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Ludwig-Maximilians-Universität München

10 ARTICLES PUBLISHED IN JoVE

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Developmental Biology

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals
Dirk Steinritz 1,2, Annette Schmidt 1,3, Frank Balszuweit 1, Horst Thiermann 1, Marwa Ibrahim 3, Birgit Bölck 3, Wilhelm Bloch 3
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.

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Behavior

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila
Sara Batelli 1, Malte Kremer 1,2, Christophe Jung 1, Ulrike Gaul 1
1Gene Center and Department of Biochemistry, Ludwig-Maximilians-University Munich, 2Janelia Farm Research Campus, Howard Hughes Medical Institute

Cells display different morphologies and establish a variety of interactions with their neighbors. This protocol describes how to reveal the morphology of single cells and to investigate cell-cell interaction by using the well-established Gal4/UAS expression system.

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Biochemistry

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy
Tanja D. Becke 1,2,3, Stefan Ness 2, Stefanie Sudhop 1,3, Hermann E. Gaub 3, Markus Hilleringmann 2, 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.

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Research

Preparing an Isotopically Pure 229Th Ion Beam for Studies of 229mTh
Lars von der Wense 1, Benedict Seiferle 1, Ines Amersdorffer 1, Peter G. Thirolf 1
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.

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Biochemistry

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy
Christophe Jung 1, Max Schnepf 1, Peter Bandilla 1, Ulrich Unnerstall 1, Ulrike Gaul 1
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.

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Behavior

Investigating the Deployment of Visual Attention Before Accurate and Averaging Saccades via Eye Tracking and Assessment of Visual Sensitivity
Luca Wollenberg 1,2, Heiner Deubel 1, Martin Szinte 1,3
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.

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Medicine

Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air-Liquid Interface
Amelie Tsoutsoulopoulos 1, Katrin Gohlsch 2, Niklas Möhle 3, Andreas Breit 2, Sebastian Hoffmann 5, Olaf Krischenowski 3,4, Harald Mückter 2, Thomas Gudermann 2, Horst Thiermann 1, Michaela Aufderheide 3,4, Dirk Steinritz 1,2
1Bundeswehr Institute of Pharmacology and Toxicology, 2Walther Straub Institute of Pharmacology and Toxicology, University of Munich, 3Cultex Laboratories GmbH, 4Cultex Technologies GmbH (formerly Cultex Laboratories GmbH), 5seh consulting + services

We present a robust, transferable and predictive in vitro exposure system for the screening and monitoring of airborne particles concerning their acute pulmonary cytotoxicity by exposing cultivated human lung cells at the air-liquid interface (ALI).

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Medicine

Implantation of Combined Telemetric ECG and Blood Pressure Transmitters to Determine Spontaneous Baroreflex Sensitivity in Conscious Mice
René D. Rötzer 1, Verena F. Brox 1, Konstantin Hennis 1, Stefan B. Thalhammer 1, Martin Biel 1,2, Christian Wahl-Schott 3, Stefanie Fenske 1,2
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.

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Medicine

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model
Manuel Lasch 1,2,3, Mykhailo Vladymyrov 4, Dominic van den Heuvel 1,5, Philipp Götz 1,3, Elisabeth Deindl 1,3, Hellen Ishikawa-Ankerhold 1,5
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.

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Biochemistry

Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics
Frederik Steiert 1,2, Tamara Heermann 1, Nikolas Hundt 3, Petra Schwille 1
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.

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