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29 ARTICLES PUBLISHED IN JoVE

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Neuroscience

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation
Dmitry A. Molotkov 1, Alexey Y. Yukin 1,2, Ramil A. Afzalov 1,2, Leonard S. Khiroug 1
1Neuroscience Center, University of Helsinki, 2Faculty of Biological and Enviromental Sciences, University of Helsinki

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

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Neuroscience

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin
Mikhail Yuryev 1, Dmitry Molotkov 1
1Neuroscience Center, University of Helsinki

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented. 

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Biology

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bis(sulfosuccinimidyl) Suberate (BS3)
Konstantin I Ivanov 1, Marta Bašić 1, Markku Varjosalo 2, Kristiina Mäkinen 1
1Department of Food and Environmental Sciences, University of Helsinki, 2Institute of Biotechnology, University of Helsinki

A method is described for efficient purification of twin-Strep-tagged fusion proteins and their specific complexes on modified streptavidin (Strep-Tactin) resin covalently cross-linked with Bis(sulfosuccinimidyl) suberate (BS3). The method has the advantages of fast speed, good target protein recovery and high purity, and is compatible with subsequent analysis by mass spectrometry.

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Medicine

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging
Mikhail Paveliev 1, Mikhail Kislin 1, Dmitry Molotkov 1, Mikhail Yuryev 1, Heikki Rauvala 1, Leonard Khiroug 1
1Neuroscience Center, University of Helsinki

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

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Engineering

Coherent anti-Stokes Raman Scattering (CARS) Microscopy Visualizes Pharmaceutical Tablets During Dissolution
Andrew L. Fussell 1, Peter Kleinebudde 2, Jennifer Herek 1, Clare J. Strachan 3, Herman L. Offerhaus 1
1Optical Sciences Group, MESA+ Institute, University of Twente, 2Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine University, 3Division of Pharmaceutical Technology, Faculty of Pharmacy, University of Helsinki

Coherent anti-Stokes Raman scattering (CARS) microscopy is combined with an intrinsic flow-through dissolution setup to allow in situ and real-time visualization of the surface of pharmaceutical tablets undergoing dissolution. Using this custom-built setup, it is possible to correlate CARS videos with drug dissolution profiles recorded using inline UV absorption spectroscopy.

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Behavior

Flat-floored Air-lifted Platform: A New Method for Combining Behavior with Microscopy or Electrophysiology on Awake Freely Moving Rodents
Mikhail Kislin 1, Ekaterina Mugantseva 1, Dmitry Molotkov 1, Natalia Kulesskaya 1, Stanislav Khirug 1, Ilya Kirilkin 2, Evgeny Pryazhnikov 1,2, Julia Kolikova 2, Dmytro Toptunov 2, Mikhail Yuryev 1, Rashid Giniatullin 3, Vootele Voikar 4, Claudio Rivera 1, Heikki Rauvala 1, Leonard Khiroug 1
1Neuroscience Center, University of Helsinki, 2Neurotar LTD, 3A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, 4Laboratory Animal Center, University of Helsinki

This method creates a tangible, familiar environment for the mouse to navigate and explore during microscopic imaging or single-cell electrophysiological recordings, which require firm fixation of the animal’s head.

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Immunology and Infection

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries
Malena E. Skogman 1, Pia M. Vuorela 1, Adyary Fallarero 1
1Pharmaceutical Design and Discovery (PharmDD), Faculty of Pharmacy, University of Helsinki

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

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Developmental Biology

A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes
Federica Cavalera 1, Mario Zanoni 1, Valeria Merico 1, Thi Thu Hien Bui 1,2, Martina Belli 1,3, Lorenzo Fassina 4, Silvia Garagna 1, Maurizio Zuccotti 1
1Laboratorio di Biologia dello Sviluppo, Dipartimento di Biologia e Biotecnologie "Lazzaro Spallanzani", University of Pavia, 2Institute of Biotechnology, University of Helsinki, 3Department of Reproductive Medicine, University of California San Diego, 4Dipartimento di Ingegneria Industriale e dell'Informazione, University of Pavia

Here, we present a protocol for non-invasive assessment of oocyte developmental competence performed during their in vitro maturation from the germinal vesicle to the metaphase II stage. This method combines time-lapse imaging with particle image velocimetry (PIV) and neural network analyses.

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Medicine

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing
Solja Kalha 1, Alison Kuony 1, Frederic Michon 1,2
1Helsinki Institute of Life Science, Institute of Biotechnology, University of Helsinki, 2School of Medicine and Institute for Science and Technology in Medicine, Keele University

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

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Immunology and Infection

Establishment and Analysis of Tumor Slice Explants As a Prerequisite for Diagnostic Testing
Ashwini S. Nagaraj 1, Jie Bao 1, Annabrita Hemmes 1, Mafalda Machado 1, Katja Närhi 1, Emmy W. Verschuren 1
1Institute for Molecular Medicine Finland (FIMM), HiLIFE, University of Helsinki

We provide a method for the generation, cultivation and systematic analysis of organotypic slices derived from murine lung tumors. We also describe how to optimize for slice thickness, and how to select drug concentrations to treat tumor slices.

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Medicine

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy
Erika Gucciardo 1, Sirpa Loukovaara 2, Ani Korhonen 1, Kaisa Lehti 1,3
1Research Programs Unit, Genome-Scale Biology, Biomedicum Helsinki, University of Helsinki, 2Unit of Vitreoretinal Surgery, Ophthalmology, University of Helsinki and Helsinki University Hospital, 3Department of Microbiology, Tumor, and Cell Biology (MTC), Karolinska Institutet

Here, we present a protocol to study the pathophysiology of proliferative diabetic retinopathy by using patient-derived, surgically-excised, fibrovascular tissues for three-dimensional native tissue characterization and ex vivo culture. This ex vivo culture model is also amenable for testing or developing new treatments.

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Biology

Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting
Svetlana Konovalova 1
1Research Programs Unit, Molecular Neurology, University of Helsinki

This protocol demonstrates the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis. Here the method applied to cultured human cells is described.

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Cancer Research

Predicting In Vivo Payloads Delivery using a Blood-brain Tumor-barrier in a Dish
Vadim Le Joncour 1, Sinem Karaman 1,2, Pirjo Maarit Laakkonen 1,3
1Research Programs Unit, Translational Cancer Medicine Research Program, University of Helsinki, 2Wihuri Research Institute, Biomedicum Helsinki, University of Helsinki, 3Laboratory Animal Center, Helsinki Institute of Life Science (HiLIFE), University of Helsinki

Drug targeting to central nervous system tumors is a major challenge. Here we describe a protocol to produce an in vitro mimic of the blood-brain tumor-barrier using murine and/or human cells and discuss their relevance for the predictability of central nervous system tumor targeting in vivo.

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Cancer Research

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay
Erika Naakka 1, Katja Tuomainen 1, Henrik Wistrand *1, Miila Palkama *1, Ilida Suleymanova 1, Ahmed Al-Samadi 1, Tuula Salo 1,2,3,4
1Department of Oral and Maxillofacial Diseases, Clinicum, Faculty of Medicine, University of Helsinki, 2Cancer and Translational Medicine Research Unit, University of Oulu, 3Medical Research Center, Oulu University Horspital, 4Helsinki University Hospital

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

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Engineering

Scattering And Absorption of Light in Planetary Regoliths
Karri Muinonen 1,2, Timo Väisänen 1, Julia Martikainen 1, Johannes Markkanen 1,3, Antti Penttilä 1, Maria Gritsevich 1,4, Jouni Peltoniemi 1,2, Jürgen Blum 5, Joonas Herranen 1, Gorden Videen 6,7, Göran Maconi 1, Petteri Helander 1, Ari Salmi 1, Ivan Kassamakov 1, Edward Haeggström 1
1Department of Physics, University of Helsinki, 2Finnish Geospatial Research Institute FGI, National Land Survey, 3Max Planck Institute for Solar System Research, 4Institute of Physics and Technology, Ural Federal University, 5Institut für Geophysik und Extraterrestrische Physik, Technische Universität Braunschweig, 6Space Science Institute, 7Army Research Laboratory

Numerical and experimental methods are presented for multiple scattering of light in discrete random media of densely-packed particles. The methods are utilized to interpret the observations of asteroid (4) Vesta and comet 67P/Churyumov-Gerasimenko.

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Behavior

Lexical Decision Task for Studying Written Word Recognition in Adults with and without Dementia or Mild Cognitive Impairment
Alexandre Nikolaev 1, Eve Higby 2,3, JungMoon Hyun 4, Sameer Ashaie 5,6
1Helsinki Collegium for Advanced Studies, University of Helsinki, 2University of California, Riverside, 3California State University, East Bay, 4Hunter College, City University of New York, 5Feinberg School of Medicine, Northwestern University, 6Shirley Ryan AbilityLab

This article describes how to implement a simple lexical decision experiment to assess written word recognition in neurologically healthy participants and in individuals with dementia and cognitive decline. We also provide a detailed description of reaction time analysis using principal components analysis (PCA) and mixed-effects modeling.

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Cancer Research

Detection of Retrotransposition Activity of Hot LINE-1s by Long-Distance Inverse PCR
Barun Pradhan 1,2, Liisa Kauppi 1,2
1Systems Oncology Research Program, University of Helsinki, 2Department of Biochemistry and Developmental Biology, Medicum, University of Helsinki

This article outlines a simple PCR-based assay to monitor the activity of an active LINE-1 retrotransposon and to map de novo retrotranspositions in a given genome. Using the MCF7 cell line, we demonstrate herein how this method can be applied to detect activity of a LINE-1 located at 22q12.1.

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Biochemistry

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors
Keni Vidilaseris *1, Niklas G. Johansson *2, Ainoleena Turku 2, Alexandros Kiriazis 2, Gustav Boije af Gennäs 2, Jari Yli-Kauhaluoma 2, Henri Xhaard 2, Adrian Goldman 1,3
1Research Program in Molecular and Integrative Biosciences, University of Helsinki, 2Drug Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, 3School of Biomedical Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds

Here we present a screening method for membrane-bound pyrophosphatase (from Thermotoga maritima) inhibitors based on the molybdenum blue reaction in a 96 well plate format.

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Genetics

LINE-1 Methylation Analysis in Mesenchymal Stem Cells Treated with Osteosarcoma-Derived Extracellular Vesicles
Snehadri Sinha *1,2, Bettina Mannerström *1,2, Riitta Seppänen-Kaijansinkko 1,2, Sippy Kaur 1,2
1Department of Oral and Maxillofacial Diseases, University of Helsinki, 2Helsinki University Hospital

Described here is the use of a methylation-specific probe amplification method to analyze methylation levels of LINE-1 elements in mesenchymal stem cells treated with osteosarcoma-derived extracellular vesicles. Ultracentrifugation, a popular procedure for separating extracellular vesicles from fetal bovine serum, is also demonstrated.

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Neuroscience

Studying Pre-formed Fibril Induced α-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons
Safak Er 1, Irena Hlushchuk 1, Mikko Airavaara 2, Piotr Chmielarz 1,3, Andrii Domanskyi 1
1Institute of Biotechnology, HiLIFE, University of Helsinki, 2Neuroscience Center, HiLIFE, University of Helsinki, 3Department of Brain Biochemistry, Maj Institute of Pharmacology, Polish Academy of Sciences

Here, we present a detailed protocol to study neuronal α-synuclein accumulation in primary mouse dopamine neurons. Phosphorylated α-synuclein aggregates in neurons are induced with pre-formed α-synuclein fibrils. Automated imaging of immunofluorescently labeled cells and unbiased image analysis make this robust protocol suitable for medium-to-high throughput screening of drugs that inhibit α-synuclein accumulation.

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Chemistry

Preparation of Silver-Palladium Alloyed Nanoparticles for Plasmonic Catalysis under Visible-Light Illumination
Erandi Peiris 1, Sébastien Hanauer 1, Kjell Knapas 1, Pedro H. C. Camargo 1
1Department of Chemistry, University of Helsinki

Presented here is a protocol for the synthesis of silver-palladium (Ag-Pd) alloy nanoparticles (NPs) supported on ZrO2 (Ag-Pd/ZrO2). This system allows for harvesting energy from visible light irradiation to accelerate and control molecular transformations. This is illustrated by nitrobenzene reduction under light irradiation catalyzed by Ag-Pd/ZrO2 NPs.

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Immunology and Infection

Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection
Zhe Zhang Ryan Lew *1, Jing Liu *1, Hsiao Hui Ong 1, Vivian Jiayi Tan 2, Annika Luukkainen 3, Yew Kwang Ong 1,4, Mark Thong 1,4, Kia Joo Puan 5, Vincent Tak Kwong Chow 2,6, Kai Sen Tan 1,2,6, De Yun Wang 1,6
1Department of Otolaryngology, Yong Loo Lin School of Medicine, National University of Singapore, 2Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 3Haartman Institute, Medicum, University of Helsinki, 4Department of Otolaryngology, Head and Neck Surgery, National University Hospital, National University Health System, 5Agency for Science, Technology and Research (A*STAR), Singapore Immunology Network (SIgN), 6NUHS Infectious Diseases Translational Research Program, Yong Loo Lin School of Medicine, National University of Singapore

This protocol details an investigation of the early interactions between virally infected nasal epithelial cells and innate cell activation. Individual subsets of immune cells can be distinguished based on their activation in response to viral infections. They can then be further investigated to determine their effects on early antiviral responses.

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Developmental Biology

Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells
Emma Juuri *1,2, Anamaria Balic *1
1Research Program in Developmental Biology, Institute of Biotechnology, University of Helsinki, 2Department of Oral and Maxillofacial diseases, Faculty of Medicine, University of Helsinki

The Trowell-type organ culture method has been used to unravel complex signaling networks that govern tooth development and, more recently, for studying regulation involved in stem cells of the continuously growing mouse incisor. Fluorescent-reporter animal models and live-imaging methods facilitate in-depth analyses of dental stem cells and their specific niche microenvironment.

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Biology

High-Resolution Respirometry to Assess Bioenergetics in Cells and Tissues Using Chamber- and Plate-Based Respirometers
Ryan Awadhpersad 1, Christopher B. Jackson 1
1Department of Biochemistry and Developmental Biology, Faculty of Medicine, University of Helsinki

Assessing oxidative phosphorylation using high-resolution respirometers has become an integral part of the functional analysis of mitochondria and cellular energy metabolism. Here, we present protocols for the analysis of cellular energy metabolism using chamber and microplate-based high-resolution respirometers and discuss the key benefits of each device.

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Neuroscience

A Flexible Platform for Monitoring Cerebellum-Dependent Sensory Associative Learning
Gerard Joey Broussard 1, Mikhail Kislin 1, Caroline Jung 1, Samuel S. -H. Wang 1
1Neuroscience Institute, Princeton University

We have developed a single platform to track animal behavior during two climbing fiber-dependent associative learning tasks. The low-cost design allows integration with optogenetic or imaging experiments directed towards climbing fiber-associated cerebellar activity.

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Neuroscience

Measuring Properties of the Membrane Periodic Skeleton of the Axon Initial Segment using 3D-Structured Illumination Microscopy (3D-SIM)
David Micinski 1, Lauri Lahti 2, Amr Abouelezz 1,3,4
1Minerva Foundation Institute for Medical Research, 2Department of Computer Science, Aalto University School of Science, 3HiLIFE - Neuroscience Center, University of Helsinki, 4HiLIFE - Institute of Biotechnology, University of Helsinki

The present protocol describes a method to visualize and measure actin rings and other components of the membrane periodic skeleton of the axon initial segment using cultured rat hippocampal neurons and 3D-structured illumination microscopy (3D-SIM).

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Biology

Zebrafish Corneal Wound Healing: From Abrasion to Wound Closure Imaging Analysis
Kaisa Ikkala 1, Sini Raatikainen 1, Frederic Michon 1,2
1Institute of Biotechnology, HiLIFE, University of Helsinki, 2Institute for Neurosciences of Montpellier, Univ Montpellier

This protocol focuses on damaging the ocular surface of zebrafish through abrasion to assess the subsequent wound closure at the cellular level. This approach exploits an ocular burr to partly remove the corneal epithelium and uses scanning electron microscopy to track changes in cell morphology during wound closure.

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Neuroscience

Quantified Assessment of Infant's Gross Motor Abilities Using a Multisensor Wearable
Elisa Taylor 1, Manu Airaksinen 1, Anastasia Gallen 1, Tuuli Immonen 2, Elina Ilén 3, Taru Palsa 1,2, Leena M. Haataja 1,2, Sampsa Vanhatalo 1,4
1BABA Center, Pediatric Research Center, Department of Clinical Neurophysiology, New Children's Hospital and HUS Imaging, Helsinki, University Hospital, 2Department of Pediatric Neurology, Children's Hospital, Helsinki University Hospital and University of Helsinki, 3Department of Materials Science and Engineering, Universitat Politècnica de Catalunya, 4Department of Physiology, University of Helsinki

This paper outlines the assessment of infants' gross motor performance with a multisensor wearable and its fully automated deep learning-based analysis pipeline. The method quantifies the posture and movement patterns of infants from lying supine until they master walking independently.

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Developmental Biology

Z-Scores for Assessing Ovarian Reserve in Young Patients Undergoing Fertility Preservation
Jasmin Hassan 1,2, Pauliina Damdimopoulou 1,2, Atte Lahtinen 3,4, Kirsi Jahnukainen 5,6, Katri Knuus 7, Ganesh Acharya 1, Richelle D. Björvang 1,8,9
1Division of Obstetrics and Gynecology, Department of Clinical Science, Intervention and Technology, Karolinska Institutet, 2Department of Gynecology and Reproductive Medicine, Karolinska University Hospital, 3Applied Tumor Genomics Research Program, Faculty of Medicine, University of Helsinki, 4Department of Medical and Clinical Genetics, Medicum, Faculty of Medicine, University of Helsinki, 5Children's Hospital, Pediatric Research Centre, University of Helsinki and Helsinki University Hospital, 6NORDFERTIL Research Lab Stockholm, Department of Women's, and Children's Health, Karolinska Institutet, and Karolinska University Hospital, 7Department of Obstetrics and Gynecology, Helsinki University Hospital, University of Helsinki, 8Department of Cell and Molecular Biology, Uppsala University, 9Department of Women's and Children's Health, Uppsala University

The present protocol describes a method for assessing ovarian reserve in patients under 25 years old who require fertility preservation through ovarian tissue cryopreservation. This method involves: (1) histological assessment of ovarian reserve in cortical samples, (2) comparison to a reference dataset, and (3) calculation of Z-scores.

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