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Utrecht University

23 ARTICLES PUBLISHED IN JoVE

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Biology

Sorting of Streptomyces Cell Pellets Using a Complex Object Parametric Analyzer and Sorter
Marloes L. C. Petrus 1, G. Jerre van Veluw 2, Han A. B. Wösten 2, Dennis Claessen 1
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.

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Neuroscience

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins
Pingan Yuanxiang 1, Sujoy Bera 1, Anna Karpova 1, Michael R. Kreutz 1, Marina Mikhaylova 1,2
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.

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Behavior

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats
Maaike Labots 1,2, Hein A. Van Lith 1,2, Frauke Ohl 1,2, Saskia S. Arndt 1,2
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.

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Bioengineering

Hybrid µCT-FMT imaging and image analysis
Felix Gremse *1, Dennis Doleschel *1, Sara Zafarnia 1, Anne Babler 2, Willi Jahnen-Dechent 2, Twan Lammers 1,3, Wiltrud Lederle 1, Fabian Kiessling 1
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.

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Medicine

Assessing Cortical Cerebral Microinfarcts on High Resolution MR Images
Susanne J. van Veluw 1, Geert Jan Biessels 1, Peter R. Luijten 2, Jaco J. M. Zwanenburg 2
1Department of Neurology, Brain Center Rudolf Magnus, University Medical Center Utrecht, 2Department of Radiology, University Medical Center Utrecht

A high resolution ex vivo 7T MR imaging protocol is presented, to perform MR-guided histopathological validation of microvascular pathology in post-mortem human brain tissue. Furthermore, guidelines are provided for the assessment of cortical microinfarcts on in vivo 7T as well as 3T MR images.

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Developmental Biology

Imaging Subcellular Structures in the Living Zebrafish Embryo
Peter Engerer 1, Gabriela Plucinska 1,2, Rachel Thong 1,3, Laura Trovò 1, Dominik Paquet 4,5,6, Leanne Godinho 1
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.

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Immunology and Infection

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins
Ryan McBride 1, James C. Paulson 1, Robert P. de Vries 1,2
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.

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Medicine

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins
Loes A. Oosterhoff 1, Hedwig S. Kruitwagen 1, Bart Spee 1, Frank G. van Steenbeek 1
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.

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Biology

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow
Arjan D. Barendrecht *1, Johan J. F. Verhoef *2, Silvia Pignatelli 1, Gerard Pasterkamp 1, Harry F. G. Heijnen 1, Coen Maas 1
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.

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Bioengineering

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids
Yu Shrike Zhang *1, Qingmeng Pi *1,2, Anne Metje van Genderen 1,3
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.

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Medicine

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model
Panithi Sukho 1,2,3, Geesien S.A. Boersema 4, Nicole Kops 5, Johan F. Lange 4, Jolle Kirpensteijn 1,6, Jan Willem Hesselink 1, Yvonne M. Bastiaansen-Jenniskens 5, Femke Verseijden 1,5
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, 6Hill's Pet Nutrition Inc.

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.

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JoVE Journal

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase
Nicholas McCaul 1,2, Hui Ying Yeoh 1, Guus van Zadelhoff 1, Naomi Lodder 1, Bertrand Kleizen 1, Ineke Braakman 1
1Cellular Protein Chemistry, Utrecht University, 2Program in Cellular and Molecular Medicine, Boston Children's Hospital

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.

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Behavior

Brain Infarct Segmentation and Registration on MRI or CT for Lesion-symptom Mapping
J. Matthijs Biesbroek 1, Hugo J. Kuijf 2, Nick A. Weaver 1, Lei Zhao 3, Marco Duering 4, , Geert Jan Biessels 1
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.

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Medicine

Characterization of Sickling During Controlled Automated Deoxygenation with Oxygen Gradient Ektacytometry
Minke A.E. Rab 1,2, Brigitte A. van Oirschot 1, Jennifer Bos 1, Celeste K. Kanne 3, Vivien A. Sheehan 3, Eduard J. van Beers 2, Richard van Wijk 1
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.

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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. Derieppe 2, Helena C. Besse 3, Gertjan J. L. Kaspers 1,2, Eelco Hoving 2, Dannis G. van Vuurden 1,2, Esther Hulleman 1,2, Mario Ries 3
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.

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Chemistry

High-Resolution Studies of Proteins in Natural Membranes by Solid-State NMR
David Beriashvili 1, Raymond D. Schellevis 1, Federico Napoli 1, Markus Weingarth 1, Marc Baldus 1
1NMR Spectroscopy, Bijvoet Centre for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University

This work details robust basic routines on how to prepare isotope-labeled membrane protein samples and analyze them at high-resolution with modern solid-state NMR spectroscopy methods.

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Cancer Research

Medium-Throughput Drug- and Radiotherapy Screening Assay using Patient-Derived Organoids
Marrit Putker *1, Rosemary Millen *2, René Overmeer *3, Else Driehuis 2,4, Maurice M. J. M. Zandvliet 5, Hans Clevers 2, Sylvia F. Boj 3, Qi-Xiang Li 6
1Crown Bioscience Netherlands B.V., 2Oncode Institute, Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and University Medical Center Utrecht, 3Hubrecht Organoid Technology (HUB), 4Pathology dept, University Medical Center Utrecht, 5Department of Clinical Sciences - Companion Animals, Faculty of Veterinary Medicine, Utrecht University, 6Crown Bioscience Inc.

We describe detailed protocols to use patient-derived organoids for medium-throughput therapy sensitivity screenings. Therapies tested include chemotherapy, radiotherapy, and chemo-radiotherapy. Adenosine triphosphate levels are used as a functional readout.

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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 Sinnige 1
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.

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Bioengineering

Quantifying Cytoskeleton Dynamics Using Differential Dynamic Microscopy
Hannah N. Verwei 1, Gloria Lee 2, Gregor Leech 2, Irene Istúriz Petitjean 3, Gijsje H. Koenderink 3, Rae M. Robertson-Anderson 2, Ryan James McGorty 2
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.

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Biology

Ultrastructural Localization of Endogenous LC3 by On-Section Correlative Light-Electron Microscopy
Jan van der Beek 1, Tineke Veenendaal 1, Cecilia de Heus 1, Suzanne van Dijk 1, Corlinda ten Brink 1, Nalan Liv 1, Judith Klumperman 1
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.

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Biology

Surgical Technique of the 3-Dimensional-printed Personalized Hip Implant for the Treatment of Canine Hip Dysplasia
Irin Kwananocha 1,2, Femke Verseijden 1, Seyed A Kamali 1, Joëll Magré 3, Koen Willemsen 3, Jacobine CM Schouten 4, Daniela Salvatori 4, Marianna A Tryfonidou 1, Björn P Meij 1
1Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, 2Research and Academic Service, Faculty of Veterinary Medicine, Kasetsart University, 3Department of Orthopedics, University Medical Center Utrecht, 4Department of Clinical Sciences, Anatomy and Physiology, Faculty of Veterinary Medicine, Utrecht University

This work describes a novel surgical technique for extracapsular implantation of a personalized, 3-dimensional-printed, joint-preserving implant. This novel treatment aims to restore hip stability in young adult dogs suffering from hip dysplasia with laxity by uniquely reproducing the anatomical shape of the acetabular rim of the hip joint.

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Biology

Polycarbonate Ultracentrifuge Tube Re-Use in Proteomic Analyses of Extracellular Vesicles
Rachel M. E. Cahalane *1,2, Mandy E. Turner *1, Cassandra L. Clift 1, Mark C. Blaser 1, Gabrielle Bogut 1, Sydney Levy 1, Taku Kasai 1, Tom A. P. Driedonks 3,4, Esther N. M. Nolte-‘t Hoen 4, Masanori Aikawa 1,5, Sasha A. Singh 1,5, Elena Aikawa 1,5
1Center for Interdisciplinary Cardiovascular Sciences, Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, 2Mechanobiology and Medical Device Research Group (MMDRG), Biomedical Engineering, College of Science and Engineering, University of Galway, 3Department of CDL Research, University Medical Center Utrecht, 4Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, 5Center 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.

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Biology

High Throughput Image-Based Phenotyping for Determining Morphological and Physiological Responses to Single and Combined Stresses in Potato
Lamis Osama Anwar Abdelhakim 1, Barbora Pleskačová 1, Natalia Yaneth Rodriguez-Granados 2, Rashmi Sasidharan 2, Lucia Sandra Perez-Borroto 3, Sophia Sonnewald 4, Kristina Gruden 5, Ute C. Vothknecht 6, Markus Teige 7, Klára Panzarová 1
1PSI (Photon Systems Instruments), spol. s r.o. Drasov, 2Plant Stress Resilience, Institute of Environmental Biology, Utrecht University, 3Plant Breeding, Wageningen University and Research, 4Department of Biology, Biochemistry, Friedrich-Alexander Universität Erlangen-Nürnberg, 5Department of Biotechnology and Systems Biology, National Institute of Biology, 6Plant Cell Biology, Institute of Cellular and Molecular Botany, University of Bonn, 7Department of Functional & Evolutionary Ecology, University of Vienna

We designed an image-based phenotyping protocol to determine the morphological and physiological responses to single and combined heat, drought, and waterlogging treatments. This approach enabled the identification of early, late, and recovery responses at a whole plant level, particularly above-ground parts, and highlighted the necessity of using multiple imaging sensors.

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