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University of Cologne

28 ARTICLES PUBLISHED IN JoVE

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Neuroscience

Studying the Neural Basis of Adaptive Locomotor Behavior in Insects
Matthias Gruhn 1, Philipp Rosenbaum 1, Hans-Peter Bollhagen 1, Ansgar Bueschges 1
1Zoological Institute, University of Cologne

We describe a method to record motor activity, timed to the electrically recorded tarsal contact signal in a tethered insect, walking on a slippery surface. This is used to study the neural basis of adaptive behavior under reduced influence of mechanical interaction between legs through the substrate.

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Immunology and Infection

Prediction of HIV-1 Coreceptor Usage (Tropism) by Sequence Analysis using a Genotypic Approach
Saleta Sierra 1, Rolf Kaiser 1, Nadine Lübke 1, Alexander Thielen 2, Eugen Schuelter 1, Eva Heger 1, Martin Däumer 3, Stefan Reuter 4, Stefan Esser 5, Gerd Fätkenheuer 6, Herbert Pfister 1, Mark Oette 7, Thomas Lengauer 2
1Institute of Virology, University of Cologne, 2Max Planck Institute for Informatics, 3Institute for Immune genetics, 4Department of Gastroenterology, Hepatology and Infectiology, University of Duesseldorf, 5Department of Dermatology, University of Essen, 6Department of Internal Medicine, University of Cologne, 7Augustinerinnen Hospital

The prediction of the coreceptor usage of HIV-1 is required for the administration of a new class of antiretroviral drugs, i.e. coreceptor antagonists. It can be performed by sequence analysis of the env gene and subsequent interpretation through an internet based interpretation system (geno2pheno[coreceptor]).

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Neuroscience

The Swimmeret System of Crayfish: A Practical Guide for the Dissection of the Nerve Cord and Extracellular Recordings of the Motor Pattern
Henriette A. Seichter 1, Felix Blumenthal 1, Carmen R. Smarandache-Wellmann 1
1Emmy Noether Group, Institute of Zoology, University of Cologne

Here we describe the dissection of the crayfish abdominal nerve cord. We also demonstrate an electrophysiological technique to record fictive locomotion from swimmeret motor neurons.

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Developmental Biology

Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation
Vaibhav Shinde *1, Stefanie Klima *2, Perumal Srinivasan Sureshkumar 1, Kesavan Meganathan 1, Smita Jagtap 1, Eugen Rempel 3, Jörg Rahnenführer 3, Jan Georg Hengstler 4, Tanja Waldmann 2, Jürgen Hescheler 1, Marcel Leist *2, Agapios Sachinidis *1
1Center of Physiology and Pathophysiology, Institute of Neurophysiology, University of Cologne, 2Department of Biology, University of Konstanz, 3Department of Statistics, Technical University of Dortmund, 4Leibniz Research Centre for Working Environment and Human Factors, Technical University of Dortmund

The protocols describe two in vitro developmental toxicity test systems (UKK and UKN1) based on human embryonic stem cells and transcriptome studies. The test systems predict human developmental toxicity hazard, and may contribute to reduce animal studies, costs and the time required for chemical safety testing.

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Immunology and Infection

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1
Elena Rahn 1, Katharina Thier 1, Philipp Petermann 1, Dagmar Knebel-Mörsdorf 1,2
1Center for Biochemistry, University of Cologne, 2Department of Dermatology, University of Cologne

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

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JoVE Journal

Experimental Research Examining How People Can Cope with Uncertainty Through Soft Haptic Sensations
Femke van Horen 1, Thomas Mussweiler 2
1Marketing Department, VU University, 2Social Psychology Department, University of Cologne

To date research has focused on cognitive strategies people adopt to cope with uncertainty. This research examines instead an experiential way of dealing with uncertainty and introduces a set of experimental methods showing how the experience of haptic softness can serve as a tool to deal with uncertainty.

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Medicine

Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera
Simona L. Schlereth 1, Stefan Kremers 1, Claus Cursiefen 1, Ludwig M. Heindl 1
1Department of Ophthalmology, University of Cologne

Human sclera tissue is mainly collagen; therefore, it is not easily usable for immunohistochemistry. To achieve the goal of performing immunohistochemistry for confocal microscopy of scleral tissue, a laminating technique was used.

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Cancer Research

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics
Juliane Mundorf 1, Mirka Uhlirova 1
1Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

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Biochemistry

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD
Christian Pichlo 1, Angelika A. Montada 1, Magdalena Schacherl 1, Ulrich Baumann 1
1Institute of Biochemistry, University of Cologne

Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein.

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Chemistry

Influence of Hybrid Perovskite Fabrication Methods on Film Formation, Electronic Structure, and Solar Cell Performance
Tobias Schnier 1, Jennifer Emara 1, Selina Olthof 1, Klaus Meerholz 1
1Department of Chemistry, University of Cologne

We present an extensive study on the effects of different fabrication methods for organic/inorganic perovskite thin films by comparing crystal structures, density of states, energy levels, and ultimately the solar cell performance.

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Behavior

Automatic Detection of Highly Organized Theta Oscillations in the Murine EEG
Ralf Müller 1, Anna Papazoglou 2, Julien Soos 2, Andreas Lundt 2, Carola Wormuth 2, Christina Henseler 2, Dan Ehninger 3, Karl Broich 4, Marco Weiergräber 2
1Department of Psychiatry and Psychotherapy, University of Cologne, 2Department of Neuropsychopharmacology, Federal Institute for Drugs and Medical Devices, 3Molecular and Cellular Cognition Lab, German Center for Neurodegenerative Diseases, 4Federal Institute for Drugs and Medical Devices

Theta activity in the hippocampus is related to specific cognitive and behavioral stages. Here, we describe an analytical method to detect highly-organized theta oscillations within the hippocampus using a time-frequency (i.e., wavelet analysis)-based approach.

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Developmental Biology

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders
Elke Gabriel 1, Jay Gopalakrishnan 1,2
1Center for Molecular Medicine Cologne, University of Cologne, 2Institute for Biochemistry I, Medical School of University of Cologne

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

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Preparation of Acute Myocardial Tissue Slices from Biopsies of Human Neonates and Infants with Congenital Heart Disease for Physiological Measurements
Moritz Haustein 1, Rewa Nazzal 1, Jan Trieschmann 2, Gerardus Bennink 3, Juergen Hescheler 2, Frank Pillekamp 4, Tobias Hannes 1,5
1Department of Pediatric Cardiology, University Hospital of Cologne, 2Institute for Neurophysiology, University of Cologne, 3Department of Cardio-thoracic Surgery, University Hospital of Cologne, 4Department of General Pediatrics, Neonatology and Pediatric Cardiology, University Children's Hospital, Heinrich Heine University, 5Children's Hospital for Wales, University Hospital of Wales

Cardiac slices are a unique model for cardiovascular research and bridge the gap between single-cell and whole-heart models. This protocol describes the preparation of viable cardiac slices from myocardial tissue samples excised during surgery for congenital heart disease.

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Medicine

Murine Short Axis Ventricular Heart Slices for Electrophysiological Studies
Gabriel Peinkofer 1,2, Juergen Hescheler 2, Marcel Halbach 1
1Department of Internal Medicine III, University of Cologne, 2Institute for Neurophysiology, University of Cologne

Here, we describe the preparation of viable ventricular slices from adult mice and their use for sharp electrode action potential recordings. These multicellular preparations provide a preserved in vivo like tissue structure, which makes them a valuable model for electrophysiological and pharmacological studies in vitro.

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Medicine

Transcanalicular Diode Laser-assisted Dacryocystorhinostomy for the Treatment of Primary Acquired Nasolacrimal Duct Obstruction
Joel M. Mor 1, Yongwei Guo 1, Konrad R. Koch 1, Ludwig M. Heindl 1
1Department of Ophthalmology, University of Cologne

The goal of this protocol is to present transcanalicular laser-assisted dacryocystorhinostomy as a minimally invasive approach in the treatment of primary acquired nasolacrimal duct obstruction.

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Genetics

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples
Rizwan Rehimi 1, Michaela Bartusel 1, Francesca Solinas 2, Janine Altmüller 2, Alvaro Rada-Iglesias 1,3
1Center for Molecular Medicine Cologne (CMMC), University of Cologne, 2Cologne Center for Genomics (CCG), University of Cologne, 3Cologne Excellence Cluster for Cellular Stress Responses in Aging-Associated Diseases (CECAD), Research Center and Systems Biology of Ageing Cologne, University of Cologne

Here, we describe a chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation protocol to generate global epigenomic profiles from low-abundance chicken embryonic samples.

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Immunology and Infection

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages
Saray Gutiérrez 1,2, Martina Wolke 2, Georg Plum 2, Nirmal Robinson 1,2
1Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, 2Cologne Cluster of Excellence in Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

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Immunology and Infection

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes
Katharina Dinger 1, Jasmine Mohr 1, Christina Vohlen 1,2, Dharmesh Hirani 1, Eva Hucklenbruch-Rother 2, Regina Ensenauer 3, Jörg Dötsch 2, Miguel A. Alejandre Alcazar 1,2
1Translational Experimental Pediatrics, Experimental Pulmonology, Department of Pediatrics and Adolescent Medicine, University of Cologne, 2Department of Pediatrics and Adolescent Medicine, University of Cologne, 3Division of Experimental Pediatrics and Metabolism, University Children's Hospital, Heinrich Heine University Düsseldorf

The incidence of obesity is rising and increases the risk of chronic lung diseases. To establish the underlying mechanisms and preventive strategies, well-defined animal models are needed. Here, we provide three methods (glucose-tolerance-test, body plethysmography, and lung fixation) to study the effect of obesity on pulmonary outcomes in mice.

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Biology

A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)
Joanna Dodzian 1,3, Sam Kean 1, Jens Seidel 1, Dario Riccardo Valenzano 1,2
1Max Planck Institute for the Biology of Ageing, 2CECAD, University of Cologne, 3International Institute of Molecular and Cell Biology in Warsaw

Laboratory housing of turquoise killifish can be scaled up to house and efficiently raise thousands of individual fish in a centralized water filtration system, employing the same infrastructure used for standard zebrafish facilities. Here we detail a list of standardized procedures that allow efficient killifish maintenance.

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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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Developmental Biology

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence
Cally Xiao 1,2,3, Frank Nitsche 4, Hisham Bazzi 2,3
1Graduate Program in Pharmacology and Experimental Therapeutics, University of Cologne, 2Department of Dermatology and Venereology, University Hospital of Cologne, 3Cologne Cluster of Excellence in Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, 4Department of General Ecology, Institute for Zoology, Biocenter Cologne, University of Cologne

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

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Neuroscience

Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice
Andreas Lundt 1,2, Julien Soos 1, Christina Henseler 1, Muhammad Imran Arshaad 1, Ralf Müller 3, Dan Ehninger 4, Jürgen Hescheler 5, Agapios Sachinidis 5, Karl Broich 6, Carola Wormuth 1,7, Anna Papazoglou 1, Marco Weiergräber 1
1Experimental Neuropsychopharmacology, Federal Institute for Drugs and Medical Devices, 2KBRwyle GmbH, 3Cognitive Neurophysiology, Department of Psychiatry and Psychotherapy, Faculty of Medicine, University of Cologne, 4Molecular and Cellular Cognition, German Center for Neurodegenerative Diseases (DZNE), 5Institute of Neurophysiology, Faculty of Medicine, University of Cologne, 6Federal Institute for Drugs and Medical Devices (BfArM), 7Thescon GmbH

Brainstem evoked response audiometry is an important tool in clinical neurophysiology. Nowadays, brainstem evoked response audiometry is also applied in the basic science and preclinical studies involving both pharmacological and genetic animal models. Here we provide a detailed description of how auditory brainstem responses can be successfully recorded and analyzed in mice.

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Medicine

Murine Cervical Aortic Transplantation Model using a Modified Non-Suture Cuff Technique
Martin Ryll 1, Julian Bucher 1, Moritz Drefs 1, Florian Bösch 1, K. Kumaraswami 2, Tobias Schiergens 1, Hanno Niess 1, Markus Schoenberg 1, Sven Jacob 1, Markus Rentsch 1, Markus Guba 1, Jens Werner 1, Joachim Andrassy 1, Michael N. Thomas 1,3
1Department of General, Visceral, and Transplant Surgery, Ludwig Maximilian University of Munich, 2Walter-Brendel-Centre of Experimental Medicine, Ludwig Maximilian University of Munich, 3Department of General, Visceral and Cancer Surgery, University Hospital of Cologne, University of Cologne

Here, we present a protocol of heterotopic aortic transplantation in mice using the non-suture cuff technique in a cervical murine model. This model can be used to study the underlying pathology of chronic allograft vasculopathy (CAV) and can help evaluate new therapeutic agents in order to prevent its formation.

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Immunology and Infection

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA
Marc Herb 1, Alina Farid 1, Alexander Gluschko 1, Martin Krönke 1,2,3,4, Michael Schramm 1
1Institute for Medical Microbiology, Immunology and Hygiene, Faculty of Medicine and University Hospital Cologne, University of Cologne, 2Center for Molecular Medicine Cologne, 3Cologne Cluster of Excellence on Cellular Stress Responses in Aging-associated Diseases (CECAD), 4German Center for Infection Research (DZIF)

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

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Medicine

Simultaneous Laryngopharyngeal and Conventional Esophageal pH Monitoring
Dolores T. Müller 1, Justus Toader 1, Benjamin Babic 1, Wolfgang Schröder 1, Jessica M. Leers 1, Christiane J. Bruns 1, Hans F. Fuchs 1
1Department of General, Visceral, Cancer and Transplant Surgery, University of Cologne

Laryngopharyngeal pH monitoring has been specifically designed to measure acid exposure above the upper esophageal sphincter and complements diagnostic evaluation in patients that present with mainly extraesophageal reflux symptoms. Patients with suspected laryngopharyngeal reflux (LPR) were evaluated using distal esophageal and laryngopharyngeal pH testing simultaneously.

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Biology

In Vitro Analysis of E3 Ubiquitin Ligase Function
Leonie Müller 1, Carl Elias Kutzner 1,2, Vishnu Balaji 1, Thorsten Hoppe 1,2
1Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases (CECAD), University of Cologne, 2Center for Molecular Medicine Cologne, University of Cologne

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

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Biology

Terminal H-reflex Measurements in Mice
Frederique Wieters *1, Matthias Gruhn *2, Ansgar Büschges 2, Gereon R. Fink 1,3, Markus Aswendt 1
1Faculty of Medicine and University Hospital Cologne, Department of Neurology, University of Cologne, 2Department for Animal Physiology, Institute for Zoology, Biocenter Cologne, University of Cologne, 3Cognitive Neuroscience, Institute of Neuroscience and Medicine (INM-3), Research Center Juelich

The clinical evaluation of spasticity based on the Hoffmann reflex (H-reflex) and using electrical stimulation of peripheral nerves is an established method. Here, we provide a protocol for a terminal and direct nerve stimulation for H-reflex quantification in the mouse forepaw.

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Biochemistry

Streptavidin-Affinity Grid Fabrication for Cryo-Electron Microscopy Sample Preparation
Trinity Cookis 1, Paul Sauer 2,3, Simon Poepsel 4,5, Bong-Gyoon Han 6, Dominik A. Herbst 1,2,6, Robert Glaeser 6, Eva Nogales 1,2,3,6
1Department of Molecular and Cell Biology, University of California, Berkeley, 2California Institute for Quantitative Biology (QB3), University of California, Berkeley, 3Howard Hughes Medical Institute, University of California, Berkeley, 4Center for Molecular Medicine Cologne (CMMC), Faculty of Medicine and University Hospital, University of Cologne, 5Cologne Excellence Cluster for Cellular Stress Responses in Ageing-Associated Diseases (CECAD), University of Cologne, 6Molecular Biophysics and Integrative Bio-Imaging Division, Lawrence Berkeley National Laboratory

A step-by-step protocol for fabricating streptavidin affinity grids is provided for use in structural studies of challenging macromolecular samples by cryo-electron microscopy.

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