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66 ARTICLES PUBLISHED IN JoVE

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JoVE Journal

Use of Human Perivascular Stem Cells for Bone Regeneration
Aaron W. James *1, Janette N. Zara *2, Mirko Corselli 2, Michael Chiang 1, Wei Yuan 2, Virginia Nguyen 1, Asal Askarinam 1, Raghav Goyal 1, Ronald K. Siu 3, Victoria Scott 1, Min Lee 3, Kang Ting 1, Bruno Péault 2,4, Chia Soo 2
1Dental and Craniofacial Research Institute and Section of Orthodontics, School of Dentistry, UCLA, 2UCLA and Orthopaedic Hospital, Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, UCLA, 3Department of Bioengineering, UCLA, 4Center for Cardiovascular Science, University of Edinburgh

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

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Biology

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations
Claire N. Medine 1, Baltasar Lucendo-Villarin 1, Wenli Zhou 1, Christopher C. West 1, David C. Hay 1
1Medical Research Council Centre for Regenerative Medicine, University of Edinburgh

This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.

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Biology

Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography
Alexander D. Leeper 1, Joanne Farrell 2, J. Michael Dixon 1, Sarah E. Wedden 2, David J. Harrison 1, Elad Katz 1
1Breakthrough Breast Cancer Research Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, 2MRC Technology

We have developed a collagen-based in vitro assay which promotes proliferation and invasion from samples of all breast cancer subtypes. Optical Projection Tomography, a three dimensional microscopy technique was utilised to visualise and quantify tumour expansion. This assay may be used to quantify drug response of individual tumour samples.

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Immunology and Infection

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells
Antoine Claessens 1, J. Alexandra Rowe 1
1Centre for Immunity, Infection and Evolution, University of Edinburgh

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

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Neuroscience

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes
Giselle Cheung 1, Michael A. Cousin 1
1Membrane Biology Group, Centre for integrative Physiology, University of Edinburgh

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

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Medicine

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence
Dana Faratian 1, Jason Christiansen 2, Mark Gustavson 2, Christine Jones 2, Christopher Scott 2, InHwa Um 1, David J. Harrison 1
1Division of Pathology, University of Edinburgh, 2HistoRx Inc.

Here we describe a method to quantify molecular heterogeneity in histological sections of tumor material using quantitative immunofluorescence, image analysis, and a statistical measure of heterogeneity. The method is intended for use in clinical biomarker development and analysis.

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Biology

Genomic Transformation of the Picoeukaryote Ostreococcus tauri
Gerben van Ooijen 1, Kirsten Knox 1, Katalin Kis 1, François-Yves Bouget 2,3, Andrew J. Millar 1
1SynthSys, University of Edinburgh , 2Centre National de la Recherche Scientifique, Université Pierre et Marie Curie, Paris 06, 3UMR 7621, Laboratoire d'Océanographie Microbienne, Observatoire Océanologique, Banyuls-sur-Mer, Université Pierre et Marie Curie, Paris 06

This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.

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Medicine

The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers
Fiach C. O'Mahony 1, Jyoti Nanda 1, Alexander Laird 1, Peter Mullen 2, Helen Caldwell 3, Ian M. Overton 4, Lel Eory 4, Marie O'Donnell 1,5, Dana Faratian 6, Thomas Powles 7, David J. Harrison 1,2, Grant D. Stewart 1
1Edinburgh Urological Cancer Group, University of Edinburgh, 2School of Medicine, University of St Andrews, 3Division of Pathology, University of Edinburgh, 4MRC Human Genetics Unit, MRC IGMM, University of Edinburgh, 5Department of Pathology, Western General Hospital, 6Breakthrough Breast Cancer Research Unit, University of Edinburgh, 7St Bartholomew's Cancer Institute, Experimental Cancer Medicine Centre, Queen Mary University of London

RPPA enables the protein expression of hundreds of samples, printed on nitrocellulose slides to be interrogated simultaneously, using fluorescently labelled antibodies. This technique has been applied to study the effect of drug treatment heterogeneity within clear cell renal carcinoma.

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Medicine

In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury
Marie-Claire Edmunds 1, Stephen Wigmore 1, David Kluth 2
1Department of Surgery, Royal Infirmary of Edinburgh, 2Department of Nephrology, Royal Infirmary of Edinburgh

Free tissue transfer is widely employed in reconstructive surgery to restore form and function following oncological resection and trauma. Preconditioning this tissue prior to surgery may improve outcome. This article describes an in situ transverse rectus abdominis myocutaneous flap (TRAM) in rats as a means for testing preconditioning strategies.

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Engineering

Sputter Growth and Characterization of Metamagnetic B2-ordered FeRh Epilayers
Chantal Le Graët 1, Mark A. de Vries 1,2, Mathew McLaren 1,3, Richard M.D. Brydson 3, Melissa Loving 4, Don Heiman 5, Laura H. Lewis 4, Christopher H. Marrows 1
1School of Physics and Astronomy, University of Leeds, 2Institute of Materials Research, University of Leeds, 3School of Chemistry, University of Edinburgh, 4Department of Chemical Engineering, Northeastern University, 5Department of Physics, Northeastern University

A method to prepare epitaxial layers of ordered alloys by sputtering is described. The B2-ordered FeRh compound is used as an example, as it displays a metamagnetic transition that depends sensitively on the degree of chemical order and the exact composition of the alloy.

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Immunology and Infection

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin
Cecile Benezech 1,2, Jorge H. Caamano 1
1School of Immunity and Infection, IBR-MRC Centre for Immune Regulation, College of Medical and Dental Sciences, University of Birmingham, 2Centre for Cardiovascular Sciences, University of Edinburgh

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

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Neuroscience

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis
Lyndsay Murray 1, Thomas H Gillingwater 2, Rashmi Kothary 1
1Regenerative Medicine Program, Ottawa Hospital Research Institute, 2Centre for Integrative Physiology, University of Edinburgh

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

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Biology

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle
William C.W. Chen 1, Arman Saparov 2,3, Mirko Corselli 4, Mihaela Crisan 5, Bo Zheng 6, Bruno Péault 7,8, Johnny Huard 9
1Stem Cell Research Center, Department of Bioengineering and Orthopedic Surgery, University of Pittsburgh, 2Department of Orthopedic Surgery, University of Pittsburgh, 3Nazarbayev University Research and Innovation System, Nazarbayev University, 4Department of Orthopaedic Surgery, UCLA Orthopaedic Hospital and the Orthopaedic Hospital Research Center, University of California at Los Angeles, 5Department of Cell Biology, Erasmus MC Stem Cell Institute, 6OHSU Center for Regenerative Medicine, Oregon Health & Science University, 7Centre for Cardiovascular Science and MRC Centre for Regenerative Medicine, Queen's Medical Research Institute and University of Edinburgh, 8David Geffen School of Medicine and the Orthopaedic Hospital Research Center, University of California at Los Angeles, 9Stem Cell Research Center, Department of Orthopedic Surgery and McGowan Institute for Regenerative Medicine, University of Pittsburgh

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

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Biology

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration
Richard L. Mort 1, Margaret Keighren 1, Leonard Hay 1, Ian J. Jackson 1
1MRC Human Genetics Unit, MRC IGMM, Western General Hospital, University of Edinburgh

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

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Neuroscience

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey
Antón Barreiro-Iglesias 1, Guixin Zhang 2, Michael E. Selzer 2,3, Michael I. Shifman 2
1Centre for Neuroregeneration, School of Biomedical Sciences, University of Edinburgh, 2Shriners Hospitals Pediatric Research Center (Center for Neural Repair and Rehabilitation), Temple University School of Medicine, 3Department of Neurology, Temple University School of Medicine

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

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Medicine

Combined In vivo Optical and µCT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice
Nicholas M. Bernthal 1, Brad N. Taylor 2, Jeffrey A. Meganck 2, Yu Wang 3, Jonathan H. Shahbazian 3, Jared A. Niska 1, Kevin P. Francis 2, Lloyd S. Miller 3,4
1Orthopaedic Hospital Research Center, Orthopaedic Hospital Department of Orthopaedic Surgery, David Geffen School of Medicine at University of California, Los Angeles (UCLA), 2PerkinElmer, 3Department of Dermatology, Johns Hopkins University School of Medicine, 4Department of Medicine, Division of Infectious Diseases, Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine

Combined optical and μCT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

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Chemistry

Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces
Baltasar Lucendo-Villarin 1, Kate Cameron 1, Dagmara Szkolnicka 1, Paul Travers 1, Ferdous Khan 2, Jeffrey G. Walton 2, John Iredale 3, Mark Bradley 2, David C. Hay 1
1MRC Centre for Regenerative Medicine, University of Edinburgh, 2School of Chemistry, University of Edinburgh, 3MRC Centre for Inflammation Research, University of Edinburgh

This article will focus on developing polymer coated surfaces for long-term, stable culture of stem cell derived human hepatocytes.

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Medicine

Renal Ischaemia Reperfusion Injury: A Mouse Model of Injury and Regeneration
Emily E. Hesketh 1, Alicja Czopek 1, Michael Clay 1, Gary Borthwick 1, David Ferenbach 1, David Kluth 1, Jeremy Hughes 1
1MRC Centre for Inflammation Research, University of Edinburgh

The mouse model of renal ischaemia reperfusion injury described here comprises of a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia that results in acute kidney injury. This model uses a midline laparotomy approach with all steps performed via one incision.

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Biology

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies
Samantha L. Eaton 1, Maica Llavero Hurtado 1, Karla J. Oldknow 2, Laura C. Graham 1, Thomas W. Marchant 1, Thomas H. Gillingwater 3,4, Colin Farquharson 2, Thomas M. Wishart 1,4
1Division of Neurobiology, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, 2Division of Developmental Biology, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, 3Centre for Integrative Physiology, University of Edinburgh, 4Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

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Medicine

Mouse Kidney Transplantation: Models of Allograft Rejection
George H. Tse *1, Emily E. Hesketh *1, Michael Clay 1, Gary Borthwick 1, Jeremy Hughes 1, Lorna P. Marson 1
1MRC Centre for Inflammation Research, The Queen's Medical Research Institute, The University of Edinburgh

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

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Immunology and Infection

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products
Chris J. C. Johnston 1, Elaine Robertson 1, Yvonne Harcus 1, John R. Grainger 2, Gillian Coakley 1, Danielle J. Smyth 1, Henry J. McSorley 1, Rick Maizels 1
1Institute of Immunology and Infection Research, University of Edinburgh, 2Manchester Collaborative Centre for Inflammation Research

Heligmosomoides polygyrus is a murine nematode with powerful immunomodulatory capabilities that closely resemble those of highly-prevalent human helminth infection. Here we describe a protocol for the long-term maintenance of the H. polygyrus lifecycle.

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Developmental Biology

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles
Stephanie Morgan 1, Lisa Campbell 1, Vivian Allison 1, Alison Murray 2, Norah Spears 1
1Centre for Integrative Physiology, University of Edinburgh, 2MRC Centre for Reproductive Health, University of Edinburgh

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

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Medicine

A Murine Model of Irreversible and Reversible Unilateral Ureteric Obstruction
Emily E. Hesketh 1, Madeleine A. Vernon 1, Peng Ding 1, Spike Clay 1, Gary Borthwick 1, Bryan Conway 1, Jeremy Hughes 1
1MRC Centre for Inflammation Research, University of Edinburgh

The murine model of irreversible unilateral ureteric obstruction (UUO) is presented together with the model of reversible UUO in which the ureteric obstruction is relieved by anastomosis of the severed ureter into the bladder. These models enable the study of renal inflammation and scarring as well as tissue remodeling.

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Chemistry

An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions
Mohameedyaseen Syedbasha 1, Janina Linnik 1,2,3, Deanna Santer 4, Daire O'Shea 5, Khaled Barakat 4,6, Michael Joyce 4, Nina Khanna 7, D. Lorne Tyrrell 4, Michael Houghton 4, Adrian Egli 1,8
1Applied Microbiology Research, Department of Biomedicine, University of Basel, 2Department of Biosystems Science and Engineering, ETH Zurich, and Swiss Institute of Bioinformatics, 3Swiss Institute of Bioinformatics, 4Li Ka Shing Institute for Virology, University of Alberta, 5Regional Infectious Diseases Unit, University of Edinburgh, 6Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, 7Infection Biology, Department of Biomedicine, University of Basel, 8Clinical Microbiology, University Hospital Basel

The presented protocols describe two enzyme-linked immunosorbent assay (ELISA) based techniques for the rapid investigation of ligand-receptor interactions: The first assay allows the determination of dissociation constant between ligand and receptor. The second assay enables a rapid screening of blocking peptides for ligand-receptor interactions.

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Neuroscience

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction
Mario Sanhueza *1, Anisha Kubasik-Thayil *2, Giuseppa Pennetta 1
1Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, 2School of Biomedical Sciences, University of Edinburgh

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

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Developmental Biology

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue
James E. Baily 1, William C.W. Chen 2,3, Nusrat Khan 4, Iain R. Murray 4, Zaniah N. González Galofre 4, Johnny Huard 5,6, Bruno Péault 4,7
1Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, 2Department of Bioengineering and Orthopaedic Surgery, University of Pittsburgh, 3Research Laboratory of Electronics and Department of Biological Engineering, Massachusetts Institute of Technology, 4MRC Centre for Regenerative Medicine, University of Edinburgh, 5Stem Cell Research Center, Department of Orthopaedic Surgery, University of Pittsburgh, 6Department of Orthopaedic Surgery, University of Texas Health Science Center at Houston, 7Department of Orthopaedic Surgery, UCLA Orthopaedic Hospital, David Geffen School of Medicine, University of California at Los Angeles

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

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Bioengineering

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices
Seshasailam Venkateswaran *1, Peter J. Gwynne *2, Mei Wu 1, Ailsa Hardman 2, Annamaria Lilienkampf 1, Salvatore Pernagallo 1, Garry Blakely 2, David G. Swann 3, Mark Bradley 1, Maurice P. Gallagher 2
1School of Chemistry, EaStCHEM, University of Edinburgh, 2School of Biological Sciences, University of Edinburgh, 3Critical Care, NHS Lothian, Royal Infirmary of Edinburgh

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

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Immunology and Infection

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)
Florence Marie-Anaïs 1, Julie Mazzolini 1, Pierre Bourdoncle 1, Florence Niedergang 1
1Inserm U1016, Institut Cochin, CNRS UMR 8104, Université Paris Descartes, Sorbonne Paris Cité

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

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Biology

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter
Louise L. Hansen 1, Gerben van Ooijen 1
1Institute for Molecular Plant Sciences, University of Edinburgh

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

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Cancer Research

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window
Carolina Rodriguez-Tirado 1, Takanori Kitamura 5, Yu Kato 1,2, Jeffery W. Pollard 1,2,5, John S. Condeelis 3,4, David Entenberg 3,4
1Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 2Department of Obstetrics/Gynecology and Woman’s Health, Albert Einstein College of Medicine, 3Department of Anatomy & Structural Biology, Albert Einstein College of Medicine, 4Gruss-Lipper Biophotonics Center Integrated Imaging Program, Albert Einstein College of Medicine, 5Medical Research Council Centre for Reproductive Health, Queen’s Medical Research Institute, University of Edinburgh

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

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Immunology and Infection

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release
Unity Jeffery 1, Robert D. Gray 2, Dana N. LeVine 3
1Department of Veterinary Microbiology and Preventative Medicine, College of Veterinary Medicine, Iowa State University, 2MRC Centre for Inflammation Research, University of Edinburgh, 3Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Iowa State University

Neutrophil extracellular traps (NETs) are networks of DNA, histones and neutrophil proteins. Although a component of the innate immune response, NETs are implicated in autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay.

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Developmental Biology

Analysis of Coronary Vessels in Cleared Embryonic Hearts
Sarah Ivins 1, Catherine Roberts 1, Bertrand Vernay 2, Peter J. Scambler 1
1Developmental Biology of Birth Defects, UCL Institute of Child Health, 2MRC Centre for Regenerative Medicine, SCRM Building, University of Edinburgh

We present a protocol for the analysis of coronary vessels in whole embryonic murine hearts up to E15.5, using standard immunological staining methods followed by optical clearance and confocal microscopy. This technique enables visualization of blood vessels throughout the entire heart without the need for time-consuming analysis of serial sections.

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Developmental Biology

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification
Dean A. Houston *1, Katherine A. Staines *1, Vicky E. MacRae 1, Colin Farquharson 1
1Developmental Biology, The Roslin Institute and R(D)SVS, The University of Edinburgh

We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

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Developmental Biology

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells
Yu Wang 1, Sharmin Alhaque 1, Kate Cameron 1, Jose Meseguer-Ripolles 1, Baltasar Lucendo-Villarin 1, Hassan Rashidi 1, David C. Hay 1
1MRC Centre for Regenerative Medicine, University of Edinburgh

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

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Developmental Biology

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification
Cui Lin 1, Dongxing Zhu 2, Greg Markby 1, Brendan M. Corcoran 3, Colin Farquharson 1, Vicky E. Macrae 1
1Developmental Biology, The Roslin Institute and R(D)SVS, University of Edinburgh, 2Guangzhou Institute of Cardiovascular Disease, The Second Affiliated Hospital, School of Basic Medical Sciences, Guangzhou Medical University, 3Clinical Sciences and R(D)SVS, University of Edinburgh

This protocol describes the isolation, culture, and calcification of rat-derived valve interstitial cells, a highly physiological in vitro model of calcific aortic valve disease (CAVD). Exploitation of this rat model facilitates CAVD research in exploring the cell and molecular mechanisms that underlie this complex pathological process.

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Immunology and Infection

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy
Bethany Mills 1, Ahsan R. Akram 1, Emma Scholefield 1, Mark Bradley 2, Kevin Dhaliwal 1
1EPSRC Proteus Hub, MRC Centre of Inflammation Research, Queen's Medical Research Institute, University of Edinburgh, 2School of Chemistry, EaStChem, University of Edinburgh

This technique describes an efficient screening process for evaluating bacteria-specific optical imaging agents within ex vivo human lung tissue, by fibered confocal fluorescence microscopy for the rapid identification of small molecule chemical probe-candidates with translatable potential.

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JoVE Journal

Experimental Procedure for Laboratory Studies of In Situ Burning : Flammability and Burning Efficiency of Crude Oil
Laurens van Gelderen 1, Grunde Jomaas 1,2
1Department of Civil Engineering, Technical University of Denmark, 2School of Engineering, BRE Centre for Fire Safety Engineering, University of Edinburgh

Here, we present a protocol to simultaneously study the flammability and burning efficiency of fresh and weathered crude oil under conditions that simulate in situ burning operations on the sea.

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Immunology and Infection

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-α
Alba Llibre *1,2, Vincent Bondet *1,2, Mathieu P. Rodero 3, David Hunt 4, Yanick J. Crow 3,5, Darragh Duffy 1,2
1Immunobiology of Dendritic Cells, Institut Pasteur, 2INSERM U1223, 3Laboratory of Neurogenetics and Neuroinflammation, INSERM UMR1163, Institut Imagine, 4MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, 5Manchester Centre for Genomic Medicine, University of Manchester

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-α subtypes in human samples.

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Neuroscience

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains
Julie Mazzolini 1, Kelda Chia 1, Dirk Sieger 1
1Centre for Discovery Brain Sciences, University of Edinburgh

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

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Biology

Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis
Caleb Sutherland *1, Yu Wang *2, Robert V. Brown *1, Julie Foley 2, Beth Mahler 2, Kyathanahalli S. Janardhan 2,3, Ramesh C. Kovi 2,4, Anton M. Jetten 1
1Immunity, Inflammation, and Disease Laboratory, Division of Intramural Research, National Institute of Environmental Health Sciences, NIH, 2Cellular and Molecular Pathology Branch, Division of the National Toxicology Program, National Institute of Environmental Health Sciences, NIH, 3Integrated Laboratory Systems Inc., 4Experimental Pathology Laboratories Inc.

Here, we describe a protocol for a reproducible laser capture microdissection (LCM) for isolating trabecular meshwork (TM) for downstream RNA analysis. The ability to analyze changes in gene expression in the TM will help in understanding the underlying molecular mechanisms of TM-related ocular diseases.

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Biology

Oral Bacterial Infection and Shedding in Drosophila melanogaster
Jonathon A. Siva-Jothy 1, Arun Prakash 1, Radhakrishnan B. Vasanthakrishnan 2, Katy M. Monteith 1, Pedro F. Vale 1,3
1Institute of Evolutionary Biology, School of Biological Sciences, University of Edinburgh, 2IGDR - CNRS UMR 6290, 3Centre for Immunity, Infection and Evolution, University of Edinburgh

This protocol describes methods to orally expose and infect the fruit fly Drosophila melanogaster with bacterial pathogens, and to measure the number of infectious bacteria shed following gut infection. We further describe the effect of immune mutants on fly survival following oral bacterial infection.

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Developmental Biology

Semi-automated Production of Hepatocyte Like Cells from Pluripotent Stem Cells
Jose Meseguer-Ripolles 1, Baltasar Lucendo-Villarin 1, Yu Wang 1, David C. Hay 1
1MRC Centre for Regenerative Medicine, University of Edinburgh

This protocol describes a semi-automated approach to produce hepatocyte-like cells from human pluripotent stem cells in a 96 well plate format. This process is rapid and cost-effective, allowing the production of quality assured batches of hepatocyte-like cells for basic and applied human research.

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JoVE Journal

A 100 KW Class Applied-field Magnetoplasmadynamic Thruster
Baojun Wang 1, Haibin Tang 2, Yibai Wang 1, Chao Lu 1, Cheng Zhou 3, Yangyang Dong 1, Ge Wang 3, Yuntian Cong 3, Daniel Luu 4, Jinbin Cao 2
1Key Laboratory of Spacecraft Design Optimization & Dynamic Simulation Technologies of Ministry of Education, School of Astronautics, Beihang University, 2Key Laboratory of Spacecraft Design Optimization & Dynamic Simulation Technologies of Ministry of Education, School of Space and Environment, Beihang University, 3Beijng Institute of Control Engineering, 4School of Aerospace Engineering and Geodesy, University of Stuttgart

The goal of this protocol is to introduce the design of a 100 kW class applied-field magnetoplasmadynamic thruster and relevant experimental methods.

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Cancer Research

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells
Takanori Kitamura *1,2, Dahlia Doughty-Shenton *3, Jeffrey W. Pollard 2, Neil O. Carragher 3,4
1Royal (Dick) School of Veterinary Studies and Roslin Institute, University of Edinburgh, 2MRC Centre for Reproductive Health, University of Edinburgh, 3Edinburgh Phenotypic Assay Centre, University of Edinburgh, 4Cancer Research UK Edinburgh Centre, MRC Institute of Genetics, Molecular Medicine, University of Edinburgh

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

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Neuroscience

Three-Dimensional Shape Modeling and Analysis of Brain Structures
Jaeil Kim 1, Maria del Carmen Valdés Hernández 2, Jinah Park 3
1School of Computer Science and Engineering, Kyungpook National University, 2Centre for Clinical Brain Sciences, University of Edinburgh, 3School of Computing and KI for Health Science and Technology (KIHST), Korea Advanced Institute of Science and Technology (KAIST)

We introduce a semi-automatic protocol for shape analysis on brain structures, including image segmentation using open software, and further group-wise shape analysis using an automated modeling package. Here, we demonstrate each step of the 3D shape analysis protocol with hippocampal segmentation from brain MR images.

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Effective Lysis of Cyanobacterial and Green Algal Single Cells for Whole Genome Amplification in Microfluidics
Yuguang Liu 1, Dirk Schulze-Makuch 2, Jean-Pierre de Vera 3, Charles Cockell 4, Thomas Leya 5, Mickael Baque 3, Marina Walther-Antonio 1,6
1Department of Surgery, Division of Surgical Research, Mayo Clinic, 2Astrobiology Group, Center of Astronomy and Astrophysics, Technical University, 3Management and Infrastructure, Astrobiological Laboratories, German Aerospace Center (DLR), Institute of Planetary Research, 4School of Physics and Astronomy, University of Edinburgh, 5Branch Bioanalytics and Bioprocesses (IZI-BB), Extremophile Research & Biobank CCCryo, Fraunhofer Institute for Cell Therapy and Immunology, 6Department of Obstetrics and Gynecology, Mayo Clinic

Here we present a protocol to lyse cyanobacteria and green algae single cells that allows for subsequent single-cell whole genome amplification in a microfluidic platform with a 100% success rate.

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Biochemistry

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting
Yu-Ting Huang 1,2, Dinja van der Hoorn *1,2, Leire M. Ledahawsky *1,2, Anna A. L. Motyl *1,2, Crispin Y. Jordan 1, Thomas H. Gillingwater 1,2, Ewout J. N. Groen 1,2
1Centre for Discovery Brain Sciences, University of Edinburgh, 2Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh

This method describes a robust and reproducible approach for the comparison of protein levels in different tissues and at different developmental timepoints using a standardized quantitative western blotting approach.

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JoVE Core

Quantifying the Relative Thickness of Conductive Ferromagnetic Materials Using Detector Coil-Based Pulsed Eddy Current Sensors
Nalika Ulapane 1, Karthick Thiyagarajan 2, David Hunt 2, Jaime Valls Miro 2
1Melbourne School of Engineering, University of Melbourne, 2Center for Autonomous Systems, University of Technology Sydney

Here, we present a protocol to quantify the relative thickness (i.e., thickness as a percentage with respect to a reference) of conductive ferromagnetic materials using detector coil-based pulsed eddy current sensors, while overcoming the calibration requirement.

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Developmental Biology

Mapping the Emergent Spatial Organization of Mammalian Cells using Micropatterns and Quantitative Imaging
Darren Wisniewski 1, Sally Lowell 1, Guillaume Blin 1
1MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh

The method presented here uses micropatterning together with quantitative imaging to reveal spatial organization within mammalian cultures. The technique is easy to establish in a standard cell biology laboratory and offers a tractable system to study patterning in vitro.

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Biology

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues
Daniels Konja 1, Cuiting Luo 1, Wai Yan Sun 1, Kangmin Yang 1, Andy W.C. Man 1, Aimin Xu 1, Paul M. Vanhoutte 1, Yu Wang 1
1The State Key Laboratory of Pharmaceutical Biotechnology and the Department of Pharmacology and Pharmacy, University of Hong Kong

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.

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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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Biochemistry

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU)
J. David Barrass 1, Jean D. Beggs 1
1Wellcome Centre for Cell Biology, School of Biological Sciences, University of Edinburgh

The use of thiolated uracil to sensitively and specifically purify newly transcribed RNA from the yeast Saccharomyces cerevisiae.

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Developmental Biology

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells
Balta Lucendo-Villarin 1, Hassan Rashidi 1,2, Sharmin Alhaque 1,3, Lena Fischer 1,4, Jose Meseguer-Ripolles 1, Yu Wang 1, Cliona O'Farrelly 4, Michael Themis 3, David C. Hay 1
1MRC Centre for Regenerative Medicine, University of Edinburgh, 2UCL Great Ormond Street Institute of Child Health, University College London, 3Division of Biosciences, Department of Life Sciences, College of Health and Life Sciences, Brunel University London, 4School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin

This protocol describes an approach to produce hepatospheres from human pluripotent stem cells using a defined culture system and cell self-assembly. This protocol is reproducible in a number of cell lines, cost effective and allows the production of stable human hepatospheres for biomedical application.

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Biology

Human Adipose Tissue Micro-fragmentation for Cell Phenotyping and Secretome Characterization
Bianca Vezzani 1,2, Mario Gomez-Salazar 1, Joan Casamitjana 1, Carlo Tremolada 3, Bruno Péault 1,4
1MRC Center for Regenerative Medicine, University of Edinburgh, 2Dept. of Morphology, Surgery and Experimental Medicine, Section of General Pathology, University of Ferrara, 3Italian Image Institute, 4Orthopaedic Hospital Research Center and Broad Stem Cell Research Center, David Geffen School of Medicine, University of California

Here, we present human adipose tissue enzyme-free micro-fragmentation using a closed system device. This new method allows the obtainment of sub-millimeter clusters of adipose tissue suitable for in vivo transplantation, in vitro culture, and further cell isolation and characterization.

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JoVE Journal

Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit
Grant A. R. Gale *1,2,3, Alejandra A. Schiavon Osorio *1,2, Anton Puzorjov *1,2, Baojun Wang 2,3, Alistair J. McCormick 1,2
1Institute of Molecular Plant Sciences, School of Biological Sciences, University of Edinburgh, 2Centre for Synthetic and Systems Biology, University of Edinburgh, 3Institute of Quantitative Biology, Biochemistry and Biotechnology, School of Biological Sciences, University of Edinburgh

Here, we present a protocol describing how to i) assemble a self-replicating vector using the CyanoGate modular cloning toolkit, ii) introduce the vector into a cyanobacterial host by conjugation, and iii) characterize transgenic cyanobacteria strains using a plate reader or flow cytometry.

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JoVE Core

Asymmetric Thermoelectrochemical Cell for Harvesting Low-grade Heat under Isothermal Operation
Kaiyu Mu *1, Xun Wang *1, Ka Ho Li 1, Yu-Ting Huang 1, Shien-Ping Feng 1
1Electrochemical Nanoengineering Group, Department of Mechanical Engineering, University of Hong Kong

Low-grade heat is abundant, but its efficient recovery is still a great challenge. We report an asymmetric thermoelectrochemical cell using graphene oxide as a cathode and polyaniline as an anode with KCl as the electrolyte. This cell works under isothermal heating, exhibiting a high heat-to-electricity conversion efficiency in low-temperature regions.

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Biochemistry

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using χCRAC
Stuart W. McKellar 1, Ivayla Ivanova 1, Robert W. van Nues 2, Ross A. Cordiner 3, Mehak Chauhan 1, Niki Christopoulou 1, Will Worboys 4, Andrew Langford 4, Torben Heick Jensen 3, Sander Granneman 1
1Centre for Engineering Biology, University of Edinburgh, 2Institute of Cell Biology, University of Edinburgh, 3Department of Molecular Biology and Genetics, Aarhus University, 4UVO3 Ltd.

Kinetic cross-linking and analysis of cDNA is a method that allows investigation of the dynamics of protein-RNA interactions in living cells at high temporal resolution. Here the protocol is described in detail, including the growth of yeast cells, UV cross-linking, harvesting, protein purification, and next generation sequencing library preparation steps.

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Immunology and Infection

Production and Characterization of Human Macrophages from Pluripotent Stem Cells
Martha Lopez-Yrigoyen 1,2, Alisha May 1, Telma Ventura 1, Helen Taylor 1, Antonella Fidanza 1, Luca Cassetta 2, Jeffrey W. Pollard 2, Lesley M. Forrester 1
1Centre for Regenerative Medicine, Scottish Centre for Regenerative Medicine, University of Edinburgh, 2Centre for Reproductive Health, The Queen's Medical Research Institute, University of Edinburgh

This protocol describes the robust generation of macrophages from human induced pluripotent stem cells, and methods for their subsequent characterization. Cell surface marker expression, gene expression, and functional assays are used to assess the phenotype and function of these iPSC-derived macrophages.

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Bioengineering

Controlling Electrospun Polymer Morphology for Tissue Engineering Demonstrated Using hepG2 Cell Line
Thomas S. R. Bate 1, Stuart J. Forbes 2, Anthony Callanan 1
1Institute for Bioengineering, School of Engineering, University of Edinburgh, 2Scottish Centre for Regenerative Medicine, University of Edinburgh

This method provides the means to test different polycaprolactone fiber morphologies and topographies for the purpose of tissue engineering. Small and large fibers are fabricated with random orientations, aligned orientations, and also porous cryogenically electrospun structures and used as platforms for cell culture.

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Biology

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers (MADM)
Robert Beattie 1, Carmen Streicher 1, Nicole Amberg 1, Giselle Cheung 1, Ximena Contreras 1, Andi H. Hansen 1, Simon Hippenmeyer 1
1Institute of Science and Technology Austria

A protocol to perform lineage tracing and functional genetic analysis of candidate genes at a single cell level using mosaic analysis with double markers (MADM) is presented. MADM clonal analysis provides a quantitative framework to measure the proliferative behavior, cellular output, and lineage relationship of individual progenitors and their daughter cells.

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Genetics

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing
Yu Wang 1, Andrew J. H. Smith 1, David C. Hay 1
1MRC Centre for Regenerative Medicine, University of Edinburgh

Here, we describe a detailed method for seamless gene editing in human pluripotent stem cells using a piggyBac-based donor plasmid and the Cas9 nickase mutant. Two point mutations were introduced into exon 8 of the hepatocyte nuclear factor 4 alpha (HNF4α) locus in human embryonic stem cells (hESCs).

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Biology

Hepatic Progenitor Specification from Pluripotent Stem Cells using a Defined Differentiation System
Jose Meseguer-Ripolles 1, Yu Wang 1, Agnes Sorteberg 1, Aishwariya Sharma 2, Nan-Linda Ding 2, Baltasar Lucendo-Villarin 1, Philipp Kramer 2, Charis-Patricia Segeritz 2, David C. Hay 1
1MRC Centre for Regenerative Medicine, University of Edinburgh, 2Research & Development, STEMCELL Technologies Inc

The goal of this article is to provide a standardized approach to induce human hepatic progenitor differentiation from pluripotent stem cells. The development of this procedure with ready-to-use media formulations offer the user a facile system to generate human liver cells for biomedical research and translation.

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Medicine

Acupuncture in a Rat Model of Asthma
Dong-Dong Zhou 1, Guang-Quan Zhang 1, Qing-Yi Zhao 1, Mi Cheng 1, Jin Lu 1, Yu Wang 1, Yu-Dong Xu 1, Yan-Jiao Chen 1, Yong-Qing Yang 1, Lei-Miao Yin 1,2
1Laboratory of Molecular Biology, Shanghai Research Institute of Acupuncture and Meridian, Shanghai University of Traditional Chinese Medicine, 2Shanghai Innovation Center of Traditional Chinese Medicine Health Service

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes methods for the fabrication of the high platform, establishes a rat model of asthma and measures changes in respiratory function using a noninvasive and real-time whole-body plethysmography (WBP) system.

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Behavior

A Behavioral Task Modeling 'Everyday Memory' in an Event Arena to Foster Allocentric Representations for Rodents
Dorothy Tse *1,2, Anna C. Norton *1, Patrick A. Spooner 1, Richard G. M. Morris 1
1Centre for Discovery Brain Sciences, Edinburgh Neuroscience, University of Edinburgh, 2Department of Psychology, Edge Hill University

The goal of this optimized 'everyday memory' protocol in an event arena was to employ a stable home-base that encourages the use of allocentric spatial representations. This animal model provides an effective test-bed for future research into the formation and retention of event memories using behavioral and physiological techniques.

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Immunology and Infection

A Mouse Model of Orotracheal Intubation and Ventilated Lung Ischemia Reperfusion Surgery
Wen-I Liao *1, Daisuke Maruyama *1, Farzaneh Kianian 1,2, Christine Tat 1, Xiaoli Tian 1, Judith Hellman 1, Jeffrey M Dodd-o 3, Arun Prakash 1
1Department of Anesthesia and Perioperative Care, University of California San Francisco and San Francisco General Hospital, 2Department of Physiology, School of Medicine, Tehran University of Medical Sciences, 3Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine

A mouse surgical model to create left lung ischemia reperfusion (IR) injury while maintaining ventilation and avoiding hypoxia.

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Developmental Biology

In Vitro Model of Fetal Human Vessel On-chip to Study Developmental Mechanobiology
Telma Ventura 1, Ewan Joshua Egan 1, Nicola Romanò 2, Antonella Fidanza 1
1Centre for Regenerative Medicine, Institute for Regeneration and Repair, University of Edinburgh, 2Centre for Discovery Brain Sciences, University of Edinburgh

Described here is a simple workflow to differentiate endothelial cells from human pluripotent stem cells followed by a detailed protocol for their mechanical stimulation. This allows for the study of the developmental mechanobiology of endothelial cells. This approach is compatible with downstream assays of live cells collected from the culture chip after mechanical stimulation.

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