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Chinese Academy of Sciences

107 ARTICLES PUBLISHED IN JoVE

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Biology

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker
Yue Zhang *1, Luv Kashyap *2, Annabel A. Ferguson 2, Alfred L. Fisher 2
1Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, 2Department of Medicine, Division of Geriatric Medicine and Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

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JoVE Core

Measurement of Bioelectric Current with a Vibrating Probe
Brian Reid 1, Min Zhao 1
1Dermatology, University of California, Davis

The manufacture, calibration and use of non-invasive vibrating probes to measure bioelectric current in various biological systems is described.

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Bioengineering

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments
Xiaoting Meng *1, Wenfei Li *2,3, Fraser Young 1, Runchi Gao 3, Laura Chalmers 3, Min Zhao 3, Bing Song 1
1School of Dentistry, Cardiff Institute of Tissue Engineering & Repair, Cardiff University , 2Shandong Qianfoshan Hospital, Shandong University School of Medicine, 3Dermatology and Ophthalmology Research, Institute for Regenerative Cures, University of California at Davis

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

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Medicine

Technical Aspects of the Mouse Aortocaval Fistula
Kota Yamamoto 1,2, Xin Li 1,3, Chang Shu 3, Tetsuro Miyata 2, Alan Dardik 1,4
1The Department of Surgery and the Interdepartmental Program in Vascular Biology and Therapeutics, Yale University, 2Department of Vascular Surgery, The University of Tokyo, 3Department of Vascular Surgery, Central South University, 4VA Connecticut Healthcare Systems

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

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Neuroscience

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury
Paul Lu 1,2, Lori Graham 2, Yaozhi Wang 2, Di Wu 2, Mark Tuszynski 1,2
1Veterans Administration Medical Center, San Diego, 2Department of Neurosciences, University of California, San Diego

Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances.

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Biology

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants
Xiaohong Zhu 1, Aaron Taylor 2, Shenyu Zhang 1, Dayong Zhang 1,3, Ying Feng 1,4, Gaimei Liang 1,5, Jian-Kang Zhu 1,6
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.

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Biology

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique
Guillaume Luxardi 1, Brian Reid 1, Fernando Ferreira 1,2, Pauline Maillard 3, Min Zhao 1,4
1Department of Dermatology, Institute for Regenerative Cures, University of California, Davis, 2Departamento de Biologia, Centro de Biologia Molecular e Ambiental, Universidade do Minho, 3Department of Neurology and Center for Neuroscience, University of California, Davis Imaging of Dementia and Aging Laboratory, 4Department of Ophthalmology, Institute for Regenerative Cures, University of California, Davis

Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.

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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 Ji 4,5, Wei Chen 6, Cheng Zhu 1
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.

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Neuroscience

Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals
Feng Liu 1, Nannan Liu 1
1Department of Entomology and Plant Pathology, Auburn University

Bed bugs rely on olfactory receptor neurons housed in their antennal olfactory sensilla to detect semiochemicals in the environment. Utilizing single sensillum recording, we demonstrate a method to evaluate bed bug response to semiochemicals and explore the coding process involved.

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JoVE Core

SSVEP-based Experimental Procedure for Brain-Robot Interaction with Humanoid Robots
Jing Zhao 1, Wei Li 1,2, Xiaoqian Mao 1, Mengfan Li 1
1School of Electrical Engineering and Automation, Tianjin University, 2Department of Computer & Electrical Engineering and Computer Science, California State University

The overall goal of this method is to establish an SSVEP-based experimental procedure by integrating multiple software programs to enable the study of brain-robot interaction with humanoid robots, which is prospective in assisting the sick and elderly as well as performing unsanitary or dangerous jobs.

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JoVE Core

Printing Fabrication of Bulk Heterojunction Solar Cells and In Situ Morphology Characterization
Feng Liu 1, Sunzida Ferdous 2, Xianjian Wan 1, Chenhui Zhu 3, Eric Schaible 3, Alexander Hexemer 3, Cheng Wang 3, Thomas P. Russell 1,2
1Materials Sciences Division, Lawrence Berkeley National Laboratory, 2Department of Polymer Science and Engineering, University of Massachusetts, Amherst, 3Advanced Light Source, Lawrence Berkeley National Laboratory

Here, we present a protocol to fabricate organic thin film solar cells using a mini-slot die coater and related in-line structure characterizations using synchrotron scattering techniques.

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Neuroscience

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons
Chun Liu 1, Christina Chan 1,2
1Department of Chemical Engineering and Materials Science, Michigan State University, 2Department of Biochemistry and Molecular Biology, Michigan State University

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

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Medicine

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis
Aoyuan Cui 1, Zhimin Hu 1, Yamei Han 1, Yi Yang 2, Yu Li 1
1CAS Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Chinese Academy of Sciences, 2Suzhou Niumag Analytical Instrument Corporation

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

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Immunology and Infection

A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines
Xi Liu 1,2, Chongfeng Xu 2, Ziyuan Duan 2
1Wuhan Institute of Virology, Chinese Academy of Sciences, 2Genetic Resources Center, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences

A simple red blood cell lysis method for establishing B-LCLs was developed with high immortalization efficiency, requiring a small amount of blood and saving time from initiation to cryopreservation.

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Medicine

Patch Angioplasty in the Rat Aorta or Inferior Vena Cava
Hualong Bai 1,2,3,4,5, Xin Li 6, Takuya Hashimoto 1,2,5, Haidi Hu 1,2,5, Trenton R. Foster 1,2,5, Jesse J. Hanisch 1,2,5, Jeans M. Santana 1,2,5, Alan Dardik 1,2,5
1Department of Surgery, Yale University, 2Vascular Biology and Therapeutics Program, Yale University, 3Department of Vascular Surgery, First Affiliated Hospital of Zhengzhou University, 4Basic Medical College of Zhengzhou University, 5VA Connecticut Healthcare Systems, West Haven, CT, 6Department of Vascular Surgery, Xiangya Second Hospital of Central South University, Changsha, China

We have established a model of pericardial patch angioplasty that can be used in either small-diameter veins or arteries. This model can be used to compare venous and arterial neointimal hyperplasia formation.

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Developmental Biology

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro
Zhaofeng Jia 1,2, Yujie Liang 3, Bin Ma 4,5, Xiao Xu 2,6, Jianyi Xiong 2, Li Duan 2, Daping Wang 1,2
1Guangzhou Medical University, 2Shenzhen Key Laboratory of Tissue Engineering, Shenzhen Laboratory of Digital Orthopeadic Engineering, Department of Orthopedics, Shenzhen Second People's Hospital (The First Hospital Affiliated to Shenzhen University), 3Department of Chemistry, The Chinese University of Hong Kong, 4School of Biomedical Engineering, Shanghai Jiao Tong University, 5Renji Hospital Clinical Stem Cell Research Center, Shanghai Jiao Tong University School of Medicine, 6Shantou University Medical College

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

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Immunology and Infection

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood
Ke Yang *1,2,3, Jiandong Wu *3,4, Ling Zhu 1, Yong Liu 1, Michael Zhang 5, Francis Lin 3,4,6,7
1Institute of Applied Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, 2University of Science and Technology of China, 3Department of Physics and Astronomy, University of Manitoba, 4Department of Biosystems Engineering, University of Manitoba, 5Seven Oaks General Hospital, 6Department of Immunology, University of Manitoba, 7Department of Biological Sciences, University of Manitoba

This article provides the detailed method of performing a rapid neutrophil chemotaxis assay by integrating the on-chip neutrophil isolation from whole blood and the chemotaxis test on a single microfluidic chip.

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Environment

Production and Measurement of Organic Particulate Matter in a Flow Tube Reactor
Yue Zhang 1,2, Pengfei Liu 1, Zhaoheng Gong 1, Franz M. Geiger 3, Scot T. Martin 1,4
1School of Engineering and Applied Sciences, Harvard University, 2Department of Environmental Science and Engineering, Gillings School of Global Public Health, University of North Carolina, Chapel Hill, 3Department of Chemistry, Northwestern University, 4Department of Earth and Planetary Sciences, Harvard University

This paper describes the operation procedure for the flow tube reactor and related data collection. It shows the protocols for setting the experiments, recording data and generating the number-diameter distribution as well as the particle mass information, which gives useful information about chemical and physical properties of the organic aerosols.

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Environment

Production and Measurement of Organic Particulate Matter in the Harvard Environmental Chamber
Yue Zhang 1,2, Zhaoheng Gong 1, Suzane de Sa 1, Adam P. Bateman 1, Yingjun Liu 1, Yongjie Li 1, Franz M. Geiger 3, Scot T. Martin 1,4
1School of Engineering and Applied Sciences, Harvard University, 2Department of Environmental Science and Engineering, Gillings School of Global Public Health, University of North Carolina, 3Department of Chemistry, Northwestern University, 4Department of Earth and Planetary Sciences, Harvard University

This paper describes operation procedures for the Harvard Environmental Chamber (HEC) and related instrumentation for measuring gaseous and particle species. The environmental chamber is used to produce and study secondary organic species produced from the organic precursors, especially related to atmospheric organic particulate matter.

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Developmental Biology

Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis
Bao Li Zhang 1,2,3, Da Yuan Gao 1,2,3, Xiao Xu Zhang 1,3, Shuo Shi 4, Winnie Shum 1
1School of Life Science and Technology, ShanghaiTech University, 2Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 3University of Chinese Academy of Sciences, 4Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University

We present a protocol that combines cell isolation and whole-cell patch-clamp recording to measure the electrical properties of the primary dissociated epithelial cells from the rat cauda epididymides. This protocol allows for investigation of the functional properties of primary epididymal epithelial cells to further elucidate the physiological role of the epididymis.

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Developmental Biology

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells
Yueying Li 2, Yong Hai 1, Jiayu Chen 3, Tie Liu 1
1Department of Orthopedics, Beijing Chao-Yang Hospital, Capital Medical University, 2Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, 3Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, School of Life Sciences and Technology, Tongji University

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

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Genetics

Protocols for C-Brick DNA Standard Assembly Using Cpf1
Shi-Yuan Li 1, Guo-Ping Zhao 1, Jin Wang 1
1Institute of Plant Physiology and Ecology, Shanghai Insititutes for Biological Sciences, Chinese Academy of Sciences

CRISPR-associated protein Cpf1 can be guided by a specially designed CRISPR RNA (crRNA) to cleave double-stranded DNA at desired sites, generating sticky ends. Based on this characteristic, a DNA assembly standard (C-Brick) was established, and a protocol detailing its use is described here.

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Developmental Biology

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells
Qing Cissy Yu 1, Wenqian Song 1, Dengwen Lai 1, Yi Arial Zeng 1
1State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

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Bioengineering

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock
Dong Wei 1,2, Jinjie Gu 1, Zhongxi Zhang 1, Chenhong Wang 1, Dexin Wang 1, Chul Ho Kim 3, Biao Jiang 1, Jiping Shi 1,4, Jian Hao 1
1Lab of Biorefinery, Shanghai Advanced Research Institute, Chinese Academy of Sciences, 2University of Chinese Academy of Sciences, 3Biorefinery Research Center, Jeonbuk Branch Institute, Korea Research Institute of Bioscience & Biotechnology (KRIBB), 4School of Life Science and Technology, ShanghaiTech University

Bamboo powder was pretreated with NaOH and enzymatically hydrolyzed. The hydrolysate of bamboo was used as the feedstock for 2,3-butanediol, R-acetoin, 2-ketogluconic acid, and xylonic acid production by Klebsiella pneumoniae.

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Neuroscience

Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat
Dongsheng Xu *1, Jingjing Cui *1, Jia Wang 1, Zhiyun Zhang 1, Chen She 1, Wanzhu Bai 1
1Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences

Here, we present a refined protocol to effectively reveal biotinylated dextran amine (BDA) labeling with a fluorescent staining method through a reciprocal neural pathway. It is suitable for analyzing the fine structure of BDA labeling and distinguishing it from other neural elements under a confocal laser scanning microscope.

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Biochemistry

An Alternative Culture Method to Maintain Genomic Hypomethylation of Mouse Embryonic Stem Cells Using MEK Inhibitor PD0325901 and Vitamin C
Cuiping Li 1, Weiyi Lai 1, Hailin Wang 1
1State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences

We described in detail two chemical-based protocols for culturing mouse embryonic stem cells. This new method utilizes synergistic mechanisms of promoting Tet-mediated oxidation (by vitamin C) and repressing de novo synthesis of 5-methylcytosine (by PD0325901) to maintain DNA hypomethylation and pluripotency of mouse embryonic stem cells.

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Education

The Modular Design and Production of an Intelligent Robot Based on a Closed-Loop Control Strategy
Libo Zhang 1, Junjie Zhu 2, Hao Ren 2, Dongdong Liu 3, Dan Meng 4, Yanjun Wu 1, Tiejian Luo 5
1Institute of Software, Chinese Academy of Sciences, 2Hunan University, 3Fuyang Normal University, 4Anhui Normal University, 5University of Chinese Academy of Sciences

We present a protocol on modular design and production of intelligent robots to help scientific and technical workers design intelligent robots with special production tasks based on personal needs and individualized design.

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Behavior

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration
Wenze Cao *1,2,3, Li Song *1,2,3, Jingjing Cheng 1,2,3, Na Yi 1,2,3, Luyi Cai 1,2,3, Fu-de Huang 4,5, Margaret Ho 1,2,3
1Research Center for Translational Medicine, Tongji University School of Medicine, 2Key Laboratory of Arrhythmias of the Ministry of Education of China, Shanghai East Hospital, Tongji University School of Medicine, 3Department of Anatomy and Neurobiology, Tongji University School of Medicine, 4Shanghai Advanced Research Institute, University of Chinese Academy of Sciences, Chinese Academy of Sciences, 5Sino-Danish College, University of Chinese Academy of Sciences, Chinese Academy of Sciences

This step-by-step protocol analyzes Drosophila negative geotaxis behavior using an automated multi-cylinder system that hosts hundreds of flies and synchronizes their action by an electric motor. Upon synchronization, fly negative geotaxis behavior is assayed, digitally recorded, and analyzed using the self-designed RflyDetection software.

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Immunology and Infection

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection
Chongfeng Xu 1, Junhong Ai 2, Qingxun Zhang 1, Ting Li 3, Xiaorong Wu 1, Zhengde Xie 2, Ziyuan Duan 1
1Genetic Resources Center, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 2MOE Key Laboratory of Major Diseases in Children, National Key Discipline of Pediatrics, National Clinical Research Center for Respiratory Diseases, Beijing Key Laboratory of Pediatric Respiratory Infection Diseases, Beijing Pediatric Research Institute, Beijing Children's Hospital, Capital Medical University, 3State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

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Developmental Biology

Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis
Junhua Lv 1,2, Feng Liu 1,2
1State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, 2University of Chinese Academy of Sciences

This protocol describes using cultured Aorta-Gonad-Mesonephros for expression analyses, colony-forming units in the culture and spleen, and long-term reconstitution to determine the effect of regulatory factors and signaling pathways on hematopoietic stem cell development. This has been demonstrated as an effective system for studying hematopoietic stem cell biology and function.

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Neuroscience

An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice
Nastasia K-H Lim 1,2, Visse Moestrup 3,4, Xiao Zhang 1, Wen-An Wang 5, Arne Møller 3,4,6, Fu-De Huang 1,4
1Shanghai Advanced Research Institute, University of Chinese Academy of Sciences, Chinese Academy of Science, 2Shanghai Institute of Materia Medica, University of Chinese Academy of Sciences, Chinese Academy of Science, 3Center of Functionally Integrative Neuroscience, Department of Clinical Medicine, Aarhus University, 4Sino-Danish Center for Education and Research (SDC), 5Department of Neurology, Xinhua Hospital Chongming Branch Affiliated to Shanghai Jiao Tong University School of Medicine, 6Department of Nuclear Medicine and PET-centre, Aarhus University Hospital

This protocol describes an improved technique for the abundant collection of cerebrospinal fluid (CSF) with no contamination from blood. With greater sample collection and purity, more analyses can be performed using CSF to further our understanding of diseases that affect the brain and spinal cord.

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Genetics

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy
Xuan Yao 1,2, Xing Wang 1,2, Junlai Liu 2,3,4, Linyu Shi 1, Pengyu Huang 3, Hui Yang 1
1Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 2College of Life Sciences, University of Chinese Academy of Sciences, 3School of Life Science and Technology, Shanghai Tech University, 4Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

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Behavior

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila
Lifen Jiang *1,2, Yinpeng Zhan *2,3, Yan Zhu 2,3
1School of Life Science, University of Science and Technology of China, 2State key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Sciences, 3University of Chinese Academy of Sciences

Quantitative food-intake assays with dyed food provide a robust and high-throughput means to evaluate feeding motivation. Combining the food consumption assay with thermogenetic and optogenetic screens is a powerful approach to investigate the neural circuits underlying appetite in adult Drosophila melanogaster.

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Cancer Research

A Time-lapse, Label-free, Quantitative Phase Imaging Study of Dormant and Active Human Cancer Cells
Jing Huang *1,2, Peng Guo *1,2, Marsha A. Moses 1,2
1Vascular Biology Program, Boston Children's Hospital, 2Department of Surgery, Harvard Medical School and Boston Children's Hospital

Dormant and active cancer cell phenotypes were characterized using quantitative phase imaging. Cell proliferation, migration, and morphology assays were integrated and analyzed in one simple method.

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Immunology and Infection

Generation of Monoclonal Antibodies Against Natural Products
Yue Zhang 1, Peng Cao 2, Fang Lu 3, Xin Yan 4, Bingqian Jiang 3, Jinjun Cheng 3, Huihua Qu 5
1School of Life Science, Beijing University of Chinese Medicine, 2Third Affiliated Hospital, Beijing University of Chinese Medicine, 3School of Basic Medical Sciences, Beijing University of Chinese Medicine, 4School of Chinese Materia Medica, Beijing University of Chinese Medicine, 5Center of Scientific Experiment, Beijing University of Chinese Medicine

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

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Biology

A Method for Islet Transplantation to the Omentum in Mouse
Ying Lu *1, Shangyou Zou *1, Suzanne Bertera *2, Rita Bottino 2, David K.C. Cooper 3, Zhengzhao Liu 1, Yi Huang 1, Chengjun Wang 1, Chungu Hong 1, Tian He 1, Hancheng Zhang 1, Qin Huo 4, Hongxing Fu 5, Zhiming Cai 1, Lisha Mou 1
1Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Institute of Translational Medicine, Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen University School of Medicine, Shenzhen University Health Science Center, 2Institute for Cellular Therapeutics, Allegheny-Singer Research Institute, 3Xenotransplantation Program/Department of Surgery, The University of Alabama at Birmingham, 4College of Life Science and Oceanography, Shenzhen University, 5School of Pharmcy, Wenzhou Medical College

A method for the omental transplantation of islets in a mouse is introduced. The isolated islets are mixed with hydrogel and the mixture is placed into the omental pouch of a diabetic mouse. Then, the blood glucose is monitored, and immuno-histochemical analysis is performed.

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Biology

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model
Zhaofeng Jia 1,2,3, Yujie Liang 4,5, Xingfu Li 2,3, Xiao Xu 1,2,3, Jianyi Xiong 2,3, Daping Wang 1,2,3, Li Duan 2,3
1Postgraduate institution, Guangzhou Medical University, 2Guangdong Provincial Research Center for Artificial Intelligence and Digital Orthopedic Technology, 3Shenzhen Key Laboratory of Tissue Engineering, Shenzhen Laboratory of Digital Orthopaedic Engineering, Shenzhen Second People's Hospital (The First Hospital Affiliated to Shenzhen University), 4Department of Chemistry, Chinese University of Hong Kong, 5Shenzhen Kangning Hospital, Shenzhen Mental Health Center

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

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Immunology and Infection

Integrate Imaging Flow Cytometry and Transcriptomic Profiling to Evaluate Altered Endocytic CD1d Trafficking
Manju Sharma 1, Xiang Zhang 1, Shouxiong Huang 1
1Department of Environmental Health, University of Cincinnati College of Medicine

Imaging flow cytometry provides an ideal approach to detect the morphological and functional alteration of cells at individual and populational levels. Disrupted endocytic function for lipid antigen presentation in pollutant-exposed human dendritic cells was demonstrated with a combined transcriptomic profiling of gene expression and morphological demonstration of protein trafficking.

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Developmental Biology

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes
Jiayin Yang *1,2,6, Lai-Yung Wong *2, Xiao-Yu Tian *3, Rui Wei 1,2, Wing-Hon Lai 2, Ka-Wing Au 2, Zhiwei Luo 4,5, Carl Ward 4,5, Wai-In Ho 2, David P. Ibañez 4,5, Hao Liu 4,5, Xichen Bao 4,5, Baoming Qin 4,5, Yu Huang 3, Miguel A. Esteban 4,5,7, Hung-Fat Tse 1,2,6,7
1Department of Medicine, University of Hong Kong-Shenzhen Hospital, 2The Cardiology Division, Department of Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong, 3School of Biomedical Sciences, Institute of Vascular Medicine, Li Ka Shing Institute of Health Sciences, Chinese University of Hong Kong, 4Key Laboratory of Regenerative Biology of the Chinese Academy of Sciences, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health and Guangzhou Medical University, 5Laboratory of RNA, Chromatin, and Human Disease, CAS Key Laboratory of Regenerative Biology and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 6Research Centre of Heart, Brain, Hormone, and Healthy Ageing, Li Ka Shing Faculty of Medicine, University of Hong Kong, 7Hong Kong-Guangdong Stem Cell and Regenerative Medicine Research Centre, University of Hong Kong and Guangzhou Institutes of Biomedicine and Health

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

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Engineering

Quantitative Analysis of Vacuum Induction Melting by Laser-induced Breakdown Spectroscopy
Tianzhuo Zhao 1,2,3, Xin Li 1,2, Qixiu Zhong 1,2, Hong Xiao 1,3, Shuzhen Nie 1,3, Fuqiang Lian 1,3, Sining Sun 4, Zhongwei Fan 1,2,3
1The Academy of Opto-electronics, Chinese Academy of Sciences, 2University of Chinese Academy of Sciences, 3National Engineering Research Center for DPSSL, 4Beijing GK Laser Technology Co., Ltd.

During vacuum induction melting, laser-induced breakdown spectroscopy is used to perform real-time quantitative analysis of the main-ingredient elements of a molten alloy.

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Biology

Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified λ DNA at the Single Molecule Level
Huijun Xue *1,2, Zhengyan Zhan *1, Kaining Zhang 1,2, Yu Vincent Fu 1,2
1State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, 2Savaid Medical School, University of Chinese Academy of Sciences

Here, we present a protocol to study DNA-protein interactions by total internal reflection fluorescence microscopy (TIRFM) using a site-specifically modified λ DNA substrate and a Quantum-dot labeled protein.

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Immunology and Infection

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice
Yongli Zhang *1,2, Hangjie Zhang *2, Wenqiang Ma *3, Kefang Liu 1,2, Min Zhao 4, Yingze Zhao 2, Xuancheng Lu 5, Fuping Zhang 6, Xiangdong Li 3, George F. Gao 1,2,4,6, William J. Liu 1,2
1School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, 2NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 3State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, 4Research Network of Immunity and Health (RNIH), Beijing Institutes of Life Science, Chinese Academy of Sciences, 5Laboratory Animal Center, Chinese Center for Disease Control and Prevention, 6CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

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JoVE Journal

A Random-displacement Measurement by Combining a Magnetic Scale and Two Fiber Bragg Gratings
Lianqing Zhu 1,2, Lidan Lu 3,4, Wei Zhuang 2,5, Zhoumo Zeng 3,4, Mingli Dong 1,2
1School of Instrument Science and Opto-electronics Engineering, Beijing Information Science and Technology University, 2Beijing Engineering Research Center of Optoelectronic Information and Instruments, Beijing Key Laboratory for Optoelectronics Measurement Technology, 3School of Precision Instrument & Opto-electronics Engineering, Tianjin University of Science and Technology, 4School of Precision Instrument and Opto-electronics Engineering, State Key Laboratory of Precision Measuring Technology and Instruments, Tianjin University, 5School of Instrument and Opto-electronics Engineering, Hefei University of Technology

A protocol to create a full-range linear displacement sensor, combining two packaged fiber Bragg grating detectors with a magnetic scale, is presented.

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Cancer Research

Synthesis and Characterization of Placental Chondroitin Sulfate A (plCSA)-Targeting Lipid-Polymer Nanoparticles
Baozhen Zhang *1, Mingbin Zheng *2,3, Lintao Cai 2, Xiujun Fan 1
1Laboratory for Reproductive Health, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (CAS), 2Guangdong Key Laboratory of Nanomedicine, CAS Key Lab for Health Informatics, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (CAS), 3Department of Chemistry, Guangdong Medical University

Here, we present a protocol for the synthesis of placental chondroitin sulfate A binding peptide (plCSA-BP)-conjugated lipid-polymer nanoparticles via single-step sonication and bioconjugate techniques. These particles constitute a novel tool for the targeted delivery of therapeutics to most human tumors and placental trophoblasts to treat cancers and placental disorders.

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Bioengineering

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods
Baozhen Zhang 1, Zhilong Chen 1,2, Jinyu Han 1,3, Mengxia Li 1, Nihar R. Nayak 4, Xiujun Fan 1
1Laboratory for Reproductive Health, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, 2College of Veterinary Medicine, Hunan Agricultural University, 3Key Laboratory of Chemical Engineering Process and Technology for High-efficiency Conversion, College of Chemistry and Material Sciences, Heilongjiang University, 4Department of Obstetrics and Gynecology, Wayne State University School of Medicine

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

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Engineering

Quantitative Analysis by Thermogravimetry-Mass Spectrum Analysis for Reactions with Evolved Gases
Rongbin Li 1, Qian Huang 2, Kai Wei 2, Hongde Xia 2
1School of Metallurgical and Ecological Engineering, University of Science and Technology Beijing, 2Institute of Engineering Thermophysics, Chinese Academy of Sciences

Precise determination of the evolved gases' flow rate is key to study the details of reactions. We provide a novel quantitative analysis method of equivalent characteristic spectrum analysis for thermogravimetry-mass spectrum analysis by establishing the calibration system of the characteristic spectrum and relative sensitivity, for obtaining the flow rate.

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Environment

Evolution of Staircase Structures in Diffusive Convection
Shuang-Xi Guo 1, Sheng-Qi Zhou 1, Xian-Rong Cen 1, Yuan-Zheng Lu 1
1State Key Laboratory of Tropical Oceanography, South China Sea Institute of Oceanology, Chinese Academy of Sciences

Diffusive convection (DC) widely occurs in natural processes and engineering applications, characterized by a series of staircases with homogeneous convecting layers and stratified interfaces. An experimental procedure is described to simulate the evolution process of the DC staircase structure, including the generation, development and disappearance, in a rectangular tank.

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Behavior

A Conditioned Place Preference Protocol for Measuring Incubation of Craving in Rats
Yongmei Sun 1, Gaowei Chen 1,2, Kuikui Zhou 1, Yingjie Zhu 1
1Brain Cognition and Brain Disease Institute, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, 2Grain College, Henan University of Technology

Here, a morphine conditioned place preference (CPP) protocol is described to measure incubation of craving in rats.

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Medicine

Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection in Rats
Hui Yang 1, Pu Zou 1, Jingyuan Chen 1, Gaoxing Shi 1, Chenlu Wu 1, Mi Wang 1, Qin Zhou 2, Shenghua Zhou 1
1Department of Cardiology, The Second Xiangya Hospital, Central South University, 2Department of General Surgery, The Second Xiangya Hospital, Central South University

Here, we present a protocol to collect blood samples from the rat subclavian vein.

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Developmental Biology

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity
Mengrou Liu *1, Chunsen Zhu *1, Shun Bai *2, Xin Li 1, Kaiqiang Fu 1, Lan Ye 1, Ke Zheng 1
1State Key Laboratory of Reproductive Medicine, Nanjing Medical University, 2Center for Reproductive Medicine, Department of Obstetrics and Gynecology, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

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Chemistry

Tuning Oxide Properties by Oxygen Vacancy Control During Growth and Annealing
Tristan Steegemans *1, Shinhee Yun *1, Carlos N. Lobato 1, Eric Brand 1, Yunzhong Chen 2, Felix Trier 1, Dennis V. Christensen 1
1Department of Energy Conversion and Storage, Technical University of Denmark, 2Beijing National Laboratory for Condensed Matter Physics and Institute of Physics, Chinese Academy of Sciences

Oxide materials show many exotic properties that can be controlled by tuning the oxygen content. Here, we demonstrate the tuning of oxygen content in oxides by varying the pulsed laser deposition parameters and by performing postannealing. As an example, electronic properties of SrTiO3-based heterostructures are tuned by growth modifications and annealing.

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Immunology and Infection

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster
Shuo Yang *1,2,3, Yaya Zhao *1,2, Junjing Yu 4, Zhiqin Fan 2, Si-tang Gong 1, Hong Tang 1,2, Lei Pan 1,2,3
1The Joint Center for Infection and Immunity, Guangzhou Institute of Pediatrics, Guangzhou Women and Children's Medical Center, 2CAS Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, 3CAS Key Laboratory of Infection and Immunity (CASKLII), Institute of Biophysics, Chinese Academy of Sciences, 4Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

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Education

Separation of Uranium and Thorium for 230Th-U Dating of Submarine Hydrothermal Sulfides
Lisheng Wang 1,2,3, Xuefeng Wang 1,2, Jun Ye 4, Zhibang Ma 1,2, Weifang Yang 5, Jule Xiao 1,2
1Key Laboratory of Cenozoic Geology and Environment, Institute of Geology and Geophysics, Chinese Academy of Sciences, 2Institutions of Earth Science, Chinese Academy of Sciences, 3University of Chinese Academy of Sciences, 4First Institute of Oceanography, State Oceanic Administration, 5Key Laboratory of Submarine Geosciences, Second Institute of Oceanography, State Oceanic Administration

The protocol describes a method to purify and separate the U and Th nuclide in submarine hydrothermal sulfide sample with Fe co-precipitation and extraction chromatography for 230Th-U disequilibrium dating.

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Behavior

A Conflict Model of Reward-seeking Behavior in Male Rats
Shaofei Jiang 1,2, Yue Zhang 1,2, Xigeng Zheng 1,2, Haoshuang Luo 1,2, Zhengkui Liu 1,2, Yunjing Bai 1,2
1CAS Key Laboratory of Mental Health, Institute of Psychology, Chinese Academy of Sciences, 2Department of Psychology, University of Chinese Academy of Sciences

This conflict model is used to measure the impairment of inhibitory control after exposure to addictive drugs, or other factors that may influence inhibitory control. A sexual stimulus and an aversive obstacle are concurrently presented, thus male rats have to conquer the obstacle to approach the sexual reward.

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Immunology and Infection

Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue
Xin Li 1, Zhengmei Mao 2, Li Yang 1, Kai Sun 1
1Center for Metabolic and Degenerative Diseases, the Brown Foundation of Institute of Molecular Medicine, University of Texas Health Science Center at Houston, 2Microscopy Core, the Brown Foundation of Institute of Molecular Medicine, University of Texas Health Science Center at Houston

New blood vessel formation and sympathetic innervation play pivotal roles in adipose tissue remodeling. However, there remain technical issues in visualizing and quantitatively measuring adipose tissue. Here we present a protocol to successfully label and quantitatively compare the densities of blood vessels and nerve fibers in different adipose tissues.

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JoVE Journal

Imaging Integrin Tension and Cellular Force at Submicron Resolution with an Integrative Tension Sensor
Yuanchang Zhao 1, Nathaniel M. Wetter 1, Xuefeng Wang 2
1Department of Physics and Astronomy, Iowa State University, 2Department of Physics and Astronomy, Molecular, Cellular, and Developmental Biology Interdepartmental Program, Iowa State University

Integrin tension plays important roles in various cell functions. With an integrative tension sensor, integrin tension is calibrated with picoNewton (pN) sensitivity and imaged at submicron resolution.

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Medicine

Blood Collection Through Subclavian Vein Puncture in Mice
Hui Yang *1, Chenlu Wu *1, Fengjiao Liu 2, Mi Wang 1, Pu Zou 1, Yuhu He 1, Qiming Liu 1, Qin Zhou 3, Shenghua Zhou 1
1Department of Cardiology, The Second Xiangya Hospital, Central South University, 2Department of Cardiology, The Xiangya Hospital, Central South University, 3Department of General Surgery, The Second Xiangya Hospital, Central South University

Here, we present a protocol to take blood samples from the subclavian vein of mice.

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JoVE Journal

Thermocapillary Convection Space Experiment on the SJ-10 Recoverable Satellite
Li Duan *1,2, Yongli Yin *3, Jia Wang *1, Qi Kang 1,2, Di Wu 1, Huan Jiang 1, Pu Zhang 1, Liang Hu 1
1National Microgravity Laboratory, Institute of Mechanics, Chinese Academy of Sciences, 2School of Engineering Sciences, University of Chinese Academy of Sciences, 3China Astronaut Research and Training Center

A protocol for the space payload design, the space experiment on thermocapillary convection, and analyses of experimental data and images are presented in this paper.

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Bioengineering

Databases to Efficiently Manage Medium Sized, Low Velocity, Multidimensional Data in Tissue Engineering
Alexander R. Ochs 1,2, Mehrsa Mehrabi 1,2, Danielle Becker 1,2, Mira N. Asad 1,2, Jing Zhao 1,2, Michael V. Zaragoza 3,4, Anna Grosberg 1,2,5,6,7
1Department of Biomedical Engineering, University of California, Irvine, 2The Edwards Lifesciences Center for Advanced Cardiovascular Technology, University of California, Irvine, 3Pediatrics-Genetics & Genomics Division-School of Medicine, University of California, Irvine, 4Biological Chemistry-School of Medicine, University of California, Irvine, 5Department of Chemical and Biomolecular Engineering, University of California, Irvine, 6Center for Complex Biological Systems, University of California, Irvine, 7The NSF-Simons Center for Multiscale Cell Fate Research (CMCF), University of California, Irvine

Many researchers generate "medium-sized", low-velocity, and multi-dimensional data, which can be managed more efficiently with databases rather than spreadsheets. Here we provide a conceptual overview of databases including visualizing multi-dimensional data, linking tables in relational database structures, mapping semi-automated data pipelines, and using the database to elucidate data meaning.

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Chemistry

Isotopic Effect in Double Proton Transfer Process of Porphycene Investigated by Enhanced QM/MM Method
Zhihui Tu 1,2, Jian Yin 3, Liangxu Xie 4
1State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, 2University of Chinese Academy of Sciences, 3XtalPi Inc. (Shenzhen Jingtai Technology Co., Ltd.), 4Institute of Bioinformatics and Medical Engineering, School of Electrical and Information Engineering, Jiangsu University of Technology

A protocol that uses enhanced QM/MM method to investigate the isotopic effect on the double proton transfer process in porphycene is presented here.

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Behavior

Analysis of Learning and Memory Ability in an Alzheimer's Disease Mouse Model using the Morris Water Maze
Huiling Tian 1, Ning Ding 2, Mengwei Guo 1, Shun Wang 1, Zidong Wang 1, Hao Liu 1, Jiayi Yang 1, Yujie Li 3, Jingyu Ren 1, Jing Jiang 4, Zhigang Li 1
1School of Acupuncture-Moxibustion and Tuina, Beijing University of Chinese Medicine, 2Guang'anmen Hospital of China Academy of Chinese Medical Science, 3The Third Affiliated Hospital of Beijng University of Chinese Medicine, 4School of Nursing, Beijing University of Chinese Medicine

Herein, a protocol to conduct the Morris water maze tests to evaluate the ability of learning and memory of Alzheimer’s Disease model mice and to assess the effect of manual acupuncture for treating them is described.

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Engineering

Graphene-Assisted Quasi-van der Waals Epitaxy of AlN Film on Nano-Patterned Sapphire Substrate for Ultraviolet Light Emitting Diodes
Xiang Zhang *1,2, Zhaolong Chen *3, Hongliang Chang 1,2, Jianchang Yan 1,2, Shenyuan Yang 2,4, Junxi Wang 1,2, Peng Gao 5, Tongbo Wei 1,2
1State Key Laboratory of Solid-State Lighting, Institute of Semiconductors, Chinese Academy of Sciences, 2Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Science, 3Center for Nanochemistry (CNC), Beijing National Laboratory for Molecular Science, College of Chemistry and Molecular Engineering, Peking University, 4State Key Laboratory of Superlattices and Microstructures, Institute of Semiconductors, Chinese Academy of Sciences, 5Electron Microscopy Laboratory, School of Physics, Peking University

A protocol for graphene-assisted growth of high-quality AlN films on nano-patterned sapphire substrate is presented.

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Neuroscience

Stimulation Location Determination using a 3D Digitizer with High-Definition Transcranial Direct Current Stimulation
Wanting Chen 1, Rui Chen 1, Qinghua He 1,2,3,4,5
1Faculty of Psychology, Southwest University, 2Key Laboratory of Cognition and Personality, Ministry of Education, Southwest University, 3Southwest University Branch, Collaborative Innovation Center of Assessment toward Basic Education Quality, Beijing Normal University, 4Key Laboratory of Mental Health, Institute of Psychology, Chinese Academy of Sciences, 5Chongqing Collaborative Innovation Center for Brain Science

Presented here is a protocol to achieve higher accuracy in determination of stimulation location combining a 3D digitizer with high-definition transcranial direct current stimulation.

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Genetics

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease
Rafael J. L. Morcillo 1, Achen Zhao 1,2, María I. Tamayo-Navarrete 3, José M. García-Garrido 3, Alberto P. Macho 1
1Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, 2University of Chinese Academy of Sciences, 3Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín (CSIC)

Here, we present a versatile method for tomato root transformation followed by inoculation with Ralstonia solanacearum to perform straightforward genetic analysis for the study of bacterial wilt disease.

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Biology

Detecting Migration and Infiltration of Neutrophils in Mice
Qingyi Lu *1, Kai Yuan *1, Xiaohong Li 1, Haixu Jiang 1, Guiyang Huo 1, Wenrui Jia 1, Guangrui Huang 1, Anlong Xu 1,2
1School of Life Sciences, Beijing University of Chinese Medicine, 2State Key Laboratory of Biocontrol, Department of Biochemistry, School of Life Sciences, Sun Yat-Sen (Zhongshan) University

Here, we present three methods to assess neutrophil migration and infiltration both in vivo and in vitro. These methods can be used to discover promising therapeutics targeting neutrophil migration.

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Developmental Biology

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue
Yiding Qi *1, Xiang Miao *2, Lian Xu 3, Mengxia Fu 3, Shi Peng 4, Kailei Shi 5, Jun Li 4, Maoqing Ye 5, Ruogu Li 1
1Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiao Tong University, 2Shanghai Institute of Nutrition and Health, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, 3Shanghai Key Laboratory of Hypertension, Shanghai Institute of Hypertension, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, 4Department of Cardiology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 5Department of Cardiology, Shanghai Key Laboratory of Clinical Geriatric Medicine, Huadong Hospital Affiliated to Fudan University

We present a protocol for the isolation, culture, and adipogenic induction of neural crest derived adipose-derived stem cells (NCADSCs) from the periaortic adipose tissue of Wnt-1 Cre+/-;Rosa26RFP/+ mice. The NCADSCs can be an easily accessible source of ADSCs for modeling adipogenesis or lipogenesis in vitro.

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Environment

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae
Bin Zhang 1,2, Xinyue Yang 2, Jing Zhao 3, Ting Xu 2,4, Daqiang Yin 2,4
1State Key Laboratory of Marine Geology, Tongji University, 2Key Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and Engineering, Tongji University, 3Shanghai Collaborative Innovation Centre for WEEE Recycling, WEEE Research Center of 10 Shanghai Polytechnic University, 4Shanghai Institute of Pollution Control and Ecological Security

A detailed experimental protocol is presented in this paper for the evaluation of neurobehavioral toxicity of environmental pollutants using a zebrafish larvae model, including the exposure process and tests for neurobehavioral indicators.

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Genetics

Inducing Hairy Roots by Agrobacterium rhizogenes-Mediated Transformation in Tartary Buckwheat (Fagopyrum tataricum)
Yaolei Mi 1,2, Zhihui Zhu 3, Guangtao Qian 3, Yu Li 4, Xiangxiao Meng 1, Jianping Xue 3, Qingfu Chen 5, Wei Sun 1, Yuhua Shi 1,2
1Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, 2Artemisinin Research Center, China Academy of Chinese Medical Sciences, 3College of Life Science, Huaibei Normal University, 4Economic Crop Research Institute Sichuan Academy of Agriculture Sciences, 5Research Center of Buckwheat Industry Technology, Guizhou Normal University

We describe a method of inducing hairy roots by Agrobacterium rhizogenes-mediated transformation in Tartary buckwheat (Fagopyrum tataricum). This can be used to investigate gene functions and production of secondary metabolites in Tartary buckwheat, be adopted for any genetic transformation, or used for other medicinal plants after improvement.

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Determining the Influence of Soil Microbial Biomass Size on Soil Organic Matter Priming and Plant Residue Decomposition
Lu-Jun Li 1,2, Xia Zhu-Barker 3, Rongzhong Ye 4, William R. Horwath 3
1National Field Observation and Research Station of Hailun Agroecosystems, Key Laboratory of Mollisols Agroecology, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, 2University of Chinese Academy of Sciences, 3Department of Land, Air and Water Resources, University of California, Davis, 4Department of Plant & Environmental Sciences, Pee Dee Research & Education Center, Clemson University

This protocol describes a method to determine the influence of ryegrass residue addition on soil organic matter mineralization (i.e., priming effect) as well as explore the changes in soil microbial biomass size induced by soil organic matter priming, which involves artificially changing the size of microbial biomass.

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Immunology and Infection

Stability and Structure of Bat Major Histocompatibility Complex Class I with Heterologous β2-Microglobulin
Di Zhang *1,2, Kefang Liu *3,4, Dan Lu *2,5, Pengyan Wang 1,2, Qingxu Zhang 1,2, Peipei Liu 2, Yingze Zhao 2, Yan Chai 4, Jianxin Lyu 1, Jianxun Qi 4, William J. Liu 1,2
1School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, 2NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 3Faculty of Health Sciences, University of Macau, 4CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, 5Savaid Medical School, University of Chinese Academy of Sciences

The protocol describes experimental methods to obtain stable major histocompatibility complex (MHC) class I through potential β2-microglobulin (β2m) substitutions from different species. The structural comparison of MHC I stabilized by homologous and heterologous β2m were investigated.

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Biochemistry

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis
Chuan-Chih Hsu 1,3, Chia-Feng Tsai 2, W. Andy Tao 3,4, Pengcheng Wang 5
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.

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Engineering

Improving the Combustion Performance of a Hybrid Rocket Engine using a Novel Fuel Grain with a Nested Helical Structure
Zezhong Wang 1,2, Xin Lin 1, Fei Li 1, Zelin Zhang 1, Xilong Yu 1,2
1Institute of Mechanics, Chinese Academy of Sciences, 2School of Engineering Science, University of Chinese Academy of Sciences

A technique utilizing a solid fuel grain with a novel nested helical structure to improve the combustion performance of a hybrid rocket engine is presented.

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Environment

Ecotoxicological Effects of Microplastics on Bird Embryo Development by Hatching without Eggshell
Liyi Wang 1,2, Nana Xue 1,2, Wenfeng Li 2, Rehemanjiang Wufuer 1, Daoyong Zhang 1,3
1Xinjiang Key Laboratory of Environmental Pollution and Bioremediation, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, 2University of Chinese Academy of Sciences, 3Key Laboratory of Microbial Technology for Industrial Pollution Control of Zhejiang Province, College of Environment, Zhejiang University of Technology

This paper introduces a method of hatching without using an eggshell for toxicological studies of particle pollutants such as microplastics.

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Neuroscience

Visualizing the Calcitonin Gene-Related Peptide Immunoreactive Innervation of the Rat Cranial Dura Mater with Immunofluorescence and Neural Tracing
Jia Wang *1, Dongsheng Xu *1, Jingjing Cui 1, Chen She 1, Hui Wang 1, Shuang Wu 1, Ling Zou 1, Jianliang Zhang 1, Wanzhu Bai 1
1Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences

Here we present a protocol to visualize spatial correlation of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers and blood vessels in the cranial dura mater using immunofluorescence and fluorescent histochemistry with CGRP and phalloidin, respectively. In addition, the origin of these nerve fibers was retrograde traced with a fluorescent neural tracer.

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JoVE Journal

Rapid Determination of Antibody-Antigen Affinity by Mass Photometry
Di Wu 1, Grzegorz Piszczek 1
1Biophysics Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health

We describe a single-molecule approach to antigen-antibody affinity measurements using mass photometry (MP). The MP-based protocol is fast, accurate, uses a very small amount of material, and does not require protein modification.

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Behavior

An Assessment Method and Toolkit to Evaluate Keyboard Design on Smartphones
Yincheng Wang 1, Ke Wang 1, Yuqi Huang 1, Di Wu 2, Jian Wu 3, Jibo He 1
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.

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Bioengineering

Microfluidic Model to Mimic Initial Event of Neovascularization
Ping Zhao 1, Xing Zhang 1, Xiao Liu 1, Li Wang 4, Haoran Su 1, Liyi Wang 1, Dongrui Zhang 1, Xiaoyan Deng 3, Yubo Fan 1,2
1Beijing Advanced Innovation Centre for Biomedical Engineering, Key Laboratory for Biomechanics and Mechanobiology of Chinese Education Ministry, School of Biological Science and Medical Engineering, Beihang University, 2School of Engineering Medicine, Beihang University, 3Artificial Intelligence Key Laboratory of Sichuan Province, School of Automation and Information Engineering, Sichuan University of Science and Engineering, 4Beijing Research Center of Urban System Engineering

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

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Chemistry

Optimization of Radiochemical Reactions using Droplet Arrays
Alejandra Rios 1,2, Travis S. Holloway 2,3, Jia Wang 2,4, R. Michael van Dam 1,2,3,4
1Physics and Biology in Medicine Interdepartmental Graduate Program, University of California Los Angeles (UCLA), 2Crump Institute of Molecular Imaging, UCLA, 3Department of Molecular & Medical Pharmacology, David Geffen School of Medicine, 4Department of Bioengineering, UCLA

This method describes the use of a novel high-throughput methodology, based on droplet chemical reactions, for the rapid and economical optimization of radiopharmaceuticals using nanomole amounts of reagents.

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Biology

Large-Scale Preparation of Synovial Fluid Mesenchymal Stem Cell-Derived Exosomes by 3D Bioreactor Culture
Li Duan 1,2, Xingfu Li 1,2, Xiao Xu 1,2, Limei Xu 1,2, Daping Wang 1,2, Kan Ouyang 1,2, Yujie Liang 1,3
1Department of Orthopedics, The First affiliated hospital of Shenzhen University, Shenzhen Second People's Hospital, 2Guangdong Provincial Research Center for Artificial Intelligence and Digital Orthopedic Technology, Shenzhen Second People's Hospital, 3Department of Child and Adolescent Psychiatry, Shenzhen Kangning Hospital, Shenzhen Mental Health Center, Shenzhen Key Laboratory for Psychological Healthcare & Shenzhen Institute of Mental Health

Here, we present a protocol to produce a large number of GMP-grade exosomes from synovial fluid mesenchymal stem cells using a 3D bioreactor.

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Neuroscience

Primary Microglia Isolation from Postnatal Mouse Brains
Siling Du *1,2, Shanshan Xiong *3, Xiangjuan Du 4, Ti-Fei Yuan 1, Bo Peng 3,4,5, Yanxia Rao 1
1Shanghai Key Laboratory of Psychotic Disorders, Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, 2Division of Biology and Biomedical Science, Washington University in St. Louis, 3Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, 4Department of Neurosurgery, Huashan Hospital, Institute for Translational Brain Research, State Key Laboratory of Medical Neurobiology, MOE Frontiers Center for Brain Science, Fudan University, 5Co-innovation Center of Neuroregeneration, Nantong University

Primary cell culture is one of the primarily used approaches for studying microglial biology in vitro. Here, we developed a method for simple and rapid microglia isolation from the mouse postnatal day 1 (P1) to P4.

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Biology

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification
Raphael Nyaruaba 1,2,3, Xiaohong Li 1, Caroline Mwaliko 2,3,4, Changchang Li 1, Matilu Mwau 5, Nelson Odiwour 1,2,3, Elishiba Muturi 1,2,3, Caroline Muema 1,2,3, Junhua Li 1, Junping Yu 1, Hongping Wei 1
1Key Laboratory of Special Pathogens and Biosafety, Center for Biosafety Mega-Science, Wuhan Institute of Virology, Chinese Academy of Sciences, 2International College, University of Chinese Academy of Sciences, 3Sino-Africa Joint Research Center, 4CAS Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, 5Center for Infectious and Parasitic Diseases Control Research, Kenya Medical Research Institute

This work summarizes steps on developing different assays for SARS-CoV-2 detection using a two color ddPCR system. The steps are elaborate and notes have been included on how to improve the assays and experiment performance. These assays may be used for multiple SARS-CoV-2 RT-ddPCR applications.

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Biochemistry

Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds
Yue Zhang *1, Peng Cao *2, Fang Lu 1, Jinjun Cheng 3, Huihua Qu 4
1School of Life Science, Beijing University of Chinese Medicine, 2Third Affiliated Hospital, Beijing University of Chinese Medicine, 3National Institute of TCM Constitution and Preventive Medicine, Beijing University of Chinese Medicine, 4Center of Scientific Experiment, Beijing University of Chinese Medicine

Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds

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Bioengineering

Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System (MMC)
Xingjin Jian *1,2, Xiaojie Guo *1,2,3, Jia Wang 4, Zheng Lin Tan 1,2,6, Xin-hui Xing 1,2,5, Liyan Wang 3, Chong Zhang 1,2,5
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., 4Biochemical Engineering Research Group, School of Chemical Engineering and Technology, Xi’an Jiaotong University, 5Center for Synthetic & Systems Biology, Tsinghua University, 6School of Life Science and Technology, Tokyo Institute of Technology

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.

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Biology

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection
Jing Zhao 1,2,3, Jie Yang 1,2,3, Lili Zhang 1,2, Rongxiang Fang 1,2, Yan Huo 1,2
1State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, 2National Plant Gene Research Center, 3University of the Chinese Academy of Sciences

The present protocol describes a method to collect sufficient saliva from piercing-sucking insects using an artificial medium. This is a convenient method for collecting insect saliva and studying salivary function on insect feeding behavior and vector-borne virus transmission.

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Biochemistry

Evaluating the Effect of Pesticides on the Larvae of the Solitary Bees
Yingying Song 1, Ruiping Li 1,2, Lili Li 1, Fang Ouyang 3, Xingyuan Men 1
1Institute of Plant Protection, Shandong Academy of Agricultural Sciences, 2College of Plant Protection, Shandong Agricultural University, 3State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences

The present protocol explains a method to feed pesticide-contaminated provisions to the larvae of the solitary bees, Osmia excavata. The procedure examines the ecotoxicity of the pesticide to the larvae of the solitary bees.

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Engineering

The Traceable Calibration of Thrust Stand by Electrostatic Force
Bin Wang 1,2, Yong Yang 3, Zhan Hu 1,2, Yelong Zheng 3
1Shanghai Engineering Center for Microsatellites, 2Innovation Academy for Microsatellites, Chinese Academy of Sciences, 3State Key Laboratory of Precision Measuring Technology and Instruments, Tianjin University

Traceability calibration of mechanical characteristics of thrust stand is an essential prerequisite to ensure traceability measurement of thrust. Here, we describe how to calibrate the thrust stand by the electrostatic force generated by the parallel plate capacitor.

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Biochemistry

Purification of Ubiquitinated p53 Proteins from Mammalian Cells
Di Wu 1, Mengfan He 1, Dawei Li 1
1Center for Translational Medicine, The Affiliated Zhangjiagang Hospital of Soochow University

The protocol describes a step-by-step method to purify ubiquitinated proteins from mammalian cells using the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified from cells under stringent nondenaturing and denaturing conditions.

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Neuroscience

Demonstrating Hairy and Glabrous Skin Innervation in a 3D Pattern Using Multiple Fluorescent Staining and Tissue Clearing Approaches
Xiaoyu Wang *1, Wanying Cao *1, Jingtao Shi *1, Xiaoning Zhang 1, Zhengyang Qu 1, Dongsheng Xu 1, Hongye Wan 1, Yangshuai Su 1, Wei He 1, Xianghong Jing 1, Wanzhu Bai 1
1Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences

The thickness of tissue sections limited the morphological study of the skin innervation. The present protocol describes a unique tissue clearing technique to visualize cutaneous nerve fibers in thick 300 µm tissue sections under confocal microscopy.

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Immunology and Infection

Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection
Fei Wang *1, Cihan Yang *1,2, Shunlong Wang 1,2, Qun Wu 1,2, Christabel Ochieng 1,2, Zhiming Yuan 1,2, Han Xia 1,2
1Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, 2University of Chinese Academy of Sciences

This methodology, which included oral feeding and intrathoracic injection infection, could effectively assess the influence of midgut and/or salivary gland barriers on arbovirus infection.

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Medicine

Clinical Application of 24 G Cannula Needle and 3-0 Polypropylene Suture in Vas Deferens Exploration
Jin Wang *1, Wenjia Li *2, Yao He 1, Yuanbin Xia 1, Lin Sun 1, Youpeng Zhang 1, Zhaohui Zhu 1
1Department of Urology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 2Department of Stomatology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology

This protocol presents the clinical application of a 24 G cannula and 3-0 polypropylene suture as a simple and effective method for the exploration of the vas deferens.

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Biology

Mosquito-Associated Virus Isolation from Field-Collected Mosquitoes
Doudou Huang *1, Haixia Ma *1, Lu Zhao 1,2, Xiaoyu Wang 1,2, Yi Huang 1,2, Fei Wang 1, Zhiming Yuan 1,2, Han Xia 1,2
1Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, 2University of Chinese Academy of Sciences

Numerous novel virus-like sequences have been found in mosquitoes due to the extensive use of sequencing technologies. We provide an effective procedure for isolating and amplifying viruses using vertebrate and mosquito cell lines, which might serve as the basis for future studies on mosquito-associated viruses, including mosquito-borne and mosquito-specific viruses.

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Biology

Induction and Diverse Assessment Indicators of Experimental Autoimmune Encephalomyelitis
Chun Wang *1, Jie Lv *1, Wei Zhuang 1,2, Ling Xie 1, Guangyu Liu 1, Kaidireya Saimaier 1, Sanxing Han 1, Changjie Shi 1, Qiuhong Hua 1, Ru Zhang 1, Guangfeng Shi 3, Changsheng Du 1
1Putuo People's Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, 2National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 3Department of Infectious Diseases, Shanghai Key Laboratory of Infectious Diseases and Biosafety Emergency Response, National Medical Center for Infectious Diseases, Huashan Hospital, Fudan University

The present protocol describes the induction of experimental autoimmune encephalomyelitis in a mouse model using myelin oligodendrocyte glycoprotein and monitoring the disease process using a clinical scoring system. Experimental autoimmune encephalomyelitis-related symptoms are analyzed using mouse femur micro-computed tomography analysis and open field test to assess the disease process comprehensively.

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Bioengineering

Developing Drosophila melanogaster Models for Imaging and Optogenetic Control of Cardiac Function
Elena Gracheva 1, Fei Wang 1, Abigail Matt 1, Hongwu Liang 1, Matthew Fishman 1,2, Chao Zhou 1
1Department of Biomedical Engineering, Washington University in St. Louis, 2Department of Computer Science and Engineering, Washington University in St. Louis

The present protocol describes the generation of Drosophila melanogaster expressing eNpHR2.0 or ReaChR opsins in the heart for OCT imaging and optogenetic heart pacing. Detailed instructions for Drosophila OCT imaging and heart beating modulation, including the simulation of restorable heart arrest, bradycardia, and tachycardia in live animals at different developmental stages, are reported.

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Neuroscience

Visualizing the Morphological Characteristics of Neuromuscular Junction in Rat Medial Gastrocnemius Muscle
Jingjing Cui *1, Shuang Wu *1, Jia Wang 1, Yuqing Wang 1, Yuxin Su 1, Dongsheng Xu 1, Yihan Liu 1, Junhong Gao 1, Xianghong Jing 1, Wanzhu Bai 1
1Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences

The protocol shows a method to examine spatial correlation among the pre-synaptic terminals, post-synaptic receptors, and peri-synaptic Schwann cells in the rat medial gastrocnemius muscle using fluorescent immunohistochemistry with different biomarkers, namely, neurofilament 200, vesicular acetylcholine transporter, alpha-bungarotoxin, and S100.

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Neuroscience

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation
Qiwei Fan *1, Xiaotan Zhang *2, Yuan Wang 3, Xiaoyu Wang 1
1Division of Histology & Embryology, Key Laboratory for Regenerative Medicine of the Ministry of Education, College of Medicine, Jinan University, 2Department of Clinical Pathology, First Affiliated Hospital of Jinan University, 3Program in Neuroscience, Department of Biomedical Sciences, College of Medicine, Florida State University

Using in ovo electroporation, we devised a method to selectively transfect the auditory inner ear and cochlear nucleus in chicken embryos to achieve a cell-group-specific knockdown of fragile X mental retardation protein during discrete periods of circuit assembly.

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Medicine

Establishment of a Simple and Effective Rat Model for Intraoperative Parathyroid Gland Imaging
Fan Chen *1,2, Chun Liu *3, Peng Guo 3, Weihui Zheng 1,2
1Key Laboratory of Head & Neck Cancer Translational Research of Zhejiang Province, Zhejiang Cancer Hospital, 2The Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital), 3Institute of Basic Medicine and Cancer (IBMC), Chinese Academy of Sciences

To date, the development of parathyroid gland (PG) identification methods is limited by the lack of animal models in preclinical research. Here, we establish a simple and effective rat model for intraoperative PG imaging and evaluate its effectiveness by using iron oxide nanoparticles as a novel PG contrast agent.

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Bioengineering

Cellular Affinity of Particle-Stabilized Emulsion to Boost Antigen Internalization
Fengqiang Cao *1,2,3, Yali Ming *3,4, Weixiang Gao 3,4, Jia Ge 3, Kenji Ogino 1,2
1Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, 2Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, 3State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, 4University of Chinese Academy of Sciences

To rationally design efficient adjuvants, we developed poly-lactic-co-glycolic acid nanoparticle-stabilized Pickering emulsion (PNPE). The PNPE possessed unique softness and a hydrophobic interface for potent cellular contact and offered high-content antigen loading, improving the cellular affinity of the delivery system to antigen-presenting cells and inducing efficient internalization of antigens.

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Developmental Biology

An Efficient Clearing Protocol for the Study of Seed Development in Tomato (Solanum lycopersicum L.)
Yixuan Feng 1,2, Tai Wang 1,2,3, Lingtong Liu 1
1Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, 2College of Life Science, University of Chinese Academy of Sciences, 3Innovative Academy of Seed Design, Chinese Academy of Sciences

The tomato seed is an important model for studying genetics and developmental biology during plant reproduction. This protocol is useful for clearing tomato seeds at different developmental stages to observe the finer embryonic structure.

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Environment

Collection of Alfalfa Root Exudates to Study the Impact of Di(2-ethylhexyl) Phthalate on Metabolite Production
Wenjie Ren 1,2, Rui Zhao 1, Ying Teng 1, Yongming Luo 1
1Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science, Chinese Academy of Sciences, 2College of Resources and Environment, University of Chinese Academy of Sciences

The secretion of root exudates is usually an external detoxification strategy for plants under stress conditions. This protocol describes how to assess the impact of xenobiotics on alfalfa via nontargeted metabolomic analysis.

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Biology

A Precise and Quantifiable Method for Collecting Hemolymph from Small Arthropods
Qing Liu 1,3, Lili Zhang 1,2, Rongxiang Fang 1,2,3, Yan Huo 1,2
1State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, 2CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, 3University of Chinese Academy of Sciences

We describe a method to collect quantifiable hemolymph efficiently from small arthropods for subsequent analysis.

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Biology

Optimizing the Rearing Procedure of Germ-Free Wasps
Zhengyu Zhu *1,2, Dandan Wang *1, Yanjun Liu 1, Ting Tang 2, Guan-Hong Wang 1
1State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, 2College of Life Sciences, Hebei University

Nasonia wasp embryos were dissected from Lucillia sericata pupae after parasitization for 12-24 h and washed with alcohol and 10% sodium hypochlorite solution to obtain germ-free embryos. After rearing the germ-free embryos and supplying them with Nasonia rearing medium to grow and develop in vitro, germ-free Nasonia adults were obtained.

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Chemistry

Multimodal Nonlinear Hyperspectral Chemical Imaging Using Line-Scanning Vibrational Sum-Frequency Generation Microscopy
Jackson C. Wagner 1, Bin Yang 1,2, Zishan Wu 1, Wei Xiong 1,3,4
1Department of Chemistry and Biochemistry, UC San Diego, 2State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 3Materials Science and Engineering Program, UC San Diego, 4Department of Electrical and Computer Engineering, UC San Diego

A multimodal, rapid hyperspectral imaging framework was developed to obtain broadband vibrational sum-frequency generation (VSFG) images, along with brightfield, second harmonic generation (SHG) imaging modalities. Due to the infrared frequency being resonant with molecular vibrations, microscopic structural and mesoscopic morphology knowledge is revealed of symmetry-allowed samples.

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Medicine

Cryopreservation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells at the Optimal Stage
Xinyue Bai *1,2,3,4,5,6,7, Ting Zhang *1,2,3,4,5,6,7, Xinyue Zhu 1,2,3,4,5,6,7, Xianyu Huang 1,2,3,4,5,6,7, Haiyun Liu 1,2,3,4,5,6,7, Xiaoyan Ding 8, Mei Jiang 1,2,3,4,5,6,7, Xiaodong Sun 1,2,3,4,5,6,7
1Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 2National Clinical Research Center for Eye Diseases, 3Shanghai Clinical Research Center for Eye Diseases, 4Shanghai Key Clinical Specialty, 5Shanghai Key Laboratory of Ocular Fundus Diseases, 6Shanghai Engineering Center for Visual Science and Photomedicine, 7Shanghai Engineering Center for Precise Diagnosis and Treatment of Eye Diseases, 8Stem Cell Core Facility, CAS Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences

This study provides a detailed protocol for the efficient cryopreservation of human stem cell-derived retinal pigment epithelial cells.

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Biology

A Magnetic Separation-Assisted High-Speed Homogenization Method for Large-Scale Production of Endosome-Derived Vesicles
Dujuan Wang 1,2, Shili Yao 2,3, Peng Guo 1,2,3,4
1Academy of Medical Engineering and Translational Medicine, Tianjin University, 2Hangzhou Institute of Medicine (HIM), Chinese Academy of Sciences, 3School of Materials Science and Engineering, Tianjin University, 4Zhejiang Cancer Hospital

Here, we describe a magnetic separation-assisted high-speed homogenization method for large-scale production of endosome-derived nanovesicles as a new type of exosome mimics (EMs) that share the same biological origin and similar structure, morphology, and protein composition of native extracellular vesicles (EVs).

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Biology

Rodent-Proof Wall: An Efficient Physical Method for Controlling Rodents and its Efficiency Statistics
Yi-Ying He 1,2, Mei-Wen Zhang 1, Yun-Lin Zhao 2, Tian Huang 3, Xun-Jun Zhou 1, Hua-Nan Huang 4
1National Field Observation and Research Station of Dongting Lake Wetland Ecosystem, Key Laboratory for Agro-Ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, 2Hunan Research Center of Engineering Technology for Utilization of Environmental and Resources Plant, Central South University of Forestry and Technology, 3Hunan Engineering Research Center of Ecological Environment lntelligent Monitoring and Disaster Prevention and Mitigation Technology in Dongting Lake, 4Datonghu Agricultural Technology Extension Center

Here, we introduce three methods for the physical control of pest rodents, four methods for counting their effectiveness against rodents, and the statistics of the effect of building a rodent-proof wall.

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Biology

Apoplast-Extraction Based Method to Improve the Purity of Plant Produced Recombinant Protein
Huiying Ji 1,2, Tianhao Zhang 2, Han Li 2,3, Chunyu Wang 1,2, Rongxiang Fang 2, Lili Zhang 1,2,4, Yan Huo 2
1Shandong Academy of Medical Sciences, Shandong First Medical University, 2State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, 3College of Veterinary Medicine, Shanxi Agricultural University, 4Beijing Life Science Academy

The purification of recombinant proteins from plant systems is usually challenged by plant proteins. This work provides a method to effectively extract and purify a secreted recombinant protein from the apoplast of Nicotiana benthamiana.

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Bioengineering

Fabrication and Characterization of Microneedle Patches for Loading and Delivery of Exosomes
Sijia Mu 1,2, Hao Chang 2, Fengli Qu 2
1Medical School, Faculty of Medicine, Tianjin University, 2Hangzhou Institute of Medicine, Chinese Academy of Sciences

Exosomes possess significant clinical potential, but their practical application is limited due to easy in vivo clearance and poor stability. Microneedles present a solution by enabling localized delivery by puncturing physiological barriers and dry-state preservation, thereby addressing the limitations of exosome administration and expanding their clinical utility.

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