Zhejiang University 28 articles published in JoVE Biology Comparison of Two Representative Methods for Differentiation of Human Induced Pluripotent Stem Cells into Mesenchymal Stromal Cells Fangqin Wang1, Langping Gao2,3, Xudong Fu4, Qintao Yan2, Lidan Hu1,2, Jianhua Mao2,3 1Department of Nephrology, The Children's Hospital, Zhejiang University School of Medicine, 3Zhejiang University School of Medicine, 4Liangzhu Laboratory, Zhejiang University This protocol describes and compares two representative methods for differentiating hiPSCs into mesenchymal stromal cells (MSCs). The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation. The embryoid bodies (EBs) method is characterized by lower time consumption. Neuroscience Modeling the Functional Network for Spatial Navigation in the Human Brain Fengxiang Zhang*1, Chenghui Zhang*1, Yi Pu2, Xiang-Zhen Kong1,3 1Department of Psychology and Behavioral Sciences, Zhejiang University, 2Department of Neuroscience, Max Planck Institute for Empirical Aesthetics, 3Department of Psychiatry of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine This paper presents an integrative approach to investigating the functional network for spatial navigation in the human brain. This approach incorporates a large-scale neuroimaging meta-analytic database, resting-state functional magnetic resonance imaging, and network modeling and graph-theoretical techniques. Neuroscience New Framework for Understanding Cross-Brain Coherence in Functional Near-Infrared Spectroscopy (fNIRS) Hyperscanning Studies Hila Zahava Gvirts Provolovski1, Mini Sharma1, Itai Gutman1, Anat Dahan2, Yafeng Pan3, Jacqueline Stotler4, Teresa Wilcox4 1The Department of Behavioral Sciences and Psychology, Ariel University, 2Braude College of Engineering, 3Department of Psychology and Behavioral Sciences, Zhejiang University, 4Department of Psychology, Florida Atlantic University Wavelet transform coherence (WTC) is a common methodology for assessing the coupling between signals that is used in functional near-infrared spectroscopy (fNIRS) hyperscanning studies. A toolbox for assessing the directionality of the signal interaction is presented in this work. Biology Hand-Rearing Method for Infant Marmosets Hao Sun1,2, Rui Li1, Yingxu Lin1,2, Xinyuan Cao1, Lingzhu Fan1, Guanglong Sun1, Min Xie1, Lin Zhu1, Chencen Yu1, Ruolan Cai1, Chenfei Lyu1, Xiaohui Wang1,2, Yuanqing Zhang1,2, Siyi Bai1,2, Runze Qi1, Binliang Tang1,3, Guoqiang Jia1,4, Xinjian Li1,2,4, Lixia Gao1,2,4 1Department of Neurology of the Second Affiliated Hospital and Interdisciplinary Institute of Neuroscience and Technology, School of Medicine, Zhejiang University, 2Key Laboratory of Biomedical Engineering of Ministry of Education, College of Biomedical Engineering and Instrument Science, Zhejiang University, 3Rehabilitation Medicine Center, Rehabilitation & Sports Medicine Research Institute of Zhejiang Province, Department of Rehabilitation Medicine, Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical College, 4NHC and CAMS Key Laboratory of Medical Neurobiology, MOE Frontier Science Center for Brain Science and Brain-Machine Integration, School of Brain Science and Brain Medicine, Zhejiang University Here, we describe a hand-rearing method for raising infant marmosets in an animal incubator. This method greatly increases the survival rate of marmoset infants, which provides the opportunity to study the development of marmoset infants with similar genetic backgrounds raised in different postnatal environments. Medicine Laminectomy for the Removal of Thoracic Ossification of the Ligamentum Flavum (TOLF) Using Ultrasonic and Conventional Osteotomes Junhui Liu1,2, Zhi Shan1,2, Yilei Chen1,2, Fengdong Zhao1,2 1Department of Orthopedics, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, 2Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province We present a methodology for using an ultrasonic osteotome combined with a conventional osteotome for laminectomy in thoracic ossification of the ligamentum flavum. This is a relatively safe and easy-to-learn method that avoids the perioperative complications associated with the conventional method. Bioengineering Quantitative Characterization of Liquid Photosensitive Bioink Properties for Continuous Digital Light Processing Based Printing Yang Li*1,2,3, Yue Wang*4, Jun Yin3, Jin Qian1,2 1School of Aeronautics and Astronautics, Zhejiang University, 2Department of Engineering Mechanics, Key Laboratory of Soft Machines and Smart Devices of Zhejiang Province, Zhejiang University, 3The State Key Laboratory of Fluid Power and Mechatronic Systems, School of Mechanical Engineering, Zhejiang University, 4Institute of Artificial Intelligence, School of Future Technology, Shanghai University This study uses temperature and material composition to control the yield stress properties of yield stress fluids. The solid-like state of the ink can protect the printing structure, and the liquid-like state can continuously fill the printing position, realizing the digital light processing 3D printing of extremely soft bioinks. Engineering Sensitivity Enhancement of Soft Capacitive Pressure Sensors Using a Solvent Evaporation-Based Porosity Control Technique Ziying Zhu1, Yunqi Cao1, Haozhen Chi1, Xiaowei Wang1, Dibo Hou1 1State Key Laboratory of Industrial Control Technology, College of Control Science and Engineering, Zhejiang University A simple and cost-efficient fabrication method based on the solvent evaporation technique is presented to optimize the performance of a soft capacitive pressure sensor, which is enabled by porosity control in the dielectric layer using different mass ratios of the molding PDMS/toluene solution. Engineering Microfluidic Channel-Based Soft Electrodes and Their Application in Capacitive Pressure Sensing Xiaowei Wang1, Peijun Shangguan1, Pingjie Huang1, Dibo Hou1 1State Key Laboratory of Industrial Control Technology, College of Control Science and Engineering, Zhejiang University Flexible electrodes have a wide range of applications in soft robotics and wearable electronics. The present protocol demonstrates a new strategy to fabricate highly stretchable electrodes with high resolution via lithographically defined microfluidic channels, which paves the way for future high-performance soft pressure sensors. Medicine Preparation of a Non-Cardiomyocyte Cell Suspension for Single-Cell RNA Sequencing from a Post-Myocardial Infarction Adult Mouse Heart Chengchen Huang1,2, Jiahe Han1,2, Shuo Sun1,2, Guosheng Fu*1,2, Min Shang*1,2 1Key Laboratory of Cardiovascular Intervention and Regenerative Medicine of Zhejiang Province, 2Department of Cardiology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University Hangzhou Here, we describe a protocol to isolate a sufficient amount of single non-cardiomyocytes with high viability from a post-myocardial infarction (MI) mouse heart. This can be used for subsequent single-cell sequencing, flow cytometry analysis, and primary cell culture. Behavior Usability Evaluation of Augmented Reality: A Neuro-Information-Systems Study Jun Wu*1, Di Zhang*1, Tao Liu2,3,4, Helen Hong Yang5, Yi Wang1, Huili Yao1, Shinan Zhao1 1Economics and Management School, Jiangsu University of Science and Technology, 2School of Health, Fujian Medical University, 3School of Management, Shanghai University, 4School of Management, Zhejiang University, 5Business School, La Trobe University This study presents an experimental paradigm for a usability test combining subjective and objective evaluations. The objective evaluation adopted Neuro-Information-Systems (NeuroIS) methods, and the subjective evaluation adopted a usability questionnaire and a NASA-Task Load Index (NASA-TLX) scale. Biology Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body Kexin Shi1, Chao Tong1 1Ministry of Education Key Laboratory of Biosystems Homeostasis and Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University The present protocol describes the induction of autophagy in the Drosophila melanogaster larval fat body via nutrient depletion and analyzes changes in autophagy using transgenic fly strains. Biology Profiling of H3K4me3 Modification in Plants using Cleavage under Targets and Tagmentation Xiaoyuan Tao*1,2, Mengtao Gao*3, Siyuan Wang*1, Xueying Guan1,4 1College of Agriculture and Biotechnology, Zhejiang University, 2Central Laboratory, State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Zhejiang Academy of Agricultural Sciences, 3State Key Laboratory of Crop Genetics and Germplasm Enhancement, Cotton Hybrid R & D Engineering Center (the Ministry of Education), College of Agriculture, Nanjing Agricultural University, 4Hainan Institute of Zhejiang University Cleavage under targets and tagmentation (CUT&Tag) is an efficient chromatin epigenomic profiling strategy. This protocol presents a refined CUT&Tag strategy for the profiling of histone modifications in plants. Biology High-Throughput Metabolic Profiling for Model Refinements of Microalgae Amnah Alzahmi1,2, Sarah Daakour1, Diana Charles El Assal3, Bushra S. Dohai3, Amphun Chaiboonchoe3, Weiqi Fu3,4, David R. Nelson1, Alexandra Mystikou1, Kourosh Salehi-Ashtiani1,3 1Center for Genomics and Systems Biology (CGSB), New York University Abu Dhabi Research Institute, 2Department of Biology, United Arab Emirates University, 3Laboratory of Algal, Systems, and Synthetic Biology (LASSB), Division of Science and Math, New York University Abu Dhabi, 4Department of Marine Science, Ocean College, Zhejiang University This protocol demonstrates the use of a phenotype microarray (PM) technology platform to define metabolic requirements of Chlamydomonas reinhardtii, a green microalga, and refine an existing metabolic network model. Environment Isolation of Quartz Grains for Optically Stimulated Luminescence (OSL) Dating of Quaternary Sediments for Paleoenvironmental Research Liliana C. Marin1, Steven L. Forman1, Victoria T. Todd1, Connor Mayhack1, Ashley Gonzalez1, Peng Liang2 1Geoluminescence Dating Research Laboratory, Dept. of Geosciences, Baylor University, 2School of Earth Sciences, Zhejiang University This protocol is for the isolation of quartz grains by size for luminescence dating of sediments. Outlined are physical cleansing and chemical digestions by soaking sequentially in H2O2, HCl, HF, and HCl again to isolate quartz grains. The quartz purity is quantified with microscopic assessment, Raman spectroscopy, and IR depletion ratio. Cancer Research Establishing a Competing Risk Regression Nomogram Model for Survival Data Lunpo Wu1,2, Chenyang Ge3, Hongjuan Zheng4, Haiping Lin5, Wei Fu6, Jianfei Fu4 1Department of Gastroenterology, Second Affiliated Hospital of Zhejiang University School of Medicine, 2Institute of Gastroenterology, Zhejiang University, 3Department of Colorectal Surgery, Afiliated Jinhua Hospital, Zhejiang University School of Medicine, 4Department of Medical Oncology, Affiliated Jinhua Hospital, Zhejiang University School of Medicine, 5Department of Hepatobiliary Pancreatic Surgery, Affiliated Jinhua Hospital, Zhejiang University School of Medicine, 6Division of Oncology, Johns Hopkins University School of Medicine Presented here is a protocol to build nomograms based on the Cox proportional hazards regression model and competing risk regression model. The competing method is a more rational method to apply when competing events are present in the survival analysis. Immunology and Infection Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR Fei-Xue Ban1, Tian-Yan Yin1, Qi Guo1, Li-Long Pan1, Yin-Quan Liu1, Xiao-Wei Wang1 1Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogen and Insects, Institute of Insect Sciences, Zhejiang University We describe an immunofluorescence and quantitative PCR method for the localization and quantification of begomoviruses in insect tissues. The immunofluorescence protocol can be used to colocalize viral and vector proteins. The quantitative PCR protocol can be extended to quantify viruses in whole whitefly bodies and virus-infected plants. Bioengineering Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks Mingjun Xie1,2, Kang Yu1,2, Yuan Sun1,2, Lei Shao1,2, Jing Nie1,2, Qing Gao1,2, Jingjiang Qiu3, Jianzhong Fu1,2, Zichen Chen1,2, Yong He1,2 1State Key Laboratory of Fluid Power and Mechatronic Systems, School of Mechanical Engineering, Zhejiang University, 2Key Laboratory of 3D Printing Process and Equipment of Zhejiang Province, School of Mechanical Engineering, Zhejiang University, 3School of Mechanics and Safety Engineering, Zhengzhou University Presented here is a method for the 3D bioprinting of gelatin methacryloyl. Neuroscience The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice Yingliang Zhuang1,2, Junchen Chen1,2, Weize Xu1, Qiang Shu1, Xuekun Li1,2 1The Children's Hospital, School of Medicine, Zhejiang University, 2The Institute of Translational Medicine, School of Medicine, Zhejiang University Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods. Neuroscience Investigating Alterations in Caecum Microbiota After Traumatic Brain Injury in Mice Liang Wen*1, Wendong You*1, Yadong Wang*1, Yuanrun Zhu1, Hao Wang1, Xiaofeng Yang1 1Emergency and Trauma Center, The International Medical Center, The First Affiliated Hospital, School of Medicine, Zhejiang University Presented here is a protocol to induce diffuse traumatic brain injury using a lateral fluid percussion device followed by the collection of the caecum content for gut microbiome analysis. Genetics Identifying Mutations by High Resolution Melting in a TILLING Population of Rice Shan Li1, Ying-Ping Yu1, Song-Mei Liu1, Hao-Wei Fu2, Jian-Zhong Huang3, Qing-Yao Shu1, Yuan-Yuan Tan4 1National Key Laboratory of Rice Biology, Zhejiang Provincial Key Laboratory of Crop Genetic Resources, Institute of Crop Science, Zhejiang University, 2Jiaxing Academy of Agricultural Sciences, 3Institute of Nuclear Agricultural Sciences, Zhejiang University, 4The New Countryside Development Institute at Zhejiang University In this article, we present the protocol that is described as high-resolution melting analysis (HRM)-based Target Induced Local Lesions In Genomes (TILLING). This method utilizes fluorescence changes during the melting of the DNA duplex and is suitable for high-throughput screening of both insertion/deletion (Indel) and single base substition (SBS). Environment Visualization of Flow Field Around a Vibrating Pipeline Within an Equilibrium Scour Hole Dawei Guan1,2, Yee-Meng Chiew2, Maoxing Wei3, Shih-Chun Hsieh4 1Key Laboratory of Coastal Disaster and Defense, Ministry of Education, Hohai University, 2School of Civil and Environmental Engineering, Nanyang Technological University, 3Ocean College, Zhejiang University, 4AXESEA Information Technology Co. LTD The goal of the protocol is to enable visualization of the detailed flow fields and determination of the near-boundary shear and normal stresses within an equilibrium scour hole induced by a vibrating pipeline. Behavior Design and Use of an Apparatus for Presenting Graspable Objects in 3D Workspace Kedi Xu1,2,3, Junjun Chen1,2, Guanghao Sun1,2, Yaoyao Hao1,3, Shaomin Zhang1,2,3, Xingchen Ran1,2, Weidong Chen1,2, Xiaoxiang Zheng1,2,3 1Qiushi Academy for Advanced Studies, Zhejiang University, 2Department of Biomedical Engineering, Zhejiang University, 3Key Laboratory of Biomedical Engineering of Ministry of Education, Zhejiang University Presented here is a protocol to build an automatic apparatus that guides a monkey to perform the flexible reach-to-grasp task. The apparatus combines a 3D translational device and turning table to present multiple objects in an arbitrary position in 3D space. Immunology and Infection Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model Ya-Wen Du*1, Qun Liu*1, Xiao-Cui Luo1, Dong-Xiao Zhao1, Jian-Bo Xue1, Pu Feng2, Robert F. Margolskee2, Hong Wang2, Liquan Huang1,2 1College of Life Sciences, Zhejiang University, 2Monell Chemical Senses Center Here we present a protocol to investigate the effect of the nullification of gustation-related genes on immune responses in a dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD) mouse model. Neuroscience Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia Yanrong Zheng*1, Zhe Shen*1, Xiaoli Wu1, Lei Jiang1, Weiwei Hu1, Zhong Chen1, Xiangnan Zhang1 1Institute of Pharmacology & Toxicology, College of Pharmaceutical Sciences, Department of Pharmacology, Key Laboratory of Medical Neurobiology of The Ministry of Health of China, Zhejiang Province Key Laboratory of Neurobiology, Zhejiang University Acidic postconditioning protects against cerebral ischemia. Here we present two models to execute APC. They are achieved respectively by transferring corticostriatal slices to acidic buffer after oxygen-glucose deprivation in vitro and by inhaling 20% CO2 after middle cerebral artery occlusion in vivo. Biology Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci Dan-Tong Zhu1, Xin-Ru Wang1, Fei-Xue Ban1, Chi Zou1, Shu-Sheng Liu1, Xiao-Wei Wang1 1Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogen and Insects, Institute of Insect Sciences, Zhejiang University Here, we present a protocol to isolate endosymbionts from the whitefly Bemisia tabaci through dissection and filtration. After amplification, the DNA samples are suitable for subsequent sequencing and study of the mutualism between endosymbionts and whitefly. Biology Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy Jingwen Yang1, Yunjun Yan1, Xiaowei Xiang1, Yuchao Xu1, Naiming Zhou2, Tianming Wang1 1National Engineering Research Center of Marine Facilities Aquaculture, Marine Science College, Zhejiang Ocean University, 2Institute of Biochemistry, College of Life Sciences, Zhejiang University This protocol describes confocal microscopy detection of G protein-coupled receptor (GPCR) internalization in mammalian cells. It includes the basic cell culture, transfection, and confocal microscopy procedure and provides an efficient and easily interpretable method to detect the subcellular localization and internalization of fusion-expressed GPCR. Bioengineering Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell Yunfeng Chen*1, Baoyu Liu*2, Lining Ju*3, Jinsung Hong*1, Qinghua Ji4,5, Wei Chen6, Cheng Zhu1 1Woodruff School of Mechanical Engineering, Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, 2Coulter Department of Biomedical Engineering, Georgia Institute of Technology, 3Charles Perkins Centre, The University of Sydney, 4Institute of Biophysics, Laboratory of RNA Biology, Chinese Academy of Sciences, 5University of Chinese Academy of Sciences, 6School of Medicine and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte. Biology Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants Xiaohong Zhu1, Aaron Taylor2, Shenyu Zhang1, Dayong Zhang1,3, Ying Feng1,4, Gaimei Liang1,5, Jian-Kang Zhu1,6 1Department of Horticulture and Landscape Architecture, Purdue University, 2Bindley Bioscience Center, Purdue University, 3Institute of Biotechnology, Jiangsu Academy of Agricultural Sciences, 4College of Environmental & Resource Science, Zhejiang University, 5Dryland Agriculture Research Centre, Shanxi Academy of Agricultural Sciences, 6Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.
