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Lund University

35 ARTICLES PUBLISHED IN JoVE

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Advanced Biology

Pure Cultures and Streak Plating: Isolation of Single Bacterial Colonies from a Mixed Sample
Tilde Andersson 1, Rolf Lood 2
1Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University

Pure Cultures and Streak Plating: Isolation of Single Bacterial Colonies from a Mixed Sample

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Advanced Biology

16S rRNA Sequencing: A PCR-based Technique to Identify Bacterial Species
Ewa Bukowska-Faniband 1, Tilde Andersson 1, Rolf Lood 1
1Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University

16S rRNA Sequencing: A PCR-based Technique to Identify Bacterial Species

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Advanced Biology

Antibiotic Susceptibility Testing: Epsilometer Tests to Determine MIC Values of Two Antibiotics and Evaluate Antibiotic Synergy
Anna Bläckberg 1, Rolf Lood 1
1Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University

Antibiotic Susceptibility Testing: Epsilometer Tests to Determine MIC Values of Two Antibiotics and Evaluate Antibiotic Synergy

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Advanced Biology

Plaque Assay: A Method to Determine Viral Titer as Plaque Forming Units (PFU)
Tilde Andersson 1, Rolf Lood 1
1Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University

Plaque Assay: A Method to Determine Viral Titer as Plaque Forming Units (PFU)

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Immunology and Infection

Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device
Susan Schlimpert 1, Klas Flärdh 2, Mark J. Buttner 1
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.

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Immunology and Infection

Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance
Birendra Singh 1,2, Maryam Mostajeran 1, Yu-Ching Su 1, Tamim Al-Jubair 1, Kristian Riesbeck 1
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.

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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. Husein 1, Friederike Reuner 1,4, Hassan Y. Naim 1, Maren von Köckritz-Blickwede 1,4
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.

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Biology

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells
Emily Tubbs 1, Jennifer Rieusset 2
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.

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Engineering

Fabrication Procedures and Birefringence Measurements for Designing Magnetically Responsive Lanthanide Ion Chelating Phospholipid Assemblies
Stéphane Isabettini 1, Mirjam E. Baumgartner 1, Peter Fischer 1, Erich J. Windhab 1, Marianne Liebi 2, Simon Kuster 1
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.

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Neuroscience

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts
Shelby Shrigley 1, Karolina Pircs 1, Roger A. Barker 1,2, Malin Parmar 1, Janelle Drouin-Ouellet 1
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.

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Immunology and Infection

Ultrasensitive Detection of Biomarkers by Using a Molecular Imprinting Based Capacitive Biosensor
Gizem Ertürk 1, Rolf Lood 1
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.

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Neuroscience

Cannula Implantation into the Cisterna Magna of Rodents
Anna L.R. Xavier 1, Natalie Linea Hauglund 1, Stephanie von Holstein-Rathlou 1, Qianliang Li 1, Simon Sanggaard 1,2, Nanhong Lou 3, Iben Lundgaard 3,4, Maiken Nedergaard 1,3
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.

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Biology

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes
Karin Dreisig 1, Frank Wojciechowski Blixt 2, Karin Warfvinge 1,2
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.

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Genetics

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations
Mikael N.E. Sommarin 1, Rebecca Warfvinge 1, Fatemeh Safi 1, Göran Karlsson 1
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.

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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
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.

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Developmental Biology

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors
Melissa Ilsley 1, Sandra Capellera-Garcia 1, Kishori Dhulipala 1, Alban Johansson 1, Johan Flygare 1
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).

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Immunology and Infection

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT
Tirthankar Mohanty 1, Pontus Nordenfelt 1
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.

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Engineering

Infrared Degenerate Four-wave Mixing with Upconversion Detection for Quantitative Gas Sensing
Rasmus L. Pedersen 1, Zhongshan Li 1
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.

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Neuroscience

In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors
Maria Pereira 1, Marcella Birtele 1, Daniella Rylander Ottosson 1
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.

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Developmental Biology

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors
Rita Silvério-Alves 1,2,3, Andreia M. Gomes 3, Ilia Kurochkin 4, Kateri A. Moore 5,6, Carlos-Filipe Pereira 1,2,3
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.

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Biochemistry

Quaternary Structure Modeling Through Chemical Cross-Linking Mass Spectrometry: Extending TX-MS Jupyter Reports
Hamed Khakzad 1,2, Swen Vermeul 3, Lars Malmström 4,5,6
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.

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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
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.

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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
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.

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Behavior

Tracking Rats in Operant Conditioning Chambers Using a Versatile Homemade Video Camera and DeepLabCut
Erik K. H. Clemensson 1, Morteza Abbaszadeh 1, Silvia Fanni 1, Elena Espa 1, M. Angela Cenci 1
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.

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Cancer Research

Decellularization of the Murine Cardiopulmonary Complex
Alejandro E. Mayorca-Guiliani 1, Maria Rafaeva 1, Oliver Willacy 1, Chris D. Madsen 2, Raphael Reuten 1, Janine T. Erler 1
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.

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Medicine

Magnetic Resonance Imaging of Multiple Sclerosis at 7.0 Tesla
Sonia Waiczies *1, Antje Els *1, Joseph Kuchling *2,3,4, Karin Markenroth Bloch 5, Anna Pankowska 6,7, Helmar Waiczies 8, Carl Herrmann 1, Claudia Chien 2,3, Carsten Finke 4,9, Friedemann Paul 2,3,4, Thoralf Niendorf 1,2,8
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.

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Neuroscience

Pre-Chiasmatic, Single Injection of Autologous Blood to Induce Experimental Subarachnoid Hemorrhage in a Rat Model
Jesper Peter Bömers 1,2, Sara Ellinor Johansson 2, Lars Edvinsson 2,4, Tiit Illimar Mathiesen 1,3,5, Kristian Agmund Haanes 2
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.

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Neuroscience

Direct Cannula Implantation in the Cisterna Magna of Pigs
Nicholas B. Bèchet 1,2, Nagesh C. Shanbhag 1,2, Iben Lundgaard 1,2
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.

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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 Magnusson 1, Sanna Nordberg 3, Matilda Dahlström 3, Phan-Kiet Tran 1
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.

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Neuroscience

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation
Sara Palma-Tortosa 1, Raquel Martínez-Curiel 1, Constanza Aretio-Medina 1, Natalia Avaliani 2, Zaal Kokaia 1
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.

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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 Siwapornchai 2, Anders P. Hakansson 4, John M. Leong 2,5, Elsa N. Bou Ghanem 1
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.

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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
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.

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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
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.

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Biology

Isolation, Behavioral Identification, and Pathogenicity Assessment of Entomopathogenic Fungi from a Forest Wood Borer
Yifei Qu *1,2, Shengxin Wu *1,2, Liqin Zhang 1,2, Jianting Fan *1, Chihang Cheng *2,3
1School of Forestry and Biotechnology, Zhejiang A&F University, 2Key Laboratory of Vector Biology and Pathogen Control of Zhejiang Province, Huzhou University, 3Department of Biology, Lund University

Here we present a protocol for obtaining entomopathogenic fungi from a forest wood borer and a substitutive way to evaluate their entomopathogenic activities using a Coleopteran model insect. This method is efficient and convenient for exploring entomopathogenic fungal resources from wood-boring insect pests in natural forests.

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Medicine

Automated Vibratome Sectioning of Agarose-Embedded Lung Tissue for Multiplex Fluorescence Imaging
Qi Wang 1,2,3, Nicholas B. Bechet 1,2,3, Sandra Lindstedt 1,2,3,4
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.

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