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Ghent University

23 ARTICLES PUBLISHED IN JoVE

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Medicine

The In ovo CAM-assay as a Xenograft Model for Sarcoma
Gwen M.L. Sys 1, Lore Lapeire 2, Nikita Stevens 1, Herman Favoreel 3, Ramses Forsyth 4, Marc Bracke 2, Olivier De Wever 2
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.

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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 Negrave 1, Thomas Van Leeuwen 3,4, Vojislava Grbic 1,2, Miodrag Grbic 1,2
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.

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JoVE Journal

Electrochemically and Bioelectrochemically Induced Ammonium Recovery
Sylvia Gildemyn 1, Amanda K. Luther 2, Stephen J. Andersen 1, Joachim Desloover 1, Korneel Rabaey 1
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.

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Medicine

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds
Marc E. Bracke 1, Bart I. Roman 2, Christian V. Stevens 2, Liselot M. Mus 1, Virinder S. Parmar 3, Olivier De Wever 1, Marc M. Mareel 1
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.

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Immunology and Infection

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein
Judith Verhelst 1,2, Dorien De Vlieger 1,2, Xavier Saelens 1,2
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.

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Immunology and Infection

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen
Srinath Govindarajan 1,2, Dirk Elewaut 1,2, Michael Drennan 1,2
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.

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JoVE Journal

Lateral Root Inducible System in Arabidopsis and Maize
Hanne Crombez *1,2, Ianto Roberts *1,2, Nick Vangheluwe *1,2, Hans Motte 1,2, Leentje Jansen 1,2, Tom Beeckman 1,2, Boris Parizot 1,2
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.

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Bioengineering

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro
Britt Van Aelst 1, Hendrik B. Feys 1, Rosalie Devloo 1, Philippe Vandekerckhove 2,3,4, Veerle Compernolle 1,2,4
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.

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Chemistry

On-line Analysis of Nitrogen Containing Compounds in Complex Hydrocarbon Matrixes
Nenad D. Ristic 1, Marko R. Djokic 1, Kevin M. Van Geem 1, Guy B. Marin 1
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.

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Bioengineering

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time
Katrijn R. Six 1,2, Rosalie Devloo 1, Britt Van Aelst 1, Philippe Vandekerckhove 2,3,4, Hendrik B. Feys 1, Veerle Compernolle 1,2,3
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.

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Immunology and Infection

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration
Lien Van Hoecke 1,2, Emma Richelle Job 1,2, Xavier Saelens 1,2, Kenny Roose 1,2
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.

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Biology

Cellular Redox Profiling Using High-content Microscopy
Tom Sieprath 1,2, Tobias Corne 1,2, Joke Robijns 1, Werner J. H. Koopman 3, Winnok H. De Vos 1,2
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).

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JoVE Journal

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange
Tim Pieters 1,2,3,4, Lieven Haenebalcke 1,2, Kenneth Bruneel 1,2,4, Niels Vandamme 1,2,4, Tino Hochepied 1,2, Jolanda van Hengel 5, Dagmar Wirth 6, Geert Berx 1,2,4, Jody J. Haigh 7, Frans van Roy 1,2,4, Steven Goossens 1,2,3,4
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.

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Cancer Research

PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator
Julie Bolcaen 1, Benedicte Descamps 2, Tom Boterberg 3, Christian Vanhove 2, Ingeborg Goethals 1
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.

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Biochemistry

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface
Marta Calatayud Arroyo 1, Tom Van de Wiele 1, Emma Hernandez-Sanabria 1
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.

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Neuroscience

Advanced Diffusion Imaging in The Hippocampus of Rats with Mild Traumatic Brain Injury
Kim Braeckman 1, Benedicte Descamps 1, Christian Vanhove 1
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.

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Cancer Research

Positron Emission Tomography-based Dose Painting Radiation Therapy in a Glioblastoma Rat Model using the Small Animal Radiation Research Platform
Sam Donche 1, Jeroen Verhoeven 1, Benedicte Descamps 2, Charlotte Bouckaert 3, Robrecht Raedt 3, Christian Vanhove 2, Ingeborg Goethals 1
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.

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Bioengineering

Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids
Irina A. Okkelman *1, Chris Vercruysse *1, Alina V. Kondrashina 2, Sergey M. Borisov 3, Ruslan I. Dmitriev 1
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.

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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 Nemegeer 1,2, Kelly Lemeire 1,2, Peter Vandenabeele 1,2, Jonathan Maelfait 1,2
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.

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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 Imschoot 1,2, Daan Verhaege 1,2, Marlies Burgelman 1,2, Roosmarijn E. Vandenbroucke 1,2
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.

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Environment

Tree Core Analysis with X-ray Computed Tomography
Tom De Mil 1, Jan Van den Bulcke 2,3
1Forest Is Life, TERRA Teaching and Research Centre, Gembloux Agro Bio-Tech, University of Liège, 2UGent-Woodlab, Laboratory of Wood Technology, Department of Environment, Ghent University, 3UGent Centre for X-ray Tomography (UGCT)

Here we show how to process tree cores with an X-ray computed tomography toolchain. Except for chemical extraction for some purposes, no further physical lab treatment is needed. The toolchain can be used for biomass estimations, for obtaining MXD/tree-ring width data as well as for obtaining quantitative wood anatomy data.

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Cancer Research

The Micronucleus Assay on Cryopreserved Whole Blood
Renu Dayal 1, Elien Beyls 1, Anne Vral 1, Ans Baeyens 1
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.

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Bioengineering

Production and Multi-Parameter Live Cell Fluorescence Lifetime Imaging Microscopy (FLIM) of Multicellular Spheroids
Angela C. Debruyne *1, Gabriele Ferrari *1, Hang Zhou *1, Nore Van Loon 1, Nina Heymans 1, Irina A. Okkelman 1,2, Ruslan I. Dmitriev 1,2
1Tissue Engineering and Biomaterials Group, Department of Human Structure and Repair, Faculty of Medicine and Health Sciences, Ghent University, 2Ghent Light Microscopy Core, Ghent University

Here, we describe different multicellular spheroid formation methods to perform follow-up multi-parameter live cell microscopy. Using fluorescence lifetime imaging microscopy (FLIM), cellular autofluorescence, staining dyes, and nanoparticles, the approach for analysis of cell metabolism, hypoxia, and cell death in live three-dimensional (3D) cancer and stem cell-derived spheroids is demonstrated.

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