Kyoto University View Institution's Website 41 articles published in JoVE 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 Toda2,3, Akira Mine1 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. Biology Generation and Maintenance of Primate Induced Pluripotent Stem Cells Derived from Urine Jessica Radmer1, Johanna Geuder1, Fiona C. Edenhofer1, Wolfgang Enard1, Mari Ohnuki1,2,3 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. Biology The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research Yuta Takase1, Kazuto Fujishima2,3, Toshio Takahashi1 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. Neuroscience Comprehensive Understanding of Inactivity-Induced Gait Alteration in Rodents Junichi Tajino1,2, Tomoki Aoyama1, Hiroshi Kuroki1, Akira Ito1 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. Neuroscience A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales Kenta Yamauchi1,2, Shinichiro Okamoto1,2,3, Megumu Takahashi1,2,4,5, Masato Koike2,3, Takahiro Furuta6, Hiroyuki Hioki1,2,7 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. 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 Maruyama1 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. 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 Ito2 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. Developmental Biology In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells Chuang-Yu Lin1, Michiko Yoshida1,2, Li-Tzu Li3, Megumu K. Saito1 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. Developmental Biology Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica Takahiro Ohde1, Toshinori Minemura1, Eiichi Hirose1, Takaaki Daimon1 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. 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 Fukushima2,3, Kazuma Hashimoto2, Yukiko Senga4, Tsubasa Sato4, Chie Katsuyama5, Yuichi Suwa1 1Department 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 Hsu1,3, Chia-Feng Tsai2, W. Andy Tao3,4, Pengcheng Wang5 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. Bioengineering Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair Takeichiro Nakane1,2,6, Mosha Abulaiti1,2, Yuko Sasaki1, William J. Kowalski3, Bradley B. Keller4,5,7, Hidetoshi Masumoto1,2 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, 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 Nishio1,3, Kenji Minatoya1, Hidetoshi Masumoto1,2 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. 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 Takahashi1 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. Neuroscience 3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury Tianshu Wang1, Akira Ito2, Junichi Tajino1,3, Hiroshi Kuroki2, Tomoki Aoyama1 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. Developmental Biology Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis Hiromi Shimojo1, Ryoichiro Kageyama1,2,3,4 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. 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 Maeda1,2, Naritaka Tamaoki1,2, Parisa Malekzadeh1, Hiroshi Kawamoto3, Nicholas P. Restifo1,2 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. Neuroscience 3D Scanning Technology Bridging Microcircuits and Macroscale Brain Images in 3D Novel Embedding Overlapping Protocol Saya Ide1, Motoki Kajiwara1, Hirohiko Imai2, Masanori Shimono1 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). Developmental Biology In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells Taiki Nakajima1, Hidetoshi Sakurai2, Makoto Ikeya2 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. 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 Osafune1 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. 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 Gashi1,2, Tomoyuki Tanaka3,4, Eriko Nango3,4, Przemyslaw Nogly1,5, Joerg Standfuss1 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. 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 Nakamura3 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. 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 Inoue5 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. 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 Maruoka1,2 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. Bioengineering Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array Yasumasa Mashimo1,2, 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 Uesugi1,5, Yong Chen1,6, Ken-ichiro Kamei1 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. 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 Ikeno1 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. Biology Establishment of a Clonal Culture of Unicellular Conjugating Algae Yuki Tsuchikane1, Takashi Hamaji2, Katsumi Ota3, Syou Kato4 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 Onda4 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. Immunology and Infection Evaluation of Blood Lactate and Plasma Insulin During High-intensity Exercise by Antecubital Vein Catheterization Minas Nalbandian1, Zsolt Radak2, Masaki Takeda3 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. 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 Anzai1,5 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. 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 Yokokawa1 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. Immunology and Infection A Recovery Cardiopulmonary Bypass Model Without Transfusion or Inotropic Agents in Rats Shingo Hirao1, Hidetoshi Masumoto1, Tatsuya Itonaga1, Kenji Minatoya1 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. Genetics An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions Akihiro Isomura1,2, Ryoichiro Kageyama1,3,4,5 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. Biology Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera Yuichi Fukutomi1, Keiji Matsumoto1,2, Noriko Funayama1, Shigeyuki Koshikawa1,3,4 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. Immunology and Infection Cell Membrane Repair Assay Using a Two-photon Laser Microscope Joshua J. A. Lee1, Rika Maruyama1, Hidetoshi Sakurai2, Toshifumi Yokota1 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. Medicine Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction Kazuyuki Nagai1,2, Shintaro Yagi2, Shinji Uemoto2, Rene H. Tolba1 1Institute 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 Schaefer1 1Department 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 Zhao2 1Department 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 Hagihara1,2, Keiko Toyama1,2, Nobuyuki Yamasaki1,3, Tsuyoshi Miyakawa1,2,4,5 1Japan 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 Takao1,2, Tsuyoshi Miyakawa2 1Genetic 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 Miyakawa1 1Genetic 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.