Toxicology and Pharmacology Department, Faculty of Veterinary medicine, Complutense University, 6Brain Institute, Federal University of Rio Grande do Norte, 7Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule (ETH) Zurich A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments. Medicine Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood Riccardo Sfriso1,2, Anjan Bongoni3, Yara Banz4, Nikolai Klymiuk5, Eckhard Wolf5, Robert Rieben1 1Department of Clinical Research, University of Bern, 2Graduate School for Cellular and Biomedical Sciences, University of Bern, 3 We present an in vitro model which allows the study and analysis of coagulation in whole, non-anticoagulated blood. Anticoagulation in the system depends on the natural anticoagulation effect of healthy endothelial cells and endothelial cell activation will result in clotting. Bioengineering 20 mJ, 1 ps Yb:YAG Thin-disk Regenerative Amplifier Ayman Alismail1,2, Haochuan Wang1,3, Jonathan Brons3, Hanieh Fattahi1,3 1Department of Physics, Ludwig Maximilian University of Munich, 2Physics and Astronomy Department, King Saud University, 3Max Planck Institute of Quantum Optics A protocol for the operation of a high-energy, high-power optical parametric chirped pulse amplifier pump source based on an Yb:YAG thin-disk regenerative amplifier is presented here. Developmental Biology Imaging Subcellular Structures in the Living Zebrafish Embryo Peter Engerer1, Gabriela Plucinska1,2, Rachel Thong1,3, Laura Trovò1, Dominik Paquet4,5,6, Leanne Godinho1 1Institute of Neuronal Cell Biology, Technische Universität München, 2Cell Biology, Department of Biology, Faculty of Science, Utrecht University, 3Faculty of Biology, Ludwig-Maximilians-Universität-München, 4Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-Universität-München, 5German Center for Neurodegenerative Diseases, 6Laboratory of Brain Development and Repair, The Rockefeller University Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy. Developmental Biology Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals Dirk Steinritz1,2, Annette Schmidt1,3, Frank Balszuweit1, Horst Thiermann1, Marwa Ibrahim3, Birgit Bölck3, Wilhelm Bloch3 1Bundeswehr Institute of Pharmacology and Toxicology, 2Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität München, 3Department of Molecular and Cellular Sports Medicine, German Sports University Cologne Investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of certain illnesses and to potentially identify novel strategies for therapeutic intervention. The following protocol describes techniques to assess cell migration that have been adapted for the investigation of EEC. Engineering Analyzing the Movement of the Nauplius 'Artemia salina' by Optical Tracking of Plasmonic Nanoparticles Silke R. Kirchner1, Michael Fedoruk1, Theobald Lohmüller1, Jochen Feldmann1 1Photonics and Optoelectronics Group, Ludwig-Maximilians-Universität We use optical tracking of plasmonic nanoparticles to probe and characterize the frequency movements of aquatic organisms. Biology Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves Regina Schweiger1, Serena Schwenkert1 1Department Biologie I, Botanik, Ludwig-Maximilians-Universität, München Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins. Bioengineering Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy Markus A. Jobst*1, Constantin Schoeler*1, Klara Malinowska1, Michael A. Nash1 1Lehrstuhl für Angewandte Physik and Center for Nanoscience, Ludwig-Maximilians-Universität Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly. Biology Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED) Samira Samtleben1, Juliane Jaepel1,2, Caroline Fecher1, Thomas Andreska1, Markus Rehberg3, Robert Blum1 1Institute for Clinical Neurobiology, University of Wuerzburg, 2Department of Synapses - Circuits - Plasticity, Max Planck Institute of Neurobiology, Martinsried, 3Walter Brendel Centre of Experimental Medicine, Ludwig-Maximilians University of Munich Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen. Immunology and Infection Purification of Pathogen Vacuoles from Legionella-infected Phagocytes Christine Hoffmann1, Ivo Finsel1, Hubert Hilbi1 1Max von Pettenkofer-Institut, Ludwig-Maximilians-Universität This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.