Kumamoto University

9 ARTICLES PUBLISHED IN JoVE

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Biology

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy
Takumi Higaki 1
1Graduate School of Frontier Sciences, The University of Tokyo

The goal of this protocol is to demonstrate how to monitor fluorescently-tagged protein dynamics on plant cell surfaces with variable-angle epifluorescence microscopy, showing blinking dots of GFP-tagged PATROL1, a membrane trafficking protein, in the cell cortex of the stomatal complex in Arabidopsis thaliana.

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Bioengineering

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device
Yuji Nashimoto 1, Yukako Teraoka 1, Ramin Banan Sadeghian 1, Akiko Nakamasu 2, Yuichiro Arima 3, Sanshiro Hanada 3, Hidetoshi Kotera 1, Koichi Nishiyama 3, Takashi Miura 2, Ryuji Yokokawa 1
1Department of Micro Engineering, Kyoto University, 2Graduate School of Medical Sciences, Kyushu University, 3International Research Center for Medical Sciences (IRCMS), Kumamoto University

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

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Neuroscience

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice
Hidenobu Mizuno 1,2,3, Shingo Nakazawa 2,3, Takuji Iwasato 2,3
1International Research Center for Medical Sciences (IRCMS), Kumamoto University, 2Division of Neurogenetics, National Institute of Genetics, 3Department of Genetics, SOKENDAI (The Graduate University for Advanced Studies)

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

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Environment

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings
Kae Akita 1, Takumi Higaki 2
1Department of Integrated Frontier Sciences, The University of Tokyo, 2International Research Organization for Advanced Science and Technology, Kumamoto University

The goal of this protocol is to demonstrate how to induce clustered stomata in cotyledons of Arabidopsis thaliana seedlings by immersion treatment with a sugar-containing medium solution and how to observe intracellular structures such as chloroplasts and microtubules in the clustered guard cells using confocal laser microscopy.

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Biology

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate
Yen Xuan Ngo 1,2,3, Kenta Haga 4, Ayako Suzuki 4, Hiroko Kato 4, Hiromi Yanagisawa 1,5, Kenji Izumi 4, Aiko Sada 1,3
1Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, 2Ph.D. Program in Human Biology, School of Integrative and Global Majors, University of Tsukuba, 3International Research Center for Medical Sciences (IRCMS), Kumamoto University, 4Division of Biomimetics, Faculty of Dentistry and Graduate School of Medical and Dental Sciences, Niigata University, 5Faculty of Medicine, University of Tsukuba

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

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Medicine

Sample Preparation for Computed Tomography-based Three-dimensional Visualization of Murine Hind-limb Vessels
Daiki Seya 1, Yuqing Xu 2,3, Toshifumi Mukunoki 4, Kenichi Tsujita 3, Osamu Nakagawa 1, Yuichiro Arima 1,2,3,4
1Department of Molecular Physiology, National Cerebral and Cardiovascular Center Research Institute, 2International Research Center for Medical Sciences (IRCMS), Kumamoto University, 3Department of Cardiovascular Medicine, Graduate School of Medical Sciences, Kumamoto University, 4X-Earth Center, Faculty of Advanced Science and Technology, Kumamoto University

Here, we describe a visualization and quantification method for murine hind-limb vessels using micro-X-ray computed tomography.

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Chemistry

Magnetometric Characterization of Intermediates in the Solid-State Electrochemistry of Redox-Active Metal-Organic Frameworks
Qi Chen 1, Zhongyue Zhang 2, Kunio Awaga 1,3
1Department of Chemistry, Graduate School of Science, Nagoya University, 2International Research Organization for Advanced Science and Technology, Kumamoto University, 3Integrated Research Consortium on Chemical Sciences, Nagoya University

Ex situ magnetic surveys can directly provide bulk and local information on a magnetic electrode to reveal its charge storage mechanism step by step. Herein, electron spin resonance (ESR) and magnetic susceptibility are demonstrated to monitor the evaluation of paramagnetic species and their concentration in a redox-active metal-organic framework (MOF).

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Medicine

Hyperglycemic Clamp and Hypoglycemic Clamp in Conscious Mice
Takashi Abe 1, Chitoku Toda 1
1Department of Neuroscience for Metabolic Control, Graduate School of Medical Science, Kumamoto University

A hyperglycemic clamp is used for measuring insulin release with a maintained higher blood glucose concentration. A hypoglycemic clamp is for measuring glucose production induced by counter-regulatory responses. Both methods use the same surgical procedure. Here, we present a clamp technique to assess systemic glucose metabolism.

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Neuroscience

In Vivo Visualization of Spontaneous Activity in Neonatal Mouse Sensory Cortex at a Single-Neuron Resolution
Nao Nakagawa-Tamagawa *1, Takamitsu Egashira *2,3, Madhura S. Rao 2,3,4, Hidenobu Mizuno 2,3
1Department of Physiology, Graduate School of Medical and Dental Sciences, Kagoshima University, 2Laboratory of Multi-Dimensional Imaging, International Research Center for Medical Sciences (IRCMS), Kumamoto University, 3Graduate School of Medical Sciences, Kumamoto University, 4Cedars-Sinai Medical Center

Primary sensory areas in the neocortex exhibit unique spontaneous activities during development. This article describes how to visualize individual neuron activities and primary sensory areas to analyze area-specific synchronous activities in neonatal mice in vivo.

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