Saarland University View Institution's Website 23 articles published in JoVE Medicine Erythrocyte Sedimentation Rate: A Physics-Driven Characterization in a Medical Context Alexis Darras1, Thomas John1, Christian Wagner1,2, Lars Kaestner1,3 1Experimental Physics, Saarland University, 2Department of Physics and Materials Science, University of Luxembourg, 3Theoretical Medicine and Biosciences, Saarland University Erythrocyte sedimentation rate (ESR) is a physical parameter, often used in routine health checks and medical diagnosis. A theoretical model that allows to extract physically-meaningful parameters from the whole sedimentation curve, based on modern colloidal knowledge, has recently been developed. Here, we present a protocol to automatically collect the ESR over time, and extract the parameters of this recent model from this automated data collection. These refined parameters are also likely to improve the medical testimony. Neuroscience Production of Human Neurogenin 2-Inducible Neurons in a Three-Dimensional Suspension Bioreactor Jeanette Wihan1, Isabell Karnatz1, Isabelle Sébastien1, Ralf Kettenhofen1, Benjamin Schmid2, Christian Clausen2, Benjamin Fischer1, Rachel Steeg3, Heiko Zimmermann1,4,5,6, Julia C. Neubauer1,4 1Fraunhofer Project Center for Stem Cell Process Engineering, Fraunhofer Institute for Biomedical Engineering IBMT, 2Bioneer A/S, 3Fraunhofer UK Research Ltd, Technology and Innovation Centre, 4Fraunhofer Institute for Biomedical Engineering IBMT, 5Department of Molecular and Cellular Biotechnology, Saarland University, 6Facultad de Ciencias del Mar, Universidad Católica del Norte This article describes a protocol for the generation of human induced pluripotent stem cell-derived neurons in a benchtop 3D suspension bioreactor. Bioengineering Metabolic Profiling to Determine Bactericidal or Bacteriostatic Effects of New Natural Products using Isothermal Microcalorimetry Katarina Cirnski*1,2, Janetta Coetzee*1,2, Jennifer Herrmann1,2, Rolf Müller1,2 1Helmholtz Institute for Pharmaceutical Research Saarland, Department of Microbial Natural Products, Helmholtz Centre for Infection Research and Department of Pharmacy, Saarland University, 2Partner Site Hannover-Braunschweig, German Centre for Infection Research (DZIF) The elucidation of the mode-of-action of a novel antibiotic is a challenging task in the drug discovery process. The goal of the method described here is the application of isothermal microcalorimetry using calScreener in antibacterial profiling to provide additional insight into drug-microbe interactions. Biology Graphene Enclosure of Chemically Fixed Mammalian Cells for Liquid-Phase Electron Microscopy Patricia Blach1,2, Sercan Keskin1, Niels de Jonge1,2 1INM-Leibniz Institute for New Materials, 2Department of Physics, Saarland University Presented here is a protocol for labeling membrane proteins in mammalian cells and coating the sample with graphene for liquid-phase scanning transmission electron microscopy. The stability of the samples against the damage caused by radiation can also studied with this protocol. Immunology and Infection P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives Carlos Victor Montefusco-Pereira*1,2, Justus C. Horstmann*1,2, Thomas Ebensen3, Christoph Beisswenger4, Robert Bals4, Carlos A. Guzmán3, Nicole Schneider-Daum1, Cristiane de Souza Carvalho-Wodarz1, Claus-Michael Lehr1,2 1Department of Drug Delivery, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), 2Department of Pharmacy, Saarland University, 3Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, 4Department of Internal Medicine V - Pulmonology, Allergology, Respiratory Intensive Care Medicine, Saarland University Hospital We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers. Chemistry Covalent Attachment of Single Molecules for AFM-based Force Spectroscopy Adrianna Kolberg1, Christiane Wenzel1, Thorsten Hugel1,3, Markus Gallei2, Bizan N. Balzer1,3 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. Medicine Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing Anouar Belkacemi1, Matthias W. Laschke2, Michael D. Menger2, Veit Flockerzi1 1Institute of Experimental and Clinical Pharmacology and Toxicology, Center for Molecular Signaling (PZMS), Saarland University, 2Institute for Clinical and Experimental Surgery, Saarland University Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing. Bioengineering Evaluation of the Storage Stability of Extracellular Vesicles Maximilian Richter1,2, Kathrin Fuhrmann1, Gregor Fuhrmann1,2 1Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Centre for Infection Research (HZI), Biogenic Nanotherapeutics group (BION), 2Department of Pharmacy, Saarland University Here we present a readily applicable protocol to assess the storage stability of extracellular vesicles, a group of naturally occurring nanoparticles produced by cells. The vesicles are loaded with glucuronidase as a model enzyme and stored under different conditions. After storage, their physicochemical parameters and the activity of the encapsulated enzyme are evaluated. Developmental Biology Thin Film Composite Silicon Elastomers for Cell Culture and Skin Applications: Manufacturing and Characterization Silviya Boyadzhieva*1,2, Sarah C.L. Fischer*1,2, Svenja Lösch1,3, Angela Rutz1, Eduard Arzt1,2, Klaus Kruttwig1 1INM - Leibniz Institute for New Materials, 2Department of Materials Science and Engineering, Saarland University, 3University of Applied Sciences Kaiserslautern A protocol for the manufacturing process of polymeric thin film composite structures possessing either different Young's moduli or thicknesses is presented. Films are produced for advanced cell culture studies or as skin adhesives. Immunology and Infection Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells Rouven Schoppmeyer1, Renping Zhao1, Markus Hoth1, Bin Qu1 1Department of Biophysics, Center for Integrative Physiology and Molecular Medicine (CIPMM), School of Medicine, Saarland University Here, we present a protocol to visualize immune cells embedded in a three-dimensional (3D) collagen matrix using light-sheet microscopy. This protocol also elaborates how to track cell migration in 3D. This protocol can be employed for other types of suspension cells in the 3D matrix. Medicine A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions Tina Histing1, Philipp Bremer1, Mika F. Rollmann1, Steven Herath1, Moritz Klein1, Tim Pohlemann1, Michael D. Menger2, Tobias Fritz1 1Department of Trauma, Hand and Reconstructive Surgery, Saarland University, 2Institute for Clinical & Experimental Surgery, Saarland University This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice. Biology A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast Björn Becker1, Manfred J. Schmitt1 1Molecular and Cell Biology, Department of Biosciences and Center of Human and Molecular Biology (ZHMB), Saarland University In the manuscript, we describe the use of a yeast-based fluorescence reporter assay to identify cellular components involved in the trafficking and killing processes of the cytotoxic A subunit of the plant toxin ricin (RTA). Bioengineering Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering Florian S. Frueh1,2, Thomas Später1, Claudia Scheuer1, Michael D. Menger1, Matthias W. Laschke1 1Institute for Clinical and Experimental Surgery, Saarland University, 2Division of Plastic Surgery and Hand Surgery, University Hospital Zurich, University of Zurich We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering. Developmental Biology Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination Henrike Julich-Haertel1, Marina Tiwari1, Christina Mehrfeld1, Elmar Krause2, Miroslaw Kornek1, Veronika Lukacs-Kornek1 1Department of Medicine II, Saarland University Medical Center, 2Department of Physiology, Center for Integrative Physiology and Molecular Medicine (CIPMM), University of Saarland Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays. Engineering Studying Dynamic Processes of Nano-sized Objects in Liquid using Scanning Transmission Electron Microscopy Justus Hermannsdörfer1, Niels de Jonge1,2 1INM-Leibniz Institute for New Materials, 2Department of Physics, University of Saarland This protocol describes the operation of a liquid flow specimen holder for scanning transmission electron microscopy of AuNPs in water, as used for the observation of nanoscale dynamic processes. Medicine An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice Tina Histing1, Michael D. Menger2, Tim Pohlemann1, Romano Matthys3, Tobias Fritz1, Patric Garcia1, Moritz Klein1 1Department of Trauma, Hand and Reconstructive Surgery, Saarland University, 2Institute for Clinical & Experimental Surgery, Saarland University, 3RISystem AG This protocol describes an osteosynthesis technique using an intramedullary locking nail for standardized fixation of femur osteotomies, which can be used to analyze normal and defective bone healing in mice. Biology Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System Emanuel G. Worst1, Matthias P. Exner2, Alessandro De Simone2, Marc Schenkelberger1, Vincent Noireaux3, Nediljko Budisa2, Albrecht Ott1 1Department of Experimental Physics, Saarland University, 2Institute of Chemistry, Technische Universität Berlin, 3School of Physics and Astronomy, University of Minnesota An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies. Engineering Experimental Methods for Investigation of Shape Memory Based Elastocaloric Cooling Processes and Model Validation Marvin Schmidt1,2, Johannes Ullrich2, André Wieczorek3, Jan Frenzel3, Gunther Eggeler3, Andreas Schütze1, Stefan Seelecke2 1Lab for Measurement Technology, Saarland University, 2Intelligent Material Systems Lab, Saarland University, 3Lab for Material Science, Ruhr Universität Bochum Experimental methods for investigation of solid state cooling processes and characterization of elastocaloric material properties of Shape Memory Alloys (SMA) are presented. A custom-built test rig has been designed for controlling and comprehensive monitoring of elastocaloric cooling processes. Furthermore, it provides a validation platform for thermomechanically coupled modeling approaches. Engineering Preparation and Friction Force Microscopy Measurements of Immiscible, Opposing Polymer Brushes Sissi de Beer1,2, Edit Kutnyanszky2, Martin H. Müser1,3, G. Julius Vancso2 1Jülich Supercomputing Centre, Forschungszentrum Jülich, 2Materials Science and Technology of Polymer, MESA+ Institute for Nanotechnology, University of Twente, 3Department of Materials Science and Engineering, Universität des Saarlandes The methodology to perform friction force microscopy experiments for contacting brushes is presented: Two polymer brushes that are grafted from (a) substrates and (b) colloidal probes are slid to show that, by using two contacting immiscible brush systems, friction in sliding contacts is reduced compared to miscible brush systems. Medicine Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia Yves Harder1, Daniel Schmauss1, Reto Wettstein2, José T. Egaña1, Fabian Weiss1, Andrea Weinzierl1, Anna Schuldt1, Hans-Günther Machens1, Michael D. Menger3, Farid Rezaeian4 1Department of Plastic Surgery and Hand Surgery, Klinikum rechts der Isar, Technische Universität München, 2Department of Plastic, Reconstructive, Aesthetic and Hand Surgery, University Hospital of Basel, 3Institute for Clinical and Experimental Surgery, University of Saarland, 4Division of Plastic and Hand Surgery, University Hospital Zurich The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses. Neuroscience Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices Christian Schauer1, Trese Leinders-Zufall1 1Department of Physiology, School of Medicine, University of Saarland, Homburg, Germany In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye. Biology Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms Sven Lang*1, Nico Schäuble*1, Adolfo Cavalié2, Richard Zimmermann1 1Medical Biochemistry and Molecular Biology, Saarland University, 2Experimental and Clinical Pharmacology and Toxicology, Saarland University The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging. Biology Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging Lars Kaestner1, Anke Scholz1, Karin Hammer1, Anne Vecerdea1, Sandra Ruppenthal1, Peter Lipp1 1Institute for Molecular Cell Biology, Universty of Saarland Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.