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59 ARTICLES PUBLISHED IN JoVE

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Biology

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays
Fardad T. Afshari 1, Jessica C. Kwok 1, James W. Fawcett 1
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.

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Biology

Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging
Derek J. Averill *1, Joel Garcia *1, Buddhima N. Siriwardena-Mahanama *1, Sashiprabha M. Vithanarachchi *1, Matthew J. Allen 1
1Department of Chemistry, Wayne State University

We demonstrate the metalation, purification, and characterization of lanthanide complexes. The complexes described here can be conjugated to macromolecules to enable tracking of these molecules using magnetic resonance imaging.

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Biology

Identification of Protein Interacting Partners Using Tandem Affinity Purification
Dalan Bailey *1, Luis Urena *1, Lucy Thorne 1, Ian Goodfellow 1
1Section of Virology, Department of Medicine, Imperial College London

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

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Biology

Preparing Individual Drosophila Egg Chambers for Live Imaging
Timothy T. Weil 1, Richard M. Parton 1, Ilan Davis 1
1Department of Biochemistry, University of Oxford

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

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Immunology and Infection

Reverse Genetics Mediated Recovery of Infectious Murine Norovirus
Armando Arias *1, Luis Ureña *1, Lucy Thorne 1, Muhammad A. Yunus 1, Ian Goodfellow 1
1Section of Virology, Imperial College London

Noroviruses are a major cause of gastroenteritis yet molecular techniques for their characterisation are still relatively new. Here we report two different reverse genetics approaches for the efficient recovery of murine norovirus (MNV), the only member of this genus which can be propagated in cell culture.

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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 Ruzsics 1, Caroline C. Friedel 3, Ulrich H. Koszinowski 1, Lars Dölken 2
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.

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Medicine

MRI and PET in Mouse Models of Myocardial Infarction
Guido Buonincontri 1, Carmen Methner 2, T. Adrian Carpenter 1, Robert C. Hawkes 1, Stephen J. Sawiak 1,3, Thomas Krieg 2
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.

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Behavior

Studying Food Reward and Motivation in Humans
Hisham Ziauddeen 1,2,3, Naresh Subramaniam 1, Victoria C. Cambridge 4, Nenad Medic 1,2, Ismaa Sadaf Farooqi 2, Paul C. Fletcher 1,2,3
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, 4West Anglia Comprehensive Local Research Network, Addenbrooke's Hospital

This article describes a set of methods for the measurement of food related motivation and food related goal values in humans.

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Biology

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo
Chih Hung Ku 1, Brian J. Ferguson 1
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.

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Biology

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics
Edward Emmott 1, Ian Goodfellow 1
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.

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Biology

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture
Amanda Andersson-Rolf 1,2, Juergen Fink 1,2, Roxana C. Mustata 2, Bon-Kyoung Koo 1,2
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.

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Immunology and Infection

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes
Martin Wagner 1, Helen Koester 1, Christian Deffge 1, Soenke Weinert 1, Johannes Lauf 1, Alexander Francke 2, Jerry Lee 3, R. C. Braun- Dullaeus 1, Joerg Herold 1
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).

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Developmental Biology

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development
Kei Miyamoto 1,2, David Simpson 1,2, John B. Gurdon 1,2
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.

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Developmental Biology

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro
Peter Baillie-Johnson 1, Susanne Carina van den Brink 1,2, Tina Balayo 1, David Andrew Turner 1, Alfonso Martinez Arias 1
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.

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Biology

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood
Mark L. Ormiston 1, Mark R. Toshner 2, Fedir N. Kiskin 1, Christopher J. Z. Huang 1, Emily Groves 1, Nicholas W. Morrell 1, Amer A. Rana 1
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.

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Chemistry

Improved Heterojunction Quality in Cu2O-based Solar Cells Through the Optimization of Atmospheric Pressure Spatial Atomic Layer Deposited
Zn1-xMgxO
Yulia Ievskaya 1, Robert L. Z. Hoye 1, Aditya Sadhanala 2, Kevin P. Musselman 2, Judith L. MacManus-Driscoll 1
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.

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Developmental Biology

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus
Jens Kieckbusch 1,2, Louise M. Gaynor 1,2, Francesco Colucci 1,2
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.

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Developmental Biology

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos
Florence A. Giger 1, Julien G. Dumortier 2, Nicolas B. David 1
1CNRS UMR8197 – INSERM U1024, IBENS, Institut de Biologie de l'École Normale Supérieure, 2Department of Physiology Development and Neuroscience, University of Cambridge

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.

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Bioengineering

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors
Laurence J. Young 1, Florian Ströhl 1, Clemens F. Kaminski 1
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.

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Biology

Generation of Marked and Markerless Mutants in Model Cyanobacterial Species
David J. Lea-Smith 1, Ravendran Vasudevan 1, Christopher J. Howe 1
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.

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Chemistry

Analyzing the Photo-oxidation of 2-propanol at Indoor Air Level Concentrations Using Field Asymmetric Ion Mobility Spectrometry
Christopher P. Ireland 1, Michael Coto 1, Lauren Brown 2, Russell Paris 2, Caterina Ducati 1
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.

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Developmental Biology

Imaging Calcium in Drosophila at Egg Activation
Christopher J. Derrick *1, Anna H. York-Andersen *1, Timothy T. Weil 1
1Department of Zoology, University of Cambridge

This article describes an adaptable ex vivo protocol for visualizing Ca2+ during egg activation in Drosophila.

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Behavior

The 4 Mountains Test: A Short Test of Spatial Memory with High Sensitivity for the Diagnosis of Pre-dementia Alzheimer's Disease
Dennis Chan 1, Laura Marie Gallaher 2, Kuven Moodley 2, Ludovico Minati 3, Neil Burgess 4, Tom Hartley 5
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.

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JoVE Journal

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii
Michal Breker 1, Kristi Lieberman 1, Frej Tulin 2, Frederick R. Cross 1
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.

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Developmental Biology

A Versatile Mounting Method for Long Term Imaging of Zebrafish Development
Estelle Hirsinger 1,3, Ben Steventon 1,2
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.

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Engineering

Monovalent Cation Doping of CH3NH3PbI3 for Efficient Perovskite Solar Cells
Mojtaba Abdi-Jalebi 1, M. Ibrahim Dar 2, Aditya Sadhanala 1, Satyaprasad P. Senanayak 1, Michael Grätzel 2, Richard H. Friend 1
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.

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Neuroscience

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration
Menghon Cheah 1, James W. Fawcett 1, Melissa R. Andrews 2,3
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.

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Neuroscience

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors
Ben Katz *1, Rita Gutorov *1, Elisheva Rhodes-Mordov 1, Roger C. Hardie 2, Baruch Minke 1
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.

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Developmental Biology

Induction of Hypoxia in Living Frog and Zebrafish Embryos
Helena Khaliullina-Skultety 1, Ngiam Zi Chao 1, William A. Harris 1
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.

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Bioengineering

A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer
Alessandra Merenda 1,2, Amanda Andersson-Rolf 1,2, Roxana C. Mustata 1, Taibo Li 1, Hyunki Kim 3, Bon-Kyoung Koo 1,2
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.

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Chemistry

Reliable Mechanochemistry: Protocols for Reproducible Outcomes of Neat and Liquid Assisted Ball-mill Grinding Experiments
Ana M. Belenguer 1, Giulio I. Lampronti 1,2, Jeremy K. M. Sanders 1
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.

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Neuroscience

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts
Shelby Shrigley 1, Karolina Pircs 1, Roger A. Barker 1,2, Malin Parmar 1, Janelle Drouin-Ouellet 1
1Department of Experimental Medical Science, Wallenberg Neuroscience Center, Division of Neurobiology and Lund Stem Cell Center, Lund University, 2John van Geest Centre for Brain Repair & Department of Neurology, Department of Clinical Neurosciences and Cambridge Stem Cell Institute, University of Cambridge

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

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Developmental Biology

Combinational Treatment of Trichostatin A and Vitamin C Improves the Efficiency of Cloning Mice by Somatic Cell Nuclear Transfer
Rika Azuma 1, Kei Miyamoto 2, Mami Oikawa 3, Masayasu Yamada 4, Masayuki Anzai 1,5
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.

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Genetics

A Fast and Quantitative Method for Post-translational Modification and Variant Enabled Mapping of Peptides to Genomes
Christoph N. Schlaffner 1,2,3, Georg J. Pirklbauer 2, Andreas Bender 3, Judith A.J. Steen 1, Jyoti S. Choudhary 2,4
1Department of Neurobiology, F. M. Kirby Neurobiology Center, Boston Children's Hospital, Harvard Medical School, 2Proteomic Mass Spectrometry, Wellcome Trust Sanger Institute, Wellcome Genome Campus, 3Centre for Molecular Informatics, Department of Chemistry, University of Cambridge, 4Functional Proteomics Group, Chester Beatty Laboratories, Institute of Cancer Research

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.

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JoVE Core

Scalable Quantum Integrated Circuits on Superconducting Two-Dimensional Electron Gas Platform
Kaveh Delfanazari 1,2, Pengcheng Ma 2, Reuben Puddy 2, Teng Yi 2, Moda Cao 2, Yilmaz Gul 3, Carly L. Richardson 4, Ian Farrer 2,5, David Ritchie 2, Hannah J. Joyce 1, Michael J. Kelly 1,2, Charles G. Smith 2
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.

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Immunology and Infection

Label-Free Identification of Lymphocyte Subtypes Using Three-Dimensional Quantitative Phase Imaging and Machine Learning
Jonghee Yoon 1, YoungJu Jo 2,3,4,7, Young Seo Kim 3,4,5, Yeongjin Yu 2,3, Jiyeon Park 6, Sumin Lee 4, Wei Sun Park 2,3, YongKeun Park 2,3,4
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.

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Immunology and Infection

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform
Michele Perni 1, Sam Casford 1, Francesco A. Aprile 1, Ellen A. Nollen 2, Tuomas P.J. Knowles 1, Michele Vendruscolo 1, Christopher M. Dobson 1
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.

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Genetics

qKAT: Quantitative Semi-automated Typing of Killer-cell Immunoglobulin-like Receptor Genes
Jyothi Jayaraman 1,2,3,4, Vitalina Kirgizova 1, Da Di 1,5, Christopher Johnson 1,6, Wei Jiang 1,7, James A. Traherne 1
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.

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Genetics

Separating Bacteria by Capsule Amount Using a Discontinuous Density Gradient
Theresa Feltwell 1, Matthew J. Dorman 1, David A. Goulding 1, Julian Parkhill 1, Francesca L. Short 1,2
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.

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Developmental Biology

Visualizing Cellular Gibberellin Levels Using the nlsGPS1 Förster Resonance Energy Transfer (FRET) Biosensor
Annalisa Rizza 1, Ankit Walia 1, Bijun Tang 1, Alexander M. Jones 1
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.

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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 Kaszas 1,4, George G. Malliaras 2,3, Adam Williamson 1,5
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.

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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. Lees 1,2, Jessica L. Forbester 1,3, Sally Forrest 2, Leanne Kane 1, David Goulding 1, Gordon Dougan 1,2
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.

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Immunology and Infection

Tuning Degradation to Achieve Specific and Efficient Protein Depletion
J. David Barrass 1, Gonzalo I. Mendoza-Ochoa 1,2, Isabella E. Maudlin 1,3, Emanuela Sani 1, Jean D. Beggs 1
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.

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Medicine

A High-Throughput In Situ Method for Estimation of Hepatocyte Nuclear Ploidy in Mice
Fátima Manzano-Núñez 1, Ruby Peters 2, Deborah J. Burks 1,3, Luke A. Noon 1,3
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.

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Biochemistry

Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy
Francesco Simone Ruggeri 1, Tomas Šneideris 1,2, Sean Chia 1, Michele Vendruscolo 1, Tuomas P. J. Knowles 1,3
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.

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Developmental Biology

Cell-cell Fusion of Genome Edited Cell Lines for Perturbation of Cellular Structure and Function
Robert Mahen 1,2, Reiner Schulte 3
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.

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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 Schierle 3, Janine Kirstein 1,4
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.

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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 Hodge 1, Pia Todtenhaupt 1, Laura Biggins 1, Eric A. Miska 2,3,4, Simon Andrews 1, Olivia Casanueva 1
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.

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Cancer Research

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone
Blake E. Hildreth III 1, Charlotte Palmer 2, Matthew J. Allen 2
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.

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Immunology and Infection

Live Imaging of Chemokine Receptors in Zebrafish Neutrophils During Wound Responses
Antonios Georgantzoglou 1, Caroline Coombs 1, Hugo Poplimont *1, Hazel A. Walker *1, Milka Sarris 1
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

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Biology

Cryo-Structured Illumination Microscopic Data Collection from Cryogenically Preserved Cells
Nina Vyas *1, Nina Perry *1, Chidinma A. Okolo 1, Ilias Kounatidis 1, Thomas M. Fish 1, Kamal L. Nahas 1,2, Archana Jadhav 1, Mohamed A. Koronfel 1, Johannes Groen 3, Eva Pereiro 3, Ian M. Dobbie 4, Maria Harkiolaki 1
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.

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Immunology and Infection

Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells
Paula Ordonez 1, Kate N. Bishop 2, Jonathan P. Stoye 1, Harriet Cordelia Theed Groom 3
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.

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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 Kieckbusch 1,2, Delia A. Hawkes 1, Francesco Colucci 1,2
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.

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Biology

High-Throughput Analysis of Non-Photochemical Quenching in Crops Using Pulse Amplitude Modulated Chlorophyll Fluorometry
Dhananjay Gotarkar 1,2, Lynn Doran 1,2, Meghan Burns 1,2, Abigail Hinkle 1, Johannes Kromdijk 1,3, Steven J. Burgess 1,2
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.

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Neuroscience

The "Brain Milking" Method for the Isolation of Neural Stem Cells and Oligodendrocyte Progenitor Cells from Live Rats
Dimitrios Dimitrakopoulos 1, Chrisitna Dimitriou 1, Freyja McClenahan 2, Robin J. M. Franklin 2,3, Ilias Kazanis 1
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.

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Medicine

A Stable Phantom Material for Optical and Acoustic Imaging
Lina Hacker 1,2, Aoife M. Ivory 3, James Joseph 4,5, Janek Gröhl 1,2, Bajram Zeqiri 3, Srinath Rajagopal 3, Sarah E. Bohndiek 1,2
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.

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Biology

Intrafemoral Injection of Human Hematopoietic Stem and Progenitor Cells into Immunocompromised Mice
Emily F. Calderbank 1, Laura Magnani 1, Elisa Laurenti 1
1Wellcome and Medical Research Council Cambridge Stem Cell Institute, Department of Haematology, University of Cambridge

Intrafemoral injections allow for the engraftment of a small number of hematopoietic stem and progenitor cells (HSPCs), by placing the cells directly in the bone marrow cavity. Here we describe an experimental protocol of intrafemoral injection of human HSPCs into immunodeficient mice.

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Neuroscience

Home-Based EEG Hyperscanning for Infant-Caregiver Social Interactions
Vaidehi Ramanarayanan *1, Qian Chern Oon *1, Amritha Varshini Devarajan 1, Stanimira Georgieva 1,2, Vanessa Reindl 1,3
1Department of Psychology, School of Social Sciences, Nanyang Technological University, 2Department of Pediatrics, University of Cambridge, 3Child Neuropsychology Section, Department of Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy, Medical Faculty, RWTH Aachen University

This protocol describes how synchronized electroencephalography, electrocardiography, and behavioral recordings were captured from infant-caregiver dyads in a home setting.

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Bioengineering

Synthetic Condensates and Cell-Like Architectures from Amphiphilic DNA Nanostructures
Layla Malouf 1,2, Diana A. Tanase 1,2, Lorenzo Di Michele 1,2,3
1Department of Chemical Engineering and Biotechnology, University of Cambridge, 2Department of Chemistry, Imperial College London, 3fabriCELL, Imperial College London

We present a protocol for preparing synthetic biomolecular condensates consisting of amphiphilic DNA nanostars starting from their constituent DNA oligonucleotides. Condensates are produced from either a single nanostar component or two components and are modified to sustain in vitro transcription of RNA from an embedded DNA template.

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