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Polish Academy of Sciences

20 ARTICLES PUBLISHED IN JoVE

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Neuroscience

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna
M. Rezaul Karim 1, Keita Endo 2, Adrian W Moore 3, Hiroaki Taniguchi 1
1Laboratory for Genetic Code, Graduate School of Life and Medical Sciences, Doshisha University, 2Laboratory for Circuit Mechanisms of Sensory Perception, RIKEN Brain Science Institute, 3Disease Mechanism Research Core, RIKEN Brain Science Institute

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

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Neuroscience

A Simple Flight Mill for the Study of Tethered Flight in Insects
Alfredo Attisano 1, James T. Murphy 2, Andrew Vickers 3, Patricia J. Moore 2
1Museum and Institute of Zoology, Polish Academy of Sciences, 2Department of Entomology, University of Georgia, 3Riventa Pool Innovation Centre

Flight in insects is influenced by a number of factors and the propensity to disperse is an important variable in understanding insect ecology and biological control strategies. We describe the construction and use of a simple, relatively inexpensive, and flexible flight mill for measuring parameters of tethered flight in insects.

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Biology

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis
Anna K. Schellhaus *1, Adriana Magalska *2, Allana Schooley 1, Wolfram Antonin 1
1Friedrich Miescher Laboratory, Max Planck Society, 2Nencki Institute of Experimental Biology, Polish Academy of Sciences

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

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Developmental Biology

An Efficient Method to Obtain Dedifferentiated Fat Cells
Hiroaki Taniguchi *1, Tomohiko Kazama *1, Kazuhiro Hagikura *1, Chii Yamamoto 1, Minako Kazama 1, Yuki Nagaoka 1, Taro Matsumoto 1
1Division of Cell Regeneration and Transplantation, School of Medicine, Nihon University

We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these cells.

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Developmental Biology

Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies
M. Rezaul Karim 1, Tomohiro Haruta 2, Taro Matsumoto 3, Hiroki Oda 2, Hiroaki Taniguchi 3,4,5
1Biotechnology and Genetic Engineering Department, Jahangirnagar University, 2JT Biohistory Research Hall, 3Division of Cell Regeneration and Transplantation, School of Medicine, Nihon University, 4Graduate School of Life and Medical Sciences, Doshisha University, 5Institute of Genetics and Animal Breeding of the Polish Academy of Sciences

Herein we describe a procedure to capture live images of Drosophila gastrulation. This has enabled us to better understand the apical constriction involved in early development and further analyze mechanisms governing cellular movements during tissue structure modification.

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Biochemistry

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis
Megumi Tsuchiya *1, M. Rezaul Karim *2, Taro Matsumoto 3, Hidesato Ogawa 1, Hiroaki Taniguchi 3,4,5
1Graduate School of Frontier Biosciences, Osaka University, 2Department of Biotechnology and Genetic Engineering, Jahangirnagar University, 3Division of Cell Regeneration and Transplantation, School of Medicine, Nihon University, 4Institute of Genetics and Animal Breeding of the Polish Academy of Sciences, 5Graduate School of Life and Medical Sciences, Doshisha University

We have established a method for the purification of coregulatory interaction proteins using the LC-MS/MS system.

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JoVE Core

Production and Characterization of Vacuum Deposited Organic Light Emitting Diodes
Daniel de Sa Pereira 1, Andrew P. Monkman 1, Przemyslaw Data 1,2,3
1Physics Department, Durham University, 2Faculty of Chemistry, Silesian University of Technology, 3Centre of Polymer and Carbon Materials, Polish Academy of Science

A protocol for the production of simple structured organic light-emitting diodes (OLEDs) is presented.

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Chemistry

Electrochemical Impedance Spectroscopy as a Tool for Electrochemical Rate Constant Estimation
Pavel Chulkin 1, Przemyslaw Data 1,2,3
1Factulty of Chemistry, Department of Physical Chemistry and Technology of Polymers, Silesian University of Technology, 2Department of Physics, Durham University, 3Centre of Polymer and Carbon Materials of the Polish Academy of Sciences

Electrochemical impedance spectroscopy (EIS) of species that undergo reversible oxidation or reduction in solution was used for determination of rate constants of oxidation or reduction.

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Chemistry

Time-resolved Photophysical Characterization of Triplet-harvesting Organic Compounds at an Oxygen-free Environment Using an iCCD Camera
Piotr Pander 1, Przemyslaw Data 1,2,3, Fernando B. Dias 1
1Department of Physics, University of Durham, 2Faculty of Chemistry, Silesian University of Technology, 3Center of Polymer and Carbon Materials, Polish Academy of Sciences

Here, we present a method of the spectroscopic characterization of organic molecules by means of time-resolved photoluminescence spectroscopy on the nanosecond-to-millisecond timescale in oxygen-free conditions. Methods to efficiently remove oxygen from the samples and, thus, limit luminescence quenching are also described.

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Chemistry

Raman and IR Spectroelectrochemical Methods as Tools to Analyze Conjugated Organic Compounds
Agata Blacha-Grzechnik 1, Krzysztof Karon 1, Przemyslaw Data 1,2,3
1Faculty of Chemistry, Department of Physical Chemistry and Technology of Polymers, Silesian University of Technology, 2Department of Physics, Durham University, 3Centre of Polymer and Carbon Materials of the Polish Academy of Sciences

A protocol of step-by-step Raman and IR spectroelectrochemical analysis is presented.

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JoVE Journal

Using Cyclic Voltammetry, UV-Vis-NIR, and EPR Spectroelectrochemistry to Analyze Organic Compounds
Sandra Pluczyk 1, Marharyta Vasylieva 1, Przemyslaw Data 1,2,3
1Faculty of Chemistry, Department of Physical Chemistry and Technology of Polymers, Silesian University of Technology, 2Department of Physics, Durham University, 3Center of Polymer and Carbon Materials of the Polish Academy of Sciences

In this article, we describe electrochemical, electron paramagnetic resonance, and ultraviolet-visible and near-infrared spectroelectrochemical methods to analyze organic compounds for application in organic electronics.

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Chemistry

Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis
Lukasz Bozycki 1, Magdalena Komiazyk 1, Saida Mebarek 2,3,4,5,6, Rene Buchet 2,3,4,5,6, Slawomir Pikula 1, Agnieszka Strzelecka-Kiliszek 1
1Laboratory of Lipid Biochemistry, Nencki Institute of Experimental Biology, Polish Academy of Sciences, 2Université de Lyon, 3Université Lyon 1, 4L'insitut National des Sciences Appliquées (INSA) de Lyon, 5Ecole Supérieure de Chimie Physique Electronique (CPE) Lyon, 6Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche (UMR), L'institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS)

We present a protocol to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. Their mineralization profiles were analyzed by Alizarin Red-S (AR-S) staining, ultraviolet (UV) light visualization, transmission electron microscopy (TEM) imaging and energy dispersive X-ray microanalysis (EDX).

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Biochemistry

Manipulating Living Cells to Construct Stable 3D Cellular Assembly Without Artificial Scaffold
Takehiro Yamazaki *1, Hiroaki Taniguchi *2, Shoto Tsuji 1, Shiho Sato 1, Takahiro Kenmotsu 1, Kenichi Yoshikawa 1, Koichiro Sadakane 1
1Faculty of Life and Medical Sciences, Doshisha University, 2The Institute of Genetics and Animal Breeding, Polish Academy of Sciences

We demonstrate a novel method for constructing a single-cell-based 3-dimensional (3D) assembly without an artificial scaffold.

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Developmental Biology

Neurogenesis Using P19 Embryonal Carcinoma Cells
Paweł Leszczyński 1, Magdalena Śmiech 1, Aamir S. Teeli 1, Aleksandra Zołocińska 2, Anna Słysz 2, Zygmunt Pojda 2, Mariusz Pierzchała 3, Hiroaki Taniguchi 1
1Department of Experimental Embryology, Institute of Genetics and Animal Breeding, Polish Academy of Sciences, 2Department of Regenerative Medicine, Maria Skłodowska-Curie Institute - Oncology Center, 3Department of Genomics and Biodiversities, Institute of Genetics and Animal Breeding, Polish Academy of Sciences

The P19 mouse embryonic carcinoma cell line (P19 cell line) is widely used for studying the molecular mechanism of neurogenesis with great simplification compared to in vivo analysis. Here, we present a protocol for retinoic acid-induced neurogenesis in the P19 cell line.

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Medicine

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products
Kinga Jaworska 1, Tomasz Huc 1, Marta Gawrys 1, Maksymilian Onyszkiewicz 1, Emilia Samborowska 2, Marcin Ufnal 1
1Department of Experimental Physiology and Pathophysiology, Laboratory of Centre for Preclinical Research, Medical University of Warsaw, 2Mass Spectrometry Laboratory, Institute of Biochemistry and Biophysics, Polish Academy of Sciences

The access of nutrients, microbiota metabolites and medicines to the circulation is controlled by the gut-blood barrier (GBB). We describe a direct method for measuring the GBB permeability in vivo, which, in contrast to commonly used indirect methods, is virtually not affected by liver and kidney functions.

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Bioengineering

Generation of Transgenic Rats using a Lentiviral Vector Approach
Paulina Koza 1, Joanna Przybyś 1, Agata Klejman 1, Gabriela Olech-Kochańczyk 1, Witold Konopka 1
1Nencki Institute of Experimental Biology, Polish Academy of Sciences

This article aims to provide the methodology for lentiviral transgenesis in rat embryos using multiple injections of a virus suspension into the zygote perivitelline space. Female rats that are mated with a fertile male strain with a different dominant fur color is used to generate pseudopregnant foster mothers.

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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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Biochemistry

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution
Lukasz S. Borowski 1,2, Karolina Kasztelan 2,3, Jolanta Czerwinska-Kostrzewska 1,2, Roman J. Szczesny 2
1Faculty of Biology, Institute of Genetics and Biotechnology, University of Warsaw, 2Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 3International Institute of Molecular and Cell Biology in Warsaw

A procedure for studying the dynamics of mitochondrial DNA (mtDNA) metabolism in cells using a multi-well plate format and automated immunofluorescence imaging to detect and quantify mtDNA synthesis and distribution is described. This can be further used to investigate the effects of various inhibitors, cellular stresses, and gene silencing on mtDNA metabolism.

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Neuroscience

Motion-Acuity Test for Visual Field Acuity Measurement with Motion-Defined Shapes
Marco Ninghetto 1, Michał Wieteska 2,3, Anna Kozak 1, Kamil Szulborski 4, Tomasz Gałecki 4, Jacek Szaflik 4, Kalina Burnat 1
1Nencki Institute of Experimental Biology, Polish Academy of Sciences, 2Mossakowski Medical Research Institute, Polish Academy of Sciences, 3Institute of Radio Electronics and Multimedia Technology, Warsaw University of Technology, 4Department of Ophthalmology, Medical University of Warsaw

A novel motion-based acuity test that allows the assessment of central and peripheral visual processing in low-vision and healthy individuals, along with goggles limiting peripheral vision compatible with MRI protocols, are described here. This method offers a comprehensive vision assessment for functional impairments and dysfunctions of the visual system.

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