Osaka University

17 ARTICLES PUBLISHED IN JoVE

Developmental Biology

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

¹Center for International Research on Integrative Biomedical System, Institute of Industrial Science, The University of Tokyo

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

Engineering

Measurement of Scattering Nonlinearities from a Single Plasmonic Nanoparticle

Hsuan Lee¹, Kuan-Yu Li¹, Yen-Ta Huang¹, Po-Ting Shen¹, Gitanjal Deka¹, Ryosuke Oketani², Yasuo Yonemaru², Masahito Yamanaka², Katsumasa Fujita², Shi-Wei Chu^1,3

¹Department of Physics, National Taiwan University, ²Department of Applied Physics, Osaka University, ³Molecular Imaging Center, National Taiwan University

Saturable and reverse saturable scattering were discovered in isolated plasmonic particles and adopted as a novel non-bleaching contrast method in super-resolution microscopy. Here the experimental procedures of detecting and extracting nonlinear scattering are explained in detail, as well as how to enhance resolution with the aid of saturated excitation microscopy.

Chemistry

Construction and Systematical Symmetric Studies of a Series of Supramolecular Clusters with Binary or Ternary Ammonium Triphenylacetates

Toshiyuki Sasaki¹, Yoko Ida¹, Tetsuharu Yuge¹, Atsushi Yamamoto¹, Ichiro Hisaki¹, Norimitsu Tohnai¹, Mikiji Miyata¹

¹Department of Material and Life Science, Graduate School of Engineering, Osaka University

This article describes construction of a series of hydrogen-bonding supramolecular clusters in crystals using primary ammonium triphenylacetates, which are recrystallized from non-polar solvents. This selective construction of the supramolecular clusters leads to effective systematical symmetric studies about a correlation between the supramolecular clusters and their components.

Biology

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

Toshitsugu Fujita¹, Hodaka Fujii¹

¹Chromatin Biochemistry Research Group, Combined Program on Microbiology and Immunology, Research Institute for Microbial Diseases, Osaka University

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

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 Matsumoto³, Hidesato Ogawa¹, 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.

Biochemistry

Real-time Tracking of DNA Fragment Separation by Smartphone

Chunxian Tao¹, Bo Yang¹, Zhenqing Li¹, Dawei Zhang¹, Yoshinori Yamaguchi^1,2,3

¹Shanghai Key Lab of Modern Optical System, University of Shanghai for Science and Technology, ²Department of Applied Physics, Graduate School of Engineering, Osaka University, ³East China University of Science and Technology, Department of Physics Faculty of Science

Traditional slab gel electrophoresis (SGE) experiments require a complicated apparatus and high chemical consumption. This work presents a protocol that describes a low-cost method to separate DNA fragments within a short timeframe.

Neuroscience

Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

Naoyuki Matsumoto^1,2, Nobuhiko Yamamoto²

¹Department of Medical Neuroscience, Graduate School of Medical Sciences, Kanazawa University, ²Neuroscience Laboratories, Graduate School of Frontier Biosciences, Osaka University

This protocol describes a method for simultaneous imaging of thalamocortical axon branching and synapse formation in organotypic cocultures of the thalamus and cerebral cortex. Individual thalamocortical axons and their presynaptic terminals are visualized by a single cell electroporation technique with DsRed and GFP-tagged synaptophysin.

Engineering

Electric-field Control of Electronic States in WS₂ Nanodevices by Electrolyte Gating

Feng Qin¹, Toshiya Ideue¹, Wu Shi², Yijin Zhang^3,4, Ryuji Suzuki¹, Masaro Yoshida⁵, Yu Saito¹, Yoshihiro Iwasa^1,5

¹Quantum-Phase Electronics Center (QPEC) and Department of Applied Physics, The University of Tokyo, ²Materials Sciences Division, Lawrence Berkeley National Laboratory, ³Institute of Scientific and Industrial Research, Osaka University, ⁴Max Planck Institute for Solid State Research, ⁵RIKEN Center for Emergent Matter Science (CEMS)

Here, we present a protocol to control the carrier number in solids by using the electrolyte.

JoVE Core

Generation and Control of Electrohydrodynamic Flows in Aqueous Electrolyte Solutions

Kentaro Doi¹, Fumika Nito¹, Ayako Yano², Ryo Nagura¹, Satoyuki Kawano¹

¹Department of Mechanical Science and Bioengineering, Graduate School of Engineering Science, Osaka University, ²Division of Mechanical Science and Technology, Faculty of Science and Technology, GUNMA University

The rectification of ion transport pathways is an effective method to generate one-directional ion-dragged electrohydrodynamic flows. By setting an ion-exchange membrane in a flow channel, an electrically polarized condition is generated and causes a liquid flow to be driven when an electric field is externally applied.

Bioengineering

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Ikki Horiguchi^1,2, Ikumi Suzuki³, Takashi Morimura³, Yasuyuki Sakai^1,4

¹Department of Chemical System Engineering, University of Tokyo, ²Department of Biotechnology, Osaka University, ³Biotech Business Unit, Fukoku Co. Ltd, ⁴Institute of Industrial Science, University of Tokyo

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

Engineering

Measuring Magnetically-Tuned Ferroelectric Polarization in Liquid Crystals

Hiroki Ueda¹, Takuya Akita², Yoshiaki Uchida², Tsuyoshi Kimura³

¹Division of Materials Physics, Graduate School of Engineering Science, Osaka University, ²Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, ³Department of Advance Materials Science, University of Tokyo

In this report, we present a protocol to examine direct magnetoelectric effects, i.e., induction of ferroelectric polarization by applying magnetic fields, in liquid crystals. This protocol provides a unique approach, supported by the softness of liquid crystals, to achieve room-temperature magnetoelectrics.

Engineering

Hydrogen Charging of Aluminum using Friction in Water

Keitaro Horikawa¹, Hidetoshi Kobayashi¹

¹Department of Mechanical Science and Bioengineering, Osaka University

In order to introduce high amounts of hydrogen in aluminum and aluminum alloys, a new method of hydrogen charging was developed, called the friction in water procedure.

Biology

High-Accuracy Correction of 3D Chromatic Shifts in the Age of Super-Resolution Biological Imaging Using Chromagnon

Atsushi Matsuda^1,2, Takako Koujin¹, Lothar Schermelleh³, Tokuko Haraguchi^1,2, Yasushi Hiraoka^1,2

¹Advanced ICT Research Institute Kobe, National Institute of Information and Communications Technology, ²Graduate School of Frontier Biosciences, Osaka University, ³Micron Advanced Bioimaging Unit, Department of Biochemistry, University of Oxford

Correction of chromatic shifts in three-dimensional (3D) multicolor fluorescence microscopy images is crucial for quantitative data analyses. This protocol is developed to measure and correct chromatic shifts in biological samples through acquisition of suitable reference images and processing with the open-source software, Chromagnon.

Bioengineering

Amplification of Escherichia coli in a Continuous-Flow-PCR Microfluidic Chip and Its Detection with a Capillary Electrophoresis System

Wenwen Dong¹, Chunxian Tao¹, Bo Yang¹, Erika Miyake², Zhenqing Li¹, Dawei Zhang¹, Yoshinori Yamaguchi^1,2

¹Shanghai Key Lab of Modern Optical System, University of Shanghai for Science and Technology, ²Graduate School of Engineering, Osaka University

This protocol describes how to build a continuous-flow-polymerase chain system based on a microfluidic chip and how to build a capillary electrophoresis system in the lab. It presents a simple method for the analysis of nucleic acids in the lab.

Neuroscience

A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales

Kenta Yamauchi^1,2, Shinichiro Okamoto^1,2,3, Megumu Takahashi^1,2,4,5, Masato Koike^2,3, Takahiro Furuta⁶, Hiroyuki Hioki^1,2,7

¹Department of Neuroanatomy, Juntendo University Graduate School of Medicine, ²Department of Cell Biology and Neuroscience, Juntendo University Graduate School of Medicine, ³Advanced Research Institute for Health Sciences, Juntendo University, ⁴Department of Neuroscience, Graduate School of Medicine, Kyoto University, ⁵Japan Society for the Promotion of Science, ⁶Department of Oral Anatomy and Neurobiology, Graduate School of Dentistry, Osaka University, ⁷Department of Multi-Scale Brain Structure Imaging, Juntendo University Graduate School of Medicine

The protocol provides a detailed method of neuronal imaging in brain slice using a tissue clearing method, ScaleSF. The protocol includes brain tissue preparation, tissue clarification, handling of cleared slices and confocal laser scanning microscopy imaging of neuronal structures from mesoscopic to microscopic levels.

Developmental Biology

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

Manabu Ozawa^*1, Chihiro Emori^*2, Masahito Ikawa^1,2

¹Laboratory of Reproductive Systems Biology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, ²Research Institute for Microbial Diseases, Osaka University

Here we present a protocol for developing genetically modified mouse models using embryonic stem cells, especially for large DNA knock-in (KI). This protocol is tuned up using CRISPR/Cas9 genome editing, resulting in significantly improved KI efficiency compared with the conventional homologous recombination-mediated linearized DNA targeting method.

Genetics

Frequency and Distribution of Crossovers in Caenorhabditis elegans Meiosis by SNP Genotyping using Real-time PCR

Miku Koishihara¹, Akihide Onishi^2,3, Akari Hirose², Takamune T. Saito^1,2

¹Graduate School of Biology-Oriented Science and Technology, Kindai University, ²Department of Genetic Engineering, Faculty of Biology-Oriented Science and Technology, Kindai University, ³Graduate School of Frontier Biosciences, Osaka University

This protocol provides a method for measuring crossover frequencies and distribution during meiosis in Caenorhabditis elegans. Single-nucleotide polymorphism genotyping with real-time PCR was used to determine the origin of the chromosomes. The 4-point mapping allowed to determine the crossover distributions of the left-arm, center, and right-arm regions of each chromosome.