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University of Liverpool

23 ARTICLES PUBLISHED IN JoVE

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Medicine

Bronchoalveolar Lavage (BAL) for Research; Obtaining Adequate Sample Yield
Andrea M. Collins 1,2, Jamie Rylance 3, Daniel G. Wootton 4, Angela D. Wright 3,5, Adam K. A. Wright 1,3, Duncan G. Fullerton 3,6, Stephen B. Gordon 3
1Biomedical Research Centre in Microbial Diseases, National Institute for Health Research, 2Respiratory Infection Group, Royal Liverpool and Broadgreen University Hospital Trust, 3Respiratory Infection Group, Liverpool School of Tropical Medicine, 4Institute of Infection and Global Health, University of Liverpool, 5Comprehensive Local Research Network, Royal Liverpool and Broadgreen University Hospital Trust, 6Department of Respiratory Research, University Hospital Aintree

We describe a research technique for fiberoptic bronchoscopy and bronchoalveolar lavage using low pressure suction. The technique is used to harvest immune cells from the lung bronchoalveolar surfaces. Local anesthetic and mild conscious sedation (midazolam) is used. Subjects tolerate the procedure well and experience minimal side effects.

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Neuroscience

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila
Rudi L. Verspoor 1, Chloe Heys 1, Thomas A. R. Price 1
1Institute of Integrative Biology, University of Liverpool

This protocol describes a simple, cost effective way to individually identify Drosophila or other insects. Demonstration data investigating mating success across three species of Drosophila show that this method is comparable or better than the use of CO2 anaesthesia.

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Bioengineering

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture
Michael P. Storm 1, Ian Sorrell 2, Rebecca Shipley 3, Sophie Regan 2, Kim A. Luetchford 1, Jean Sathish 2, Steven Webb 4, Marianne J. Ellis 1
1Department of Chemical Engineering and Centre for Regenerative Medicine, University of Bath, 2MRC Centre for Drug Safety Science and Institute of Translational Medicine, University of Liverpool, 3Mechanical Engineering, University College London, 4Department of Applied Mathematics, Liverpool John Moores University

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

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Biology

Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition
Josephine C. Moran 1, Emma L. Crank 1, Hanaa A. Ghabban 1, Malcolm J. Horsburgh 1
1Institute of Integrative Biology, University of Liverpool

The deferred growth inhibition assay can be used to assess the competition effect by one bacterial isolate on another. Inhibition is quantified by measuring the zone of clearing around the inhibitor-producing isolate, or qualitatively assessed by determining the visible extent of inhibition.

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Developmental Biology

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans
Ana Soriano-Arroquia 1, Peter D. Clegg 1, Andrew P. Molloy 1,2, Katarzyna Goljanek-Whysall 1
1Institute of Ageing and Chronic Disease, University of Liverpool, 2Aintree University Hospital

This protocol describes a robust, reproducible and simple method of isolation and culture of myoblast progenitor cells from the skeletal muscle of adult and aged people. The muscles used here include foot and leg muscles. This approach enables the isolation of an enriched population of primary myoblasts for functional studies.

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Medicine

Contractility Measurements of Human Uterine Smooth Muscle to Aid Drug Development
Sarah Arrowsmith *1, Peter Keov 2, Markus Muttenthaler 3,4, Christian W. Gruber *2,5
1Harris-Wellbeing Preterm Birth Research Centre, Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, 2School of Biomedical Sciences, The University of Queensland, 3Faculty of Chemistry, Institute of Biological Chemistry, University of Vienna, 4Institute for Molecular Bioscience, University of Queensland, 5Center for Physiology and Pharmacology, Medical University of Vienna

This article describes experimental protocols to study ex vivo contractions of human myometrium and their application in drug discovery. This technique is used to improve the understanding of myometrial physiology and pathophysiology as well as to validate pharmacological data from novel research probes or drug leads.

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Bioengineering

Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies
I-Ning Lee *1,2, Joseph Hosford *1, Shuai Wang 3, John A. Hunt 4, Judith M. Curran 2, William P. Heath 3, Lu Shin Wong 1
1Manchester Institute of Biotechnology & School of Chemistry, University of Manchester, 2School of Engineering, University of Liverpool, 3School of Electrical and Electronic Engineering, University of Manchester, 4School of Science and Technology, Nottingham Trent University

Here we present a protocol for wide-area scanning probe nanolithography enabled by the iterative alignment of probe arrays, as well as the utilization of lithographic patterns for cell-surface interaction studies.

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Genetics

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations
Alec Kittredge 1, Changyi Ji 1, Yu Zhang  1, Tingting Yang 1
1Department of Pharmacology and Physiology, University of Rochester, School of Medicine and Dentistry

Here we present a protocol to differentiate retinal pigment epithelium (RPE) cells from human pluripotent stem cells bearing patient-derived mutations. The mutant cell lines may be used for functional analyses including immunoblotting, immunofluorescence, and patch clamp. This disease-in-a-dish approach circumvents the difficulty of obtaining native human RPE cells.

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Biochemistry

Expression and Purification of Mammalian Bestrophin Ion Channels
Alec Kittredge 1, Nancy Ward 1, Austin Hopiavuori 1, Yu Zhang  1, Tingting Yang 1
1Department of Pharmacology and Physiology, University of Rochester, School of Medicine and Dentistry

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

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Immunology and Infection

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy
Kyle Taruc 1, Charles Yin 1, Daniel G. Wootton 2,3, Bryan Heit 1
1Department of Microbiology and Immunology and the Center for Human Immunology, University of Western Ontario, 2Institute of Infection and Global Health, University of Liverpool, 3Department of Respiratory Research, Aintree University Hospital NHS Foundation Trust

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

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Medicine

Transdermal Measurement of Glomerular Filtration Rate in Mice
Lauren Scarfe 1,2, Daniel Schock-Kusch 3, Lorenzo Ressel 4, Jochen Friedemann 3, Yury Shulhevich 3, Patricia Murray 2, Bettina Wilm 2, Mark de Caestecker 1
1Division of Nephrology, Department of Medicine, Vanderbilt University Medical Center, 2Department of Cellular and Molecular Physiology, University of Liverpool, 3MediBeacon GmbH, 4Department of Veterinary Pathology and Public Health, Institute of Veterinary Science, University of Liverpool

Here we describe a protocol to measure glomerular filtration rate (GFR) in conscious, freely moving mice using a transdermal GFR monitor.

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Medicine

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo
Na Jia *1, Haoan Wu *2, Juanli Duan 1, Chunxue Wei 1, Kaixuan Wang 2, Yu Zhang  2, Xiaohua Mao 1
1Key Laboratory of Ministry of Education for Developmental Genes & Human Diseases, Institute of Life Sciences and School of Medicine, Southeast University, 2State Key Laboratory of Bioelectronics, Jiangsu Key Laboratory for Biomaterials & Devices, School of Biological Science & Medical Engineering, Southeast University

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

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JoVE Journal

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis
Yu Zhang  *1, Guan Wang *1, Jia Lu 1, Li-ming Xu 2, Jun-yu Xiong 1
1Anesthesiology Department, The Second Hospital of Dalian Medical University, 2Scientific Research Center, The Second Hospital of Dalian Medical University

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.

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Chemistry

Reactive Inkjet Printing and Propulsion Analysis of Silk-based Self-propelled Micro-stirrers
David A. Gregory 1, Piyush Kumar 1, Ana Jimenez-Franco 1, Yi Zhang 1, Yu Zhang  2, Stephen J. Ebbens 1, Xiubo Zhao 1
1Department of Chemical and Biological Engineering, University of Sheffield, 2School of Engineering, University of Liverpool

This protocol demonstrates the ability to utilize reactive inkjet printing to print self-motile biocompatible and environmentally friendly micro-stirrers for use in biomedical and environmental applications.

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Immunology and Infection

Pan-lyssavirus Real Time RT-PCR for Rabies Diagnosis
Denise A. Marston 1, Daisy L. Jennings 1, Nikki C. MacLaren 1, Daniel Dorey-Robinson 1, Anthony R. Fooks 1,2, Ashley C. Banyard 1, Lorraine M. McElhinney 1,2
1Wildlife Zoonoses & Vector-Borne Diseases Research Group, Animal and Plant Health Agency, 2Institute of Infection and Global Health, University of Liverpool

This real-time RT-PCR using dsDNA intercalating dye is suitable to diagnose lyssavirus infections. The method begins with RNA extracted from rabies suspected ante-mortem or post-mortem samples, detailing master mix preparation, RNA addition, setup of the real-time machine and correct interpretation of results.

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Medicine

Electrochemotherapy in 3D Ocular Melanoma Spheroids using a Customized Electrode
Miltiadis Fiorentzis 1,2, Arne Viestenz 1, Berthold Seitz 3, Sarah E Coupland 4, Joana Heinzelmann 1
1Department of Ophthalmology, University Hospital Halle (Saale), Martin-Luther University Halle-Wittenberg, 2Department of Ophthalmology, University Hospital Essen, Duisburg Essen University, 3Department of Ophthalmology, Saarland University Medical Center, 4Liverpool Ocular Oncology Research Group, Department of Molecular and Clinical Cancer Medicine, Institute of Translational Medicine, University of Liverpool

Here, we present a protocol for the development of 3D conjunctival and uveal melanoma spheroids and the use of hand-held customized electrodes for in vitro electrochemotherapy of 3D spheroids in a culture well. This offers new perspectives in the use of electrochemotherapy in ocular melanoma treatment.

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Biochemistry

An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter
Florence B Kineavy 1, Alex A Davies 1, Molly R Mitchell 1, Daniel Lay 1, Matt J Dominguez 2, Elliott J Stollar 1
1School of Life Sciences, University of Liverpool, 2Health Sciences Center, Texas Tech University

A multi-column plate adapter allows chromatography columns to be interfaced with multi-well collection plates for parallel affinity or ion exchange purification providing an economical high throughput protein purification method. It can be used under gravity or vacuum yielding milligram quantities of protein via affordable instrumentation.

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Chemistry

Synthesis of Metal Nanoparticles Supported on Carbon Nanotube with Doped Co and N Atoms and its Catalytic Applications in Hydrogen Production
Pui-Ching Poon *1, Ka-Man Lee *1, Yuanhao Wang 2, William Wai Yan Lam 1, Peter Shu-Wai Leung 1, Xiao-Ying Lu 1, Weiqun Li 3, B. Layla Mehdi 3, Yang Lu 4, Chi-Wing Tsang 1, Eugene Yin Cheung Wong 2
1Faculty of Science and Technology, Technological and Higher Education Institute of Hong Kong (THEi), 2Department of Supply Chain and Information Management, The Hang Seng University of Hong Kong, 3Department of Mechanical, Materials and Aerospace Engineering, University of Liverpool, 4Laboratory of Building Environment and New Energy Resources, School of Civil Engineering, Faculty of Infrastructure Engineering, Dalian University of Technology

Here, we present a protocol to synthesize Co nanoparticles supported on carbon nanotubes with Co- and N- dopants for hydrogen productions.

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Biology

Determining Temperature Preference of Mosquitoes and Other Ectotherms
Amirah Haziqah-Rashid 1, Kinga Stobierska 1, Lorna Glenn 1, Soeren Metelmann 1,2, Ken Sherlock 3, Ewa Chrostek 1, Marcus S. C. Blagrove 1
1Department of Evolution, Ecology, and Behavior, Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, 2COVID-19 Outbreak Surveillance Team, UK Health Security Agency, 3Department of Livestock and One Health, Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool

Insects have an optimal environmental temperature range which they seek to remain within, and many external and internal factors can alter this preference. Here, we describe a cost-effective and simple method to study temperature choice, which allows insects to freely exhibit their natural behaviors.

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Environment

Dissolved Solute Sampling Across an Oxic-Anoxic Soil-Water Interface Using Microdialysis Profilers
Sha Zhang 1,2, Zhaofeng Yuan 2, Yujia Cai 2, Hao Liu 2, Ziyan Liu 2, Zheng Chen 2
1Department of Geography and Planning, University of Liverpool, 2Department of Health and Environmental Sciences, Xi’an Jiaotong-Liverpool University

A microdialysis profiler is described to sample dissolved porewater solutes across an oxic-anoxic soil-water interface in situ with minimal disturbance. This device is designed to capture rapid changes in concentration-depth profiles induced by disturbances at the soil-water interface and beyond.

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Biology

Understanding the Impact of Temperate Bacteriophages on Their Lysogens Through Transcriptomics
Revathy Krishnamurthi 1, Enrique González-Tortuero 2, Grace Plahe 2, Ian B. Goodhead 2, Joanne L. Fothergill 1, Chloë E. James 2, Heather E. Allison 1
1Department of Clinical Infection, Microbiology and Immunology, Institute of Infection, Veterinary and Ecological Sciences (IVES), University of Liverpool, 2School of Science, Engineering, and Environment, University of Salford

This protocol enables the impact of prophages on their hosts to be revealed. Bacterial cultures are synchronized using conditions that best support the lysogenic state, limiting spontaneous induction. RT-qPCR unequivocally distinguishes prophage-restricted genes and those uncoupled from phage control from those that are expressed during the lytic replication cycle.

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Engineering

Microembossing: A Convenient Process for Fabricating Microchannels on Nanocellulose Paper-Based Microfluidics
Wenwen Yuan 1,2,3, Hang Yuan 1, Sixuan Duan 1,2, Ruiqi Yong 1, Jia Zhu 1,2,4, Eng Gee Lim 1,2, Ivona Mitrovic 2, Pengfei Song 1,2
1School of Advanced Technology, Xi’an Jiaotong - Liverpool University, 2Department of Electrical Engineering and Electronics, University of Liverpool, 3State Key Laboratory for Manufacturing Systems Engineering, Xi'an Jiaotong University, 4School of Intelligent Manufacturing and Smart Transportation, Suzhou City University

This protocol describes a straightforward process that utilizes convenient plastic micro-molds for simple microembossing operations to fabricate microchannels on nanofibrillated cellulose paper, achieving a minimum width of 200 µm.

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Biology

Galleria mellonella as an Antimicrobial Screening Model
Thomas E. Barton 1, Liberty Duignan 2, Aras Kadioglu 2, Joanne L. Fothergill 2, Daniel R. Neill 1
1Division of Molecular Microbiology, School of Life Sciences, University of Dundee, 2Department of Clinical Infection, Microbiology and Immunology, University of Liverpool

This study presents a standardized framework for optimizing G. mellonella infection models for use in preclinical antimicrobial assessment. The application of a G. mellonella model as part of a preclinical antimicrobial development pipeline could decrease the number of ineffective compounds progressing to clinical trials.

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