University of Tokyo View Institution's Website 47 articles published in JoVE Cancer Research Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma Hiroki Kobayashi1,2,3,4, Krystyna A. Gieniec1,2, Jia Q. Ng1,2, Jarrad Goyne1,2, Tamsin R. M. Lannagan1,2, Elaine M. Thomas1,2, Georgette Radford1,2, Tongtong Wang1,2, Nobumi Suzuki1,2,5, Mari Ichinose1,2, Josephine A. Wright2, Laura Vrbanac1,2, Alastair D. Burt6, Masahide Takahashi3,4,7, Atsushi Enomoto3, Daniel L. Worthley2, Susan L. Woods1,2 1Adelaide Medical School, University of Adelaide, 2South Australian Health and Medical Research Institute (SAHMRI), 3Department of Pathology, Nagoya University Graduate School of Medicine, 4Division of Molecular Pathology, Center for Neurological Disease and Cancer, Nagoya University Graduate School of Medicine, 5Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, 6Translational and Clinical Research Institute, Newcastle University, 7International Center for Cell and Gene Therapy, Fujita Health University Portal vein injection of colorectal cancer (CRC) organoids generates stroma-rich liver metastasis. This mouse model of CRC hepatic metastasis represents a useful tool to study tumor-stroma interactions and develop novel stroma-directed therapeutics such as adeno-associated virus-mediated gene therapies. Engineering Electroantennography-based Bio-hybrid Odor-detecting Drone using Silkmoth Antennae for Odor Source Localization Daigo Terutsuki1, Tomoya Uchida2, Chihiro Fukui3, Yuji Sukekawa1, Yuki Okamoto4, Ryohei Kanzaki1 1Research Center for Advanced Science and Technology, The University of Tokyo, 2Department of Mechano-Informatics, Graduate School of Information Science and Technology, The University of Tokyo, 3Department of Applied Biological Science, Graduate School of Science and Technology, Tokyo University of Science, 4Sensing System Research Center, National Institute of Advanced Industrial Science and Technology This study introduces experimental protocols for a bio-hybrid odor-detecting drone based on silkmoth antennae. The operation of an experimental electroantennogram device with silkmoth antennae is presented, in addition to the structure of a bio-hybrid drone designed for odor source localization using the spiral-surge algorithm. Engineering Theoretical Calculation and Experimental Verification for Dislocation Reduction in Germanium Epitaxial Layers with Semicylindrical Voids on Silicon Motoki Yako1, Yasuhiko Ishikawa2, Eiji Abe1, Kazumi Wada1,3 1Department of Materials Engineering, The University of Tokyo, 2Department of Electrical and Electronic Information Engineering, Toyohashi University of Technology, 3Department of Materials Science and Engineering, Massachusetts Institute of Technology Theoretical calculation and experimental verification are proposed for a reduction of threading dislocation (TD) density in germanium epitaxial layers with semicylindrical voids on silicon. Calculations based on the interaction of TDs and surface via image force, TD measurements, and transmission electron microscope observations of TDs are presented. Genetics Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice Hirofumi Nishizono*1,2,3, Mohamed Darwish*4,5, Hideki Uosaki6,7, Nanami Masuyama8,9,10, Motoaki Seki8,11, Hiroyuki Abe3, Nozomu Yachie8,9,10,12,13, Ryohei Yasuda1 1Max Planck Florida Institute for Neuroscience, 2Life Science Research Center, University of Toyama, 3Department of Biochemical Engineering, Graduate School of Science and Engineering, Yamagata University, 4Graduate School of Innovative Life Science, University of Toyama, 5Department of Biochemistry, Faculty of Pharmacy, Cairo University, 6Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, 7Division of Stem Cell Research and Drug Development, Center for Development of Advanced Medical Technology, Jichi Medical University, 8Synthetic Biology Division, Research Center for Advanced Science and Technology, University of Tokyo, 9Institute for Advanced Biosciences, Keio University, 10Graduate School of Media and Governance, Keio University, 11Department of Molecular Oncology, Graduate School of Medicine, Chiba University, 12Department of Biological Sciences, School of Science, University of Tokyo, 13College of Arts and Sciences, University of Tokyo Here, we present a modified method for cryopreservation of one-cell embryos as well as a protocol that couples the use of freeze-thawed embryos and electroporation for the efficient generation of genetically modified mice. Environment Visualization of Low-Level Gamma Radiation Sources Using a Low-Cost, High-Sensitivity, Omnidirectional Compton Camera Hiroshi Muraishi1, Ryoji Enomoto2, Hideaki Katagiri3, Mika Kagaya4, Takara Watanabe5, Naofumi Narita3, Daisuke Kano5 1School of Allied Health Sciences, Kitasato University, 2Institute for Cosmic Ray Research, University of Tokyo, 3College of Science, Ibaraki University, 4National Institute of Technology, Sendai College, 5National Cancer Center Hospital East We present experimental protocols for visualizing various low-level gamma radiation sources in the ambient environment using a low-cost, high-sensitivity, omnidirectional, gamma-ray imaging Compton camera. Environment Microfocus X-ray CT (microCT) Imaging of Actinia equina (Cnidaria), Harmothoe sp. (Annelida), and Xenoturbella japonica (Xenacoelomorpha) Akiteru Maeno1, Hisanori Kohtsuka2, Kensuke Takatani3, Hiroaki Nakano3 1Mammalian Genetics Laboratory, National Institute of Genetics, 2Misaki Marine Biological Station, The University of Tokyo, 3Shimoda Marine Research Center, University of Tsukuba Here, protocols for performing microfocus X-ray computed tomography (microCT) imaging of three marine invertebrate animals are explained in detail. This study describes steps such as sample fixation, staining, mounting, scanning, image reconstruction, and data analyses. Suggestions on how the protocol can be adjusted for different samples are also provided. Medicine Murine Model of Central Venous Stenosis using Aortocaval Fistula with an Outflow Stenosis Toshihiko Isaji1,2,3, Shun Ono1,2,4,5, Takuya Hashimoto1,2,3, Kota Yamamoto1,2,3, Ryosuke Taniguchi1,2,3, Haidi Hu1,2, Tun Wang1,2, Jun Koizumi4, Toshiya Nishibe5, Katsuyuki Hoshina3, Alan Dardik1,2,6 1Department of Surgery, Yale University, 2Vascular Biology and Therapeutics Program, Yale University, 3Department of Vascular Surgery, University of Tokyo, 4Department of Diagnostic Radiology, Tokai University School of Medicine, 5Department of Cardiovascular Surgery, Tokyo Medical University, 6Department of Vascular Surgery, VA Connecticut Healthcare Systems An aortocaval fistula was created by puncturing the murine infra-renal aorta through both walls into the inferior vena cava and was followed by creation of a stenosis in its outflow via partial ligation of the inferior vena cava. This reproducible model can be used to study central venous stenosis. Chemistry An Electrochemical Cholesteric Liquid Crystalline Device for Quick and Low-Voltage Color Modulation Shoichi Tokunaga*1, Mengyan Zeng*1,2, Yoshimitsu Itoh1, Fumito Araoka3, Takuzo Aida3 1Department of Chemistry and Biotechnology, The University of Tokyo, 2Department of Chemistry, Tsinghua University, 3RIKEN Center for Emergent Matter Science A protocol for the fabrication of a reflective cholesteric liquid crystalline display device containing a redox-responsive chiral dopant allowing quick and low-voltage operation is presented. Environment An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings Kae Akita1, Takumi Higaki2 1Department of Integrated Frontier Sciences, The University of Tokyo, 2International Research Organization for Advanced Science and Technology, Kumamoto University The goal of this protocol is to demonstrate how to induce clustered stomata in cotyledons of Arabidopsis thaliana seedlings by immersion treatment with a sugar-containing medium solution and how to observe intracellular structures such as chloroplasts and microtubules in the clustered guard cells using confocal laser microscopy. Biology A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging Shigeyuki Betsuyaku1,2,3,4, Nobuhiko Nomura3,4, Hiroo Fukuda2 1Japan Science and Technology Agency (JST), PRESTO, 2Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 3Faculty of Life and Environmental Sciences, University of Tsukuba, 4Microbiology Research Center for Sustainability, University of Tsukuba We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses. Biology TChIP-Seq: Cell-Type-Specific Epigenome Profiling Mari Mito1,2, Mitsutaka Kadota3, Shinichi Nakagawa1,4, Shintaro Iwasaki2,5 1RNA Biology Laboratory, RIKEN, 2RNA Systems Biochemistry Laboratory, RIKEN Cluster for Pioneering Research, 3Laboratory for Phyloinformatics, RIKEN Center for Biosystems Dynamics Research, 4RNA Biology Laboratory, Faculty of Pharmaceutical Sciences, Hokkaido University, 5Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, University of Tokyo We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification. Chemistry Spatiotemporally Controlled Nuclear Translocation of Guests in Living Cells Using Caged Molecular Glues as Photoactivatable Tags Rina Mogaki1, Kou Okuro1, Akio Arisaka1, Takuzo Aida1,2 1Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, 2Riken Center for Emergent Matter Science This protocol describes light-triggered nuclear translocation of guests in living cells using caged molecular glue tags. This method is promising for site-selective nuclear-targeting drug delivery. Bioengineering An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates Ikki Horiguchi1,2, Ikumi Suzuki3, Takashi Morimura3, Yasuyuki Sakai1,4 1Department of Chemical System Engineering, University of Tokyo, 2Department of Biotechnology, Osaka University, 3Biotech Business Unit, Fukoku Co. Ltd, 4Institute of Industrial Science, University of Tokyo Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels. Neuroscience In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes Kaoru Yamada1 1Department of Neuropathology, Graduate School of Medicine, University of Tokyo In vivo microdialysis has enabled collection of molecules present in brain interstitial fluid (ISF) from awake, freely-behaving animals. In order to analyze relatively large molecules in ISF, the current article specifically focuses on the microdialysis protocol using probes with high molecular weight cut off membranes. Biochemistry Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee Shota Suenami1,2, Ryo Miyazaki1, Takeo Kubo2 1Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, 2Department of Biological Sciences, Graduate School of Science, The University of Tokyo To test the inhibitory effects of pharmacologic agents on phospholipase C (PLC) in different regions of the honeybee brain, we present a biochemical assay to measure PLC activity in those regions. This assay could be useful for comparing PLC activity among tissues, as well as among bees exhibiting different behaviors. Engineering Measuring Magnetically-Tuned Ferroelectric Polarization in Liquid Crystals Hiroki Ueda1, Takuya Akita2, Yoshiaki Uchida2, Tsuyoshi Kimura3 1Division of Materials Physics, Graduate School of Engineering Science, Osaka University, 2Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, 3Department of Advance Materials Science, University of Tokyo In this report, we present a protocol to examine direct magnetoelectric effects, i.e., induction of ferroelectric polarization by applying magnetic fields, in liquid crystals. This protocol provides a unique approach, supported by the softness of liquid crystals, to achieve room-temperature magnetoelectrics. Chemistry Preparation of Polyoxometalate-based Photo-responsive Membranes for the Photo-activation of Manganese Oxide Catalysts Akira Yamaguchi1,5, Toshihiro Takashima2, Kazuhito Hashimoto1,6, Ryuhei Nakamura3,4 1Department of Applied Chemistry, The University of Tokyo, 2Clean Energy Research Center, University of Yamanashi, 3Biofunctional Catalyst Research Team, RIKEN Center for Sustainable Resource Science, 4Earth-Life Science Institute (ELSI), Tokyo Institute of Technology, 5Department of Materials Science and Engineering, Tokyo Institute of Technology, 6National Institute for Materials Science Here, we present a protocol to prepare charge transfer chromophores based on a polyoxometalate/polymer composite membrane. Biology Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-β Signaling Hiroshi I. Suzuki1, Masafumi Horie2,3, Hajime Mihira4, Akira Saito2 1David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 2Department of Respiratory Medicine, Graduate School of Medicine, The University of Tokyo, 3Hastings Center for Pulmonary Research, Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, Keck School of Medicine, University of Southern California, 4Department of Molecular Pathology, Graduate School of Medicine, The University of Tokyo A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-β in MS-1 endothelial cells. Environment Self-standing Electrochemical Set-up to Enrich Anode-respiring Bacteria On-site Akihiro Okamoto1, Annette Rowe2, Xiao Deng3, Kenneth H. Nealson2 1International Center for Materials Nanoarchitectonics, National Institute for Materials Science, 2Department of Earth Sciences, University of Southern California, 3Department of Applied Chemistry, The University of Tokyo On-site microbial enrichment or in situ cultivation techniques can facilitate the isolation of difficult-to-culture microbial taxa, especially from low-biomass or geochemically extreme environments. Here, we describe an electrochemical set-up without using an external power source to enrich microbial strains that are capable of extracellular electron transport (EET). Engineering Experimental Methods for Spin- and Angle-Resolved Photoemission Spectroscopy Combined with Polarization-Variable Laser Kenta Kuroda1, Koichiro Yaji1, Ayumi Harasawa1, Ryo Noguchi1, Takeshi Kondo1, Fumio Komori1, Shik Shin1 1Institute for Solid State Physics, University of Tokyo Here, we combine polarization-variable 7-eV laser with spin- and angle-resolved photoemission technique to visualize the spin-orbital coupling effect in solid states. Developmental Biology Zygotic Fluorescence Recovery After Photo-bleaching Analysis for Chromatin Looseness That Allows Full-term Development Masatoshi Ooga1,3, Satoshi Funaya2, Fugaku Aoki2, Teruhiko Wakayama1,3 1Faculty of Life and Environmental Sciences, Department of Biotechnology, University of Yamanashi, 2Department of Integrated Bioscience, Graduate School of Frontier Sciences, University of Tokyo, 3Advanced Biotechnology Center, University of Yamanashi Chromatin looseness appears to be involved in the developmental potential of blastomeres. However, it is not known whether chromatin looseness can be used as a reliable index for the developmental potential for embryos. Here, an experimental system in which chromatin looseness-evaluated zygotes can develop to full term has been described. Biochemistry Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1 Yoshihide Tokunou1, Kazuhito Hashimoto2, Akihiro Okamoto2 1Department of Applied Chemistry, University of Tokyo, 2Global Research Center for Environment and Energy based on Nanomaterials Science, National Institute for Materials Science Here we present a protocol of whole-cell electrochemical experiments to study the contribution of proton transport to the rate of extracellular electron transport via the outer-membrane cytochromes complex in Shewanella oneidensis MR-1. Engineering Electric-field Control of Electronic States in WS2 Nanodevices by Electrolyte Gating Feng Qin1, Toshiya Ideue1, Wu Shi2, Yijin Zhang3,4, Ryuji Suzuki1, Masaro Yoshida5, Yu Saito1, Yoshihiro Iwasa1,5 1Quantum-Phase Electronics Center (QPEC) and Department of Applied Physics, The University of Tokyo, 2Materials Sciences Division, Lawrence Berkeley National Laboratory, 3Institute of Scientific and Industrial Research, Osaka University, 4Max Planck Institute for Solid State Research, 5RIKEN Center for Emergent Matter Science (CEMS) Here, we present a protocol to control the carrier number in solids by using the electrolyte. Neuroscience Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat Yu Shikano1, Takuya Sasaki1, Yuji Ikegaya1,2 1Graduate School of Pharmaceutical Sciences, The University of Tokyo, 2Center for Information and Neural Networks This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs. Cancer Research Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology Kenji Amemiya1,2, Yosuke Hirotsu1, Toshio Oyama2, Masao Omata1,3 1Genome Analysis Center, Yamanashi Central Hospital, 2Pathology Division, Laboratory Department, Yamanashi Central Hospital, 3The University of Tokyo Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens. Behavior Recording Horizontal Saccade Performances Accurately in Neurological Patients Using Electro-oculogram Yasuo Terao1, Hideki Fukuda2, Yusuke Sugiyama3, Satomi Inomata-Terada1, Shin-ichi Tokushige4, Masashi Hamada3, Yoshikazu Ugawa5 1Department of Cell Physiology, Kyorin University, 2Segawa Memorial Neurological Clinic for Children, 3Department of Neurology, University of Tokyo, 4Department of Neurology, Kyorin University, 5Department of Neurology, Fukushima Medical University The article describes a practical method for recording horizontal eye movements with high accuracy by electro-oculogram in neurological patients, using a cup Ag-AgCl electrode with a wide plastic fringe. Stable measurement requires proper selection and fixation of electrodes, taking sufficient time for light adaptation to occur, and re-calibration as needed. Biochemistry Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag Genta Ito1, Taisuke Tomita1,2 1Laboratory of Brain and Neurological Disorders, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 2Laboratory of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo The present study describes a simple method of detecting endogenous levels of Rab10 phosphorylation by leucine-rich repeat kinase 2. Biology Establishment of an Extracellular Acidic pH Culture System Ayano Kondo1, Tsuyoshi Osawa2 1Innovative Technology Laboratories, Kyowa Hakko Kirin Co., Ltd, 2Laboratory for Systems Biology and Medicine, RCAST, The University of Tokyo The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems. Neuroscience Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes Naoki Ihara1, Yuji Ikegaya1,2, Haruki Takeuchi1,3 1Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 2Center for Information and Neural Networks, National Institute of Information and Communications Technology, 3PRESTO, Japan Science and Technology Agency (JST) Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons. Bioengineering Methods for the Self-integration of Megamolecular Biopolymers on the Drying Air-LC Interface Kosuke Okeyoshi1, Kensuke Osada2, Maiko K. Okajima1, Tatsuo Kaneko1 1Japan Advanced Institute of Science and Technology, 2Department of Bioengineering, School of Engineering, The University of Tokyo A method for the drying-induced self-integration of megamolecular biopolymers on the air-liquid crystalline interface is provided here. This methodology will be useful not only for understanding the macroscopic potentials of biopolymers, but also as an evaluation method for soft materials in biomedical and environmental fields. Engineering Gain-compensation Methodology for a Sinusoidal Scan of a Galvanometer Mirror in Proportional-Integral-Differential Control Using Pre-emphasis Techniques Tomohiko Hayakawa1, Takanoshin Watanabe2, Taku Senoo1, Masatoshi Ishikawa1 1Department of Creative Informatics, University of Tokyo, 2Hitachi Industry & Control Solutions, Ltd. We propose a method to extend the corresponding frequency by using a pre-emphasis technique. This method compensates for the gain reduction of a galvanometer mirror in sine-wave path tracking using proportional-integral-differential control. Bioengineering A Simple and Scalable Fabrication Method for Organic Electronic Devices on Textiles Usein Ismailov1, Esma Ismailova1, Seiichi Takamatsu2 1Department of Bioelectronics, Ecole Nationale Supérieure des Mines de Saint-Étienne, CMP-EMSE, MOC, 2Graduate School of Frontier Sciences, The University of Tokyo In this paper, we present a protocol to selectively deposit organic materials on textiles, which allows for the direct integration of organic electronic devices with wearables. The fabricated devices can be fully integrated in textiles, respecting their mechanical appearance and enabling sensing capabilities. Biochemistry Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion Yuno Natsume1, Hsin-i Wen2, Tong Zhu2, Kazumi Itoh1, Li Sheng2, Kensuke Kurihara2,3,4 1 Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials. Chemistry Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy Takashi Hiroi1, Mitsuhiro Shibayama2 1Department of Chemistry, School of Science, University of Tokyo, 2Institute for Solid State Physics, University of Tokyo A protocol for the direct measurement of particle size distribution in concentrated solutions using dynamic light scattering microscopy is presented. Medicine 2-Methacryloyloxyethyl Phosphorylcholine Polymer Treatment of Complete Dentures to Inhibit Denture Plaque Deposition Kenji Ikeya1, Miya Fukunishi1, Fuminori Iwasa1, Yuuki Inoue2, Kazuhiko Ishihara2, Kazuyoshi Baba1 1Department of Prosthodontics, School of Dentistry, Showa University, 2Department of Materials Engineering, School of Engineering, The University of Tokyo Removable poly(methyl methacrylate) (PMMA) dentures are prone to bacterial adherence and plaque formation. Denture plaque-associated infection is a source of serious dental and medical complications in the elderly. This paper introduces a novel protocol to treat PMMA dentures with 2-methacryloyloxyethyl phosphorylcholine polymer, poly(MPC-co-BMA-co-MPAz), to suppress plaque deposition on PMMA dentures. Neuroscience Insect-controlled Robot: A Mobile Robot Platform to Evaluate the Odor-tracking Capability of an Insect Noriyasu Ando1, Shuhei Emoto1, Ryohei Kanzaki1 1Research Center for Advanced Science and Technology, The University of Tokyo The capability to localize an odor source is necessary for insect survival and is expected to be applicable to artificial odor-tracking. The insect-controlled robot is driven by an actual silkmoth and enables us to evaluate the odor-tracking capability of insects through a robotic platform. Neuroscience In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation Daisuke Ino1,2, Masamitsu Iino1 1Department of Pharmacology, Graduate School of Medicine, The University of Tokyo, 2Laboratory for Cell Polarity Regulation, Quantitative Biology Center, RIKEN Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs. Neuroscience Preparation of Single-cohort Colonies and Hormone Treatment of Worker Honeybees to Analyze Physiology Associated with Role and/or Endocrine System Takayuki Ueno1,2, Kiyoshi Kawasaki2, Takeo Kubo1 1Department of Biological Sciences, Graduate School of Sciences, The University of Tokyo, 2 Here we describe our detailed protocol for the preparation of single-cohort honeybee colonies – a useful tool for analyzing the role-associated worker physiology. We also describe detailed protocols for treating workers with juvenile hormone and ecdysone to evaluate the involvement of these hormones in the regulation of worker behavior and/or physiology. Biology Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions Gen Kaneko1,4, Tatsuki Yoshinaga2, Kristin E. Gribble3, David M. Welch3, Hideki Ushio1 1Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 2School of Marine Biosciences, Department of Marine Biosciences, Kitasato University, 3Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, 4School of Arts and Sciences, University of Houston-Victoria Rotifers are microscopic zooplankton used as models in ecotoxicological and aging studies. Here we provide a protocol for powerful and reproducible measurement of survival time in Brachionus rotifers. Synchronization of culture conditions over several generations is of particular importance because maternal condition affects life history of offspring. Medicine Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium Takuya Hashimoto1,2,3, Kota Yamamoto1,2,3, Trenton Foster1, Hualong Bai1, Kunihiro Shigematsu4, Alan Dardik1,3 1Department of Surgery and the Vascular Biology and Therapeutics Program, Yale University, 2Department of Vascular Surgery, University of Tokyo, 3Department of Vascular Surgery, VA Connecticut Healthcare Systems, 4Department of Vascular Surgery, International University of Health and Welfare Mita Hospital After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route. Bioengineering Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation Keiji Itaka1, Satoshi Uchida1, Akitsugu Matsui1, Kayoko Yanagihara1, Masaru Ikegami1, Taisuke Endo2, Takehiko Ishii3, Kazunori Kataoka2 1Graduate School of Medicine, Laboratory of Clinical Biotechnology, The University of Tokyo, 2Graduate School of Engineering, Department of Materials Engineering, The University of Tokyo, 3Graduate School of Engineering, Department of Bioengineering, The University of Tokyo This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells. Developmental Biology Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle Ikki Horiguchi1, Yasuyuki Sakai1 1Center for International Research on Integrative Biomedical System, Institute of Industrial Science, The University of Tokyo We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche. Medicine Three-dimensional Co-culture Model for Tumor-stromal Interaction Masafumi Horie1,2, Akira Saito1,3, Yoko Yamaguchi4, Mitsuhiro Ohshima5, Takahide Nagase1 1Department of Respiratory Medicine, Graduate School of Medicine, The University of Tokyo, 2Department of Clinical Laboratory, Graduate School of Medicine, The University of Tokyo, 3Division for Health Service Promotion, The University of Tokyo, 4Department of Biochemistry, Nihon University School of Dentistry, 5Department of Biochemistry, Ohu University School of Pharmaceutical Sciences Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts. Neuroscience Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion Teruyuki Matsunaga1, Akira Fushiki1, Akinao Nose1,2, Hiroshi Kohsaka1 1Department of Complexity Science and Engineering, Graduate School of Frontier Sciences, The University of Tokyo, 2Department of Physics, Graduate School of Science, The University of Tokyo Here we describe a protocol for optogenetic manipulation of motoneuronal activity while monitoring changes in motor output (muscle contraction) in semi-intact Drosophila larvae using lasers within a conventional confocal microscope. This technique enables researchers to achieve local perturbation of neural activity in a few neuromeres to elucidate the dynamics of motor circuits. Bioengineering Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells Robert DeRose1, Christopher Pohlmeyer1, Nobuhiro Umeda1,2, Tasuku Ueno1,2, Tetsuo Nagano2, Scot Kuo1,3, Takanari Inoue1 1Department of Cell Biology, Center for Cell Dynamics, Johns Hopkins University, 2Graduate School of Pharmaceutical Sciences, University of Tokyo, 3Biomedical Engineering, Johns Hopkins University A method for spatio-temporal control of small GTPase activity by light is described. This method is based on rapamycin-induced FKBP-FRB heterodimerization and photo-caging systems. Optimization of light-irradiation enables the spatio-temporally controlled activation of small GTPases at the subcellular level. Immunology and Infection Expansion of Human Peripheral Blood γδ T Cells using Zoledronate Makoto Kondo1,2, Takamichi Izumi1,2, Nao Fujieda1,2, Atsushi Kondo1,2, Takeharu Morishita1,2, Hirokazu Matsushita1, Kazuhiro Kakimi1 1Department of Immunotherapeutics (Medinet), University of Tokyo Hospital, 2MEDINET Co., Ltd A method to expand γδ T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived γδ T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of γδ T cells can be applied to autologous cellular immunotherapy of cancer. Biology In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy Akihiro Asai1, Nita Sahani1, Yasuyoshi Ouchi2, Jeevendra Martyn1, Shingo Yasuhara1 1Department of Anesthesiology and Critical Care, Shriners Hospital for Children, Massachusetts General Hospital, and Harvard Medical School, 2Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.