Utrecht University 19 articles published in JoVE Biology Polycarbonate Ultracentrifuge Tube Re-Use in Proteomic Analyses of Extracellular Vesicles Rachel M. E. Cahalane*1,2, Mandy E. Turner*1, Cassandra L. Clift1, Mark C. Blaser1, Gabrielle Bogut1, Sydney Levy1, Taku Kasai1, Tom A. P. Driedonks3,4, Esther N. M. Nolte-‘t Hoen4, Masanori Aikawa1,5, Sasha A. Singh1,5, Elena Aikawa1,5 1Center for Excellence in Vascular Biology, Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School A detailed protocol is provided for cleaning and re-using polycarbonate ultracentrifuge tubes to perform extracellular vesicle isolation suitable for proteomics experiments. Biology Ultrastructural Localization of Endogenous LC3 by On-Section Correlative Light-Electron Microscopy Jan van der Beek1, Tineke Veenendaal1, Cecilia de Heus1, Suzanne van Dijk1, Corlinda ten Brink1, Nalan Liv1, Judith Klumperman1 1Center for Molecular Medicine-Cell Biology, University Medical Center Utrecht, Utrecht University Here, we present a protocol for optimized on-section correlative light-electron microscopy based on endogenous, fluorescent labeling as a tool to investigate the localization of rare proteins in relation to cellular ultrastructure. The power of this approach is demonstrated by ultrastructural localization of endogenous LC3 in starved cells without Bafilomycin treatment. Bioengineering Quantifying Cytoskeleton Dynamics Using Differential Dynamic Microscopy Hannah N. Verwei1, Gloria Lee2, Gregor Leech2, Irene Istúriz Petitjean3, Gijsje H. Koenderink3, Rae M. Robertson-Anderson2, Ryan James McGorty2 1Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, 2Department of Physics and Biophysics, University of San Diego, 3Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology Differential dynamic microscopy (DDM) combines features of dynamic light scattering and microscopy. Here, the process of using DDM to characterize reconstituted cytoskeleton networks by quantifying the subdiffusive and caged dynamics of particles in vimentin networks and the ballistic motion of active myosin-driven actin-microtubule composites is presented. Biochemistry Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans Jelle Molenkamp*1, Anna den Outer*1, Vera van Schijndel*1, Tessa Sinnige1 1Bijvoet Centre for Biomolecular Research, Utrecht University Here, a method is presented for the analysis of protein aggregation kinetics in the nematode Caenorhabditis elegans. Animals from an age-synchronized population are imaged at different time points, followed by semiautomated inclusion counting in CellProfiler and fitting to a mathematical model in AmyloFit. Neuroscience A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents Rianne Haumann*1,2, Elvin ’t Hart*2, Marc P. P. Derieppe2, Helena C. Besse3, Gertjan J. L. Kaspers1,2, Eelco Hoving2, Dannis G. van Vuurden1,2, Esther Hulleman1,2, Mario Ries3 1Amsterdam UMC, Vrije Universiteit Amsterdam, Pediatric Oncology, Cancer Center Amsterdam, 2Princess Máxima Center for Pediatric Oncology, 3Imaging Division, Utrecht University The blood-brain barrier (BBB) can be temporarily disrupted with microbubble-mediated focused ultrasound (FUS). Here, we describe a step-by-step protocol for high-throughput BBB opening in vivo using a modular FUS system accessible for non-ultrasound experts. Medicine Characterization of Sickling During Controlled Automated Deoxygenation with Oxygen Gradient Ektacytometry Minke A.E. Rab1,2, Brigitte A. van Oirschot1, Jennifer Bos1, Celeste K. Kanne3, Vivien A. Sheehan3, Eduard J. van Beers2, Richard van Wijk1 1Laboratory of Clinical Chemistry and Hematology, University Medical Center Utrecht, Utrecht University, 2Van Creveldkliniek, University Medical Center Utrecht, Utrecht University, 3Department of Pediatrics, Division of Hematology/Oncology, Baylor College of Medicine Here, we present oxygen gradient ektacytometry, a rapid and reproducible method to measure red blood cell deformability in samples from patients with sickle cell disease under controlled deoxygenation and reoxygenation. This technique provides a way to study red blood cell sickling and to monitor sickle cell disease treatment efficacy. Behavior Brain Infarct Segmentation and Registration on MRI or CT for Lesion-symptom Mapping J. Matthijs Biesbroek1, Hugo J. Kuijf2, Nick A. Weaver1, Lei Zhao3, Marco Duering4, Meta VCI Map Consortium, Geert Jan Biessels1 1Department of Neurology and Neurosurgery, UMC Utrecht Brain Center, University Medical Center Utrecht, Utrecht University, 2Image Sciences Institute, University Medical Center Utrecht, 3BrainNow Research Institute, 4Institute for Stroke and Dementia Research, University Hospital, LMU Munich Provided here is a practical tutorial for an open-access, standardized image processing pipeline for the purpose of lesion-symptom mapping. A step-by-step walkthrough is provided for each processing step, from manual infarct segmentation on CT/MRI to subsequent registration to standard space, along with practical recommendations and illustrations with exemplary cases. Biochemistry Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase Nicholas McCaul1,2, Hui Ying Yeoh1, Guus van Zadelhoff1, Naomi Lodder1, Bertrand Kleizen1, Ineke Braakman1 1Cellular Protein Chemistry, Utrecht University, 2 Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells. Bioengineering Fabrication of Decellularized Cartilage-derived Matrix Scaffolds Kim E.M. Benders1, Margo L. Terpstra1, Riccardo Levato1, Jos Malda1,2 1Department of Orthopedics, University Medical Center Utrecht, The Netherlands, 2Department of Veterinary Medicine, Equine Surgery, University of Utrecht, The Netherlands Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings. Medicine Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model Panithi Sukho1,2,3, Geesien S.A. Boersema4, Nicole Kops5, Johan F. Lange4, Jolle Kirpensteijn1,6, Jan Willem Hesselink1, Yvonne M. Bastiaansen-Jenniskens5, Femke Verseijden1,5 1Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, 2Department of Otorhinolaryngology, Erasmus MC University Medical Center, 3Department of Clinical Sciences and Public Health, Faculty of Veterinary Science, Mahidol University, 4Department of Surgery, Erasmus MC University Medical Center, 5Department of Orthopaedics, Erasmus MC University Medical Center, 6 The preparation and transplantation of adipose tissue derived stem cell (ASC) sheets onto insufficiently sutured colorectal anastomoses in a rat model is presented. This novel application shows that ASC sheets can reduce colorectal anastomosis leakage. Bioengineering Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids Yu Shrike Zhang*1, Qingmeng Pi*1,2, Anne Metje van Genderen1,3 1Division of Engineering in Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 2Department of Plastic and Reconstructive Surgery, Renji Hospital, Shanghai Jiao Tong University School of Medicine, 3Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids. Biology Live-cell Imaging of Platelet Degranulation and Secretion Under Flow Arjan D. Barendrecht*1, Johan J. F. Verhoef*2, Silvia Pignatelli1, Gerard Pasterkamp1, Harry F. G. Heijnen1, Coen Maas1 1Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, 2Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University This work describes a fluorescence microscopy-based method for the study of platelet adhesion, spreading, and secretion under flow. This versatile platform enables the investigation of platelet function for mechanistic research on thrombosis and hemostasis. Medicine Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins Loes A. Oosterhoff1, Hedwig S. Kruitwagen1, Bart Spee1, Frank G. van Steenbeek1 1Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis. Immunology and Infection A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins Ryan McBride1, James C. Paulson1, Robert P. de Vries1,2 1Department of Cell and Molecular Biology, Chemical Physiology and Microbial Science, The Scripps Research Institute, 2Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors. 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. Bioengineering Hybrid µCT-FMT imaging and image analysis Felix Gremse*1, Dennis Doleschel*1, Sara Zafarnia1, Anne Babler2, Willi Jahnen-Dechent2, Twan Lammers1,3, Wiltrud Lederle1, Fabian Kiessling1 1Experimental Molecular Imaging, RWTH Aachen University, 2Institute for Biomedical Engineering - Biointerface Laboratory, RWTH Aachen University, 3Utrecht Institute for Pharmaceutical Sciences, Utrecht University We describe a protocol for hybrid imaging, combining fluorescence-mediated tomography (FMT) with micro computed tomography (µCT). After fusion and reconstruction, we perform interactive organ segmentation to extract quantitative measurements of the fluorescence distribution. Behavior The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats Maaike Labots1,2, Hein A. Van Lith1,2, Frauke Ohl1,2, Saskia S. Arndt1,2 1Department of Animals in Science and Society, Utrecht University, 2Brain Center Rudolf Magnus This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities. Neuroscience Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins Pingan Yuanxiang1, Sujoy Bera1, Anna Karpova1, Michael R. Kreutz1, Marina Mikhaylova1,2 1RG Neuroplasticity, Leibniz Institute for Neurobiology, 2Department of Cell Biology, Utrecht University We provide a detailed protocol for induction of long-term potentiation in the CA1 region of the hippocampus and the subsequent isolation of nuclear enriched fractions from the tetanized area of the slice. This approach can be used to determine activity dependent nuclear protein import in cellular models of learning and memory. Biology Sorting of Streptomyces Cell Pellets Using a Complex Object Parametric Analyzer and Sorter Marloes L. C. Petrus1, G. Jerre van Veluw2, Han A. B. Wösten2, Dennis Claessen1 1Microbial Biotechnology, Institute Biology Leiden, Leiden University, 2Microbiology, Kluyver Centre for Genomics of Industrial Fermentation, Utrecht University Liquid-grown Streptomyces cultures are characterized by mycelial pellets that are heterogeneous in size. We here describe a method to analyze and sort such pellets in a high-throughput manner. These pellets can be used for further analyses, which will provide leads to understand and control growth heterogeneity.
