Ghent University 23 articles published in JoVE Cancer Research The Micronucleus Assay on Cryopreserved Whole Blood Renu Dayal1, Elien Beyls1, Anne Vral1, Ans Baeyens1 1Radiobiology group, Department of Human Structure and Repair, Ghent University Here we present an optimized protocol for the cytokinesis-block micronucleus assay on cryopreserved whole blood samples. This optimized method of cryopreservation of whole blood for micronucleus analysis is a reliable technique for large-scale sampling and multi-center studies and can be used for other blood-related assays as well. Neuroscience Microdissection and Whole Mount Scanning Electron Microscopy Visualization of Mouse Choroid Plexus Elien Van Wonterghem*1,2, Lien Van Hoecke*1,2, Griet Van Imschoot1,2, Daan Verhaege1,2, Marlies Burgelman1,2, Roosmarijn E. Vandenbroucke1,2 1VIB Center for Inflammation Research, VIB, 2Department of Biomedical Molecular Biology, Ghent University The choroid plexus (CP), an understudied tissue in neuroscience, plays a key role in health and disease of the central nervous system. This protocol describes a microdissection technique for isolating the CP and the use of scanning electron microscopy to obtain an overall view of its cellular structure. Immunology and Infection Tyramide Signal Amplification for the Immunofluorescent Staining of ZBP1-Dependent Phosphorylation of RIPK3 and MLKL After HSV-1 Infection in Human Cells Josephine Nemegeer1,2, Kelly Lemeire1,2, Peter Vandenabeele1,2, Jonathan Maelfait1,2 1VIB-UGent Center for Inflammation Research, 2Department of Biomedical Molecular Biology, Ghent University Tyramide signal amplification during immunofluorescent staining enables the sensitive detection of phosphorylated RIPK3 and MLKL during ZBP1-induced necroptosis after HSV-1 infection. Bioengineering Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids Irina A. Okkelman*1, Chris Vercruysse*1, Alina V. Kondrashina2, Sergey M. Borisov3, Ruslan I. Dmitriev1 1Tissue Engineering and Biomaterials Group, Department of Human Structure and Repair, Faculty of Medical and Health Sciences, Ghent University, 2Health and Happiness (H&H) Group, National Food Innovation Hub, 3Institute of Analytical Chemistry and Food Chemistry, Graz University of Technology The protocol describes high-throughput spheroid generation for bioprinting using the multi-parametric analysis of their oxygenation and cell death on a standard fluorescence microscope. This approach can be applied to control the spheroids viability and perform standardization, which is important in modeling 3D tissue, tumor microenvironment, and successful (micro)tissue biofabrication. Cancer Research Positron Emission Tomography-based Dose Painting Radiation Therapy in a Glioblastoma Rat Model using the Small Animal Radiation Research Platform Sam Donche1, Jeroen Verhoeven1, Benedicte Descamps2, Charlotte Bouckaert3, Robrecht Raedt3, Christian Vanhove2, Ingeborg Goethals1 1Department of Diagnostic Sciences, Ghent University, 2Department of Electronics and Information Systems, Ghent University, 3Department of Head and Skin, Ghent University Here we present a protocol to perform preclinical positron emission tomography-based radiotherapy in a rat glioblastoma model using algorithms developed in-house to optimize the accuracy and efficiency. Neuroscience Advanced Diffusion Imaging in The Hippocampus of Rats with Mild Traumatic Brain Injury Kim Braeckman1, Benedicte Descamps1, Christian Vanhove1 1Infinity lab, Medical Imaging and Signal Processing Group, Ghent University The overall goal of this procedure is to obtain quantitative microstructural information of the hippocampus in a rat with mild traumatic brain injury. This is done using an advanced diffusion-weighted magnetic resonance imaging protocol and region-of-interest based analysis of parametric diffusion maps. Biochemistry Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface Marta Calatayud Arroyo1, Tom Van de Wiele1, Emma Hernandez-Sanabria1 1Center for Microbial Ecology and Technology (CMET), Ghent University Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations' survivability. Cancer Research PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator Julie Bolcaen1, Benedicte Descamps2, Tom Boterberg3, Christian Vanhove2, Ingeborg Goethals1 1Department of Nuclear Medicine, Ghent University Hospital, 2IBiTech-MEDISIP, Department of Electronics and Information Systems, Ghent University, 3Department of Radiation Oncology, Ghent University Hospital In the past, small animal irradiation was usually performed without the ability to target a well-delineated tumor volume. The goal was to mimic the treatment of human glioblastoma in rats. Using a small animal irradiation platform, we performed MRI-guided 3D conformal irradiation with PET-based sub-volume boosting in a preclinical setting. Biology Cellular Redox Profiling Using High-content Microscopy Tom Sieprath1,2, Tobias Corne1,2, Joke Robijns1, Werner J. H. Koopman3, Winnok H. De Vos1,2 1Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, 2Cell Systems and Imaging Research Group (CSI), Department of Molecular Biotechnology, Ghent University, 3Department of Biochemistry , Radboud Institute for Molecular Life Sciences, Radboud University Medical Center This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology – jointly referred to as mitochondrial morphofunction – in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM). Immunology and Infection Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration Lien Van Hoecke1,2, Emma Richelle Job1,2, Xavier Saelens1,2, Kenny Roose1,2 1UGent Center for Medical Biotechnology, VIB, 2Department of Biomedical Molecular Biology, Ghent University (UGent) The health status of the lung is reflected by the type and number of immune cells that are present in the bronchioles of the lung. We describe a bronchoalveolar lavage technique that allows the isolation and study of nonadherent cells and soluble factors from the lower respiratory tract of mice. Developmental Biology Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange Tim Pieters1,2,3,4, Lieven Haenebalcke1,2, Kenneth Bruneel1,2,4, Niels Vandamme1,2,4, Tino Hochepied1,2, Jolanda van Hengel5, Dagmar Wirth6, Geert Berx1,2,4, Jody J. Haigh7, Frans van Roy1,2,4, Steven Goossens1,2,3,4 1Department of Biomedical Molecular Biology, Ghent University, 2Inflammation Research Center, VIB, 3Center for Medical Genetics, Ghent University Hospital, 4Cancer Research Institute Ghent (CRIG), 5Department of Basic Medical Sciences, Faculty of Medicine and Health Sciences, Ghent University, 6Helmholtz Center for Infection Research, 7Mammalian Functional Genetics Laboratory, Division of Blood Cancers, Australian Centre for Blood Diseases, Department of Clinical Haematology, Monash University and Alfred Health Alfred Centre Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein. Bioengineering A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time Katrijn R. Six1,2, Rosalie Devloo1, Britt Van Aelst1, Philippe Vandekerckhove2,3,4, Hendrik B. Feys1, Veerle Compernolle1,2,3 1Transfusion Research Center, Belgian Red Cross-Flanders, 2Faculty of Medicine and Health Sciences, Ghent University, 3Blood Service, Belgian Red Cross-Flanders, 4Department of Public Health and Primary Care, KULeuven - University of Leuven This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion. Chemistry On-line Analysis of Nitrogen Containing Compounds in Complex Hydrocarbon Matrixes Nenad D. Ristic1, Marko R. Djokic1, Kevin M. Van Geem1, Guy B. Marin1 1Laboratory for Chemical Technology, Faculty of Engineering and Architecture, Ghent University A method combining comprehensive two-dimensional gas chromatography with nitrogen chemiluminescence detection has been developed and applied to on-line analysis of nitrogen containing compounds in a complex hydrocarbon matrix. Bioengineering Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro Britt Van Aelst1, Hendrik B. Feys1, Rosalie Devloo1, Philippe Vandekerckhove2,3,4, Veerle Compernolle1,2,4 1Transfusion Research Center, Belgium Red Cross-Flanders, 2Blood Service, Belgium Red Cross-Flanders, 3Department of Public Health and Primary Care, Catholic University of Leuven, 4Faculty of Medicine and Health Sciences, University of Ghent Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro. Biology Lateral Root Inducible System in Arabidopsis and Maize Hanne Crombez*1,2, Ianto Roberts*1,2, Nick Vangheluwe*1,2, Hans Motte1,2, Leentje Jansen1,2, Tom Beeckman1,2, Boris Parizot1,2 1Department of Plant Systems Biology, VIB, Ghent, 2Department of Plant Biotechnology and Bioinformatics, Ghent University The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize. Immunology and Infection An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen Srinath Govindarajan1,2, Dirk Elewaut1,2, Michael Drennan1,2 1Department of Rheumatology, Laboratory for Molecular Immunology and Inflammation, Ghent University Hospital, 2VIB Inflammation Research Center, Ghent University Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents. Medicine Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds Marc E. Bracke1, Bart I. Roman2, Christian V. Stevens2, Liselot M. Mus1, Virinder S. Parmar3, Olivier De Wever1, Marc M. Mareel1 1Department of Radiation Oncology and Experimental Cancer Research, University of Ghent, 2Department of Sustainable Organic Chemistry and Technology, University of Ghent, 3Department of Chemistry, University of Delhi Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro. Immunology and Infection Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein Judith Verhelst1,2, Dorien De Vlieger1,2, Xavier Saelens1,2 1Inflammation Research Center, VIB, 2Department of Biomedical Molecular Biology, Ghent University This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein. Engineering Laser-induced Forward Transfer for Flip-chip Packaging of Single Dies Kamal S. Kaur1, Geert Van Steenberge1 1Center for Microsystems Technology (CMST), Ghent University-imec We demonstrate the use of the Laser-induced Forward Transfer (LIFT) technique for flip-chip assembly of optoelectronic components. This approach provides a simple, cost-effective, low-temperature, fast and flexible solution for fine-pitch bumping and bonding on chip-scale for achieving high-density circuits for optoelectronic applications. Chemistry Electrochemically and Bioelectrochemically Induced Ammonium Recovery Sylvia Gildemyn1, Amanda K. Luther2, Stephen J. Andersen1, Joachim Desloover1, Korneel Rabaey1 1Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, 2Department of Environmental Sciences, Rutgers University We demonstrate the extraction of ammonium from an ammonium-rich stream using an electrochemical and a bioelectrochemical system. The reactor setup, operation and data analysis are discussed. Environment Application of Two-spotted Spider Mite Tetranychus urticae for Plant-pest Interaction Studies Marc Cazaux*1,2, Marie Navarro*1,2, Kristie A. Bruinsma*1, Vladimir Zhurov*1, Tara Negrave1, Thomas Van Leeuwen3,4, Vojislava Grbic1,2, Miodrag Grbic1,2 1Department of Biology, The University of Western Ontario, 2Instituto de Ciencias de la Vid y el Vino, 3Department of Crop Protection, Ghent University, 4Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam Protocols for efficient preparation of homogenous samples of spider mites, infestation of experimental plants, and assessment of plant damage, as required for studies of plant-pest interaction were developed. Environment Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry Colin Ruprecht1, Takayuki Tohge1, Alisdair Fernie1, Cara L. Mortimer2, Amanda Kozlo2, Paul D. Fraser2, Norma Funke1, Igor Cesarino3,4, Ruben Vanholme3,4, Wout Boerjan3,4, Kris Morreel3,4, Ingo Burgert5,6, Notburga Gierlinger5,6, Vincent Bulone7, Vera Schneider8, Andrea Stockero8, Juan Navarro-Aviñó9, Frank Pudel10, Bart Tambuyser11, James Hygate12, Jon Bumstead13, Louis Notley13, Staffan Persson1,14 1Max Planck Institute for Molecular Plant Physiology, 2School of Biological Sciences, Plant Molecular Science, Centre for Systems and Synthetic Biology, Royal Holloway, University of London, 3Department of Plant Systems Biology, VIB, 4Department of Plant Biotechnology and Bioinformatics, UGhent, 5Institute for Building Materials, ETH Zurich, 6Applied Wood Materials, EMPA, 7Division of Glycoscience, School of Biotechnology, AlbaNova University Center, Royal Institute of Technology (KTH), 8European Research and Project Office GmbH, 9ABBA Gaia S.L., 10Pflanzenöltechnologie, 11Capax Environmental Services, 12Green Fuels, 13Neutral Consulting Ltd, 14Plant Cell Biology Research Centre, School of Botany, University of Melbourne Plant biomass offers a renewable resource for multiple products, including fuel, feed, food, and a variety of materials. In this paper we investigate the properties of tobacco tree (Nicotiana glauca) and poplar as suitable sources for a biorefinery pipeline. Medicine The In ovo CAM-assay as a Xenograft Model for Sarcoma Gwen M.L. Sys1, Lore Lapeire2, Nikita Stevens1, Herman Favoreel3, Ramses Forsyth4, Marc Bracke2, Olivier De Wever2 1Department of Orthopaedic Surgery and Traumatology, Ghent University Hospital, 2Department of Radiation Oncology and Experimental Cancer Research, Ghent University, 3Department of Virology, Parasitology, and Immunology, Ghent University, 4Pathlicon The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.
