Tsinghua University 28 articles published in JoVE Neuroscience The Ex vivo Preparation of Spinal Cord Slice for the Whole-Cell Patch-Clamp Recording in Motor Neurons During Spinal Cord Stimulation Qingyu Yao1, Xuesong Luo1, Jie Liu2,3, Luming Li1,2 1National Engineering Research Center of Neuromodulation, School of Aerospace Engineering, Tsinghua University, 2IDG/McGovern Institute for Brain Research, Tsinghua University, 3School of Life Sciences, Tsinghua University This protocol describes a method using a patch-clamp to study the electrical responses of motor neurons to spinal cord stimulation (SCS) with high spatiotemporal resolution, which can help researchers improve their skills in separating the spinal cord and maintaining cell viability simultaneously. Bioengineering Large-Scale Cell Production Based on GMP-Grade Dissolvable Porous Microcarriers Yuyang Chen*1, Huanye Xu*2, Yuanyuan Zhang*2, Leilei Guo2, Miaomiao Lan2, Yanping Yang2, Wei Liu2, Xiaojun Yan2, Yanan Du1 1Department of Biomedical Engineering, School of Medicine, Tsinghua University, 2Beijing CytoNiche Biotechnology Co. Ltd. Here, we present a protocol to achieve the large-scale manufacturing of adherent cells through a fully closed system based on GMP-grade dissolvable microcarriers. The cultivation of human mesenchymal stem cells, HEK293T cells, and Vero cells was validated and met both quantity demands and quality criteria for the cell and gene therapy industry. Behavior Evaluating Flight Performance and Eye Movement Patterns Using Virtual Reality Flight Simulator Luoma Ke1, Zhejun Zhang2, Yidan Ma1, Yi Xiao3, Shengjun Wu4, Xiuchao Wang4, Xufeng Liu4, Jibo He1 1Psychology Department, School of Social Sciences, Tsinghua University, 2Faculty of Psychology, Beijing Normal University, 3National Key Laboratory of Human Factors Engineering, the China Astronaut Research and Training Center, 4Department of Military Medical Psychology, Air Force Medical University A new virtual reality flight simulator was built, which enables efficient and low-cost evaluation of flight performance and eye movement patterns. It also provides a high-potential research tool for ergonomics and other research. Biology Direct Synthesis of EM-Visible Gold Nanoparticles in Cells for Protein Localization Analysis with Well-Preserved Ultrastructure Zhaodi Jiang1,2 1National Institute of Biological Sciences, Beijing, 2Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University The present protocol describes a clonable electron microscopy labeling technology for detecting metallothionein-tagged proteins in cells using a novel autonucleation suppression mechanism-based gold nanoparticle synthesis technique. Biology De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data Yanan Zhu*1, Fajin Li*2,3, Xuerui Yang2,3, Zhengtao Xiao1 1School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, 2MOE Key Laboratory of Bioinformatics, Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University, 3Joint Graduate Program of Peking-Tsinghua-National Institute of Biological Science Translating ribosomes decode three nucleotides per codon into peptides. Their movement along mRNA, captured by ribosome profiling, produces the footprints exhibiting characteristic triplet periodicity. This protocol describes how to use RiboCode to decipher this prominent feature from ribosome profiling data to identify actively translated open reading frames at the whole-transcriptome level. Bioengineering Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System (MMC) Xingjin Jian*1,2, Xiaojie Guo*1,2,3, Jia Wang4, Zheng Lin Tan1,2,6, Xin-hui Xing1,2,5, Liyan Wang3, Chong Zhang1,2,5 1Department of Chemical Engineering, Institute of Biochemical Engineering, Tsinghua University, 2Key Laboratory of Industrial Biocatalysis, Ministry of Education, Tsinghua University, 3Luoyang TMAXTREE Biotechnology Co., Ltd., 4 This protocol describes how to use the Microbial Microdroplet Culture system (MMC) to conduct automated microbial cultivation and adaptive evolution. MMC can cultivate and sub-cultivate microorganisms automatically and continuously and monitor online their growth with relatively high throughput and good parallelization, reducing labor and reagent consumption. Behavior Interaction between Phonological and Semantic Processes in Visual Word Recognition using Electrophysiology Yuling Wang1, Minghu Jiang1, Xinyi Xu1, Yunlong Huang2 1Center for Psychology and Cognitive Science, Lab of Computational Linguistics, Tsinghua University, 2Advanced Innovation Center for Future Education, Beijing Normal University We present a protocol to explore the relative activation sequence of phonology and semantics in visual word recognition. The results show that consistent with interactive accounts, semantic and phonological representations may be processed interactively, and higher-level linguistic representations may affect early processing. Bioengineering 3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting Tiankun Liu*1,2, Yongchun Shao*1,2, Zitong Wang1,2, Yuqiu Chen1,2, Yuan Pang1,2, Ding Weng1,2, Wei Sun1,2,3 1Biomanufacturing Center, Dept. of Mechanical Engineering, Tsinghua University, 2Biomanufacturing and Rapid Forming Technology Key Laboratory of Beijing, 3Department of Mechanical Engineering, Drexel University Presented here is a mild 3D printing technique driven by alternating viscous-inertial forces to enable the construction of hydrogel microcarriers. Homemade nozzles offer flexibility, allowing easy replacement for different materials and diameters. Cell binding microcarriers with a diameter of 50-500 µm can be obtained and collected for further culturing. Biochemistry Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy Feng Ge1, Jianfeng Xue1, Yan He1 1Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Tsinghua University Here, we show the use of traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on cell membrane. The location and orientation of single AuNRs are detected using ImageJ and MATLAB, and the diffusive states of AuNRs are characterized by single particle tracking analysis. Behavior Tactile Vibrating Toolkit and Driving Simulation Platform for Driving-Related Research Ao Zhu1, Annebella Tsz Ho Choi1, Ko-Hsuan Ma1, Shi Cao2, Han Yao1, Jian Wu3, Jibo He1 1Psychology Department, School of Social Sciences, Tsinghua University, 2Department of Systems Design Engineering, University of Waterloo, 3Haier Innovation Design Center, Haier Company This protocol describes a driving simulation platform and a tactile vibrating toolkit for the investigation of driving-related research. An exemplar experiment exploring the effectiveness of tactile warnings is also presented. Behavior An Assessment Method and Toolkit to Evaluate Keyboard Design on Smartphones Yincheng Wang1, Ke Wang1, Yuqi Huang1, Di Wu2, Jian Wu3, Jibo He1 1Department of Psychology, School of Social Sciences, Tsinghua University, 2Department of Computer Science, Beijing Normal University, 3Haier Innovation Design Center, Haier Company The presented protocol integrates various evaluation methods and demonstrates a method to evaluate the keyboard design on smartphones. Pairs matched by English characters are proposed as the input material, and the transition time between two keys is used as the dependent variable. Genetics Transcriptome-Wide Profiling of Protein-RNA Interactions by Cross-Linking and Immunoprecipitation Mediated by FLAG-Biotin Tandem Purification Xianju Bi1,2, Xuechun Zhang1, Xiaohua Shen1 1Tsinghua-Peking Center for Life Sciences, School of Medicine and School of Life Sciences, Tsinghua University, 2Department of Molecular Biology, University of Texas Southwestern Medical Center Here we present a modified CLIP-seq protocol called FbioCLIP-seq with FLAG-biotin tandem purification to determine the RNA targets of RNA-binding proteins (RBPs) in mammalian cells. Immunology and Infection Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics (DCAF) Xue Bai*1,2, Lin Zhang*3, Jin Hu2, Xiuxiu Zhao2, Jinheng Pan2, Haiteng Deng3, Shan Feng1,2 1Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, 2Mass Spectrometry Core Facility, School of Life Sciences, Westlake University, 3MOE Key Laboratory of Bioinformatics, Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University Development of a dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF) is novel for the elimination of harmful antibodies. Here, we describe a detailed protocol for the synthesis of DCAF1 molecule, which can selectively block 4G2 antibody to eliminate antibody dependent enhancement effect during Dengue virus infection. Engineering Optimized Sealing Process and Real-Time Monitoring of Glass-to-Metal Seal Structures Zhichun Fan1, Kangjia Hu2, Zhiyong Huang1, Yong Zhang2, He Yan1 1Institute of Nuclear and New Energy Technology, Key Laboratory of Advanced Reactor Engineering and Safety of Ministry of Education, Collaborative Innovation Center for Advanced Nuclear Energy Technology, Tsinghua University, 2Institute of Nuclear and New Energy Technology, Beijing Key Laboratory of Fine Ceramics, Tsinghua University Key procedures to optimize the sealing process and achieve real-time monitoring of the metal-to-glass seal (MTGS) structure are described in detail. The embedded fiber Bragg grating (FBG) sensor is designed to achieve online monitoring of temperature and high-level residual stress in the MTGS with simultaneous environmental pressure monitoring. Immunology and Infection Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy Jiahuan Chen1, Ying Xu2, Yan Shi2, Tie Xia2 1State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, 2Institute for Immunology, School of Medicine, Tsinghua University We present a protocol to functionalize atomic force microscope (AFM) cantilevers with a single T cell and bead particle for immunological studies. Procedures to probe single-pair T cell-dendritic cell binding by AFM and to monitor the real-time cellular response of macrophages to a single solid particle by AFM with fluorescence imaging are shown. Behavior Dissociation of the Confounding Influences of Expectancy and Integrative Difficulty Residing in Anomalous Sentences in Event-related Potential Studies Yunlong Huang1, Minghu Jiang1, Qian Guo2, Yuling Wang1, Fan-Pei Gloria Yang3 1Center for Psychology and Cognitive Science, School of Humanities, Tsinghua University, 2Department of Foreign Languages and Literatures, School of Humanities, Tsinghua University, 3Center for Cognition and Mind Sciences, Lab for Language and Cognitive Neurology, National TsingHua University We present a protocol to dissociate the intertwining factors of integrative difficulty and unexpectedness in semantically anomalous sentences by applying multiple repetitions to enhance participant's expectancy for anomalous sentences. The dissociation helps to investigate the major contributor of elicited event-related potentials (ERP) effects such as N400 in language studies. 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. Cancer Research Establishing Cell Lines Overexpressing DR3 to Assess the Apoptotic Response to Anti-mitotic Therapeutics Xin Wang *1, Jiamin Zhou*2, Chen Qi*1, Gelin Wang1,3 1School of Pharmaceutical Sciences, Tsinghua University, 2Department of Radiology, University of California, San Francisco, 3Beijing Advanced Innovation Center for Structural Biology Establishing a stable cell line overexpressing a gene of interest to study gene function can be done by stable transfection-picking single clones after transfecting them via retroviral infection. Here we show that HT29-DR3 cell lines generated in this way elucidate the mechanisms by which death receptor 3 (DR3) contributes to antimitotics-induced apoptosis. Behavior Using the Visual World Paradigm to Study Sentence Comprehension in Mandarin-Speaking Children with Autism Peng Zhou*1, Weiyi Ma*2, Likan Zhan*3, Huimin Ma*4 1Department of Foreign Languages and Literatures, Tsinghua University, 2School of Human Environmental Sciences, University of Arkansas, 3Institute for Speech Pathology and the Brain Science, Beijing Language and Culture University, 4Department of Electronic Engineering, Tsinghua University We present a protocol to examine the use of morphological cues during real-time sentence comprehension by children with autism. Engineering Generation of Size-controlled Poly (ethylene Glycol) Diacrylate Droplets via Semi-3-Dimensional Flow Focusing Microfluidic Devices Yan Wu1, Xiang Qian1, Shengli Mi1, Min Zhang1, Shuqing Sun1, Xiaohao Wang1,2 1Graduate School at Shenzhen, Tsinghua University, 2The State Key Laboratory of Precision Measurement Technology and Instruments, Tsinghua University Here, we present a protocol to illustrate the fabrication processes and verifying experiments of a semi-three-dimensional (semi-3D) flow-focusing microfluidic chip for droplet formation. Immunology and Infection Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model Shouming Zhang1,2,3, Xueping Gao1,2,3, Genhui Xiao1,2,3,4, Chengping Lu1,2,3, Huochun Yao1,2,3, Hongjie Fan1,2,3,5, Zongfu Wu1,2,3 1College of Veterinary Medicine, Nanjing Agricultural University, 2Key Lab of Animal Bacteriology, Ministry of Agriculture, 3OIE Reference Lab for Swine Streptococcosis, 4School of Medicine, Tsinghua University, 5Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses Here, we describe the intracranial subarachnoidal route of infection in mice to study roles of biofilms in Streptococcus suis meningitis. This infection model is also suitable for studying the pathogenesis of other bacterial meningitis and the efficacy of new drugs against bacterial meningitis. Bioengineering Microhoneycomb Monoliths Prepared by the Unidirectional Freeze-drying of Cellulose Nanofiber Based Sols: Method and Extensions Zheng-Ze Pan1,2, Hirotomo Nishihara3, Wei Lv1, Cong Wang1,2, Yi Luo1,2, Liubing Dong1,2, Houfu Song1,4, Wenjie Zhang2, Feiyu Kang1,2,4, Takashi Kyotani3, Quan-Hong Yang1,4,5 1Engineering Laboratory for Functionalized Carbon Materials and Shenzhen Key Laboratory for Graphene-based Materials, Graduate School at Shenzhen, Tsinghua University, 2School of Materials Science and Engineering, Tsinghua University, 3Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 4Tsinghua-Berkeley Shenzhen Institute (TBSI), Tsinghua University, 5School of Chemical Engineering and Technology, Tianjin University Here, we present a general protocol to prepare a variety of microhoneycomb monoliths (MHMs) in which fluid can pass through with an extremely low pressure drop. MHMs obtained are expected to be used as filters, catalyst supports, flow-type electrodes, sensors and scaffolds for biomaterials. Chemistry Fabricating High-viscosity Droplets using Microfluidic Capillary Device with Phase-inversion Co-flow Structure Jiang Li1, Jia Man2, Zhongnan Li2, Haosheng Chen2 1School of Mechanical Engineering, University of Science and Technology Beijing, 2State Key Laboratory of Tribology, Tsinghua University A phase-inversion co-flow device is demonstrated to generate monodisperse high-viscosity droplets above 1 Pas, which is difficult to realize in droplet microfluidics. Bioengineering Visual Detection of Multiple Nucleic Acids in a Capillary Array Jianwei Chen*1,2,3, Ning Shao*1,2,3, Jiaying Hu*4, Rong Li4, Yuanshou Zhu1,2,3, Dabing Zhang4,5, Shujuan Guo1,2,3, Junhou Hui6, Peng Liu6, Litao Yang4, Sheng-Ce Tao1,2,3 1Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, 2State Key Laboratory of Oncogenes and Related Genes, 3School of Biomedical Engineering, Shanghai Jiao Tong University, 4Collaborative Innovation Center for Biosafety of GMOs, National Center for the Molecular Characterization of Genetically Modified Organisms, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 5Key Laboratory of Crop Marker-Assisted Breeding of Huaian Municipality, Jiangsu Collaborative Innovation Center of Regional Modern Agriculture and Environmental Protection, 6Department of Biomedical Engineering, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School of Medicine, Tsinghua University This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets. Chemistry Synthesis of High Purity Nonsymmetric Dialkylphosphinic Acid Extractants Junlian Wang1, Meiying Xie1, Xinyu Liu1, Shengming Xu2 1School of Civil and Resource Engineering, University of Science and Technology Beijing, 2Institute of Nuclear and New Energy Technology, Tsinghua University A protocol for the synthesis of high purity nonsymmetric dialkylphosphinic acid extractants is presented, taking (2,3-dimethylbutyl)(2,4,4'-trimethylpentyl)phosphinic acid as an example. Bioengineering 3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening Yaqian Li*1,2, Xiaojun Yan*1, Wei Liu*1, Lyu Zhou1,3, Zhifeng You1, Yanan Du1,2 1Department of Biomedical Engineering, School of Medicine, Tsinghua University, 2Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, 3School of Life Sciences, Tsinghua University This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening. Chemistry Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture Yongsan Li1, Yaling Zhang2, Yen Wei1, Lei Tao1 1The Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, 2Institute of Chemical Materials, China Academy of Engineering Physics Here we describe a facile preparation of chitosan-based injectable hydrogels using dynamic imine chemistry. Methods to adjust the hydrogel’s mechanical strength and its application in 3D cell culture are presented. Chemistry Self-assembly of Complex Two-dimensional Shapes from Single-stranded DNA Tiles Bryan Wei1, Michelle K. Vhudzijena2, Joanna Robaszewski2, Peng Yin2,3 1Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, 2Wyss Institute for Biologically Inspired Engineering, Harvard University, 3Department of Systems Biology, Harvard Medical School DNA tiling is an effective approach to make programmable nanostructures. We describe the protocols to construct complex two-dimensional shapes by the self-assembly of single-stranded DNA tiles.
