Karolinska Institutet View Institution's Website 52 articles published in JoVE Biology Functional Characterization and Visualization of Esophageal Fibroblasts Using Organoid Co-Cultures Evelien Eenjes1, David Grommisch1, Maria Genander1 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. Behavior A Computer-Based Platform for Aiding Clinicians in Eating Disorder Analysis and Diagnosis Ulf Brodin2, Modjtaba Zandian1, Billy Langlet1, Per Södersten1, Anna Anvret2, Jennie Sjöberg2, Cecilia Bergh1,2 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. Developmental Biology Murine Neural Plate Targeting by In Utero Nano-Injection (NEPTUNE) at Embryonic Day 7.5 Katrin Mangold1, Jingyan He1, Sanne Stokman1, Emma R. Andersson1 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. Developmental Biology Rapid Isolation of Single Cells from Mouse and Human Teeth Jan Krivanek1, Josef Lavicky1, Thibault Bouderlique2, Igor Adameyko2,3 1Department of Histology and Embryology, Faculty of Medicine, Masaryk University, 2Department of Molecular Neuroimmunology, Centre for Brain Research, Medical University of Vienna, 3Department of Physiology and Pharmacology, Karolinska Institute The current protocol presents a fast, efficient, and gentle method for isolating single cells suitable for single-cell RNA-seq analysis from a continuously growing mouse incisor, mouse molar, and human teeth. Neuroscience Cryo-section Dissection of the Adult Subependymal Zone for Accurate and Deep Quantitative Proteome Analysis Christian Friess1, Magdalena Götz1,2,3, Jacob Kjell1,2,4 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. Medicine DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis Simona Hankeova*1, Jakub Salplachta*2, Noemi Van Hul1, Michaela Kavkova2, Afshan Iqbal1, Tomas Zikmund2, Jozef Kaiser2, Emma R. Andersson1 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. 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. Adzemovic3, Manuel Zeitelhofer4, Ute Schaefer1, Franz Fazekas2, Sonja Hochmeister2 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. Neuroscience How to Calculate and Validate Inter-brain Synchronization in a fNIRS Hyperscanning Study Yinying Hu1, Zixuan Wang1, Bei Song2, Yafeng Pan3, Xiaojun Cheng4, Yi Zhu1, Yi Hu1 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. Biochemistry Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay Lisha Wang1, Liza Bergkvist1, Rajnish Kumar1,2, Bengt Winblad1,3, Pavel F. Pavlov1 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. Biochemistry Practical Aspects of Sample Preparation and Setup of 1H R1ρ Relaxation Dispersion Experiments of RNA Hannes Feyrer1, Judith Schlagnitweit1, Katja Petzold1 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 R1ρ relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy. The focus of this protocol lies in high-purity sample preparation and setup of NMR experiments. 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. Biochemistry A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1 Miriam Yagüe-Capilla1, Sean G. Rudd1 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. Genetics An Approach to Study Shape-Dependent Transcriptomics at a Single Cell Level Payam Haftbaradaran Esfahani1, Ralph Knöll1,2 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. Immunology and Infection Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection Akhirunnesa Mily1,2, Sadaf Kalsum1, Marco Giulio Loreti1, Rokeya Sultana Rekha3, Jagadeeswara Rao Muvva1, Magda Lourda1,4, Susanna Brighenti1 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. Biology Histological-Based Stainings Using Free-Floating Tissue Sections Emily M. Potts1, Giuseppe Coppotelli1, Jaime M. Ross1,2 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. Biology Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging Jérôme Gilleron1, Cindy Meziat1, André Sulen2, Stoyan Ivanov3, Jennifer Jager1, David Estève4, Catherine Muller4, Jean-Francois Tanti1, Mireille Cormont1 1Université Côte d'Azur, Inserm UMR1065, C3M, Team "Cellular and Molecular Pathophysiology of Obesity", Nice, France, 2Integrated Cardio Metabolic Center, Department of Medicine, Karolinska Institutet, Stockholm, Sweden, 3Université Côte d'Azur, Inserm UMR1065, C3M, Team "Haematometabolism in Diseases", Nice, France, 4Institut de Pharmacologie et de Biologie Structurale (IPBS), Université de Toulouse, CNRS, UPS, Toulouse, France Here, we describe a simple, inexpensive and fast clearing method to resolve the 3D structure of both mouse and human white adipose tissue using a combination of markers to visualize vasculature, nuclei, immune cells, neurons, and lipid-droplet coat proteins by fluorescent imaging. Neuroscience Induction of Leptomeningeal Cells Modification Via Intracisternal Injection Margherita Zamboni1, Giuseppe Santopolo1, Jonas Frisén1 1Department of Cell and Molecular Biology, Karolinska Institute We describe an intracisternal injection that employs a needle bent at the tip that can be stabilized to the skull, thus eliminating the risk of damage to the underlying parenchyma. The approach can be used for genetic fate mapping and manipulations of leptomeningeal cells and for tracking cerebrospinal fluid movement. Developmental Biology Generation of a Human iPSC-Based Blood-Brain Barrier Chip Srikanth Jagadeesan1,2,3, Michael J. Workman4, Anna Herland5,6, Clive N. Svendsen4, Gad D. Vatine1,2,3 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. Cancer Research Fibroblast-Derived 3D Matrix System Applicable to Endothelial Tube Formation Assay Cristina Galindo-Pumariño1, Alberto Herrera2, Alberto Muñoz3, Alfredo Carrato1, Mercedes Herrera4, Cristina Peña1 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). Neuroscience Meta-analysis of Voxel-Based Neuroimaging Studies using Seed-based d Mapping with Permutation of Subject Images (SDM-PSI) Anton Albajes-Eizagirre1,2, Aleix Solanes1,2, Miquel Angel Fullana2,3, John P. A. Ioannidis4, Paolo Fusar-Poli5,6,7, Carla Torrent1,2,3,8, Brisa Solé1,2,3,8, Caterina Mar Bonnín1,2,3,8, Eduard Vieta1,2,3,8, David Mataix-Cols9, Joaquim Radua1,2,5,9 1 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). Developmental Biology Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues Baoyi Zhou1, Marketa Kaucka1, Andrei S. Chagin1,2, Phillip T. Newton1,3 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. Bioengineering A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression Donghee Lee1, Alek Erickson2, Andrew T. Dudley1, Sangjin Ryu3,4 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. Biochemistry Fluorescent Silver Staining of Proteins in Polyacrylamide Gels Alex Y. H. Wong1, Sheng Xie1, Ben Zhong Tang2, Sijie Chen1 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. Medicine An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy Erika Gucciardo1, Sirpa Loukovaara2, Ani Korhonen1, Kaisa Lehti1,3 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. Biochemistry Using High Content Imaging to Quantify Target Engagement in Adherent Cells Hanna Axelsson1,2, Helena Almqvist1,2, Brinton Seashore-Ludlow1,3 1Chemical Biology Consortium Sweden, Science for Life Laboratory, Karolinska Institutet, Solna, 2Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Solna, 3Science for Life Laboratory, Department of Oncology-Pathology, Karolinska Institutet, Stockholm Measurements of drug target engagement are central to effective drug development and chemical probe validation. Here, we detail a protocol for measuring drug-target engagement using high content imaging in a microplate-compatible adaption of the cellular thermal shift assay (CETSA). 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 Hadzidimitriou1, Cristina Tresoldi4, Anton W. Langerak5, Chrysoula Belessi6, Frederic Davi7, Richard Rosenquist2,3, Kostas Stamatopoulos1,2, Paolo Ghia8 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). Immunology and Infection Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture Andrea Introini1,2, Christophe Vanpouille2, Wendy Fitzgerald2, Kristina Broliden1, Leonid Margolis2 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. Biology Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions Xiaofeng Zheng1,2, Calvin Qing Wei Ho1, Xiaowei Zheng3, Kian Leong Lee4, Katarina Gradin5, Teresa S. Pereira3, Per-Olof Berggren1,2,3, Yusuf Ali1,2 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. Behavior Control of Eating Behavior Using a Novel Feedback System Maryam Esfandiari*1, Vasileios Papapanagiotou*2, Christos Diou2, Modjtaba Zandian1, Jenny Nolstam1, Per Södersten1, Cecilia Bergh1 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. Medicine Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis Filippo Locri1, Monica Aronsson1, Ophélie Beaujean1, Anders Kvanta1, Helder André1 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. 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 Aronsson1, Sofie Westman1, Fredrik Lanner2, Anders Kvanta1 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. Developmental Biology Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix Elias Uhlin1, Ana Marin Navarro1,2, Harriet Rönnholm1, Kelly Day1, Malin Kele1, Anna Falk1 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. Medicine Improving Strength, Power, Muscle Aerobic Capacity, and Glucose Tolerance through Short-term Progressive Strength Training Among Elderly People Eva A. Andersson1,2, Per Frank1,3, Marjan Pontén1, Björn Ekblom1, Maria Ekblom1,2, Marcus Moberg1, Kent Sahlin1 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). Biochemistry Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy Ramakrishnan B. Kumar1, Lin Zhu2, Hans Hebert1,2, Caroline Jegerschöld1,2 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. Biochemistry A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing Irena Roci1,2, Hector Gallart-Ayala3, Jeramie Watrous4, Mohit Jain4, Craig E. Wheelock3, Roland Nilsson1,2 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. Immunology and Infection Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy Elina Staaf1, Sunitha Bagawath-Singh1, Sofia Johansson1 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. Immunology and Infection Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry Faezzah Baharom1, Gregory Rankin2, Saskia Scholz1, Jamshid Pourazar2, Clas Ahlm3, Anders Blomberg2, Anna Smed-Sörensen1 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. Immunology and Infection In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Naïve CD4+ T Cells Using a TGF-β-containing Protocol Angelika Schmidt1, Szabolcs Éliás*1, Rubin N. Joshi*1, Jesper Tegnér1 1Unit of Computational Medicine, Center for Molecular Medicine, Department of Medicine Solna, Karolinska Institutet, Karolinska University Hospital, & Science for Life Laboratory This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from naïve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols. Neuroscience Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections Milena Z. Adzemovic1,2, Manuel Zeitelhofer1,3, Marianne Leisser2, Ulricke Köck2, Angela Kury2, Tomas Olsson1 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. 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 Bollampalli1, Susanne Nylén1, Antonio Gigliotti Rothfuchs1 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. Bioengineering Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair Clara I. Chamorro1, Said Zeiai1,2, Gisela Reinfeldt Engberg1,2, Magdalena Fossum1,2 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. Immunology and Infection A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection Clara Braian1, Mattias Svensson2, Susanna Brighenti2, Maria Lerm1, Venkata R. Parasa1,2 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. Medicine Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis Agnieszka Martowicz*1,4, Johann Kern*1,2, Eberhard Gunsilius1, Gerold Untergasser1,3 1Department of Internal Medicine V, Innsbruck Medical University, 2Oncotyrol GmbH, 3Tyrolean Cancer Research Institute, 4Division of Vascular Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis. Immunology and Infection A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide Abraham B. Roos1, Tove Berg1, Kerstin M. Ahlgren1, Johan Grunewald1, Magnus Nord1,2 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. Biology Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale Valentina Gandin1, Kristina Sikström2, Tommy Alain3, Masahiro Morita3, Shannon McLaughlan1, Ola Larsson2, Ivan Topisirovic1 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. Medicine Inducing Myointimal Hyperplasia Versus Atherosclerosis in Mice: An Introduction of Two Valid Models Mandy Stubbendorff*1,2, Xiaoqin Hua*1,2, Tobias Deuse1,2,3, Ziad Ali4,5, Hermann Reichenspurner2,3, Lars Maegdefessel6, Robert C. Robbins7, Sonja Schrepfer1,2,3,4 1Transplant and Stem Cell Immunobiology Lab, Cardiovascular Research Center, University Hospital Hamburg, 2Cardiovascular Research Center (CVRC) and DZHK University Hamburg, 3Department of Cardiovascular Surgery, University Heart Center Hamburg, 4Center for Interventional Vascular Therapy, Division of Cardiology, Columbia University, 5Cardiovascular Research Foundation, New York, 6Karolinska Institute, Stockholm, 7Department of Cardiothoracic Surgery, Stanford University School of Medicine, Falk Cardiovascular Research Center This video shows two models of intimal plaque development in murine arteries and emphasizes the differences in myointimal hyperplasia and atherosclerosis. Neuroscience Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury Nicolas Guerout1,2, Alexandre Paviot1,3, Nicolas Bon-Mardion1,3, Axel Honoré1, Rais OBongo1,4, Célia Duclos1, Jean-Paul Marie1,3 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. 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. Abdulreda1,2, Alejandro Caicedo1,3,4, Per-Olof Berggren1,5 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. Medicine Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract Konstantin Galichanin1,2, Nooshin Talebizadeh2, Per Söderberg2 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. Biology Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET Elvira Hukasova1, Helena Silva Cascales1, Shravan R. Kumar1, Arne Lindqvist1 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. Biology Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry Jaime M. Ross1,2 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. 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 Johansson1, Gabriella B. S. Lundkvist1 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.
