Uppsala University View Institution's Website 23 articles published in JoVE Biochemistry Design and Construction of an Experimental Setup to Enhance Mineral Weathering through the Activity of Soil Organisms Tullia Calogiuri1,2, Mathilde Hagens2, Jan Willem Van Groenigen1, Thomas Corbett3, Jens Hartmann4, Rick Hendriksen5, Iris Janssens6, Ivan A. Janssens7, Guillermo Ledesma Dominguez7, Grant Loescher4, Steven Mortier6, Anna Neubeck3, Harun Niron7, Reinaldy P. Poetra4, Lukas Rieder4, Eric Struyf7, Michiel Van Tendeloo8, Tom De Schepper6, Tim Verdonck9, Siegfried E. Vlaeminck8, Sara Vicca7, Alix Vidal1 1Soil Biology Group, Wageningen University & Research, 2Soil Chemistry and Chemical Soil Quality, Wageningen University & Research, 3Department of Earth Sciences, Uppsala University, 4Institute for Geology, Center for Earth System Research and Sustainability, University of Hamburg, 5Tupola, Wageningen University & Research, 6IDLab - Department of Computer Science, University of Antwerp - imec, 7Plants and Ecosystems (PLECO), Biology Department, University of Antwerp, 8Research Group of Sustainable Energy, Air and Water Technology, University of Antwerp, 9Department of Mathematics, University of Antwerp - imec Here we present the construction and operation of an experimental setup to enhance mineral weathering through the activity of soil organisms while concurrently manipulating abiotic variables known to stimulate weathering. Representative results from the functioning of the setup and sample analyses are discussed together with points for improvement. Cancer Research A Biomimetic Model for Liver Cancer to Study Tumor-Stroma Interactions in a 3D Environment with Tunable Bio-Physical Properties Carlemi Calitz1, Nataša Pavlović1, Jenny Rosenquist2, Claudia Zagami1, Ayan Samanta2, Femke Heindryckx1 1Department of Medical Cell Biology, Uppsala University, 2Polymer Chemistry, Department of Chemistry-Ångström Laboratory, Uppsala University This protocol presents a 3D biomimetic model with accompanying fibrotic stromal compartment. Prepared with physiologically relevant hydrogels in ratios mimicking the bio-physical properties of the stromal extracellular matrix, an active mediator of cellular interactions, tumor growth and metastasis. Behavior Two Different Real-Time Place Preference Paradigms Using Optogenetics within the Ventral Tegmental Area of the Mouse Zisis Bimpisidis1, Niclas König1, Åsa Wallén-Mackenzie1 1Department of Organismal Biology, Unit of Comparative Physiology, Uppsala University Here we present two easy-to-follow step-by-step protocols for place preference paradigms using optogenetics in mice. Using these two different setups, preference and avoidance behaviors can be solidly assessed within the same apparatus with high spatial and temporal selectivity, and in a straightforward manner. Medicine A Fluorescence-based Assay for Characterization and Quantification of Lipid Droplet Formation in Human Intestinal Organoids Jorik M. van Rijn1,2,3, Marliek van Hoesel1,2, Sabine Middendorp1,2 1Division of Pediatrics, Department of Pediatric Gastroenterology, Wilhelmina Children's Hospital, University Medical Center Utrecht, Utrecht University, 2Regenerative Medicine Center, University Medical Center Utrecht, Utrecht University, 3Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University This protocol describes an assay for the characterization of lipid droplet (LD) formation in human intestinal organoids upon stimulation with fatty acids. We discuss how this assay is used for quantification of LD formation, and how it can be used for high throughput screening for drugs that affect LD formation. Immunology and Infection Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry Zhengkang Luo1, Lina Thorvaldson1, Martin Blixt1, Kailash Singh1 1Department of Medical Cell Biology, Uppsala University Herein, we present a protocol to prepare single cells from murine thymus, pancreatic draining lymph node and spleen to further study these cells using flow cytometry. In addition, this protocol was used for determining the subsets of regulatory T cells using flow cytometry. 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). Biochemistry Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method Narendra Padhan1 1Uppsala University, Dept. Immunology, Genetics and Pathology, Rudbeck Laboratory, 751 85, Uppsala, Sweden Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner. Biochemistry High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB) Xiaoying Qi*1, Yunyun Zhang*2, Yuan Zhang1, Tianhui Ni3, Wenfeng Zhang2, Chunhua Yang1, Jia Mi1,4, Jiandi Zhang2,3, Geng Tian1 1Medicine and Pharmacy Research Center, Binzhou Medical University, 2Yantai Zestern Biotechnique Co. LTD, 3Precision Medicine research center, Binzhou Medical University, 4Department of Chemistry - BMC, Uppsala University Here we demonstrate a detailed process of quantitative dot blot analysis (QDB) by determining the absolute content of a targeted protein, capping actin protein, gelsolin-like (CAPG), in three different mouse tissues. We demonstrate a high throughput, convenient, quantitative immunoblot technique for biomarker validation at the cellular and tissue level. Environment Rearing and Long-Term Maintenance of Eristalis tenax Hoverflies for Research Studies Sarah Nicholas1, Malin Thyselius2, Marissa Holden1, Karin Nordström1,2 1Centre for Neuroscience, Flinders University, 2Department of Neuroscience, Uppsala University The overall goal of these procedures is to establish, maintain and refresh a captive population of Eristalis tenax in a research setting. Chemistry Measurements of Long-range Electronic Correlations During Femtosecond Diffraction Experiments Performed on Nanocrystals of Buckminsterfullerene Rebecca A. Ryan1, Sophie Williams1, Andrew V. Martin1, Ruben A. Dilanian1, Connie Darmanin2, Corey T. Putkunz1, David Wood3, Victor A. Streltsov4, Michael W.M. Jones5, Naylyn Gaffney6, Felix Hofmann7, Garth J. Williams8, Sebastien Boutet9, Marc Messerschmidt10, M. Marvin Seibert11, Evan K. Curwood11, Eugeniu Balaur2, Andrew G. Peele5, Keith A. Nugent2, Harry M. Quiney1, Brian Abbey2 1ARC Centre of Excellence in Advanced Molecular Imaging, School of Physics, University of Melbourne, 2Australian Research Council (ARC) Centre of Excellence in Advanced Molecular Imaging, Department of Chemistry and Physics, La Trobe Institute for Molecular Sciences, La Trobe University, 3Department of Physics, Imperial College London, 4Florey Institute of Neuroscience and Mental Health, 5Science and Engineering Faculty, Queensland University of Technology, 6Swinburne University of Technology, 7Department of Engineering Science, University of Oxford, 8Brookhaven National Laboratory, 9Linac Coherent Light Source, SLAC National Accelerator Laboratory, 10BioXFEL Science and Technology Center, 11Laboratory of Molecular Biophysics, Department of Cell and Molecular Biology, Uppsala University, 12Australian Synchrotron We describe an experiment designed to probe the electronic damage induced in nanocrystals of Buckminsterfullerene (C60) by intense, femtosecond pulses of X-rays. The experiment found that, surprisingly, rather than being stochastic, the X-ray induced electron dynamics in C60 are highly correlated, extending over hundreds of unit cells within the crystals1. Developmental Biology Dissection and Culture of Mouse Embryonic Kidney Bejan Aresh1, Christiane Peuckert2 1Department of Neuroscience, Uppsala University, 2Department of Organismal Biology, Evolutionary Biology Center, Uppsala University This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos. Immunology and Infection Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils Farzaneh Assadian1, Karl Sandström2, Göran Laurell2, Catharina Svensson1, Göran Akusjärvi1, Tanel Punga1 1Department of Medical Biochemistry and Microbiology, Uppsala Biomedical Center, Uppsala University, 2Department of Surgical Sciences, Otolaryngology and Head & Neck Surgery, Akademiska sjukhuset Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis. Developmental Biology Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments Shahrzad Shirazi Fard1, Maria Blixt1, Finn Hallböök1 1Department of Neuroscience, Biomedical Center, Uppsala University This protocol describes a method to dissect, experimentally manipulate and culture whole retinal explants from chicken embryos. The explant cultures are useful when high success rate, efficacy and reproducibility are needed to test the effects of plasmids for electroporation and/or reagent substances, i.e., enzymatic inhibitors. Chemistry Transport of Surface-modified Carbon Nanotubes through a Soil Column Prabhakar Sharma1,2, Fritjof Fagerlund2 1School of Ecology and Environmental Studies, Nalanda University, 2Department of Earth Sciences, Uppsala University Surface properties of a nanoparticle are important for their interaction with the surrounding medium. Therefore the surface modification of carbon nanotubes can be critical for their transport and retention through porous media. Here, lab scale column experiments are used to understand the possible transport and retention of these nanoparticles. Immunology and Infection A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium Sofia Nordling1, Bo Nilsson1, Peetra U. Magnusson1 1Department of Immunology, Genetics and Pathology, Uppsala University The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy). Biology Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam Stefano Rubino1,4, Petter Melin3, Paul Spellward2, Klaus Leifer1 1Department of Engineering Sciences, Uppsala University, 2Gatan Inc., 3Department of Microbiology, Swedish University of Agricultural Sciences, 4Physics Department, University of Oslo Cryo Electron Microscopes, either Scanning (SEM) or Transmission (TEM), are widely used for characterization of biological samples or other materials with a high water content1. A SEM/Focused Ion Beam (FIB) is used to identify features of interest in samples and extract a thin, electron-transparent lamella for transfer to a cryo-TEM. Biology Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival Per Wessman1, Sebastian Håkansson1, Klaus Leifer2, Stefano Rubino2 1Department of Microbiology, Uppsala Biocenter, Swedish University of Agricultural Sciences, 2Department of Engineering Sciences, Uppsala University Freeze-drying is often an easy and convenient way to obtain dry products of viable bacterial cells. An issue of the process is cell survival. We detail here a procedure to investigate how cell survival during freeze-drying is influenced by the properties of the formulation used. 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 Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas Caroline Kampf1, IngMarie Olsson1, Urban Ryberg1, Evelina Sjöstedt1, Fredrik Pontén1 1Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University Tissue microarrays allows for an efficient method to gain concurrent information from a multitude of tissues. Representative parts of tissues are assembled into a single paraffin block. Sections from the block are used for immunohistochemistry and analysis of protein expression patterns. Digital scanning generates corresponding images for distribution of data. Medicine MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease Jörg Hanrieder1,2, Anna Ljungdahl1, Malin Andersson1 1Department of Pharmaceutical Biosciences, Uppsala University, 2Department of Chemical and Biological Engineering, Chalmers University of Technology Dopamine replacement pharmacotherapy using L-DOPA is the most commonly used symptomatic treatment of Parkinson’s disease, but is accompanied by side effects including involuntary abnormal movements, termed dyskinesia 1. Here, a protocol for MALDI imaging mass spectrometry is presented that detects changes in rat brain neuropeptide levels related to dyskinesia. Biology Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast Pernilla Bjerling1, Ida Olsson1,2, Xi'nan Meng1 1Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, University of Uppsala, 2Department of Microbiology, Swedish University of Agricultural Sciences The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors. Neuroscience GABA-activated Single-channel and Tonic Currents in Rat Brain Slices Zhe Jin1, Yang Jin1, Bryndis Birnir1 1Department of Neuroscience, Uppsala University, Sweden We use the patch-clamp technique to measure GABA-activated single-channel currents (GABAA channels, GABAA receptors) and the synaptic and tonic currents they generate in neurons. Activation of the channels decreases neuronal excitability in health and disease 1,2,3,4. Biology Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species Anna Karlgren1, Jenny Carlsson2, Niclas Gyllenstrand2, Ulf Lagercrantz1, Jens F. Sundström2 1Department of Evolutionary Functional Genomics, Evolutionary Biology Center, Uppsala University, 2Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.