Lund University

31 articles published in JoVE

• Bioengineering

  
  An Intra-Tissue Radiometry Microprobe for Measuring Radiance In Situ in Living Tissue Amanda L. Holt^1,3, Yakir Luc Gagnon^2,4, Alison M. Sweeney^{1,3 1}Department 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 Bäckström^{1,2 1}The 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 Kokaia^{1 1}Laboratory 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. Leong^2,5, Elsa N. Bou Ghanem^{1 1}Department 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 Khakzad^1,2, Swen Vermeul³, Lars Malmström^{4,5,6 1}Equipe 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 Stenmark^1,2, Edin Omerbašić^1,2, Måns Magnusson¹, Sanna Nordberg³, Matilda Dahlström³, Phan-Kiet Tran^{1 1}Cardiac 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 Bömers^1,2, Sara Ellinor Johansson², Lars Edvinsson^2,4, Tiit Illimar Mathiesen^1,3,5, Kristian Agmund Haanes^{2 1}Department 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. Bèchet^1,2, Nagesh C. Shanbhag^1,2, Iben Lundgaard^{1,2 1}Department 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. Erler^{1 1}Biotech 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 Pankowska^6,7, Helmar Waiczies⁸, Carl Herrmann¹, Claudia Chien^2,3, Carsten Finke^4,9, Friedemann Paul^2,3,4, Thoralf Niendorf^{1,2,8 1}Berlin 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 Cenci^{1 1}Basal 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 Nilsson^1,2, Dan Holmberg^1,2, Anja Schmidt-Christensen^{1,2 1}Department 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)26Sor^tm4-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 Nordenfelt^{1 1}Department 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-Alves^1,2,3, Andreia M. Gomes³, Ilia Kurochkin⁴, Kateri A. Moore^5,6, Carlos-Filipe Pereira^{1,2,3 1}Molecular 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 Jagau^1,2, Ina-Kristin Behrens^1,3, Michael Steinert^1,4, Simone Bergmann^{1 1}Institut 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 Ottosson^{1 1}Department 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 Li^{1 1}Combustion 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 Gerckens^1,2,3, Hani N. Alsafadi^4,5,6, Darcy E. Wagner^4,5,6, Michael Lindner^2,7, Gerald Burgstaller*^1,2,3, Melanie Königshoff*^{1,2,3,8 1}Comprehensive 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 Flygare^{1 1}Department 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 Karlsson^{1 1}Division 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 Singh^1,2, Maryam Mostajeran¹, Yu-Ching Su¹, Tamim Al-Jubair¹, Kristian Riesbeck^{1 1}Clinical 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 Warfvinge^{1,2 1}Department 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 Sanggaard^1,2, Nanhong Lou³, Iben Lundgaard^3,4, Maiken Nedergaard^{1,3 1}Center 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 Lood^{1 1}Department 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. Barker^1,2, Malin Parmar¹, Janelle Drouin-Ouellet^{1 1}Department 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 Kuster^{1 1}Laboratory 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 Reuner^1,4, Hassan Y. Naim¹, Maren von Köckritz-Blickwede^{1,4 1}Department 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 Rieusset^{2 1}Lund 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. Buttner^{1 1}Department 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 Jovinge^{1,2 1}Strategic 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 Oxburgh^{1 1}Department 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.