Lund University View Institution's Website 32 articles published in JoVE Medicine Automated Vibratome Sectioning of Agarose-Embedded Lung Tissue for Multiplex Fluorescence Imaging Qi Wang1,2,3, Nicholas B. Bechet1,2,3, Sandra Lindstedt1,2,3,4 1Wallenberg Centre for Molecular Medicine, Lund University, 2Department of Clinical Sciences, Lund University, 3Lund Stem Cell Centre, Lund University, 4Department of Cardiothoracic Surgery and Transplantation, Skåne University Hospital We have developed a tissue-processing technique utilizing a vibratome and agarose-embedded lung tissue to generate lung sections, thereby permitting the acquisition of high-resolution images of lung architecture. We employed immunofluorescence staining to observe spatial protein expression using specific lung structural markers. Bioengineering An Intra-Tissue Radiometry Microprobe for Measuring Radiance In Situ in Living Tissue Amanda L. Holt1,3, Yakir Luc Gagnon2,4, Alison M. Sweeney1,3 1Department of Physics, Yale University, 2Lund Vision Group, Department of Biology, Lund University, 3Department of Physics and Astronomy, University of Pennsylvania, 4Department of Biology, Duke University In this paper, a method for measuring radiance in situ in living tissue is described. This work includes details of the construction of micro-scale probes for different measurements of radiance and irradiance, provides guidance for mounting tissue for the characterization of radiance, and outlines computational methods for analyzing the resulting data. Immunology and Infection A Rapid, Simple, and Standardized Homogenization Method to Prepare Antigen/Adjuvant Emulsions for Inducing Experimental Autoimmune Encephalomyelitis B. Thomas Bäckström1,2 1The Rausing Laboratory, Autoimmunity Section, Division of Neurosurgery, Department of Clinical Sciences, Lund University, 2Department of Autoimmunity, BTB Emulsions To induce experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, mice are immunized with a water-in-oil emulsion containing an autoantigen and complete Freund's adjuvant. While several protocols exist for the preparation of these emulsions, a rapid, simple, and standardized homogenization protocol for emulsion preparation is presented here. Neuroscience Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation Sara Palma-Tortosa1, Raquel Martínez-Curiel1, Constanza Aretio-Medina1, Natalia Avaliani2, Zaal Kokaia1 1Laboratory of Stem Cells and Restorative Neurology, Lund Stem Cell Center, Lund University, 2Lund Stem Cell Center, Lund University This protocol describes long-term organotypic cultures of adult human cortex combined with ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors, which present a novel methodology to further test stem cell-based therapies for human neurodegenerative disorders. Immunology and Infection A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness Alexsandra Lenhard*1, Basma H. Joma*2,3, Nalat Siwapornchai2, Anders P. Hakansson4, John M. Leong2,5, Elsa N. Bou Ghanem1 1Department of Microbiology and Immunology, University at Buffalo School of Medicine, 2Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 3Graduate Program in Immunology, Tufts Graduate School of Biomedical Sciences, 4Department of Translational Medicine, Lund University, 5Stuart B. Levy Center for the Integrated Management of Antimicrobial Resistance, Tufts University This paper describes a novel mouse model for the transition of pneumococcus from an asymptomatic colonizer to a disease-causing pathogen during viral infection. This model can be readily adapted to study polymicrobial and host-pathogen interactions during the different phases of disease progression and across various hosts. Biochemistry Quaternary Structure Modeling Through Chemical Cross-Linking Mass Spectrometry: Extending TX-MS Jupyter Reports Hamed Khakzad1,2, Swen Vermeul3, Lars Malmström4,5,6 1Equipe Signalisation Calcique et Infections Microbiennes, Ecole Normale Supérieure Paris-Saclay, 2Institut National de la Santé et de la Recherche Médicale, 3Scientific IT Services, ETH Zurich, 4Institute for Computational Science, University of Zurich, 5S3IT, University of Zurich, 6Division of Infection Medicine, Department of Clinical Sciences Lund, Faculty of Medicine, Lund University Targeted cross-linking mass spectrometry creates quaternary protein structure models using mass spectrometry data acquired using up to three different acquisition protocols. When executed as a simplified workflow on the Cheetah-MS web server, the results are reported in a Jupyter Notebook. Here, we demonstrate the technical aspects of how the Jupyter Notebook can be extended for a more in-depth analysis. Medicine Robotized Testing of Camera Positions to Determine Ideal Configuration for Stereo 3D Visualization of Open-Heart Surgery Maj Stenmark1,2, Edin Omerbašić1,2, Måns Magnusson1, Sanna Nordberg3, Matilda Dahlström3, Phan-Kiet Tran1 1Cardiac Surgery Unit, Children’s Heart Centre, Skåne University Hospital, 2Department of Clinical Sciences, Lund University, 3Student at Lund University The human depth perception of 3D stereo videos depends on the camera separation, point of convergence, distance to, and familiarity of the object. This paper presents a robotized method for rapid and reliable test data collection during live open-heart surgery to determine the ideal camera configuration. Neuroscience Pre-Chiasmatic, Single Injection of Autologous Blood to Induce Experimental Subarachnoid Hemorrhage in a Rat Model Jesper Peter Bömers1,2, Sara Ellinor Johansson2, Lars Edvinsson2,4, Tiit Illimar Mathiesen1,3,5, Kristian Agmund Haanes2 1Department of Neurosurgery, Rigshospitalet, 2Department of Clinical Experimental Research, Glostrup Research Institute, Rigshospitalet, 3Department of Clinical Medicine, University of Copenhagen, 4Department of Clinical Sciences, Division of Experimental Vascular Research, Lund University, 5Department of Clinical Neuroscience, Karolinska Institutet Subarachnoid hemorrhage continues to carry a high burden of mortality and morbidity in man. To facilitate further research into the condition and its pathophysiology, a pre-chiasmatic, single injection model is presented. Neuroscience Direct Cannula Implantation in the Cisterna Magna of Pigs Nicholas B. Bèchet1,2, Nagesh C. Shanbhag1,2, Iben Lundgaard1,2 1Department of Experimental Medical Science, Lund University, 2Wallenberg Centre for Molecular Medicine, Lund University This article presents a step-by-step protocol for the direct cannula implantation in the cisterna magna of pigs. Cancer Research Decellularization of the Murine Cardiopulmonary Complex Alejandro E. Mayorca-Guiliani1, Maria Rafaeva1, Oliver Willacy1, Chris D. Madsen2, Raphael Reuten1, Janine T. Erler1 1Biotech Research and Innovation Centre (BRIC), University of Copenhagen (UCPH), 2Department of Laboratory Medicine, Division of Translational Cancer Research, Lund University This protocol aims to decellularize the heart and lungs of mice. The resulting extracellular matrix (ECM) scaffolds can be immunostained and imaged to map the location and topology of their components. Medicine Magnetic Resonance Imaging of Multiple Sclerosis at 7.0 Tesla Sonia Waiczies*1, Antje Els*1, Joseph Kuchling*2,3,4, Karin Markenroth Bloch5, Anna Pankowska6,7, Helmar Waiczies8, Carl Herrmann1, Claudia Chien2,3, Carsten Finke4,9, Friedemann Paul2,3,4, Thoralf Niendorf1,2,8 1Berlin Ultrahigh Field Facility (B.U.F.F.), Max Delbrück Center for Molecular Medicine in the Helmholtz Association, 2Experimental and Clinical Research Center, Max Delbrueck Center for Molecular Medicine and Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, 3NeuroCure Clinical Research Center, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, 4Department of Neurology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, 5The Swedish National 7T Facility, Lund University Bioimaging Center, Lund University, 6Department of Radiography, Medical University of Lublin, 7ECOTECH-COMPLEX, Maria Curie-Skłodowska University, 8MRI.TOOLS GmbH, 9Berlin School of Mind and Brain, Humboldt-Universität zu Berlin Here, we present a protocol to acquire magnetic resonance (MR) images of multiple sclerosis (MS) patient brains at 7.0 Tesla. The protocol includes preparation of the setup including the radio-frequency coils, standardized interview procedures with MS patients, subject positioning in the MR scanner and MR data acquisition. Behavior Tracking Rats in Operant Conditioning Chambers Using a Versatile Homemade Video Camera and DeepLabCut Erik K. H. Clemensson1, Morteza Abbaszadeh1, Silvia Fanni1, Elena Espa1, M. Angela Cenci1 1Basal Ganglia Pathophysiology Unit, Dept. Experimental Medical Science, Lund University This protocol describes how to build a small and versatile video camera, and how to use videos obtained from it to train a neural network to track the position of an animal inside operant conditioning chambers. This is a valuable complement to standard analyses of data logs obtained from operant conditioning tests. Medicine Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber Julia Nilsson1,2, Dan Holmberg1,2, Anja Schmidt-Christensen1,2 1Department of Experimental Medical Science, Lund University, 2Lund University Diabetes Centre The goal of this protocol is to continuously monitor the dynamics of the human pancreatic islet engraftment process and the contributing host versus donor cells. This is accomplished by transplanting human islets into the anterior chamber of the eye (ACE) of an NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mouse recipient followed by repeated 2-photon imaging. Immunology and Infection Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT Tirthankar Mohanty1, Pontus Nordenfelt1 1Department of Clinical Sciences, Division of Infection Medicine, Lund University Here, we present a protocol for generating neutrophil extracellular traps (NETs) and operating NETQUANT, a fully automatic software option for quantification of NETs in immunofluorescence images. Developmental Biology Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors Rita Silvério-Alves1,2,3, Andreia M. Gomes3, Ilia Kurochkin4, Kateri A. Moore5,6, Carlos-Filipe Pereira1,2,3 1Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, 2Wallenberg Center for Molecular, Lund University, 3Center for Neuroscience and Cell Biology, University of Coimbra, 4Skolkovo Institute of Science and Technology, 5Department of Cell, Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, 6Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells. Immunology and Infection Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow Hilger Jagau1,2, Ina-Kristin Behrens1,3, Michael Steinert1,4, Simone Bergmann1 1Institut für Mikrobiologie, Technische Universität Braunschweig, 2Devision of Infection Medicine, Department of Clinical Science, Lund University, 3Medical Microbiology and Hospital Epidemiology, Max von Pettenkofer Institute, Ludwig Maximilians University, 4Department of Molecular Infection Biology, Helmholtz Center for Infection Research This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures. Neuroscience In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors Maria Pereira1, Marcella Birtele1, Daniella Rylander Ottosson1 1Department of Experimental Medical Sciences and Lund Stem Cell Center, BMC, Lund University This protocol aims at generating directly reprogrammed interneurons in vivo, using an AAV-based viral system in the brain and a FLEX synapsin-driven GFP reporter, which allows for cell identification and further analysis in vivo. Engineering Infrared Degenerate Four-wave Mixing with Upconversion Detection for Quantitative Gas Sensing Rasmus L. Pedersen1, Zhongshan Li1 1Combustion Physics, Department of Physics, Lund University Here, we present a protocol to perform sensitive, spatially resolved gas spectroscopy in the mid-infrared region, using degenerate four-wave mixing combined with upconversion detection. Medicine Generation of Human 3D Lung Tissue Cultures (3D-LTCs) for Disease Modeling Michael Gerckens1,2,3, Hani N. Alsafadi4,5,6, Darcy E. Wagner4,5,6, Michael Lindner2,7, Gerald Burgstaller*1,2,3, Melanie Königshoff*1,2,3,8 1Comprehensive Pneumology Center, Ludwig-Maximilians-Universität and Helmholtz Zentrum Munich, 2German Center of Lung Research (DZL), 3Translational Lung Research and CPC-M bioArchive, Helmholtz Zentrum München, Comprehensive Pneumology Center Munich DZL/CPC-M, 4Department of Experimental Medical Science, Lung Bioengineering and Regeneration, Lund University, 5Wallenberg Center for Molecular Medicine, Lund University, 6Stem Cell Centre, Lund University, 7Asklepios Fachkliniken Munich-Gauting, 8Department of Medicine, Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Here, we present a protocol for the preparation of agarose-filled human precision-cut lung slices from resected patient tissue that are suitable for generating 3D lung tissue cultures to model human lung diseases in biological and biomedical studies. Developmental Biology Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors Melissa Ilsley1, Sandra Capellera-Garcia1, Kishori Dhulipala1, Alban Johansson1, Johan Flygare1 1Department of Molecular Medicine and Gene Therapy, Lund University Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR). Genetics A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations Mikael N.E. Sommarin1, Rebecca Warfvinge1, Fatemeh Safi1, Göran Karlsson1 1Division of Molecular Hematology, Lund Stem Cell Center, Lund University Bulk gene expression measurements cloud individual cell differences in heterogeneous cell populations. Here, we describe a protocol for how single-cell gene expression analysis and index sorting by Florescence Activated Cell Sorting (FACS) can be combined to delineate heterogeneity and immunophenotypically characterize molecularly distinct cell populations. Immunology and Infection Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance Birendra Singh1,2, Maryam Mostajeran1, Yu-Ching Su1, Tamim Al-Jubair1, Kristian Riesbeck1 1Clinical Microbiology, Department of Translational Medicine, Lund University, 2Department of Molecular Biology, Umea University This report describes protocols for characterizing interactions between bacterial outer membrane proteins and the human complement regulator vitronectin. The protocols can be used to study the binding reactions and biological function of vitronectin in any bacterial species. Biology Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes Karin Dreisig1, Frank Wojciechowski Blixt2, Karin Warfvinge1,2 1Department of Clinical Experimental Research, Glostrup Research Institute, 2Department of Clinical Sciences, Division of Experimental Vascular Research, Lund University We demonstrate three different tissue preparation techniques for immunohistochemical visualization of rat retinal microvascular pericytes, i.e., cryo-sections, whole-mounts, and hypotonic isolation of the vascular network. Neuroscience Cannula Implantation into the Cisterna Magna of Rodents Anna L.R. Xavier1, Natalie Linea Hauglund1, Stephanie von Holstein-Rathlou1, Qianliang Li1, Simon Sanggaard1,2, Nanhong Lou3, Iben Lundgaard3,4, Maiken Nedergaard1,3 1Center for Translational Neuromedicine, Division of Glial Therapeutics, University of Copenhagen, 2Department of Anesthesiology, Yale School of Medicine, 3Center for Translational Neuromedicine, Division of Glial Therapeutics, University of Rochester Medical Center, 4Department of Experimental Medical Science, Wallenberg Center for Molecular Medicine, Lund University Here we describe a protocol to perform cisterna magna cannulation (CMc), a minimally invasive way to deliver tracers, substrates and signaling molecules into the cerebrospinal fluid (CSF). Combined with different imaging modalities, CMc enables glymphatic system and CSF dynamics assessment, as well as brain-wide delivery of various compounds. Immunology and Infection Ultrasensitive Detection of Biomarkers by Using a Molecular Imprinting Based Capacitive Biosensor Gizem Ertürk1, Rolf Lood1 1Department of Clinical Sciences Lund, Division of Infection Medicine, Lund University Here, we present a protocol for the detection and quantification of low abundant molecules in complex solutions using molecular imprinting in combination with a capacitance biosensor. Neuroscience Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts Shelby Shrigley1, Karolina Pircs1, Roger A. Barker1,2, Malin Parmar1, Janelle Drouin-Ouellet1 1Department of Experimental Medical Science, Wallenberg Neuroscience Center, Division of Neurobiology and Lund Stem Cell Center, Lund University, 2John van Geest Centre for Brain Repair & Department of Neurology, Department of Clinical Neurosciences and Cambridge Stem Cell Institute, University of Cambridge Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors. Engineering Fabrication Procedures and Birefringence Measurements for Designing Magnetically Responsive Lanthanide Ion Chelating Phospholipid Assemblies Stéphane Isabettini1, Mirjam E. Baumgartner1, Peter Fischer1, Erich J. Windhab1, Marianne Liebi2, Simon Kuster1 1Laboratory of Food Process Engineering, ETH Zurich, 2MAX IV Laboratory, Lund University Fabrication procedures for highly magnetically responsive lanthanide ion chelating polymolecular assemblies are presented. The magnetic response is dictated by the assembly size, which is tailored by extrusion through nanopore membranes. The assemblies' magnetic alignability and temperature-induced structural changes are monitored by birefringence measurements, a complimentary technique to nuclear magnetic resonance and small angle neutron scattering. Immunology and Infection Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps Graham Brogden*1,2, Ariane Neumann*1,3, Diab M. Husein1, Friederike Reuner1,4, Hassan Y. Naim1, Maren von Köckritz-Blickwede1,4 1Department of Physiological Chemistry, University of Veterinary Medicine Hannover, 2Fish Disease Research Unit, University of Veterinary Medicine, 3Department of Clinical Sciences, Biomedical Center, Lund University, 4Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover Lipids are known to play an important role in cellular functions. Here, we describe a method to determine the lipid composition of neutrophils, with emphasis on the cholesterol level, by using both HPTLC and HPLC to gain a better understanding of the underlying mechanisms of neutrophil extracellular trap formation. Biology Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells Emily Tubbs1, Jennifer Rieusset2 1Lund University Diabetes Centre, Lund University, 2INSERM UMR-1060, CarMeN Laboratory, Lyon 1 University, INRA U1235, INSA of Lyon, Rockefeller and Charles Merieux Lyon-Sud Medical Universities Here, we describe a procedure to visualize and quantify with high sensitivity the endogenous interactions between the endoplasmic reticulum and mitochondria in fixed cells. The protocol features an optimized in situ proximity ligation assay targeting the inositol 1,4,5-triphosphate receptor/glucose-regulated protein 75/voltage-dependent anion channel/cyclophilin D complex at the mitochondria-associated membrane interface. Immunology and Infection Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device Susan Schlimpert1, Klas Flärdh2, Mark J. Buttner1 1Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, 2Department of Biology, Lund University Streptomyces are characterized by a complex life cycle that has been experimentally challenging to study by cell biological means. Here we present a protocol to perform fluorescence time-lapse microscopy of the complete life cycle by growing Streptomyces venezuelae in a microfluidic device. Medicine Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue Olaf Bergmann1, Stefan Jovinge1,2 1Strategic Research Center for Stem Cell Biology and Cell Therapy, University of Lund, 2Department of Cardiology Lund University Hospital, University of Lund Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry. Biology Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney Aaron C. Brown1, Ulrika Blank2, Derek C. Adams1, Michele J. Karolak1, Jennifer L. Fetting1, Beth L. Hill1, Leif Oxburgh1 1Department of Molecular Medicine, Maine Medical Center Research Institute, 2Molecular Medicine and Gene Therapy, Lund University Hospital In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.