Leiden University View Institution's Website 12 articles published in JoVE Biology MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T Remco van Schadewijk1, Julia R. Krug2,3,4, Andrew Webb5, Henk Van As2,4, Aldrik H. Velders3,4, Huub J. M. de Groot1, A. Alia1,6 1Solid-state NMR, Leiden Institute of Chemistry, Faculty of Science, Leiden University, 2Laboratory of Biophysics, Wageningen University & Research, 3Laboratory of BioNanoTechnology, Wageningen University & Research, 4MAGNEtic resonance Facility, Wageningen University & Research, 5C.J. Gorter Center for High Field MRI, Radiology department, Leiden University Medical Centre, Leiden University, 6Institute for Medical Physics and Biophysics, Leipzig University A protocol to study biological tissue at high spatial resolution using ultra-high field magnetic resonance microscopy (MRM) using microcoils is presented. Step-by-step instructions are provided for characterizing the microcoils. Finally, optimization of imaging is demonstrated on plant roots. Chemistry Capillary Electrophoresis Mass Spectrometry Approaches for Characterization of the Protein and Metabolite Corona Acquired by Nanomaterials Andrew J. Chetwynd*1, Wei Zhang*2, Klaus Faserl*3, James A. Thorn4, Iseult Lynch1, Rawi Ramautar2, Herbert H. Lindner3 1School of Geography Earth and Environmental Sciences, University of Birmingham, 2Biomedical Microscale Analytics, Leiden University, 3Institute of Medical Biochemistry, Medical University of Innsbruck, 4AB Sciex UK Ltd Here we present a protocol to characterize the complete biomolecular corona, proteins, and metabolites, acquired by nanomaterials from biofluids using a capillary electrophoresis – mass spectrometry approach. Bioengineering Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro Vincent van Duinen1,2, Wendy Stam1, Viola Borgdorff3, Arie Reijerkerk3, Valeria Orlova4, Paul Vulto5, Thomas Hankemeier2, Anton Jan van Zonneveld1 1Department of Internal Medicine (Nephrology) and the Einthoven Laboratory for Vascular and Regenerative Medicine, Leiden University Medical Center, 2Division of Systems Biomedicine and Pharmacology, Department of Analytical BioSciences and Metabolomics, Leiden University, 3Ncardia, 4Department of Anatomy and Embryology, Leiden University Medical Center, 5Mimetas This method describes the culture of iPSC-derived endothelial cells as 40 perfused 3D microvessels in a standardized microfluidic platform. This platform enables the study of gradient-driven angiogenic sprouting in 3D, including anastomosis and stabilization of the angiogenic sprouts in a scalable and high-throughput manner. Bioengineering A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection Xiaolin Zhang1,2, Leonie de Boer1, Oliver W. Stockhammer1, Dirk W. Grijpma2,3, Herman P. Spaink4, Sebastian A.J. Zaat1 1Department of Medical Microbiology, Amsterdam UMC, 2Technical Medical Center, Department of Biomaterials Science and Technology, University of Twente, 3Department of Biomedical Engineering, W.J. Kolff Institute, University Medical Center Groningen, University of Groningen, 4Institute of Biology, Leiden University The present study describes a zebrafish embryo model for in vivo visualization and intravital analysis of biomaterial-associated infection over time based on fluorescence microscopy. This model is a promising system complementing mammalian animal models such as mouse models for studying biomaterial-associated infections in vivo. Behavior Creating Virtual-hand and Virtual-face Illusions to Investigate Self-representation Ke Ma1, Dominique P. Lippelt1, Bernhard Hommel1 1Cognitive Psychology Unit, Leiden University Here, we describe virtual-hand and virtual-face illusion paradigms that can be used to study body-related self-perception/-representation. They have already been used in various studies to demonstrate that, under specific conditions, a virtual hand or face can be incorporated into one's body representation, suggesting that body representations are rather flexible. Biochemistry Sheathless Capillary Electrophoresis–Mass Spectrometry for Metabolic Profiling of Biological Samples Wei Zhang1, M. Can Gulersonmez1, Thomas Hankemeier1, Rawi Ramautar1 1Division of Analytical Biosciences, Leiden Academic Center for Drug Research, Leiden University A protocol for metabolic profiling of biological samples by capillary electrophoresis–mass spectrometry using a sheathless porous tip interface design is presented. Immunology and Infection Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery Wouter J. Veneman*1, Rubén Marín-Juez*2, Jan de Sonneville3, Anita Ordas1, Susanne Jong-Raadsen2, Annemarie H. Meijer1, Herman P. Spaink1 1Institute of Biology, Leiden University, 2ZF-screens BV, 3Life Science Methods BV This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism. 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. Biology Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein Ruth Greussing1, Hermann Unterluggauer1, Rafal Koziel1, Andrea B. Maier2, Pidder Jansen-Dürr1 1Molecular and Cell Biology, Institute for Biomedical Aging Research, 2Department of Gerontology and Geriatrics, Netherlands Consortium for Healthy Aging, Leiden University Medical Center A method to monitor ubiquitin-proteasome activity in living cells is described. A degron-destabilized GFP- (GFP-dgn) and a stable GFP-dgnFS fusion protein are generated and transduced into the cell using a lentiviral expression vector. This technique allows to generate a stable GFP-dgn/GFP-dgnFS expressing cell line in which ubiquitin-proteasome activity can be easily assessed using epifluorescence or flow cytometry. Immunology and Infection Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens Erica L. Benard1, Astrid M. van der Sar2, Felix Ellett3, Graham J. Lieschke3, Herman P. Spaink1, Annemarie H. Meijer1 1Department of Molecular Cell Biology, Institute of Biology, Leiden University, 2Department of Medical Microbiology and Infection Control, VU University Medical Center, 3Australian Regenerative Medicine Institute, Monash University Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models. Medicine Spheroid Assay to Measure TGF-β-induced Invasion Hildegonda P.H. Naber1, Eliza Wiercinska1, Peter ten Dijke1, Theo van Laar1 1Department of Molecular Cell Biology and Centre for Biomedial Genetics, Leiden University Medical Centre An assay to quantitatively measure Transforming Growth Factor (TGF)-β-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion. Medicine Accurate and Simple Evaluation of Vascular Anastomoses in Monochorionic Placenta using Colored Dye Enrico Lopriore1, Femke Slaghekke2, Johanna M. Middeldorp2, Frans J. Klumper2, Jan M. van Lith3, Frans J. Walther1, Dick Oepkes2 1Division of Neonatology, Department of Pediatrics, Leiden University Medical Center, 2Division of Fetal Therapy, Department of Obstetrics, Leiden University Medical Center, 3Department of Obstetrics, Leiden University Medical Center Twin-to-twin transfusion syndrome and twin anemia polycythemia sequence are two potentially devastating problems in perinatal medicine. Both disorders occur only in monochorionic twins and result from unbalanced blood flow through placental vascular anastomoses. We provide a simple protocol to accurately evaluate the presence of vascular anastomoses using colored dye injection of placental vessels after birth.