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Kyoto University

45 ARTICLES PUBLISHED IN JoVE

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Biology

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse
Li Liu 1, Karen Duff 1
1Department of Pathology, Columbia University

Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why Aβ or tau levels in CSF change as the disease progresses in human patients. Here, we demonstrate a refined cisterna magna puncture technique for serial CSF sampling from the mouse.

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JoVE Journal

Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution
Li Liu 1, Lauren Boldon 1, Melissa Urquhart 1, Xiangyu Wang 1
1Department of Mechanical, Aerospace, and Nuclear Engineering, Rensselaer Polytechnic Institute

The demonstration of the small and wide angle X-ray scattering (SWAXS) procedure has become instrumental in the study of biological macromolecules. Through the use of the instrumentation and procedures of specific angle methods and preparation, the experimental data from the SWAXS displays the atomic and nano-scale characterization of macromolecules.

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Biology

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.
Cui Cao 1, Wen J. Wang 1, Ying Y. Huang 1, Hong L. Yao 1, Louis P. Conway 1, Li Liu 1, Josef Voglmeir 1
1Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

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Immunology and Infection

Evaluation of Blood Lactate and Plasma Insulin During High-intensity Exercise by Antecubital Vein Catheterization
Minas Nalbandian 1, Zsolt Radak 2, Masaki Takeda 3
1Graduate School of medicine, Kyoto University, 2Research Institute of Sports Science, University of Physical Education, 3Graduate School of Sports and Health Science, Doshisha University

Here we present a protocol to obtain several blood samples from the antecubital vein during high-intensity interval training. This protocol may be useful for the measurement of blood metabolites and endocrinal markers during exercise.

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Biology

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera
Yuichi Fukutomi 1, Keiji Matsumoto 1,2, Noriko Funayama 1, Shigeyuki Koshikawa 1,3,4
1Graduate School of Science, Kyoto University, 2Graduate School of Science, Osaka City University, 3The Hakubi Center for Advanced Research, Kyoto University, 4Graduate School of Environmental Science, Hokkaido University

Protocols for staging pupal periods and measurement of wing pigmentation of Drosophila guttifera are described. Staging and quantification of pigmentation provide a solid basis for studying developmental mechanisms of adult traits and enable interspecific comparison of trait development.

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Immunology and Infection

A Recovery Cardiopulmonary Bypass Model Without Transfusion or Inotropic Agents in Rats
Shingo Hirao 1, Hidetoshi Masumoto 1, Tatsuya Itonaga 1, Kenji Minatoya 1
1Department of Cardiovascular Surgery, Graduate School of Medicine, Kyoto University

Here, we present a protocol to describe a simple recovery cardiopulmonary bypass model without transfusion or inotropic agents in a rat. This model allows the study of the long-term multiple organ sequelae of cardiopulmonary bypass.

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Immunology and Infection

Cell Membrane Repair Assay Using a Two-photon Laser Microscope
Joshua J. A. Lee 1, Rika Maruyama 1, Hidetoshi Sakurai 2, Toshifumi Yokota 1
1Department of Medical Genetics, University of Alberta Faculty of Medicine and Dentistry, 2Center for iPS Cell Research and Application, Kyoto University

Cell membrane wounding via two-photon laser is a widely used method for assessing membrane resealing ability and can be applied to multiple cell types. Here, we describe a protocol for in vitro live-imaging of membrane resealing in dysferlinopathy patient cells following two-photon laser ablation.

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Developmental Biology

Combinational Treatment of Trichostatin A and Vitamin C Improves the Efficiency of Cloning Mice by Somatic Cell Nuclear Transfer
Rika Azuma 1, Kei Miyamoto 2, Mami Oikawa 3, Masayasu Yamada 4, Masayuki Anzai 1,5
1Division of Biological Science, Graduate School of Biology-Oriented Science and Technology, Kindai University, 2Faculty of Biology-Oriented Science and Technology, Kindai University, 3Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Zoology, University of Cambridge, 4Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, 5Institute of Advanced Technology, Kindai University

We describe a dramatically improved method for mouse cloning using trichostatin A, vitamin C, and deionized bovine serum albumin. We show a simplified, reproducible protocol that supports efficient development of cloned embryos. Hence, this method could become a standardized procedure for mouse cloning.

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Genetics

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions
Akihiro Isomura 1,2, Ryoichiro Kageyama 1,3,4,5
1Institute for Frontier Life and Medical Sciences, Kyoto University, 2Japan Science and Technology Agency, PRESTO, 3Institute for Integrated Cell-Material Sciences, Kyoto University, 4Graduate School of Medicine, Kyoto University, 5Graduate School of Biostudies, Kyoto University

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

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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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Bioengineering

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array
Yasumasa Mashimo 1,2, Momoko Yoshioka 1, Yumie Tokunaga 1, Christopher Fockenberg 1, Shiho Terada 1, Yoshie Koyama 1, Teiko Shibata-Seki 2, Koki Yoshimoto 1, Risako Sakai 1, Hayase Hakariya 1, Li Liu 1, Toshihiro Akaike 3, Eiry Kobatake 2, Siew-Eng How 4, Motonari Uesugi 1,5, Yong Chen 1,6, Ken-ichiro Kamei 1
1Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, 2Department of Life Science and Technology, School of Life Science and Technology, Tokyo Institute of Technology, 3Biomaterials Center for Regenerative Medical Engineering, Foundation for Advancement of International Science, 4Faculty of Science and Natural Resources, Universiti Malaysia Sabah, 5Institute for Chemical Research, Kyoto University, 6Ecole Normale Supérieure

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

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Environment

Measuring the Flight Ability of the Ambrosia Beetle, Platypus Quercivorus (Murayama), Using a Low-Cost, Small, and Easily Constructed Flight Mill
Ryuichi Okada 1, Duy Long Pham 2, Yasuto Ito 3, Michimasa Yamasaki 2, Hidetoshi Ikeno 1
1School of Human Science and Environment, University of Hyogo, 2Laboratory of Forest Biology, Division of Forest and Biomaterials Science, Graduate School of Agriculture, Kyoto University, 3Hyogo Prefectural Technology Center for Agriculture, Forestry and Fisheries

We developed a low cost and small flight mill, constructed with commonly available items and easily used in experimentation. Using this apparatus, we measured the flight ability of an ambrosia beetle, Platypus quercivorus.

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Chemistry

Novel Techniques for Observing Structural Dynamics of Photoresponsive Liquid Crystals
Masaki Hada 1, Shohei Saito 2, Ryuma Sato 3, Kiyoshi Miyata 4, Yasuhiko Hayashi 1, Yasuteru Shigeta 3, Ken Onda 4
1Graduate School of Natural Science and Technology, Okayama University, 2Graduate School of Science, Kyoto University, 3Center for Computational Sciences, University of Tsukuba, 4Graduate School of Science, Kyushu University

Here, we present the protocols of differential-detection analyses of time-resolved infrared vibrational spectroscopy and electron diffraction which enable observations of the deformations of local structures around photoexcited molecules in a columnar liquid crystal, giving an atomic perspective on the relationship between the structure and the dynamics of this photoactive material.

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Developmental Biology

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures
Taro Toyoda 1, Azuma Kimura 1, Hiromi Tanaka 1, Kenji Osafune 1
1Center for iPS Cell Research and Application (CiRA), Kyoto University

Here, we present a detailed protocol to differentiate human pluripotent stem cells (hPSCs) into pancreas/duodenum homeobox protein 1+ (PDX1+) cells for the generation of pancreatic lineages based on the non-colony type monolayer growth of dissociated single cells. This method is suitable for producing homogenous hPSC-derived cells, genetic manipulation and screening.

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Biology

Establishment of a Clonal Culture of Unicellular Conjugating Algae
Yuki Tsuchikane 1, Takashi Hamaji 2, Katsumi Ota 3, Syou Kato 4
1Department of Chemical and Biological Sciences, Faculty of Science, Japan Women's University, 2Department of Botany, Graduate School of Science, Kyoto University, 3Katsumi Ota Translation Office, 4Wetlands International Japan

In this article, we demonstrate the establishment of clonal cultures of unicellular conjugating algal species collected from a natural field site.

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Biochemistry

Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+
Ya-Min Du 1, Shen-Li Zheng 1, Li Liu 1, Josef Voglmeir 1, Gabriel Yedid 2
1Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, 2College of Life Science, Nanjing Agricultural University

We describe a simple and rapid method for the preparation and analysis of N-glycans from different cultivars of radish (Raphanus sativus).

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Neuroscience

Ethanol-Induced Cervical Sympathetic Ganglion Block Applications for Promoting Canine Inferior Alveolar Nerve Regeneration Using an Artificial Nerve
Yoshiki Shionoya 1, Katsuhisa Sunada 2, Gentarou Tsujimoto 2, Keiji Shigeno 3, Tatsuo Nakamura 3
1Department of Dental Anesthesia, Nippon Dental University Hospital at Tokyo, 2Department of Dental Anesthesiology, Nippon Dental University School of Life Dentistry at Tokyo, 3Department of Bioartificial Organs, Institute for Frontier Medical Science, Kyoto University

We evaluated the effect of cervical sympathetic ganglion block on nerve repair using artificial nerve conduits. Male beagle dogs were each implanted with an artificial nerve across a 10-mm gap in the left inferior alveolar nerve; left cervical sympathetic ganglion was blocked by injecting 99.5% ethanol via lateral thoracotomy.

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JoVE Journal

Chemoselective Preparation of 1-Iodoalkynes, 1,2-Diiodoalkenes, and 1,1,2-Triiodoalkenes Based on the Oxidative Iodination of Terminal Alkynes
Youzhi Li *1, Daya Huang *1, Ju Huang 1, Yan Liu 1, Keiji Maruoka 1,2
1School of Chemical Engineering and Light Industry, Guangdong University of Technology, 2Department of Chemistry, Graduate School of Science, Kyoto University

Herein, detailed protocols for the oxidative iodination of terminal alkynes using hypervalent-iodine reagents are presented, which chemoselectively afford 1-iodoalkynes, 1,2-diiodoalkenes, and 1,1,2-triiodoalkenes.

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Neuroscience

Analyzing Neural Activity and Connectivity Using Intracranial EEG Data with SPM Software
Wataru Sato *1, Takanori Kochiyama *2, Shota Uono 3, Naotaka Usui 4, Akihiko Kondo 5, Kazumi Matsuda 5, Keiko Usui 6, Motomi Toichi 7, Yushi Inoue 5
1Kokoro Research Center, Kyoto University, 2Brain Activity Imaging Center, Advanced Telecommunications Research Institute International, 3Department of Neurodevelopmental Psychiatry, Habilitation and Rehabilitation, Graduate School of Medicine, Kyoto University, 4National Epilepsy Center, 5Shizuoka Institute of Epilepsy and Neurological Disorders, 6Department of System Neuroscience, Sapporo Medical University, 7Faculty of Human Health Science, Graduate School of Medicine, Kyoto University

We present two analytical protocols that can be used to analyze intracranial electroencephalography data using the Statistical Parametric Mapping (SPM) software: time-frequency statistical parametric mapping analysis for neural activity, and dynamic causal modeling of induced responses for intra- and inter-regional connectivity.

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Neuroscience

3D Scanning Technology Bridging Microcircuits and Macroscale Brain Images in 3D Novel Embedding Overlapping Protocol
Saya Ide 1, Motoki Kajiwara 1, Hirohiko Imai 2, Masanori Shimono 1
1Graduate School of Medicine and Faculty of Medicine, Kyoto University, 2Graduate School of Informatics, Kyoto University

This article introduces an experimental protocol using 3D scanning technology bridging two spatial scales: the macroscopic spatial scale of whole-brain anatomy imaged by MRI at >100 μm and the microscopic spatial scale of neuronal distributions using immunohistochemistry staining and a multielectrode array system and other methods (~10 μm).

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Chemistry

Improving High Viscosity Extrusion of Microcrystals for Time-resolved Serial Femtosecond Crystallography at X-ray Lasers
Daniel James 1, Tobias Weinert 1, Petr Skopintsev 1, Antonia Furrer 1, Dardan Gashi 1,2, Tomoyuki Tanaka 3,4, Eriko Nango 3,4, Przemyslaw Nogly 1,5, Joerg Standfuss 1
1Division of Biology and Chemistry - Laboratory for Biomolecular Research, Paul Scherrer Institut, 2Photon Science Division - SwissFEL, Paul Scherrer Institut, 3RIKEN SPring-8 Center, 4Department of Cell Biology, Graduate School of Medicine, Kyoto University, 5Department of Biology, ETH Zürich

The success of a time-resolved serial femtosecond crystallography experiment is dependent on efficient sample delivery. Here, we describe protocols to optimize the extrusion of bacteriorhodopsin microcrystals from a high viscosity micro-extrusion injector. The methodology relies on sample homogenization with a novel three-way coupler and visualization with a high-speed camera.

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Engineering

Evaluating Targeting Accuracy in the Focal Plane for an Ultrasound-guided High-intensity Focused Ultrasound Phased-array System
Ke Li 1,2, Jingfeng Bai 1,2, Yazhu Chen 1,2, Xiang Ji 1,2
1Biomedical Instrument Institute, School of Biomedical Engineering, Shanghai Jiao Tong University, 2Shanghai Med-X Engineering Center for Medical Equipment and Technology, Shanghai Jiao Tong University

This study describes a protocol to evaluate the targeting accuracy in the focal plane of an ultrasound-guided high-intensity focused ultrasound phased-array system.

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Developmental Biology

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells
Taiki Nakajima 1, Hidetoshi Sakurai 2, Makoto Ikeya 2
1Department of Life Science Frontiers, Center for iPS Cells Research and Application, Kyoto University, 2Department of Clinical Application, Center for iPS Cells Research and Application, Kyoto University

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

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Bioengineering

A Converging Strategy for the Generation of a Virtually Sequenced cDNA Library from Unreferenced Pacific Oysters
Yong M. Lyu 1, Yu Q. Li 1, Hui B. Song 1, Juan Guo 1, Ting Wang 1, Li Liu 1, Gabriel Yedid 2, Josef Voglmeir 1
1Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, 2College of Life Science, Nanjing Agricultural University

We describe a strategy for how to use RNA samples from unreferenced Pacific oyster specimens, and evaluate the genetic material by comparison with publicly available genome data to generate a virtually sequenced cDNA library.

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Environment

Measurement of the Potential Rates of Dissimilatory Nitrate Reduction to Ammonium Based on 14NH4+/15NH4+ Analyses via Sequential Conversion to N2O
Megumi Kuroiwa 1, Keitaro Fukushima 2,3, Kazuma Hashimoto 2, Yukiko Senga 4, Tsubasa Sato 4, Chie Katsuyama 5, Yuichi Suwa 1
1Department of Biological Sciences, Faculty of Science and Engineering, Chuo University, 2Faculty & Graduate School of Urban Environmental Sciences, Tokyo Metropolitan University, 3Center for Ecological Research, Kyoto University, 4Department of Chemistry, Faculty of Science, Toho University, 5Graduate School of Integrated Arts and Sciences, Hiroshima University

A series of methods to determine the potential DNRA rate based on 14NH4+/15NH4+ analyses is provided in detail. NH4+ is converted into N2O via several steps and analyzed using quadrupole gas chromatography–mass spectrometry.

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Developmental Biology

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition
Ryo Ohta *1, Ryohichi Sugimura *1, Akira Niwa 1, Megumu K. Saito 1
1Center for iPS cell research and application (CiRA)

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

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Cancer Research

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells
Meghan L. Good *1,2, Raul Vizcardo *1,2, Takuya Maeda 1,2, Naritaka Tamaoki 1,2, Parisa Malekzadeh 1, Hiroshi Kawamoto 3, Nicholas P. Restifo 1,2
1Surgery Branch, National Cancer Institute, NIH, 2Center for Cell-Based Therapy, National Cancer Institute, NIH, 3Laboratory of Immunology, Institute for Frontier Life and Medical Sciences, Kyoto University

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8αβ+ single positive T cells using OP9/DLL1 co-culture system.

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Neuroscience

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury
Tianshu Wang 1, Akira Ito 2, Junichi Tajino 1,3, Hiroshi Kuroki 2, Tomoki Aoyama 1
1Department of Development and Rehabilitation of Motor Function, Human Health Sciences, Graduate School of Medicine, Kyoto University, 2Department of Motor Function Analysis, Human Health Sciences, Graduate School of Medicine, Kyoto University, 3Department of Otolaryngology, The Ohio State University Wexner Medical Center

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

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Education

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis
Hiromi Shimojo 1, Ryoichiro Kageyama 1,2,3,4
1Institute for Frontier Life and Medical Sciences, Kyoto University, 2Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, 3Kyoto University Graduate School of Medicine, 4Kyoto University Graduate School of Biostudies

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

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Medicine

A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure
Hiroomi Nishio 1,3, Kenji Minatoya 1, Hidetoshi Masumoto 1,2
1Department of Cardiovascular Surgery, Graduate School of Medicine, Kyoto University, 2Clinical Translational Research Program, RIKEN Center for Biosystems Dynamics Research, 3Department of Cardiovascular Surgery, Takamatsu Red Cross Hospital

The present protocol aims to experimentally create venous intimal hyperplasia by subjecting veins to arterial blood pressure for developing strategies to attenuate venous intimal hyperplasia following revascularization surgery using vein grafts.

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Medicine

Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes
Yun Liu *1, Yin Liang *1, Mengxue Wang *1, Chen Wang 1, Heng Wei 2, Keiji Naruse 1, Ken Takahashi 1
1Department of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University

We present a model of ischemic heart disease using cardiomyocytes derived from human induced pluripotent stem cells, together with a method for quantitative evaluation of tissue damage caused by ischemia. This model can provide a useful platform for drug screening and further research on ischemic heart disease.

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Bioengineering

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair
Takeichiro Nakane 1,2,6, Mosha Abulaiti 1,2, Yuko Sasaki 1, William J. Kowalski 3, Bradley B. Keller 4,5,7, Hidetoshi Masumoto 1,2
1Clinical Translational Research Program, RIKEN Center for Biosystems Dynamics Research, 2Department of Cardiovascular Surgery, Graduate School of Medicine, Kyoto University, 3Laboratory of Stem Cell and Neuro-Vascular Biology, Cell and Developmental Biology Center, National Institutes of Health, 4Kosair Charities Pediatric Heart Research Program, Cardiovascular Innovation Institute, University of Louisville, 5Department of Pediatrics, School of Medicine, University of Louisville, 6Department of Cardiovascular Surgery, Mitsubishi Kyoto Hospital, 7Cincinnati Children's Heart Institute

The present protocol generates mesh-shaped engineered cardiac tissues containing cardiovascular cells derived from human induced pluripotent stem cells to allow the investigation of cell implantation therapy for heart diseases.

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Developmental Biology

In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells
Chuang-Yu Lin 1, Michiko Yoshida 1,2, Li-Tzu Li 3, Megumu K. Saito 1
1Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, 2Department of Pediatrics, Kyoto Prefectural University of Medicine, 3Graduate Institute of Clinical Medicine, Taipei Medical University

Here we provide a protocol to generate in vitro NMJs from human induced pluripotent stem cells (iPSCs). This method can induce NMJs with mature morphology and function in 1 month in a single well. The resulting NMJs could potentially be used to model related diseases, to study pathological mechanisms or to screen drug compounds for therapy.

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Biochemistry

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis
Chuan-Chih Hsu 1,3, Chia-Feng Tsai 2, W. Andy Tao 3,4, Pengcheng Wang 5
1Department of Plant Biology, Carnegie Institute for Science, 2Graduate School of Pharmaceutical Sciences, Kyoto University, 3Department of Biochemistry, Purdue University, 4Department of Chemistry, Purdue University, 5Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences

Presented here is a phosphoproteomic approach, namely stop and go extraction tip based phosphoproteomic, which provides high-throughput and deep coverage of Arabidopsis phosphoproteome. This approach delineates the overview of osmotic stress signaling in Arabidopsis.

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Developmental Biology

Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica
Takahiro Ohde 1, Toshinori Minemura 1, Eiichi Hirose 1, Takaaki Daimon 1
1Department of Applied Biosciences, Graduate School of Agriculture, Kyoto University

We provide a detailed protocol for rearing, microinjection of eggs and for efficient mating of the firebrat Thermobia domestica to generate and maintain mutant strains after genome editing.

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Medicine

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee
Xiang Ji 1, Akihiro Nakahata 2, Zixi Zhao 2, Hiroshi Kuroki 2, Tomoki Aoyama 1, Akira Ito 2
1Department of Development and Rehabilitation of Motor Function, Human Health Sciences, Graduate School of Medicine, Kyoto University, 2Department of Motor Function Analysis, Human Health Sciences, Graduate School of Medicine, Kyoto University

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

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Environment

A Standardized Procedure for Monitoring Harmful Algal Blooms in Chile by Metabarcoding Analysis
Kyoko Yarimizu 1, So Fujiyoshi 1, Mikihiko Kawai 2, Jacquelinne J. Acuña 3, Joaquin-Ignacio Rilling 3, Marco Campos 3, Jonnathan Vilugrón 4, Henry Cameron 5, Karen Vergara 6, Gonzalo Gajardo 6, Oscar Espinoza-González 4, Leonardo Guzmán 4, Satoshi Nagai 7, Carlos Riquelme 5, Milko A. Jorquera 3, Fumito Maruyama 1
1Office of Research and Academia-Government-Community Collaboration, Hiroshima University, 2Graduate School of Human and Environmental Studies, Kyoto University, 3Scientific and Biotechnological Bioresource Nucleus, Universidad de La Frontera, 4Centro de Estudios de Algas Nocivas, Instituto de Fomento Pesquero, 5Ciencias del Mar y Recursos Biologicos, Universidad de Antofagasta, 6Departamento de Ciencias Biológicas y Biodiversidad, Universidad de Los Lagos, 7Japan Fisheries Research and Education Agency, Fisheries Resources Institute

This protocol introduces steps of metabarcoding analysis, targeting 16S rRNA and 18S rRNA genes, for monitoring harmful algal blooms and their associated microbiome in seawater samples. It is a powerful molecular-based tool but requires several procedures, which are visually explained here step-by-step.

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Medicine

Interphase Fluorescence in situ Hybridization of Bone Marrow Smears of Multiple Myeloma
Yalan Yu *1, Hui Shen *1, Li Liu 1, Ping Luo 1, Sanyun Wu 1, Jing He 1,2, Xiqin Tong 1, Yufeng Shang 1, Liang Shao 1, Fuling Zhou 1
1Department of Hematology, Zhongnan Hospital of Wuhan University, 2Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology

Here, we present a protocol for improving the success of interphase fluorescence in situ hybridization detection on bone marrow smears from multiple myeloma patients.

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Neuroscience

Comprehensive Understanding of Inactivity-Induced Gait Alteration in Rodents
Junichi Tajino 1,2, Tomoki Aoyama 1, Hiroshi Kuroki 1, Akira Ito 1
1Department of Motor Function Analysis, Human Health Sciences, Graduate School of Medicine, Kyoto University, 2Otolaryngology - Head & Neck Surgery, Ohio State Wexner Medical Center

The present protocol describes three-dimensional motion tracking/evaluation to depict gait motion alteration of rats after exposure to a simulated disuse environment.

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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 Furuta 6, 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.

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Biology

Generation and Maintenance of Primate Induced Pluripotent Stem Cells Derived from Urine
Jessica Radmer 1, Johanna Geuder 1, Fiona C. Edenhofer 1, Wolfgang Enard 1, Mari Ohnuki 1,2,3
1Faculty of Biology, Ludwig Maximilian University of Munich, 2Institute for the Advanced Study of Human Biology, Kyoto University, 3Hakubi Center, Kyoto University

The present protocol describes a method to isolate, expand, and reprogram human and non-human primate urine-derived cells to induced pluripotent stem cells (iPSCs), as well as instructions for feeder-free maintenance of the newly generated iPSCs.

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Biology

The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research
Yuta Takase 1, Kazuto Fujishima 2,3, Toshio Takahashi 1
1Suntory Foundation for Life Sciences, Bioorganic Research Institute, 2Institute for Integrated Cell-Material Sciences (KUIAS-iCeMS), Kyoto University, 3Faculty of Medicine, Osaka Medical and Pharmaceutical University

We describe a protocol to isolate murine small intestinal crypts and culture intestinal 3D organoids from the crypts. Additionally, we describe a method to generate organoids from a single intestinal stem cell in the absence of a sub-epithelial cellular niche.

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Immunology and Infection

Direct Observation and Automated Measurement of Stomatal Responses to Pseudomonas syringae pv. tomato DC3000 in Arabidopsis thaliana
Rikako Hirata *1, Momoko Takagi *2, Yosuke Toda 2,3, Akira Mine 1
1Graduate School of Agriculture, Kyoto University, 2Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, 3Phytometrics Co., Ltd.

Here, we present a simple method for direct observation and automated measurement of stomatal responses to bacterial invasion in Arabidopsis thaliana. This method leverages a portable stomatal imaging device, together with an image analysis pipeline designed for leaf images captured by the device.

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Medicine

Vascular Reconstruction with the Cuff Technique in Mouse Orthotopic Liver Transplantation
Kosuke Tanaka 1, Yoichiro Uchida 1, Shoichi Kageyama 1, Kojiro Nakamura 1, Hirofumi Hirao 1, Kentaro Kadono 1, Hiroshi Kawamoto 1, Kenichi Saga 1, Yuki Kidoguchi 1, Takeshi Watanabe 2, Etsuro Hatano 1
1Department of Surgery, Graduate School of Medicine, Kyoto University, 2Institute for Frontier Life and Medical Sciences, Kyoto University

This protocol provides technical information for vessel reconstruction using the cuff technique in mouse orthotopic liver transplantation.

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Neuroscience

Electroencephalography Measurements in Awake Marmosets Listening to Conspecific Vocalizations
Naho Konoike 1,2, Miki Miwa 2, Kosuke Itoh 3, Katsuki Nakamura 2
1Hakubi Center, Kyoto University, 2Center for the Evolutionary Origins of Human Behavior, Kyoto University, 3Center for Integrated Human Brain Science, Brain Research Institute, University of Niigata

To study the evolution of language, comparing brain mechanisms in humans with those in nonhuman primates is important. We developed a method to noninvasively measure the electroencephalography (EEG) of awake animals. It allows us to directly compare EEG data between humans and animals for the long term without harming them.

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