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University of Freiburg

28 ARTICLES PUBLISHED IN JoVE

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Biology

ES Cell-derived Neuroepithelial Cell Cultures
Shreeya Karki 1, Jan Pruszak 1, Ole Isacson 1, Kai C Sonntag 1
1McLean Hospital, Harvard Medical School

Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA).

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Biology

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates
Gina J. Fiala 1,2,3, Wolfgang W. A. Schamel *2,3, Britta Blumenthal *3
1Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, 2Centre for Biological Signalling Studies (bioss) and Biology III, Faculty of Biology, University of Freiburg, 3Department of Molecular Immunology, Max-Planck-Institute of Immunology and Epigenetics

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

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Biology

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations
Dominik Schnerch 1, Marie Follo 1, Julia Felthaus 1, Monika Engelhardt 1, Ralph Wäsch 1
1Department of Hematology, Oncology and Stem Cell Transplantation, University Medical Center Freiburg

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

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Neuroscience

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy
Ludovico Silvestri 1, Alessandro Bria 2,3, Irene Costantini 1, Leonardo Sacconi 1,4, Hanchuan Peng 5, Giulio Iannello 2, Francesco Saverio Pavone 1,4,6,7
1European Laboratory for Non-linear Spectroscopy (LENS), 2Integrated Research Centre, University Campus Bio-medico of Rome, 3DAEMI, University of Cassino, 4National Institute of Optics (CNR-INO), 5Allen Institute for Brain Science, 6Department of Physics, University of Florence, 7ICON Foundation, Sesto Fiorentino, Italy

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

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Neuroscience

Laser Nanosurgery of Cerebellar Axons In Vivo
Anna L. Allegra Mascaro 1, Leonardo Sacconi 1,2, Francesco Saverio Pavone 1,2,3,4
1European Laboratory for Non-Linear Spectroscopy, University of Florence, 2National Institute of Optics, National Research Council, 3Department of Physics and Astronomy, University of Florence, 4International Center for Computational Neurophotonics (ICON Foundation)

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

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Neuroscience

Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types
Vishal Menon 1, Ria Thomas 1,2, Arun R. Ghale 1,3, Christina Reinhard 1, Jan Pruszak 1,4
1Emmy Noether-Group for Stem Cell Biology, Department of Molecular Embryology, Institute of Anatomy and Cell Biology, University of Freiburg, 2Spemann Graduate School of Biology and Medicine and Faculty of Biology, University of Freiburg, 3School of Life Sciences, Keele University, 4Center for Biological Signaling Studies (BIOSS), University of Freiburg

We provide a detailed description of a protocol for flow cytometric analysis of surface antigens and/or intracellular antigens in neural cell types. Critical aspects of experimental planning, step-by-step methodological procedures, and fundamental principles of flow cytometry are explained in order to enable neurobiologists to exploit this powerful technology.

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Engineering

Investigating Single Molecule Adhesion by Atomic Force Spectroscopy
Frank W. S. Stetter 1,2, Sandra Kienle 1,2, Stefanie Krysiak 1,2, Thorsten Hugel 1,2
1Physik-Department E22a, Technische Universität München, 2IMETUM, Technische Universität München

A protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. Procedures and examples to determine the adhesion force and free energy of these molecules on solid supports and bio-interfaces are provided.

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Neuroscience

Non-invasive Assessment of Changes in Corticomotoneuronal Transmission in Humans
Wolfgang Taube 1, Christian Leukel 1,2, Jens Bo Nielsen 3,4, Jesper Lundbye-Jensen 3,4
1Department of Medicine, Movement and Sport Science, University of Fribourg (Switzerland), 2Department of Sport Science, University of Freiburg (Germany), 3Department of Neuroscience and Pharmacology, University of Copenhagen, 4Department of Nutrition, Exercise and Sports, University of Copenhagen

The aim of the present study was to assess changes in transmission at the corticomotoneuronal synapses in humans after repetitive transcranial magnetic stimulation. For this purpose, an electrophysiological method is introduced that allows assessment of pathway specific corticospinal transmission, i.e. differentiation of fast, direct corticospinal pathways from polysynaptic connections.

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Immunology and Infection

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice
Nadine Herr 1, Maximilian Mauler 1,2, Christoph Bode 1, Daniel Duerschmied 1
1Department of Cardiology and Angiology I, Heart Center, University of Freiburg, 2Faculty of Biology, University of Freiburg

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

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Behavior

Force and Position Control in Humans - The Role of Augmented Feedback
Benedikt Lauber 1,2, Martin Keller 2, Christian Leukel 1,3, Albert Gollhofer 1, Wolfgang Taube 2
1Department of Sport Science, University of Freiburg, 2Department of Medicine, Movement and Sport Science, University of Fribourg, 3Bernsteincenter Freiburg

Controlling an identical movement with position or force feedback results in different neural activation and motor behavior. This protocol describes how to investigate behavioral changes by looking at neuromuscular fatigue and how to evaluate motor cortical (inhibitory) activity using subthreshold TMS with respect to the interpretation of augmented feedback.

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Immunology and Infection

Standardized Colon Ascendens Stent Peritonitis in Rats - a Simple, Feasible Animal Model to Induce Septic Acute Kidney Injury
Natalie Burkard 1, Wolfgang Baar 2,3, Sven Flemming 1, Nicolas Schlegel 1, Jakob Wollborn 2,3, Reinhard Schneider 4, Robert W Brock 5, Christian Wunder 6, Martin Alexander Schick 2,3
1Department of General, Visceral, Transplantation, Vascular and Paediatric Surgery, Department of Surgery I, University of Würzburg, 2Department of Anesthesiology and Critical Care, Medical Center-University of Freiburg, 3Faculty of Medicine, University of Freiburg, 4Department of Internal Medicine I, Division of Nephrology, University of Würzburg, 5Department of Physiology and Pharmacology, West Virginia University School of Medicine, Robert C. Byrd Health Science Center, 6Department of Anesthesiology and Intensive Care Medicine, Robert-Bosch-Krankenhaus

Acute kidney injury (AKI) is a severe complication in critically ill patients and is related with an increased mortality. Here, we present a reliable and reproducible in vivo model to mimic AKI under inflammatory conditions that might contribute towards understanding the pathogenesis of septic AKI.

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Biology

LED Thermo Flow — Combining Optogenetics with Flow Cytometry
Kathrin Brenker 1,2,3, Kerstin Osthof 1,4, Jianying Yang 1,3, Michael Reth 1,3,5
1Max-Planck Institute for Immunobiology und Epigenetics, 2Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, 3Centre for Biological Signaling Studies, BIOSS, University of Freiburg, 4Albert-Ludwigs-Universität, 5Institute for Biology III (Mol. Immunology), Albert-Ludwigs-Universität

Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

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Bioengineering

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation
Richard Bruch *1, André Kling *1,3, Gerald A. Urban 1,2, Can Dincer 1,2
1Department of Microsystems Engineering, University of Freiburg, 2Freiburg Materials Research Center, University of Freiburg, 3Department of Biosystems Science and Engineering, ETH Zurich

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

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Analysis of Spinal Cord Blood Supply Combining Vascular Corrosion Casting and Fluorescence Microsphere Technique: A Feasibility Study in an Aortic Surgical Large Animal Model
Babak E. Saravi 1, Karin Wittmann 1, Sonja Krause 1, Luisa Puttfarcken 1, Matthias Siepe 1, Ulrich Göbel 2, Friedhelm Beyersdorf 1, Fabian A. Kari 1
1Cardiovascular Surgery, Heart Center Freiburg University, Faculty of Medicine, University of Freiburg, 2Anesthesiology and Intensive Care, University Medical Center Freiburg, Faculty of Medicine, University of Freiburg

This study combines fluorescence microsphere technique and vascular corrosion casting to simultaneously investigate spinal cord blood flow and visualize spinal cord feeding arteries in a large animal model. This model can be employed to investigate morphological vascular alterations and hemodynamic parameters in the same tissue.

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JoVE Journal

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark
Patrick Bovio 1,2, Deborah Roidl 1, Stefanie Heidrich 1, Tanja Vogel 1, Henriette Franz 1
1Institute for Anatomy and Cell Biology, Department of Molecular Embryology, Faculty of Medicine, University of Freiburg, 2Faculty of Biology, University of Freiburg

We present an effective and reproducible method to isolate and culture neural progenitor cells from embryonic and postnatal brain tissue for chromatin immunoprecipitation (ChIP) of histone 3 lysine 79 dimethylation (H3K79me2) - a histone mark located within the globular domain of histone 3.

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Biochemistry

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions
Markus Götz 1, Philipp Wortmann 1, Sonja Schmid 1,2, Thorsten Hugel 1
1Institute of Physical Chemistry, University of Freiburg, 2Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology

Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

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Neuroscience

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice
Jonas-Frederic Sauer 1, Michael Strüber 1, Marlene Bartos 1
1Institute of Physiology I, University of Freiburg

This protocol describes the recording of local field potentials with multi-shank linear silicon probes. Conversion of the signals using current source density analysis allows the reconstruction of local electrical activity in the mouse hippocampus. With this technique, spatially restricted brain oscillations can be studied in freely moving mice.

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Medicine

Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease
Louise Benning 1, Samuel Robinson 1,2, Marie Follo 3, Lukas Andreas Heger 1, Daniela Stallmann 1, Daniel Duerschmied 1, Christoph Bode 1, Ingo Ahrens 1,4, Marcus Hortmann 1
1Department of Cardiology and Angiology I, Heart Center Freiburg University, Faculty of Medicine, University of Freiburg, 2Department of Medicine, Monash University, 3Department of Medicine I, Lighthouse Core Facility, Medical Center, University of Freiburg, 4Department of Cardiology, Augustinerinnen Hospital, Academic Teaching Hospital, University of Cologne

Circulating microRNAs have shown promise as biomarkers for cardiovascular diseases and acute myocardial infarctions. In this study, we describe a protocol for miRNA extraction, reverse transcription, and digital PCR for the absolute quantification of miRNAs in the serum of patients with cardiovascular disease.

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Medicine

Electromechanical Assessment of Optogenetically Modulated Cardiomyocyte Activity
Ramona A. Kopton 1,2,3, Cinthia Buchmann 1,2, Robin Moss 1,2, Peter Kohl 1,2, Rémi Peyronnet 1,2, Franziska Schneider-Warme 1,2
1Institute for Experimental Cardiovascular Medicine, University Heart Center Freiburg-Bad Krozingen, Medical Center-University of Freiburg, 2Faculty of Medicine, University of Freiburg, 3Faculty of Biology, University of Freiburg

We present a protocol for evaluating the electromechanical effects of GtACR1 activation in rabbit cardiomyocytes. We provide detailed information on cell isolation, culturing and adenoviral transduction, and on functional experiments with the patch-clamp and carbon-fiber techniques.

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Chemistry

Covalent Attachment of Single Molecules for AFM-based Force Spectroscopy
Adrianna Kolberg 1, Christiane Wenzel 1, Thorsten Hugel 1,3, Markus Gallei 2, Bizan N. Balzer 1,3
1Institute of Physical Chemistry, Albert-Ludwigs-Universität Freiburg, 2Chair in Polymer Chemistry, Saarland University, 3Cluster of Excellence livMatS at FIT - Freiburg Center for Interactive Materials and Bioinspired Technologies, University of Freiburg

Covalent attachment of probe molecules to atomic force microscopy (AFM) cantilever tips is an essential technique for the investigation of their physical properties. This allows us to determine the stretching force, desorption force and length of polymers via AFM-based single molecule force spectroscopy with high reproducibility.

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Chemistry

Directed Assembly of Elastin-like Proteins into defined Supramolecular Structures and Cargo Encapsulation In Vitro
Andreas Schreiber *1,2, Lara G. Stühn *1,2, Süreyya E. Geissinger 1,2, Matthias C. Huber 1,2, Stefan M. Schiller 1,2,3,4,5,6
1Center for Biological Systems Analysis, University of Freiburg, 2Faculty of Biology, University of Freiburg, 3Freiburg Institute for Advanced Studies (FRIAS), University of Freiburg, 4BIOSS Centre for Biological Signalling Studies, University of Freiburg, 5IMTEK Department of Microsystems Engineering, University of Freiburg, 6Cluster of Excellence livMatS at FIT - Freiburg Center for Interactive Materials and Bioinspired Technologies, University of Freiburg

At the interface of organic and aqueous solvents, tailored amphiphilic elastin-like proteins assemble into complex supramolecular structures such as vesicles, fibers and coacervates triggered by environmental parameters. The described assembly protocols yield Protein Membrane-Based Compartments (PMBCs) with tunable properties, enabling the encapsulation of various cargo.

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Medicine

Advanced Cardiac Rhythm Management by Applying Optogenetic Multi-Site Photostimulation in Murine Hearts
Laura Diaz-Maue *1,2, Janna Steinebach *1, Michael Schwaerzle 8,9, Stefan Luther 1,3,4,6, Patrick Ruther 8,9, Claudia Richter 1,5,6,7
1Research Group Biomedical Physics, Max Planck Institute for Dynamics and Self-Organization, 2Research Electronics Department, Max Planck Institute for Dynamics and Self-Organization, 3Department of Pharmacology and Toxicology, University Medical Center Goettingen, 4Institute for Nonlinear Dynamics, Georg-August-University Goettingen, 5Department of Cardiology and Pneumology, University Medical Center Goettingen, 6German Center for Cardiovascular Research, DZHK e.V., partner site Goettingen, 7Laboratory Animal Science Unit, German Primate Center Leibniz Institute for Primate Research, 8Department of Microsystems Engineering (IMTEK), University of Freiburg, 9Cluster of Excellence BrainLinks-BrainTools, University of Freiburg

This work reports a method for controlling the cardiac rhythm of intact murine hearts of transgenic channelrhodopsin-2 (ChR2) mice using local photostimulation with a micro-LED array and simultaneous optical mapping of epicardial membrane potential.

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Medicine

Organotypic Slice Cultures as Preclinical Models of Tumor Microenvironment in Primary Pancreatic Cancer and Metastasis
Rüdiger Braun 1, Olha Lapshyna 1, Susanne Eckelmann 2,3, Kim Honselmann 1, Louisa Bolm 1, Meike ten Winkel 1, Steffen Deichmann 1, Oliver Schilling 4, Charli Kruse 2,3, Tobias Keck 1, Ulrich Wellner 1, Peter Bronsert 4,5,6, Matthias Brandenburger 3
1Department of Surgery, University Medical Center Schleswig-Holstein, 2Institute of Medical and Marine Biotechnology, University of Lübeck, 3Fraunhofer Research and Development Center for Marine and Cellular Biotechnology, 4Institute for Surgical Pathology, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, 5Tumorbank Comprehensive Cancer Center Freiburg, Medical Center - University of Freiburg, 6Core Facility Histopathology and Digital Pathology Freiburg, Medical Center - University of Freiburg

This protocol describes the preparation of organotypic slice cultures (OTSCs). This technique facilitates the ex vivo cultivation of intact multicellular tissue. OTSCs can be used immediately to test for their respective response to drugs in a multicellular environment.

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Behavior

Animal Models of Depression - Chronic Despair Model (CDM)
Stefan Vestring 1,2, Tsvetan Serchov 3,4, Claus Normann 1,5
1Department of Psychiatry and Psychotherapy, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, 2Berta-Ottenstein-Programme for Clinician Scientists, Faculty of Medicine, University of Freiburg, 3Centre National de la Recherche Scientifique (CNRS), Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, 4Department of Stereotactic and Functional Neurosurgery, Medical Center - University Freiburg, Faculty of Medicine, University of Freiburg, 5Center for Basics in Neuromodulation, Faculty of Medicine, University of Freiburg

The chronic despair mouse model (CDM) of depression consists of repetitive forced swim sessions and another delayed swim phase as a read-out. It represents a suitable model for induction of a chronic depressive-like state stable for at least 4 weeks, amendable to evaluate subchronic and acute treatment interventions.

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Biology

Mesoscopic Optical Imaging of Whole Mouse Heart
Francesco Giardini *1, Erica Lazzeri *1, Camilla Olianti *1, Giada Beconi 1, Irene Costantini 1,2, Ludovico Silvestri 1,3,4, Elisabetta Cerbai 1,5, Francesco S. Pavone 1,3,4, Leonardo Sacconi 1,3,6
1European Laboratory for Non-Linear Spectroscopy, 2Department of Biology, University of Florence, 3National Institute of Optics, National Research Council, 4Department of Physics and Astronomy, University of Florence, 5Department of Neurosciences, Psychology, Drugs and Child Health, University of Florence, 6Institute for Experimental Cardiovascular Medicine, Faculty of Medicine, University of Freiburg

We report a method for mesoscopic reconstruction of the whole mouse heart by combining new advancements in tissue transformation and staining with the development of an axially scanned light-sheet microscope.

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Biochemistry

Absolute Quantitation of Inositol Pyrophosphates by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry
Danye Qiu 1, Verena B. Eisenbeis 1, Adolfo Saiardi 2, Henning J. Jessen 1
1Institute of Organic Chemistry, University of Freiburg, 2Medical Research Council, Laboratory for Molecular Cell Biology, University College London

A procedure for capillary electrophoresis electrospray ionization mass spectrometry for the absolute quantitation of inositol pyrophosphates from mammalian cell extracts is described.

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Genetics

Isolation and Processing of Murine White Adipocytes for Transcriptome and Epigenome Analyses
Chih-Hsiang Yang 1,2, John Longinotto 2, Ilaria Panzeri 1,2, Laura Arrigoni 2, Vanessa Wegert 1, Steffen Heyne 2, Gabriel Seifert 3, Adelheid Lempradl 1,2, Ulrike Bönisch 2, John Andrew Pospisilik 1,2
1Van Andel Institute, 2Max Planck Institute of Immunobiology and Epigenetics, 3Department of General and Visceral Surgery, Medical Center-University of Freiburg, Faculty of Medicine, University of Freiburg

The present protocol summarizes a universal method for isolating, purifying, and upstream processing of murine white adipocytes optimized for downstream total RNA sequencing, Nuclei Extraction by SONication (NEXSON), and ChIP-seq.

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Medicine

Investigating Angiogenesis on a Functional and Molecular Level by Leveraging the Scratch Wound Migration Assay and the Spheroid Sprouting Assay
Julian Rapp *1,2, Jan Ness *1,3, Paula Liang 1, Martin J. Hug 4, Hansjürgen Agostini 1, Günther Schlunck 1, Clemens Lange 5, Felicitas Bucher 1
1Eye Center, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, 2Department of Medicine I, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, 3Institute of Pharmaceutical Sciences, Faculty of Chemistry and Pharmacy, University of Freiburg, 4Pharmacy, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, 5Ophtha-Lab, Department of Ophthalmology, St. Franziskus Hospital Muenster

This study presents the two-dimensional (2D) scratch wound migration assay and the three-dimensional (3D) spheroid sprouting assay, along with their respective downstream analysis methods, including RNA extraction and immunocytochemistry, as suitable assays to study angiogenesis in vitro.

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