JoVE Logo

Sign In

Karolinska Institutet

51 ARTICLES PUBLISHED IN JoVE

image

Neuroscience

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus
Sergey A. Savelyev *1, Karin C. Larsson *1, Anne-Sofie Johansson 1, Gabriella B. S. Lundkvist 1
1Swedish Medical Nanoscience Center, Department of Neuroscience, Karolinska Institutet

The procedure of preparing slices containing the adult mouse hypothalamic suprachiasmatic nucleus (SCN), and a rapid way to culture the SCN tissue in organotypic culture condition, are reported. Further, the measurement of oscillatory clock gene protein expression using dynamic luciferase reporter technology is described.

image

Biology

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry
Jaime M. Ross 1,2
1Department of Neuroscience, Karolinska Institutet, 2National Institute on Drug Abuse (NIDA)

The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

image

Biology

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET
Elvira Hukasova 1, Helena Silva Cascales 1, Shravan R. Kumar 1, Arne Lindqvist 1
1Department of Cell and Molecular Biology, Karolinska Institutet

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

image

Medicine

Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract
Konstantin Galichanin 1,2, Nooshin Talebizadeh 2, Per Söderberg 2
1St. Erik's Eye Hospital, Karolinska Institutet, 2Gullstrand lab, Section for Ophthalmology, Department of Neuroscience, Uppsala University

Cataract is the leading cause of blindness in the world. Solar ultraviolet radiation (UVR) is the main risk factor for cataract development. An animal model of far UVR-B induced cataract was developed. In this article we describe methods for investigation of cataract formation: exposure to UVR, quantitative RT-PCR and immunohistochemistry.

image

Neuroscience

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections
Milena Z. Adzemovic 1,2, Manuel Zeitelhofer 1,3, Marianne Leisser 2, Ulricke Köck 2, Angela Kury 2, Tomas Olsson 1
1Department of Clinical Neuroscience, Neuroimmunology Unit, Center for Molecular Medicine, Karolinska Institutet, 2Department of Neuroimmunology, Center for Brain Research, Medical University of Vienna, 3Department of Medical Biochemistry and Biophysics, Vascular Biology Unit, Karolinska Institutet

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

image

Medicine

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time
Midhat H. Abdulreda 1,2, Alejandro Caicedo 1,3,4, Per-Olof Berggren 1,5
1Diabetes Research Institute, University of Miami Miller School of Medicine, 2Department of Surgery, University of Miami Miller School of Medicine, 3Department of Medicine, University of Miami Miller School of Medicine, 4Department of Physiology & Biophysics, University of Miami Miller School of Medicine, 5The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

image

Neuroscience

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury
Nicolas Guerout 1,2, Alexandre Paviot 1,3, Nicolas Bon-Mardion 1,3, Axel Honoré 1, Rais OBongo 1,4, Célia Duclos 1, Jean-Paul Marie 1,3
1UPRES EA3830, Institute for Research and Innovation in Biomedicine, University of Rouen, 2Neuroscience, Karolinska Institutet, 3Otorhinolaryngology, Head and Neck Surgery Department, Rouen University Hospital, 4Otorhinolaryngology, Head and Neck Surgery Department, Amiens University Hospital

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

image

Biology

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale
Valentina Gandin 1, Kristina Sikström 2, Tommy Alain 3, Masahiro Morita 3, Shannon McLaughlan 1, Ola Larsson 2, Ivan Topisirovic 1
1Lady Davis Institute and Department of Oncology, McGill University, 2Department of Oncology-Pathology, Karolinska Institutet, 3Goodman Cancer Centre and Department of Biochemistry, McGill University

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

image

Immunology and Infection

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide
Abraham B. Roos 1, Tove Berg 1, Kerstin M. Ahlgren 1, Johan Grunewald 1, Magnus Nord 1,2
1Department of Medicine, Solna and CMM, Respiratory Medicine Unit, Karolinska Institutet, 2Safety Science, Global Regulator Affairs & Patient Safety, AstraZeneca Global Medicines Development

We describe a method for inducing neutrophilic pulmonary inflammation by challenge to aerosolized lipopolysaccharide by nebulization, to model acute lung injury. In addition, basic surgical techniques for lung isolation, tracheal intubation and bronchoalveolar lavage are also described.

image

Bioengineering

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair
Clara I. Chamorro 1, Said Zeiai 1,2, Gisela Reinfeldt Engberg 1,2, Magdalena Fossum 1,2
1Department of Women's and Children's Health, Center for Molecular Medicine, Karolinska Institutet, 2Department of Pediatric Surgery, Urology Section, Astrid Lindgren Children's Hospital, Karolinska University Hospital

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

image

Immunology and Infection

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection
Clara Braian 1, Mattias Svensson 2, Susanna Brighenti 2, Maria Lerm 1, Venkata R. Parasa 1,2
1Department of Clinical and Experimental Medicine, Linköping University, 2Department of Medicine, Karolinska Institute

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

image

Immunology and Infection

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Guérin
Vishnu Priya Bollampalli 1, Susanne Nylén 1, Antonio Gigliotti Rothfuchs 1
1Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Guérin.

image

JoVE Journal

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy
Elina Staaf 1, Sunitha Bagawath-Singh 1, Sofia Johansson 1
1Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet

A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

image

Biochemistry

A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing
Irena Roci 1,2, Hector Gallart-Ayala 3, Jeramie Watrous 4, Mohit Jain 4, Craig E. Wheelock 3, Roland Nilsson 1,2
1Department of Medicine, Unit of Computational Medicine, Karolinska Institutet, 2Center for Molecular Medicine, Karolinska Institutet, 3Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry 2, Karolinska Institutet, 4Department of Medicine, University of California San Diego

This article describes a method for studying cellular metabolism in complex communities of multiple cell types, using a combination of stable isotope tracing, cell sorting to isolate specific cell types, and mass spectrometry.

image

JoVE Journal

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy
Ramakrishnan B. Kumar 1, Lin Zhu 2, Hans Hebert 1,2, Caroline Jegerschöld 1,2
1Department of Biosciences and Nutrition, Karolinska Institutet, 2School of Technology and Health, KTH Royal Institute of Technology

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

image

Immunology and Infection

Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry
Faezzah Baharom 1, Gregory Rankin 2, Saskia Scholz 1, Jamshid Pourazar 2, Clas Ahlm 3, Anders Blomberg 2, Anna Smed-Sörensen 1
1Immunology and Allergy Unit, Department of Medicine Solna, Karolinska Institutet, 2Division of Medicine, Department of Public Health and Clinical Medicine, Umeå University, 3Divison of Infectious Diseases, Department of Clinical Microbiology, Umeå University

Lung-resident immune cells, including dendritic cells (DCs) in humans, are critical for defense against inhaled pathogens and allergens. However, due to the scarcity of human lung tissue, studies are limited. This work presents protocols to process human mucosal endobronchial biopsies for studying lung DCs using immunohistochemistry and flow cytometry.

image

Developmental Biology

Dissection of Larval Zebrafish Gonadal Tissue
Xinjian Wang *1, Sijie Chen *1, Wei Zhang 1, Yiyuan Ren 1, Quan Zhang 1, Gang Peng 1
1Institutes of Brain Science, State Key Laboratory of Medical Neurobiology and Collaborative Innovation Center for Brain Science, Fudan University

Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.

image

JoVE Core

Improving Strength, Power, Muscle Aerobic Capacity, and Glucose Tolerance through Short-term Progressive Strength Training Among Elderly People
Eva A. Andersson 1,2, Per Frank 1,3, Marjan Pontén 1, Björn Ekblom 1, Maria Ekblom 1,2, Marcus Moberg 1, Kent Sahlin 1
1Åstrand Laboratory of Work Physiology, The Swedish School of Sport and Health Sciences, GIH, 2Department of Neuroscience, Karolinska Institutet, 3Department of Physiology and Pharmacology, Karolinska Institutet

The effect of short-term resistance training on elderly people was investigated through the simultaneous use of several methods. Compared to a control group, many improvements were seen, including on muscle aerobic capacity, glucose tolerance, strength, power, and muscle quality (i.e., protein involved in cell signaling and muscle fiber type composition).

image

Developmental Biology

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix
Elias Uhlin 1, Ana Marin Navarro 1,2, Harriet Rönnholm 1, Kelly Day 1, Malin Kele 1, Anna Falk 1
1Department of Neuroscience, Karolinska Institutet, 2Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

image

Biology

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy
Sandra Petrus-Reurer *1,2, Hammurabi Bartuma *1, Monica Aronsson 1, Sofie Westman 1, Fredrik Lanner 2, Anders Kvanta 1
1Clinical Neuroscience, Section for Ophtalmology and Vision, Karolinska Institutet, 2Clinical Sciences, Intervention and Technology, Division of Obstetrics and Gynecology, Karolinska Institutet

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

image

Immunology and Infection

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture
Andrea Introini 1,2, Christophe Vanpouille 2, Wendy Fitzgerald 2, Kristina Broliden 1, Leonid Margolis 2
1Department of Medicine Solna, Center for Molecular Medicine, Karolinska University Hospital, Karolinska Institutet, 2Section of Intercellular Interactions, Eunice Shriver National Institute of Child Health and Human Development, National Institutes of Health

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

image

Medicine

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis
Filippo Locri 1, Monica Aronsson 1, Ophélie Beaujean 1, Anders Kvanta 1, Helder André 1
1Department of Clinical Neuroscience, Section of Eye and Vision, St Erik Eye Hospital, Karolinska Institutet

Iris neovascularization, a common complication of ischemic retinal disease, may lead to sight-threatening neovascular glaucoma. Here, we describe a murine protocol for inducing experimental iris neovascularization that may be used for noninvasive evaluation of angiogenesis-modulating substances.

image

Behavior

Control of Eating Behavior Using a Novel Feedback System
Maryam Esfandiari *1, Vasileios Papapanagiotou *2, Christos Diou 2, Modjtaba Zandian 1, Jenny Nolstam 1, Per Södersten 1, Cecilia Bergh 1
1Mandometer Clinic, Karolinska Institutet, 2Department of Electrical and Computer Engineering, Aristotle University of Thessaloniki

Subjects eat food from a plate placed on a scale connected to a computer that records the weight loss of the plate during the meal. Feedback on the computer screen allows the subject to adapt her/his eating behavior to reference curves thus normalizing body weight.

image

Cancer Research

Immunoglobulin Gene Sequence Analysis In Chronic Lymphocytic Leukemia: From Patient Material To Sequence Interpretation
Andreas Agathangelidis *1, Lesley Ann Sutton *2,3, Anastasia Hadzidimitriou 1, Cristina Tresoldi 4, Anton W. Langerak 5, Chrysoula Belessi 6, Frederic Davi 7, Richard Rosenquist 2,3, Kostas Stamatopoulos 1,2, Paolo Ghia 8
1Institute of Applied Biosciences, Centre for Research and Technology Hellas, 2Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, 3Department of Molecular Medicine and Surgery, Karolinska Institutet, 4Division of Immunology, Transplantation and Infectious, IRCCS San Raffaele Scientific Institute, 5Department of Immunology, Laboratory for Medical Immunology, Erasmus University Medical Center, 6Hematology Department, Nikea General Hospital, 7Assistance publique - Hôpitaux de Paris (AP-HP), Hopital Pitié-Salpêtrière, Department of Hematology, and UPMC University Paris 06, UMRS 1138, 8Division of Experimental Oncology, IRCCS Istituto Scientifico San Raffaele and Università Vita-Salute San Raffaele

Herein, we present a protocol that details the technical aspects and essential requirements to ensure robust IG gene sequence analysis in patients with chronic lymphocytic leukemia (CLL), based on the accumulated experience of the European Research initiative on CLL (ERIC).

image

Biology

Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions
Xiaofeng Zheng 1,2, Calvin Qing Wei Ho 1, Xiaowei Zheng 3, Kian Leong Lee 4, Katarina Gradin 5, Teresa S. Pereira 3, Per-Olof Berggren 1,2,3, Yusuf Ali 1,2
1Lee Kong Chian School of Medicine, Nanyang Technological University, 2Singapore Eye Research Institute (SERI), Singapore General Hospital, 3The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Karolinska University Hospital, 4Cancer and Stem Cell Biology Program, Duke-NUS Medical School, 5Department of Cell and Molecular Biology, Karolinska Institutet

Here we describe a co-immunoprecipitation protocol to study protein-protein interactions between endogenous nuclear proteins under hypoxic conditions. This method is suitable for demonstration of the interactions between transcription factors and transcriptional co-regulators at hypoxia.

image

Neuroscience

Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines
Muammer Üçal *1, Michaela Tanja Haindl *2, Milena Z. Adzemovic 3, Manuel Zeitelhofer 4, Ute Schaefer 1, Franz Fazekas 2, Sonja Hochmeister 2
1Research Unit of Experimental Neurotraumatology, Department of Neurosurgery, Medical University Graz, 2Department of Neurology, Medical University Graz, 3Centre for Molecular Medicine, Department of Clinical Neuroscience, Karolinska Institutet, 4Division of Vascular Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet

The protocol presented here allows the reproduction of a widespread grey matter demyelination of both cortical hemispheres in adult male Dark Agouti rats. The method comprises of intracerebral implantation of a catheter, subclinical immunization against myelin oligodendrocyte glycoprotein, and intracerebral injection of a pro-inflammatory cytokine mixture through the implanted catheter.

image

Biochemistry

Fluorescent Silver Staining of Proteins in Polyacrylamide Gels
Alex Y. H. Wong 1, Sheng Xie 1, Ben Zhong Tang 2, Sijie Chen 1
1Ming Wai Lau Centre for Reparative Medicine, Karolinska Institutet, 2Department of Chemistry, Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction, Institute of Molecular Functional Materials, State Key Laboratory of Neuroscience, Division of Biomedical Engineering, and Division of Life Science, Hong Kong University of Science and Technology

Here, we describe a detailed protocol outlining a new fluorescent staining technique for total protein detection in polyacrylamide gels. The protocol utilizes a silver ion-specific fluorescence turn-on probe, which detects Ag+-protein complexes, and eliminates certain limitations of traditional chromogenic silver stains.

image

Medicine

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy
Erika Gucciardo 1, Sirpa Loukovaara 2, Ani Korhonen 1, Kaisa Lehti 1,3
1Research Programs Unit, Genome-Scale Biology, Biomedicum Helsinki, University of Helsinki, 2Unit of Vitreoretinal Surgery, Ophthalmology, University of Helsinki and Helsinki University Hospital, 3Department of Microbiology, Tumor, and Cell Biology (MTC), Karolinska Institutet

Here, we present a protocol to study the pathophysiology of proliferative diabetic retinopathy by using patient-derived, surgically-excised, fibrovascular tissues for three-dimensional native tissue characterization and ex vivo culture. This ex vivo culture model is also amenable for testing or developing new treatments.

image

Bioengineering

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression
Donghee Lee 1, Alek Erickson 2, Andrew T. Dudley 1, Sangjin Ryu 3,4
1Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, 2Department of Physiology and Pharmacology, Karolinska Institutet, 3Department of Mechanical and Materials Engineering, University of Nebraska-Lincoln, 4Nebraska Center for Materials and Nanoscience, University of Nebraska-Lincoln

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

image

Neuroscience

Meta-analysis of Voxel-Based Neuroimaging Studies using Seed-based d Mapping with Permutation of Subject Images (SDM-PSI)
Anton Albajes-Eizagirre 1,2, Aleix Solanes 1,2, Miquel Angel Fullana 2,3, John P. A. Ioannidis 4, Paolo Fusar-Poli 5,6,7, Carla Torrent 1,2,3,8, Brisa Solé 1,2,3,8, Caterina Mar Bonnín 1,2,3,8, Eduard Vieta 1,2,3,8, David Mataix-Cols 9, Joaquim Radua 1,2,5,9
1Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), 2Mental Health Research Networking Center (CIBERSAM), 3Institute of Neurosciences, Hospital Clinic de Barcelona, 4Departments of Medicine, of Health Research and Policy, and of Biomedical Data Science, Stanford University School of Medicine, and Department of Statistics, Stanford University School of Humanities and Sciences, 5Department of Psychosis Studies, Institute of Psychiatry, Psychology and Neuroscience, King's College London, 6OASIS Service, South London and Maudsley NHS Foundation Trust, 7Department of Nervous System and Behavioral Sciences, University of Pavia, 8University of Barcelona, 9Centre for Psychiatric Research and Education, Department of Clinical Neuroscience, Karolinska Institutet

We detail how to conduct a meta-analysis of voxel-based neuroimaging studies using Seed-based d Mapping with Permutation of Subject Images (SDM-PSI).

image

Cancer Research

Fibroblast-Derived 3D Matrix System Applicable to Endothelial Tube Formation Assay
Cristina Galindo-Pumariño 1, Alberto Herrera 2, Alberto Muñoz 3, Alfredo Carrato 1, Mercedes Herrera 4, Cristina Peña 1
1Medical Oncology Department, Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), CIBERONC, 2Department of Medical Oncology, Hospital Universitario Puerta de Hierro de Majadahonda, 3Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas - Universidad Autónoma de Madrid, CIBERONC, 4Department of Oncology & Pathology, Karolinska Institutet

The aim of this method is to obtain fibroblast-derived 3D matrices as a natural scaffold for subsequent cellular assays. Fibroblasts are seeded in a pre-treated culture plate and stimulated with ascorbic acid for matrix generation. Matrices are decellularized and blocked to culture relevant cells (e.g., endothelial cells).

image

Developmental Biology

Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues
Baoyi Zhou 1, Marketa Kaucka 1, Andrei S. Chagin 1,2, Phillip T. Newton 1,3
1Department of Physiology and Pharmacology, Karolinska Institutet, 2Institute for Regenerative Medicine, Sechenov First Moscow State Medical University (Sechenov University), 3Department of Women's and Children's Health, Karolinska Institutet and Pediatric Endocrinology Unit, Karolinska University Hospital

This method describes the use of the R26R-Confetti (Confetti) mouse model to study mineralized tissues, covering all steps from the breeding strategy to the image acquirements. Included is a general protocol that can be applied to all soft tissues and a modified protocol that can be applied to mineralized tissues.

image

Developmental Biology

Generation of a Human iPSC-Based Blood-Brain Barrier Chip
Srikanth Jagadeesan 1,2,3, Michael J. Workman 4, Anna Herland 5,6, Clive N. Svendsen 4, Gad D. Vatine 1,2,3
1The Department of Physiology and Cell Biology, Faculty of Health Sciences, Ben-Gurion University of the Negev, 2The Regenerative Medicine and Stem Cell (RMSC) Research Center, Ben-Gurion University of the Negev, 3The Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, 4The Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, 5Division of Micro and Nanosystems, KTH Royal Institute of Technology, 6AIMES, Department of Neuroscience, Karolinska Institutet

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

image

Genetics

An Approach to Study Shape-Dependent Transcriptomics at a Single Cell Level
Payam Haftbaradaran Esfahani 1, Ralph Knöll 1,2
1Department of Medicine, Integrated Cardio Metabolic Centre (ICMC), Heart and Vascular Theme, Karolinska Institutet, 2Bioscience Cardiovascular, Research and Early Development, Cardiovascular, Renal and Metabolism (CVRM), BioPharmaceuticals R&D, AstraZeneca

This paper presents methods for growing cardiac myocytes with different shapes, which represent different pathologies, and sorting these adherent cardiac myocytes based on their morphology at a single cell level. The proposed platform provides a novel approach to high throughput and drug screening for different types of heart failure.

image

Biology

Histological-Based Stainings Using Free-Floating Tissue Sections
Emily M. Potts 1, Giuseppe Coppotelli 1, Jaime M. Ross 1,2
1George & Anne Ryan Institute for Neuroscience, College of Pharmacy, Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, 2Department of Neuroscience, Biomedicum, Karolinska Institutet

The free-floating technique allows researchers to perform histological-based stainings including immunohistochemistry on fixed tissue sections to visualize biological structures, cell type, and protein expression and localization. This is an efficient and reliable histochemical technique that can be useful for investigating a multitude of tissues, such as brain, heart, and liver.

image

Immunology and Infection

Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection
Akhirunnesa Mily 1,2, Sadaf Kalsum 1, Marco Giulio Loreti 1, Rokeya Sultana Rekha 3, Jagadeeswara Rao Muvva 1, Magda Lourda 1,4, Susanna Brighenti 1
1Center for Infectious Medicine (CIM), Department of Medicine Huddinge, ANA Futura, Karolinska Institutet, 2Infectious Diseases Division, International Centre for Diarrhoeal Disease Research, Bangladesh, 3Clinical Microbiology, Department of Laboratory Medicine (Labmed), ANA Futura, Karolinska Institutet, 4Childhood Cancer Research Unit, Department of Women's and Children's Health, Karolinska Institutet

This protocol provides a method to study Mycobacterium tuberculosis infection in human M1- or M2-polarized macrophages based on differentiation of peripheral-blood-monocytes to macrophage-like cells that are infected with the GFP-labeled virulent strain H37Rv, and analyzed with flow cytometry using a 10-color panel including expression of selected M1/M2 markers.

image

Cancer Research

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice
Lisha Wang 1, Zhiming Wang 1, Junyi Guo 2, Huayu Lin 1, Shuqiong Wen 2, Qiao Liu 1, Yiding Li 3, Qing Wu 1, Leiqiong Gao 1, Xiangyu Chen 1, Luoyingzi Xie 1, Qin Tian 1, Jianfang Tang 1, Zhirong Li 1, Li Hu 1, Juan Wang 4, Lifan Xu 1, Qizhao Huang 5, Lilin Ye 1
1Institute of Immunology, Third Military Medical University, 2Guanghua School of Stomatology, Guangdong Provincial Key Laboratory of Stomatology, Stomatological Hospital, Sun Yat-Sen University, 3Shigatse Branch, Xinqiao Hospital, Third Military Medical University, 4Department of Emergency Medicine, Southwest Hospital, Third Military Medical University, 5Cancer Center, The General Hospital of Western Theater Command

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.

image

Biochemistry

Practical Aspects of Sample Preparation and Setup of 1H R Relaxation Dispersion Experiments of RNA
Hannes Feyrer 1, Judith Schlagnitweit 1, Katja Petzold 1
1Department of Medical Biochemistry and Biophysics, Karolinska Institutet

We present a protocol to measure micro- to millisecond dynamics on 13C/15N-labeled and unlabeled RNA with 1H R relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy. The focus of this protocol lies in high-purity sample preparation and setup of NMR experiments.

image

Biochemistry

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1
Miriam Yagüe-Capilla 1, Sean G. Rudd 1
1Science for Life Laboratory, Department of Oncology-Pathology, Karolinska Institutet

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase with critical roles in human health and disease. Here we present a versatile enzyme-coupled SAMHD1 activity assay, deployed in a 384-well microplate format, that allows for the evaluation of small molecules and nucleotide analogues as SAMHD1 substrates, activators, and inhibitors.

image

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.

image

Biochemistry

Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay
Lisha Wang 1, Liza Bergkvist 1, Rajnish Kumar 1,2, Bengt Winblad 1,3, Pavel F. Pavlov 1
1Department of Neuroscience Care and Society, Division of Neurogeriatrics, Karolinska Institutet, 2Department of Pharmaceutical Engineering & Technology, Indian Institute of Technology (BHU), 3Theme Inflammation and Aging, Karolinska University Hospital

This protocol presents a technique for probing protein-protein interactions using glutathione-linked donor beads with GST-fused TPR-motif co-chaperones and acceptor beads coupled with an Hsp90-derived peptide. We have used this technique to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.

image

Neuroscience

How to Calculate and Validate Inter-brain Synchronization in a fNIRS Hyperscanning Study
Yinying Hu 1, Zixuan Wang 1, Bei Song 2, Yafeng Pan 3, Xiaojun Cheng 4, Yi Zhu 1, Yi Hu 1
1Institute of Brain and Education Innovation, School of Psychology and Cognitive Science, East China Normal University, 2Department of Musicology, Harbin Conservatory of Music, 3Department of Clinical Neuroscience, Karolinska Institutet, 4School of Psychology, Shenzhen University

The dynamics between coupled brains of individuals have been increasingly represented by inter-brain synchronization (IBS) when they coordinate with each other, mostly using simultaneous-recording signals of brains (namely hyperscanning) with fNIRS. In fNIRS hyperscanning studies, IBS has been commonly assessed through the wavelet transform coherence (WTC) method because of its advantage on expanding time series into time-frequency space where oscillations can be seen in a highly intuitive way. The observed IBS can be further validated via the permutation-based random pairing of the trial, partner, and condition. Here, a protocol is presented to describe how to obtain brain signals via fNIRS technology, calculate IBS through the WTC method, and validate IBS by permutation in a hyperscanning study. Further, we discuss the critical issues when using the above methods, including the choice of fNIRS signals, methods of data preprocessing, and optional parameters of computations. In summary, using the WTC method and permutation is a potentially standard pipeline for analyzing IBS in fNIRS hyperscanning studies, contributing to both the reproducibility and reliability of IBS.

image

Medicine

DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis
Simona Hankeova *1, Jakub Salplachta *2, Noemi Van Hul 1, Michaela Kavkova 2, Afshan Iqbal 1, Tomas Zikmund 2, Jozef Kaiser 2, Emma R. Andersson 1
1Karolinska Institutet, 2Central European Institute of Technology

Double resin casting micro-computed tomography, or DUCT, enables visualization, digitalization, and segmentation of two tubular systems simultaneously to facilitate 3D analysis of organ architecture. DUCT combines ex vivo injection of two radiopaque resins followed by micro-computed tomography scanning and segmentation of the tomographic data.

image

Neuroscience

Cryo-section Dissection of the Adult Subependymal Zone for Accurate and Deep Quantitative Proteome Analysis
Christian Friess 1, Magdalena Götz 1,2,3, Jacob Kjell 1,2,4
1Division of Physiological Genomics, Biomedical Center, Ludwig Maximilian University of Munich, 2Institute for Stem Cell Research, Helmholtz Zentrum München, 3SYNERGY, Excellence Cluster Systems Neurology, University of Munich, 4Department of Clinical Neuroscience, Karolinska Institutet

Cryo-section-dissection allows fresh, frozen preparation of the largest neurogenic niche in the murine brain for deep quantitative proteome analysis. The method is precise, efficient, and causes minimal tissue perturbation. Therefore, it is ideally suited for studying the molecular microenvironment of this niche, as well as other organs, regions, and species.

image

Developmental Biology

Murine Neural Plate Targeting by In Utero Nano-Injection (NEPTUNE) at Embryonic Day 7.5
Katrin Mangold 1, Jingyan He 1, Sanne Stokman 1, Emma R. Andersson 1
1Department of Cell and Molecular Biology, Karolinska Institutet

In this protocol, we describe how to inject the mouse amniotic cavity at E7.5 with lentivirus, leading to uniform transduction of the entire neural plate, with minimal detrimental effects on survival or embryonic development.

image

Behavior

A Computer-Based Platform for Aiding Clinicians in Eating Disorder Analysis and Diagnosis
Ulf Brodin 2, Modjtaba Zandian 1, Billy Langlet 1, Per Södersten 1, Anna Anvret 2, Jennie Sjöberg 2, Cecilia Bergh 1,2
1Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, 2Mandometer Clinic

Diagnosing eating disorders in healthcare is challenging. Therefore, the present protocol develops an algorithm based on 949 patient responses to a questionnaire, with the diagnosis displayed on an easy-to-use web-based interface. This system facilitates the accurate diagnosis of eating disorders while excluding those believed to have an eating disorder.

image

Pedicle Screw Placement Using an Augmented Reality Head-Mounted Display in a Porcine Model

Pedicle Screw Placement Using an Augmented Reality Head-Mounted Display in a Porcine Model
Henrik Frisk 1, Gustav Burström 1, Juliane Weinzierl 2, Linda Westernhagen 2, Florentin Tranchant 2, Erik Edström 1,3, Adrian Elmi-Terander 1,3
1Department of Clinical Neuroscience, Karolinska Institutet, 2Brainlab AG, 3Capio Spine Center Stockholm, Löwenströmska Hospital

The augmented reality head-mounted display, Magic Leap, was used in combination with a conventional navigation system to place pedicle screws in a porcine model by adhering to a novel workflow. With a median insertion time of <2.5 min, submillimeter technical accuracy and 100% clinical accuracy were achieved according to Gertzbein.

image

Biology

Functional Characterization and Visualization of Esophageal Fibroblasts Using Organoid Co-Cultures
Evelien Eenjes 1, David Grommisch 1, Maria Genander 1
1Department of Cell and Molecular Biology, Karolinska Institutet

Organoid-fibroblast co-cultures provide a model to study the in vivo stem cell niche. Here, a protocol for esophageal organoid-fibroblast co-cultures is described. Additionally, whole mount imaging is used to visualize the fibroblast-organoid interaction.

image

Immunology and Infection

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy
Maryam Sahi 1, Sarah Andersson 1, Cecilia Mattson 1, Matilda Dale 1, Sofia Kagiolglou 1, Camilla Hofström 2, Helena Persson 2, Jonas Klingström 3,4, Francesca Chiodi 5, Claudia Fredolini 1
1Affinity Proteomics-Stockholm Unit, SciLifeLab, Division of Affinity Proteomics, Department of Protein Science, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), KTH Royal Institute of Technology, 2Human Antibody Therapeutics Unit, SciLifeLab, Division of Drug Discovery and Development, Department of Protein Science, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), KTH Royal Institute of Technology, 3Center for Infectious Medicine, Department of Medicine Huddinge, Karolinska Institutet, 4Public Health Agency of Sweden, 5Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet

Here, we describe a newly developed multiplex fluorescent immunoassay that uses a dual-reporter flow cytometric system to concurrently detect two unique spike protein epitopes on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles that had been captured by angiotensin-converting enzyme-2-coupled magnetic microspheres.

image

Developmental Biology

Z-Scores for Assessing Ovarian Reserve in Young Patients Undergoing Fertility Preservation
Jasmin Hassan 1,2, Pauliina Damdimopoulou 1,2, Atte Lahtinen 3,4, Kirsi Jahnukainen 5,6, Katri Knuus 7, Ganesh Acharya 1, Richelle D. Björvang 1,8,9
1Division of Obstetrics and Gynecology, Department of Clinical Science, Intervention and Technology, Karolinska Institutet, 2Department of Gynecology and Reproductive Medicine, Karolinska University Hospital, 3Applied Tumor Genomics Research Program, Faculty of Medicine, University of Helsinki, 4Department of Medical and Clinical Genetics, Medicum, Faculty of Medicine, University of Helsinki, 5Children's Hospital, Pediatric Research Centre, University of Helsinki and Helsinki University Hospital, 6NORDFERTIL Research Lab Stockholm, Department of Women's, and Children's Health, Karolinska Institutet, and Karolinska University Hospital, 7Department of Obstetrics and Gynecology, Helsinki University Hospital, University of Helsinki, 8Department of Cell and Molecular Biology, Uppsala University, 9Department of Women's and Children's Health, Uppsala University

The present protocol describes a method for assessing ovarian reserve in patients under 25 years old who require fertility preservation through ovarian tissue cryopreservation. This method involves: (1) histological assessment of ovarian reserve in cortical samples, (2) comparison to a reference dataset, and (3) calculation of Z-scores.

image

Bioengineering

Surgical Model for Single-Staged Tissue-Engineered Urothelial Tubes in Minipigs
Nikolai Juul 1,2, Oliver Willacy 1,2, Anastasia Buch Kjeldgaard 1,2, Dennis Rootsi 3, Karsten Hammelev 4, Clara Ibel Chamorro 3, Magdalena Fossum 1,2,3
1Division of Pediatric Surgery, Department of Surgery and Transplantation, Copenhagen University Hospital - Rigshospitalet, 2Laboratory of Tissue Engineering, Department of Clinical Medicine, University of Copenhagen, 3Laboratory of Tissue Engineering, Department of Women's and Children's Health, Karolinska Institutet, 4Department of Experimental Medicine, University of Copenhagen

Tissue-engineered implants for reconstructive surgery rarely progress beyond preclinical trials due to laborious ex vivo culturing, which includes complex and expensive scaffold components. Here, we present a single-staged procedure designed for urinary diversion with an accessible collagen-based tubular scaffold containing autologous micrografts.

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved