University of Cambridge View Institution's Website 58 articles published in JoVE Neuroscience The "Brain Milking" Method for the Isolation of Neural Stem Cells and Oligodendrocyte Progenitor Cells from Live Rats Dimitrios Dimitrakopoulos1, Chrisitna Dimitriou1, Freyja McClenahan2, Robin J. M. Franklin2,3, Ilias Kazanis1 1Laboratory of Developmental Biology, Department of Biology, University of Patras, 2Wellcome Trust-MRC Cambridge Stem Cell Institute, University of Cambridge, 3Altos Labs, Cambridge Institute of Science A method for the isolation of neural stem cells and oligodendrocyte progenitor cells from the brains of live rats is presented here in experimental detail. It allows multiple collections of these cells from the same animals without compromising their well-being. Medicine A Stable Phantom Material for Optical and Acoustic Imaging Lina Hacker1,2, Aoife M. Ivory3, James Joseph4,5, Janek Gröhl1,2, Bajram Zeqiri3, Srinath Rajagopal3, Sarah E. Bohndiek1,2 1Department of Physics, University of Cambridge, 2Cancer Research UK Cambridge Institute, University of Cambridge, 3Ultrasound and Underwater Acoustics Group, Department of Medical, Marine and Nuclear Physics, National Physical Laboratory, 4School of Science and Engineering, University of Dundee, 5Centre for Medical Engineering and Technology, University of Dundee This protocol describes the fabrication of a stable, biologically relevant phantom material for optical and acoustic biomedical imaging applications, featuring independently tunable acoustic and optical properties. Biology High-Throughput Analysis of Non-Photochemical Quenching in Crops Using Pulse Amplitude Modulated Chlorophyll Fluorometry Dhananjay Gotarkar1,2, Lynn Doran1,2, Meghan Burns1,2, Abigail Hinkle1, Johannes Kromdijk1,3, Steven J. Burgess1,2 1Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 2Department of Plant Biology, Morrill Hall, University of Illinois at Urbana-Champaign, 3Environmental Plant Physiology group, Department of Plant Sciences, University of Cambridge The protocol introduces a high-throughput method for measuring the relaxation of non-photochemical quenching by pulse amplitude modulated chlorophyll fluorometry. The method is applied to field-grown Glycine max and can be adapted to other species to screen for genetic diversity or breeding populations. Immunology and Infection Isolation of Uterine Innate Lymphoid Cells for Analysis by Flow Cytometry Delphine M. Depierreux*1,2, Emily Seshadri*1,2, Evgeniya V. Shmeleva*1,2, Jens Kieckbusch1,2, Delia A. Hawkes1, Francesco Colucci1,2 1Department of Obstetrics and Gynaecology, National Institute for Health Research Cambridge Biomedical Research Centre, University of Cambridge School of Clinical Medicine, 2Centre for Trophoblast Research, University of Cambridge This is a method to isolate uterine lymphoid cells from both pregnant and non-pregnant mice. This method can be used for multiple downstream applications such as FACS phenotyping, cell sorting, functional assays, RNA-seq, and proteomics. The protocol here demonstrates how to phenotype group 1 uterine innate lymphoid cells by flow cytometry. Immunology and Infection Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells Paula Ordonez1, Kate N. Bishop2, Jonathan P. Stoye1, Harriet Cordelia Theed Groom3 1Retrovirus-Host Interactions Laboratory, The Francis Crick Institute, 2Retroviral Replication Laboratory, The Francis Crick Institute, 3Sidney Sussex College, Department of Medicine, University of Cambridge Described here is an established method to determine the extent of HIV-1 restriction by the cellular inhibitory protein SAMHD1. Human myeloid lineage U937 cells are transduced with a SAMHD1 expression vector co-expressing YFP, differentiated and then challenged with HIV-RFP. The level of restriction is determined by flow cytometry analysis. Biology Cryo-Structured Illumination Microscopic Data Collection from Cryogenically Preserved Cells Nina Vyas*1, Nina Perry*1, Chidinma A. Okolo1, Ilias Kounatidis1, Thomas M. Fish1, Kamal L. Nahas1,2, Archana Jadhav1, Mohamed A. Koronfel1, Johannes Groen3, Eva Pereiro3, Ian M. Dobbie4, Maria Harkiolaki1 1Harwell Science and Innovation Campus, Beamline B24, Diamond Light Source, 2Division of Virology, Department of Pathology, University of Cambridge, 3ALBA Synchrotron, Beamline 09 - MISTRAL, 4Micron Advanced Imaging Consortium, Department of Biochemistry, University of Oxford This protocol demonstrates how to image biological cryo-preserved samples using cryo-structured illumination microscopy. We demonstrate the methodology by imaging the cytoskeleton of U2OS cells. Immunology and Infection Live Imaging of Chemokine Receptors in Zebrafish Neutrophils During Wound Responses Antonios Georgantzoglou1, Caroline Coombs1, Hugo Poplimont*1, Hazel A. Walker*1, Milka Sarris1 1Department of Physiology, Development and Neuroscience, University of Cambridge Here we describe protocols to perform live imaging and quantitative analysis of chemoattractant receptor dynamics in zebrafish neutrophils Cancer Research Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone Blake E. Hildreth III1, Charlotte Palmer2, Matthew J. Allen2 1Department of Pathology and O'Neal Comprehensive Cancer Center, University of Alabama at Birmingham, 2Surgical Discovery Centre, Department of Veterinary Medicine, University of Cambridge Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth. Genetics High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology Laetitia Chauve*1, Jérémie Le Pen*2,3,5, Francesca Hodge1, Pia Todtenhaupt1, Laura Biggins1, Eric A. Miska2,3,4, Simon Andrews1, Olivia Casanueva1 1Babraham Institute, 2Gurdon Institute, University of Cambridge, 3Department of Genetics, University of Cambridge, 4Wellcome Trust Genome Campus, Wellcome Trust Sanger Institute, 5Laboratory of Virology and Infectious Disease, The Rockefeller University In this article a high-throughput protocol for fast and reliable determination of gene expression levels in single or bulk C. elegans samples is described. This protocol does not require RNA isolation and produces cDNA directly from samples. It can be used together with high-throughput multiplexed nanofluidic real-time qPCR platforms. Medicine A High-Throughput In Situ Method for Estimation of Hepatocyte Nuclear Ploidy in Mice Fátima Manzano-Núñez1, Ruby Peters2, Deborah J. Burks1,3, Luke A. Noon1,3 1Centro de Investigación Príncipe Felipe (CIPF), 2Department of Physiology, Development and Neuroscience, University of Cambridge, 3Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM) We present a robust, cost-effective, and flexible method for measuring changes in hepatocyte number and nuclear ploidy within fixed/cryopreserved tissue samples that does not require flow cytometry. Our approach provides a powerful sample-wide signature of liver cytology ideal for tracking the progression of liver injury and disease. Biochemistry Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging Maria Lucia Pigazzini*1,2, Christian Gallrein*1, Manuel Iburg*1, Gabriele Kaminski Schierle3, Janine Kirstein1,4 1Leibniz Research Institute for Molecular Pharmacology im Forschungsverbund Berlin, 2NeuroCure Cluster of Excellence, Charité - Universitätsmedizin Berlin, 3Molecular Neuroscience Group, Department of Chemical Engineering and Biotechnology, University of Cambridge, 4Cell Biology, University of Bremen Fluorescence lifetime imaging monitors, quantifies and distinguishes the aggregation tendencies of proteins in living, aging, and stressed C. elegans disease models. Developmental Biology Cell-cell Fusion of Genome Edited Cell Lines for Perturbation of Cellular Structure and Function Robert Mahen1,2, Reiner Schulte3 1Photonics Group, Department of Physics, Imperial College London, 2The Medical Research Council Cancer Unit, Hutchison/MRC Research Centre, Cambridge Biomedical Campus, University of Cambridge, 3Cambridge Institute for Medical Research, Cambridge Biomedical Campus, University of Cambridge The purpose of this protocol is to fuse two different cell types to create hybrid cells. Fluorescence microscopy analysis of fused cells is used to track the cell of origin of cellular organelles. This assay can be used to explore how cellular structure and function respond to perturbation by cell fusion. Biochemistry Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy Francesco Simone Ruggeri1, Tomas Šneideris1,2, Sean Chia1, Michele Vendruscolo1, Tuomas P. J. Knowles1,3 1Centre for Misfolding Diseases, Department of Chemistry, University of Cambridge, 2Institute of Biotechnology, Life Sciences Center, Vilnius University, 3Cavendish Laboratory, Department of Physics, University of Cambridge We describe the application of infrared nanospectroscopy and high-resolution atomic force microscopy to visualize the process of protein self-assembly into oligomeric aggregates and amyloid fibrils, which is closely associated with the onset and development of a wide range of human neurodegenerative disorders. Engineering Scalable Quantum Integrated Circuits on Superconducting Two-Dimensional Electron Gas Platform Kaveh Delfanazari1,2, Pengcheng Ma2, Reuben Puddy2, Teng Yi2, Moda Cao2, Yilmaz Gul3, Carly L. Richardson4, Ian Farrer2,5, David Ritchie2, Hannah J. Joyce1, Michael J. Kelly1,2, Charles G. Smith2 1Centre for Advanced Photonics and Electronics, Engineering Department, University of Cambridge, 2Department of Physics, Cavendish Laboratory, University of Cambridge, 3Department of Electronic and Electrical Engineering, University College London, 4Department of Materials Science and Metallurgy, University of Cambridge, 5Department of Electronic and Electrical Engineering, University of Sheffield Quantum integrated circuits (QICs) consisting of array of planar and ballistic Josephson junctions (JJs) based on In0.75Ga0.25As two-dimensional electron gas (2DEG) is demonstrated. Two different methods for fabrication of the two-dimensional (2D) JJs and QICs are discussed followed by the demonstration of quantum transport measurements in sub-Kelvin temperatures. Immunology and Infection Tuning Degradation to Achieve Specific and Efficient Protein Depletion J. David Barrass1, Gonzalo I. Mendoza-Ochoa1,2, Isabella E. Maudlin1,3, Emanuela Sani1, Jean D. Beggs1 1Wellcome Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, 2Department of Plant Sciences, University of Cambridge, 3Sir William Dunn School of Pathology, University of Oxford Here, we present a protocol to effectively and specifically deplete a protein of interest in the yeast Saccharomyces cerevisiae using the β-est AID system. Neuroscience Electrophoretic Delivery of γ-aminobutyric Acid (GABA) into Epileptic Focus Prevents Seizures in Mice Andrea Slezia*1,5, Christopher M. Proctor*2,3, Attila Kaszas1,4, George G. Malliaras2,3, Adam Williamson1,5 1Aix Marseille Université, Institut de Neurosciences des Systèmes (INS), 2Electrical Engineering Division, University of Cambridge, 3Department of Bioelectronics, Centre Microélectronique de Provence - Ecole Nationale Supérieure des Mines de Saint-Étienne (CMP-EMSE), 4Institut de Neurosciences de la Timone, Centre National de la Recherche Scientifique (CNRS) UMR 7289 & Aix- Marseille Université, 5Neuroengineering Research Group, Interdisciplinary Excellence Center, Department of Medical Microbiology and Immunobiology, University of Szeged The challenge of epilepsy research is to develop novel treatments for patients where classical therapy is inadequate. Using a new protocol—with the help of an implantable drug delivery system—we are able to control seizures in anesthetized mice by the electrophoretic delivery of GABA into the epileptic focus. Immunology and Infection Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens Emily A. Lees1,2, Jessica L. Forbester1,3, Sally Forrest2, Leanne Kane1, David Goulding1, Gordon Dougan1,2 1Wellcome Trust Sanger Institute, 2Department of Medicine, University of Cambridge, 3University of Cardiff Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids offer exciting opportunities to model enteric diseases in vitro. We demonstrate the differentiation of hiPSCs into intestinal organoids (iHOs), the stimulation of these iHOs with cytokines, and the microinjection of Salmonella Typhimurium into the iHO lumen, enabling the study of an epithelial invasion by this pathogen. Genetics qKAT: Quantitative Semi-automated Typing of Killer-cell Immunoglobulin-like Receptor Genes Jyothi Jayaraman1,2,3,4, Vitalina Kirgizova1, Da Di1,5, Christopher Johnson1,6, Wei Jiang1,7, James A. Traherne1 1Department of Pathology, University of Cambridge, 2Department of Physiology, Development and Neuroscience, University of Cambridge, 3Department of Obstetrics and Gynaecology, University of Cambridge School of Medicine, NIHR Cambridge Biomedical Research Centre, 4Centre for Trophoblast Research, University of Cambridge, 5Department of Genetics & Evolution, University of Geneva, 6Royal Papworth Hospital, 7Department of Plant Sciences, University of Cambridge Quantitative killer cell immunoglobulin-like receptor (KIR) semi-automated typing (qKAT) is a simple, high-throughput, and cost-effective method to copy number type KIR genes for their application in population and disease association studies. Developmental Biology Visualizing Cellular Gibberellin Levels Using the nlsGPS1 Förster Resonance Energy Transfer (FRET) Biosensor Annalisa Rizza1, Ankit Walia1, Bijun Tang1, Alexander M. Jones1 1Sainsbury Laboratory, University of Cambridge Gibberellin Perception Sensor 1 (GPS1) is the first Förster resonance energy transfer-based biosensor for measuring the cellular levels of gibberellin phytohormones with a high spatiotemporal resolution. This protocol reports on the method to visualize and quantify cellular gibberellin levels using the genetically encoded nlsGPS1 biosensor in Arabidopsis hypocotyls and root tips. Genetics Separating Bacteria by Capsule Amount Using a Discontinuous Density Gradient Theresa Feltwell1, Matthew J. Dorman1, David A. Goulding1, Julian Parkhill1, Francesca L. Short1,2 1Wellcome Sanger Institute, Wellcome Genome Campus, 2Department of Medicine, University of Cambridge We demonstrate the use of discontinuous density gradients to separate bacterial populations based on capsule production. This method is used to compare capsule amount between cultures, isolate mutants with a specific capsule phenotype, or to identify capsule regulators. Described here is the optimization and running of the assay. Immunology and Infection Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform Michele Perni1, Sam Casford1, Francesco A. Aprile1, Ellen A. Nollen2, Tuomas P.J. Knowles1, Michele Vendruscolo1, Christopher M. Dobson1 1Centre for Misfolding Diseases, Department of Chemistry, University of Cambridge, 2European Research Institute for the Biology of Aging, University Medical Centre Groningen We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion. Immunology and Infection Label-Free Identification of Lymphocyte Subtypes Using Three-Dimensional Quantitative Phase Imaging and Machine Learning Jonghee Yoon1, YoungJu Jo2,3,4,7, Young Seo Kim3,4,5, Yeongjin Yu2,3, Jiyeon Park6, Sumin Lee4, Wei Sun Park2,3, YongKeun Park2,3,4 1Department of Physics, University of Cambridge, 2Department of Physics, Korea Advanced Institute of Science and Technology, 3KAIST Institute for Health Science and Technology, Korea Advanced Institute of Science and Technology, 4Tomocube, Inc., 5Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, 6Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 7Department of Applied Physics, Stanford University We describe a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and a machine learning algorithm. Measurements of 3D refractive index tomograms of lymphocytes present 3D morphological and biochemical information for individual cells, which is then analyzed with a machine-learning algorithm for identification of cell types. Chemistry Analyzing the Photo-oxidation of 2-propanol at Indoor Air Level Concentrations Using Field Asymmetric Ion Mobility Spectrometry Christopher P. Ireland1, Michael Coto1, Lauren Brown2, Russell Paris2, Caterina Ducati1 1Department of Materials Science and Metallurgy, University of Cambridge, 2Owlstone Nanotechnology A protocol for determining the effectiveness of photocatalysts in degrading indoor air concentration (ppb) model volatile organic carbons such as 2-propanol is described. Genetics A Fast and Quantitative Method for Post-translational Modification and Variant Enabled Mapping of Peptides to Genomes Christoph N. Schlaffner1,2,3, Georg J. Pirklbauer2, Andreas Bender3, Judith A.J. Steen1, Jyoti S. Choudhary2,4 1 Here we present the proteogenomic tool PoGo and protocols for fast, quantitative, post-translational modification and variant enabled mapping of peptides identified through mass spectrometry onto reference genomes. This tool is of use to integrate and visualize proteogenomic and personal proteomic studies interfacing with orthogonal genomics data. Developmental Biology Combinational Treatment of Trichostatin A and Vitamin C Improves the Efficiency of Cloning Mice by Somatic Cell Nuclear Transfer Rika Azuma1, Kei Miyamoto2, Mami Oikawa3, Masayasu Yamada4, Masayuki Anzai1,5 1Division of Biological Science, Graduate School of Biology-Oriented Science and Technology, Kindai University, 2Faculty of Biology-Oriented Science and Technology, Kindai University, 3Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Zoology, University of Cambridge, 4Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, 5Institute of Advanced Technology, Kindai University We describe a dramatically improved method for mouse cloning using trichostatin A, vitamin C, and deionized bovine serum albumin. We show a simplified, reproducible protocol that supports efficient development of cloned embryos. Hence, this method could become a standardized procedure for mouse cloning. Neuroscience Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts Shelby Shrigley1, Karolina Pircs1, Roger A. Barker1,2, Malin Parmar1, Janelle Drouin-Ouellet1 1Department of Experimental Medical Science, Wallenberg Neuroscience Center, Division of Neurobiology and Lund Stem Cell Center, Lund University, 2John van Geest Centre for Brain Repair & Department of Neurology, Department of Clinical Neurosciences and Cambridge Stem Cell Institute, University of Cambridge Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors. Chemistry Reliable Mechanochemistry: Protocols for Reproducible Outcomes of Neat and Liquid Assisted Ball-mill Grinding Experiments Ana M. Belenguer1, Giulio I. Lampronti1,2, Jeremy K. M. Sanders1 1Department of Chemistry, University of Cambridge, 2Department of Earth Sciences, University of Cambridge We present detailed procedures to produce experimental equilibrium curves of the phase composition as a function of solvent concentration in a solid state system under milling conditions. Bioengineering A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer Alessandra Merenda1,2, Amanda Andersson-Rolf1,2, Roxana C. Mustata1, Taibo Li1, Hyunki Kim3, Bon-Kyoung Koo1,2 1Wellcome Trust - Medical Research Council Stem Cell Institute, University of Cambridge, 2Department of Genetics, University of Cambridge, 3Department of Pathology, Yonsei University College of Medicine This protocol describes the steps for cloning multiple single guide RNAs into one guide RNA concatemer vector, which is of particular use in creating multi-gene knockouts using CRISPR/Cas9 technology. The generation of double knockouts in intestinal organoids is shown as a possible application of this method. Developmental Biology Induction of Hypoxia in Living Frog and Zebrafish Embryos Helena Khaliullina-Skultety1, Ngiam Zi Chao1, William A. Harris1 1Department of Physiology, Development and Neuroscience, University of Cambridge We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia. Neuroscience Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors Ben Katz*1, Rita Gutorov*1, Elisheva Rhodes-Mordov1, Roger C. Hardie2, Baruch Minke1 1Department of Medical Neurobiology, Faculty of Medicine and the Edmond and Lily Safra Center for Brain Sciences (ELSC), Hebrew University, 2Department of Physiology, Development and Neuroscience, University of Cambridge Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel. Neuroscience Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration Menghon Cheah1, James W. Fawcett1, Melissa R. Andrews2,3 1John van Geest Center for Brain Repair, University of Cambridge, 2School of Medicine, University of St. Andrews, 3Department of Biological Sciences, University of Southampton This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration. Biology Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells Arianna Psichas*1, Gwen Tolhurst*1, Cheryl A. Brighton1, Fiona M. Gribble1, Frank Reimann1 1Metabolic Research Laboratories and MRC Metabolic Diseases Unit, Wellcome Trust-MRC Institute of Metabolic Science, University of Cambridge This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques. Engineering Monovalent Cation Doping of CH3NH3PbI3 for Efficient Perovskite Solar Cells Mojtaba Abdi-Jalebi1, M. Ibrahim Dar2, Aditya Sadhanala1, Satyaprasad P. Senanayak1, Michael Grätzel2, Richard H. Friend1 1Cavendish Laboratory, University of Cambridge, 2Institute of Chemical Sciences and Engineering, École Polytechnique Fédérale de Lausanne Here, we present a protocol to adjust the properties of solution-processed CH3NH3PbI3 through the incorporation of monovalent cation additives in order to achieve highly efficient perovskite solar cells. Developmental Biology A Versatile Mounting Method for Long Term Imaging of Zebrafish Development Estelle Hirsinger1,3, Ben Steventon1,2 1Department of Developmental and Stem Cell Biology, Institut Pasteur, 2Department of Genetics, University of Cambridge, 3IBPS- Laboratoire de Biologie du Developpement (LBD), CNRS, UPMC, UMR 7622, INSERM ERL U1156 Here, we present a versatile mounting method that allows for the long-term time-lapse imaging of the posterior body development of live zebrafish embryos without perturbing normal development. Genetics High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii Michal Breker1, Kristi Lieberman1, Frej Tulin2, Frederick R. Cross1 1Laboratory of Cell Cycle Genetics, The Rockefeller University, 2Sainsbury Laboratory, University of Cambridge Temperature-sensitive (ts) lethal mutants are valuable tools to identify and analyze essential functions. Here we describe methods to generate and classify ts lethal mutants in high throughput. Behavior The 4 Mountains Test: A Short Test of Spatial Memory with High Sensitivity for the Diagnosis of Pre-dementia Alzheimer's Disease Dennis Chan1, Laura Marie Gallaher2, Kuven Moodley2, Ludovico Minati3, Neil Burgess4, Tom Hartley5 1Department of Clinical Neurosciences, University of Cambridge, 2Clinical Imaging Sciences Centre, Brighton and Sussex Medical School, 3U.O. Direzione Scientifica, Fondazione IRCCS Istituto Neurologico Carlo Besta, 4Institute of Cognitive Neuroscience, University College London, 5Department of Psychology, University of York This article describes the 4 Mountains Test (4MT), a hippocampus-dependent test of working allocentric spatial memory. The hippocampus is affected early in Alzheimer's disease (AD) and this article outlines the 4MT methodology and results of patient testing, which demonstrates the value of the 4MT in the diagnosis of pre-dementia AD. Developmental Biology Imaging Calcium in Drosophila at Egg Activation Christopher J. Derrick*1, Anna H. York-Andersen*1, Timothy T. Weil1 1Department of Zoology, University of Cambridge This article describes an adaptable ex vivo protocol for visualizing Ca2+ during egg activation in Drosophila. Chemistry Improved Heterojunction Quality in Cu2O-based Solar Cells Through the Optimization of Atmospheric Pressure Spatial Atomic Layer Deposited Zn1-xMgxO Yulia Ievskaya1, Robert L. Z. Hoye1, Aditya Sadhanala2, Kevin P. Musselman2, Judith L. MacManus-Driscoll1 1Department of Materials Science and Metallurgy, University of Cambridge, 2Cavendish Laboratory, University of Cambridge Here we present a protocol for synthesizing Zn1-xMgxO/Cu2O heterojunctions in open-air at low temperature via atmospheric pressure spatial atomic layer deposition (AP-SALD) of Zn1-xMgxO on cuprous oxide. Such high quality conformal metal oxides can be grown on a variety of substrates including plastics by this cheap and scalable method. Bioengineering A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors Laurence J. Young1, Florian Ströhl1, Clemens F. Kaminski1 1Department of Chemical Engineering and Biotechnology, University of Cambridge This article provides an in depth guide for the assembly and operation of a structured illumination microscope operating with total internal reflection fluorescence illumination (TIRF-SIM) to image dynamic biological processes with optical super-resolution in multiple colors. Biology Generation of Marked and Markerless Mutants in Model Cyanobacterial Species David J. Lea-Smith1, Ravendran Vasudevan1, Christopher J. Howe1 1Department of Biochemistry, University of Cambridge Introducing multiple genomic alterations into cyanobacteria is an essential tool in the development of strains for industrial and basic research purposes. We describe a system for generating unmarked mutants in the model cyanobacterial species Synechocystis sp. PCC6803 and marked mutants in Synechococcus sp. PCC7002. Developmental Biology Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos Florence A. Giger1, Julien G. Dumortier2, Nicolas B. David1 1 Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration. Biology Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood Mark L. Ormiston1, Mark R. Toshner2, Fedir N. Kiskin1, Christopher J. Z. Huang1, Emily Groves1, Nicholas W. Morrell1, Amer A. Rana1 1Department of Medicine, University of Cambridge, 2Papworth Hospital This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells. Developmental Biology Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus Jens Kieckbusch1,2, Louise M. Gaynor1,2, Francesco Colucci1,2 1Department of Obstetrics and Gynaecology, School of Clinical Medicine, University of Cambridge, 2Centre for Trophoblast Research, University of Cambridge This protocol describes a technique to assess changes in the maternal vasculature during pregnancy in mice. Using stereological methods, remodeling of the decidual spiral arteries is assessed quantitatively and the results confirmed qualitatively using immunohistochemistry. Developmental Biology Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro Peter Baillie-Johnson1, Susanne Carina van den Brink1,2, Tina Balayo1, David Andrew Turner1, Alfonso Martinez Arias1 1Department of Genetics, University of Cambridge, 2Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences We have developed a protocol to generate aggregates of mouse embryonic stem cells that display self-organization, symmetry breaking and elongation paralleling axial development. This technique allows the study of axial developmental processes and the generation of cell types that are otherwise difficult to perform in monolayer culture. Developmental Biology Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development Kei Miyamoto1,2, David Simpson1,2, John B. Gurdon1,2 1Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, 2Department of Zoology, University of Cambridge We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development. Medicine Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice Maria E. Laucho-Contreras1, Katherine L. Taylor1, Ravi Mahadeva2, Steve S. Boukedes3, Caroline A. Owen1,4 1 The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice. Immunology and Infection Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes Martin Wagner1, Helen Koester1, Christian Deffge1, Soenke Weinert1, Johannes Lauf1, Alexander Francke2, Jerry Lee3, R. C. Braun- Dullaeus1, Joerg Herold1 1Department for Cardiology, Angiology and Pneumology, Otto von Guericke University Magdeburg, 2Herzzentrum Dresden, Universitätsklinikum an der Technischen Universität Dresden, Technische Universität Dresden, 3Department of Public Health and Primary Care, University of Cambridge Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS). Biology Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo Chih Hung Ku1, Brian J. Ferguson1 1Department of Pathology, University of Cambridge The aim of the protocol is to use optimal methods for stimulating the cytoplasmic DNA sensing pathways in cells and in vivo. This is achieved by improving the generation of long, double-stranded DNA during blunt-end ligation. Cells or mice are then transfected using a lipid transfection reagent. Biology A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture Amanda Andersson-Rolf1,2, Juergen Fink1,2, Roxana C. Mustata2, Bon-Kyoung Koo1,2 1Department of Genetics, University of Cambridge, 2Wellcome Trust - Medical Research Council Stem Cell Institute, University of Cambridge This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system. Biology Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics Edward Emmott1, Ian Goodfellow1 1Division of Virology, Department of Pathology, University of Cambridge SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions. Immunology and Infection Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE Matteo Donegà1, Elena Giusto1, Chiara Cossetti1, Julia Schaeffer1, Stefano Pluchino1,2 1Department of Clinical Neurosciences, John Van Geest Centre for Brain Repair, University of Cambridge, UK, 2NIHR Biomedical Research Center, University of Cambridge, UK The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system. Behavior Studying Food Reward and Motivation in Humans Hisham Ziauddeen1,2,3, Naresh Subramaniam1, Victoria C. Cambridge4, Nenad Medic1,2, Ismaa Sadaf Farooqi2, Paul C. Fletcher1,2,3 1Department of Psychiatry, University of Cambridge, 2Metabolic Research Laboratories, Wellcome Trust-MRC Institute of Metabolic Science, University of Cambridge, 3Cambridgeshire & Peterborough NHS Foundation Trust, University of Cambridge, 4 This article describes a set of methods for the measurement of food related motivation and food related goal values in humans. Medicine MRI and PET in Mouse Models of Myocardial Infarction Guido Buonincontri1, Carmen Methner2, T. Adrian Carpenter1, Robert C. Hawkes1, Stephen J. Sawiak1,3, Thomas Krieg2 1Wolfson Brain Imaging Centre, Department of Clinical Neurosciences, Unversity of Cambridge, 2Department of Medicine, University of Cambridge, 3Behavioural and Clinical Neurosciences Institute, University of Cambridge We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure. Biology Robust 3D DNA FISH Using Directly Labeled Probes Daniel J. Bolland1, Michelle R. King2, Wolf Reik2,3, Anne E. Corcoran1, Christel Krueger2 1Nuclear Dynamics Programme, The Babraham Institute, 2Epigenetics Programme, The Babraham Institute, 3Centre for Trophoblast Research, University of Cambridge We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes. Biology Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture Bernd Rädle*1, Andrzej J. Rutkowski*2, Zsolt Ruzsics1, Caroline C. Friedel3, Ulrich H. Koszinowski1, Lars Dölken2 1Max von Pettenkofer Institute, 2Department of Medicine, University of Cambridge, 3Institute for Informatics, Ludwig-Maximilians-University Munich Total cellular RNA provides a poor template for studying short-term changes in RNA synthesis and decay as well as the kinetics of RNA processing. Here, we describe metabolic labeling of newly transcribed RNA with 4-thiouridine followed by thiol-specific biotinylation and purification of newly transcribed RNA allowing to overcome these limitations. Neuroscience Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique Samsudeen Ponissery Saidu*1, Michele Dibattista*1, Hugh R. Matthews2, Johannes Reisert1 1Monell Chemical Senses Center, 2Department of Physiology, Development & Neuroscience, Physiological Laboratory, University of Cambridge Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs. Immunology and Infection Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging Joel R. Meyerson1,2, Tommi A. White1, Donald Bliss3, Amy Moran3, Alberto Bartesaghi1, Mario J. Borgnia1, M. Jason V. de la Cruz1, David Schauder1, Lisa M. Hartnell1, Rachna Nandwani1,4, Moez Dawood5, Brianna Kim6, Jun Hong Kim7, John Sununu8, Lisa Yang9, Siddhant Bhatia10, Carolyn Subramaniam1, Darrell E. Hurt11, Laurent Gaudreault12, Sriram Subramaniam1 1Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 2The Medical Research Council Mitochondrial Biology Unit, University of Cambridge, 3National Library of Medicine, National Institutes of Health, 4Massachusetts Institute of Technology, 5William Fremd High School, 6University of Virginia, 7Duke University, 8Yale University, 9University of Notre Dame, 10Washington University in St. Louis, 11Bioinformatics and Computational Biosciences Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12Thomas Jefferson High School for Science and Technology The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis. Biology Analysis of Schwann-astrocyte Interactions Using In Vitro Assays Fardad T. Afshari1, Jessica C. Kwok1, James W. Fawcett1 1Cambridge Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.