Kyoto University

38 articles published in JoVE

• Neuroscience

  
  Comprehensive Understanding of Inactivity-Induced Gait Alteration in Rodents Junichi Tajino^1,2, Tomoki Aoyama¹, Hiroshi Kuroki¹, Akira Ito^{1 1}Department 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.

• 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 1}Department 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.

• Environment

  
  A Standardized Procedure for Monitoring Harmful Algal Blooms in Chile by Metabarcoding Analysis Kyoko Yarimizu¹, So Fujiyoshi¹, Mikihiko Kawai², Jacquelinne J. Acuña³, Joaquin-Ignacio Rilling³, Marco Campos³, Jonnathan Vilugrón⁴, Henry Cameron⁵, Karen Vergara⁶, Gonzalo Gajardo⁶, Oscar Espinoza-González⁴, Leonardo Guzmán⁴, Satoshi Nagai⁷, Carlos Riquelme⁵, Milko A. Jorquera³, Fumito Maruyama^{1 1}Office 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.

• Medicine

  
  A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee Xiang Ji¹, Akihiro Nakahata², Zixi Zhao², Hiroshi Kuroki², Tomoki Aoyama¹, Akira Ito^{2 1}Department 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.

• Developmental Biology

  
  In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells Chuang-Yu Lin¹, Michiko Yoshida^1,2, Li-Tzu Li³, Megumu K. Saito^{1 1}Department 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.

• Developmental Biology

  
  Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica Takahiro Ohde¹, Toshinori Minemura¹, Eiichi Hirose¹, Takaaki Daimon^{1 1}Department 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.

• Environment

  
  Measurement of the Potential Rates of Dissimilatory Nitrate Reduction to Ammonium Based on ¹⁴NH₄⁺/¹⁵NH₄⁺ Analyses via Sequential Conversion to N₂O Megumi Kuroiwa¹, Keitaro Fukushima^2,3, Kazuma Hashimoto², Yukiko Senga⁴, Tsubasa Sato⁴, Chie Katsuyama⁵, Yuichi Suwa^{1 1}Department of Applied Chemistry, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 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.

• Biochemistry

  
  Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis Chuan-Chih Hsu^1,3, Chia-Feng Tsai², W. Andy Tao^3,4, Pengcheng Wang^{5 1}Department 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.

• 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 Sasaki¹, William J. Kowalski³, Bradley B. Keller^4,5,7, Hidetoshi Masumoto^{1,2 1}Clinical 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, ⁷ 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.

• 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 Minatoya¹, Hidetoshi Masumoto^{1,2 1}Department 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.

• 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 Wang¹, Heng Wei², Keiji Naruse¹, Ken Takahashi^{1 1}Department 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.

• Neuroscience

  
  3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury Tianshu Wang¹, Akira Ito², Junichi Tajino^1,3, Hiroshi Kuroki², Tomoki Aoyama^{1 1}Department 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.

• Developmental Biology

  
  Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis Hiromi Shimojo¹, Ryoichiro Kageyama^{1,2,3,4 1}Institute 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.

• 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 Malekzadeh¹, Hiroshi Kawamoto³, Nicholas P. Restifo^{1,2 1}Surgery 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.

• Neuroscience

  
  3D Scanning Technology Bridging Microcircuits and Macroscale Brain Images in 3D Novel Embedding Overlapping Protocol Saya Ide¹, Motoki Kajiwara¹, Hirohiko Imai², Masanori Shimono^{1 1}Graduate 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).

• Developmental Biology

  
  In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells Taiki Nakajima¹, Hidetoshi Sakurai², Makoto Ikeya^{2 1}Department 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.

• Developmental Biology

  
  Efficient Generation of Pancreas/Duodenum Homeobox Protein 1⁺ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures Taro Toyoda¹, Azuma Kimura¹, Hiromi Tanaka¹, Kenji Osafune^{1 1}Center 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.

• Chemistry

  
  Improving High Viscosity Extrusion of Microcrystals for Time-resolved Serial Femtosecond Crystallography at X-ray Lasers Daniel James¹, Tobias Weinert¹, Petr Skopintsev¹, Antonia Furrer¹, Dardan Gashi^1,2, Tomoyuki Tanaka^3,4, Eriko Nango^3,4, Przemyslaw Nogly^1,5, Joerg Standfuss^{1 1}Division 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.

• Neuroscience

  
  Ethanol-Induced Cervical Sympathetic Ganglion Block Applications for Promoting Canine Inferior Alveolar Nerve Regeneration Using an Artificial Nerve Yoshiki Shionoya¹, Katsuhisa Sunada², Gentarou Tsujimoto², Keiji Shigeno³, Tatsuo Nakamura^{3 1}Department 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.

• Neuroscience

  
  Analyzing Neural Activity and Connectivity Using Intracranial EEG Data with SPM Software Wataru Sato*¹, Takanori Kochiyama*², Shota Uono³, Naotaka Usui⁴, Akihiko Kondo⁵, Kazumi Matsuda⁵, Keiko Usui⁶, Motomi Toichi⁷, Yushi Inoue^{5 1}Kokoro 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.

• Chemistry

  
  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 Huang¹, Yan Liu¹, Keiji Maruoka^{1,2 1}School 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.

• Bioengineering

  
  Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array Yasumasa Mashimo^1,2, Momoko Yoshioka¹, Yumie Tokunaga¹, Christopher Fockenberg¹, Shiho Terada¹, Yoshie Koyama¹, Teiko Shibata-Seki², Koki Yoshimoto¹, Risako Sakai¹, Hayase Hakariya¹, Li Liu¹, Toshihiro Akaike³, Eiry Kobatake², Siew-Eng How⁴, Motonari Uesugi^1,5, Yong Chen^1,6, Ken-ichiro Kamei^{1 1}Institute 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.

• Environment

  
  Measuring the Flight Ability of the Ambrosia Beetle, Platypus Quercivorus (Murayama), Using a Low-Cost, Small, and Easily Constructed Flight Mill Ryuichi Okada¹, Duy Long Pham², Yasuto Ito³, Michimasa Yamasaki², Hidetoshi Ikeno^{1 1}School 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.

• Biology

  
  Establishment of a Clonal Culture of Unicellular Conjugating Algae Yuki Tsuchikane¹, Takashi Hamaji², Katsumi Ota³, Syou Kato^{4 1} In this article, we demonstrate the establishment of clonal cultures of unicellular conjugating algal species collected from a natural field site.

• Chemistry

  
  Novel Techniques for Observing Structural Dynamics of Photoresponsive Liquid Crystals Masaki Hada¹, Shohei Saito², Ryuma Sato³, Kiyoshi Miyata⁴, Yasuhiko Hayashi¹, Yasuteru Shigeta³, Ken Onda^{4 1}Graduate 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.

• Immunology and Infection

  
  Evaluation of Blood Lactate and Plasma Insulin During High-intensity Exercise by Antecubital Vein Catheterization Minas Nalbandian¹, Zsolt Radak², Masaki Takeda^{3 1}Graduate 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.

• Developmental Biology

  
  Combinational Treatment of Trichostatin A and Vitamin C Improves the Efficiency of Cloning Mice by Somatic Cell Nuclear Transfer Rika Azuma¹, Kei Miyamoto², Mami Oikawa³, Masayasu Yamada⁴, Masayuki Anzai^{1,5 1}Division 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.

• Bioengineering

  
  Perfusable Vascular Network with a Tissue Model in a Microfluidic Device Yuji Nashimoto¹, Yukako Teraoka¹, Ramin Banan Sadeghian¹, Akiko Nakamasu², Yuichiro Arima³, Sanshiro Hanada³, Hidetoshi Kotera¹, Koichi Nishiyama³, Takashi Miura², Ryuji Yokokawa^{1 1}Department 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.

• Immunology and Infection

  
  A Recovery Cardiopulmonary Bypass Model Without Transfusion or Inotropic Agents in Rats Shingo Hirao¹, Hidetoshi Masumoto¹, Tatsuya Itonaga¹, Kenji Minatoya^{1 1}Department 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.

• 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 1}Institute 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.

• Biology

  
  Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera Yuichi Fukutomi¹, Keiji Matsumoto^1,2, Noriko Funayama¹, Shigeyuki Koshikawa^{1,3,4 1}Graduate 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.

• Immunology and Infection

  
  Cell Membrane Repair Assay Using a Two-photon Laser Microscope Joshua J. A. Lee¹, Rika Maruyama¹, Hidetoshi Sakurai², Toshifumi Yokota^{1 1}Department 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.

• Medicine

  
  Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction Kazuyuki Nagai^1,2, Shintaro Yagi², Shinji Uemoto², Rene H. Tolba^{1 1}Institute for Laboratory Animal Science and Experimental Surgery, RWTH-Aachen University, ²Department of Hepato-Biliary-Pancreatic Surgery and Transplantation, Graduate School of Medicine, Kyoto University Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

• Medicine

  
  Orthotopic Small Bowel Transplantation in Rats Koji Kitamura*^1,2, Martin W. von Websky*¹, Ichiro Ohsawa¹, Azin Jaffari¹, Thomas C. Pech¹, Tim Vilz¹, Sven Wehner¹, Shinji Uemoto², Joerg C. Kalff¹, Nico Schaefer^{1 1}Department of Surgery, University of Bonn, Germany, ²Department of Surgery, Kyoto University Hospital Small bowel transplantation has become an accepted treatment option for patients with irreversible intestinal failure. Our experimental model of orthotopic small bowel transplantation in rats serves as a reliable tool to address underlying immunologic and inflammatory processes that complicate intestinal transplantation.

• Medicine

  
  In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model Debabrata Saha¹, Henry Dunn², Heling Zhou², Hiroshi Harada³, Masahiro Hiraoka³, Ralph P. Mason², Dawen Zhao^{2 1}Department of Radiation Oncology, University of Texas Southwestern Medical Center, ²Department of Radiology, University of Texas Southwestern Medical Center, ³Department of Radiation Oncology, Kyoto University Graduate School of Medicine Bioluminescence imaging of hypoxia inducible factor-1α activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

• Biology

  
  Dissection of Hippocampal Dentate Gyrus from Adult Mouse Hideo Hagihara^1,2, Keiko Toyama^1,2, Nobuyuki Yamasaki^1,3, Tsuyoshi Miyakawa^{1,2,4,5 1}Japan Science and Technology Agency, Core Research for Evolutionary Science and Technology (CREST), ²Division of Systems Medical Science, Institute for Comprehensive Medical Science, Fujita Health University, ³Department of Psychiatry, Graduate School of Medicine, Kyoto University, ⁴Genetic Engineering and Functional Genomics Group, Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University, ⁵Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, National Institutes of Natural Sciences A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

• Biology

  
  Elevated Plus Maze for Mice Munekazu Komada¹, Keizo Takao^1,2, Tsuyoshi Miyakawa^{2 1}Genetic Engineering and Functional Genomics Unit, Frontier Technology Center, Graduate School of Medicine, Kyoto University, ²Institute for Comprehensive Medical Science Division of Systems Medicine, Fujita Health University The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

• Biology

  
  Light/dark Transition Test for Mice Keizo Takao¹, Tsuyoshi Miyakawa^{1 1}Genetic Engineering and Functional Genomics Unit, Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.