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Nankai University

19 ARTICLES PUBLISHED IN JoVE

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Medicine

Programmed Electrical Stimulation in Mice
Na Li 1, Xander H.T Wehrens 1,2
1Department of Molecular Physiology and Biophysics, Baylor College of Medicine (BCM), 2The Margaret M. and Albert B. Alkek Department of Medicine, Baylor College of Medicine (BCM)

Programmed electrical stimulation provides the ability to determine conduction properties of the heart, and the possibility to induce and terminate cardiac arrhythmias using various pacing protocols. Using a transvenous catheter, intracardiac electrogram recordings can be obtained in mice following programmed electrical stimulation protocols to identify arrhythmogenic substrates.

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Biology

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy
Takashi Sakurai 1,2, Anthony Lanahan 2, Melissa J. Woolls 2, Na Li 2, Daniela Tirziu 2, Masahiro Murakami 2
1Max-Planck-Institute for Molecular Biomedicine and Institute of Cell Biology, 2Department of Internal Medicine, Yale Cardiovascular Research Center and Section of Cardiovascular Medicine

This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

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Cancer Research

How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases
Mercedes Rodriguez-Teja 1, Claudia Breit 2, Mitchell Clarke 3, Kamil Talar 3, Kai Wang 3, Mohammad A. Mohammad 3, Sage Pickwell 3, Guillermina Etchandy 1, Graeme J. Stasiuk 3, Justin Sturge 3
1Departamento de Genética, Facultad de Medicina, Universidad de la República (UDELAR), 2Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, 3School of Biological, Biomedical & Environmental Sciences, University of Hull

Here we explain a protocol for modelling the biophysical microenvironment where crosslinking and increased stiffness of the basement membrane (BM) induced by advanced glycation endproducts (AGEs) has pathological relevance.

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Bioengineering

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior
Kai Wang 1, Kam W. Leong 2, Yong Yang 1
1Department of Chemical and Biomedical Engineering, West Virginia University, 2Department of Biomedical Engineering, Columbia University

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

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Bioengineering

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice
Kai Wang *1, Xi Wang *1, Cheng-sheng Han *2, Liang-yi Chen 2, Ying Luo 1
1Department of Biomedical Engineering, Peking University, 2Institute of Molecular Medicine, Peking University

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

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Bioengineering

Surface Engineering of Pancreatic Islets with a Heparinized StarPEG Nanocoating
Jingyi Yang 1, Shaofeng Lou 1, Deling Kong 1, Chen Li 1
1Tianjin Key Laboratory of Biomaterial Research, Institute of Biomedical Engineering, Chinese Academy of Medical Science & Peking Union Medical College

This protocol aims to achieve surface engineering of pancreatic islets using a heparin-incorporated starPEG nanocoating via pseudo-bioorthogonal chemistry between the N-hydroxysuccinimide groups of the nanocoating and the amine groups of islet cell membrane.

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Biology

Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model
Kang Qin 1, Yifan Wu 1, Yiwa Pan 1, Kai Wang 1, Deling Kong 1, Qiang Zhao 1
1State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials (Ministry of Education), College of Life Sciences, Nankai University

Here, we present a modified electrospinning method to fabricate PCL vascular grafts with thick fibers and large pores, and describe a protocol to evaluate the in vivo performance in a rat model of abdominal aorta replacement.

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Biology

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay
Courtney M. Anderson 1, Annelies Laeremans 1, Xiao-Ming Mindy Wang 1, Xingyong Wu 1, Bingqing Zhang 1, Emerald Doolittle 1, Jeffrey Kim 1, Na Li 1, Helly Xiao Yan Pimentel 1, Emily Park 1, Xiao-Jun Ma 1
1Advanced Cell Diagnostics, Inc

Here, we describe an in situ hybridization assay which enables sensitive and specific detection of sequences as short as 50 nucleotides with single-nucleotide resolution at the single-cell level. The assay, which can be performed manually or automatically, can enable visualization of splice variants, short sequences, and mutations within the tissue context.

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Engineering

Femtosecond Laser Filaments for Use in Sub-Diffraction-Limited Imaging and Remote Sensing
Matthews M. Springer 1, Benjamin D. Strycker 1,2, Kai Wang 1, Alexei V. Sokolov 1,2, Marlan O. Scully 1,2
1Texas A&M University, 2Baylor University

High-intensity femtosecond pulses of laser light can undergo cycles of Kerr self-focusing and plasma defocusing, propagating an intense sub-millimeter-diameter beam over long distances. We describe a technique for generating and using these filaments to perform remote imaging and sensing beyond the classical diffraction limits of linear optics.

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Behavior

Behavioral Assessments of Spontaneous Locomotion in a Murine MPTP-induced Parkinson's Disease Model
Pei-En Jiang 1, Qiu-Han Lang 1, Qin-Yi Yu 1, Xin-Yu Tang 1, Qian-Qian Liu 1, Xin-Yu Li 1, Xi-Zeng Feng 1
1State Key Laboratory of Medicinal Chemical Biology, The Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Science, Nankai University

We describe establishment of a murine model for Parkinson's disease using MPTP, and behavioral assessments using cylinder and open field tests to measure motor function. We then use L-DOPA as one example to show how to apply this model in the study of PD drugs.

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Biology

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana
Kai Wang 1, Kayin Law 1, Manhin Leung 1, Waishing Wong 1, Ning Li 1,2
1Division of Life Science, Energy Institute, Institute for the Environment, The Hong Kong University of Science and Technology, 2Shenzhen Research Institute, The Hong Kong University of Science and Technology

A gentle touch-force loading machine is built from human hair brushes, robotic arms and a controller. The hair brushes are driven by robotic arms installed on the machine and move periodically to apply touch-force on plants. The strength of machine-driven hair touches is comparable to that of manually applied touches.

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Medicine

Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis
Kaiyue Zhang 1, Chen Wang 1, Ran Wang 2, Shang Chen 1, Zongjin Li 1
1Nankai University School of Medicine, 2State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University

This protocol describes the establishment of a tumor-bearing mouse model to monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging.

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Cancer Research

Preparation of Mitochondria from Ovarian Cancer Tissues and Control Ovarian Tissues for Quantitative Proteomics Analysis
Xianquan Zhan 1,2,3,4, Huanni Li 5, Shehua Qian 1,2,3, Xiaohan Zhan 1,2,3, Na Li 1,2,3
1Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, 2Hunan Engineering Laboratory for Structural Biology and Drug Design, Xiangya Hospital, Central South University, 3State Local Joint Engineering Laboratory for Anticancer Drugs, Xiangya Hospital, Central South University, 4National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, 5Department of Obstetrics and Gynecology, Xiangya Hospital, Central South University

This article presents a protocol of differential-speed centrifugation in combination with density gradient centrifugation to separate mitochondria from human ovarian cancer tissues and control ovarian tissues for quantitative proteomics analysis, resulting in a high-quality mitochondrial sample and high-throughput and high-reproducibility quantitative proteomics analysis of a human ovarian cancer mitochondrial proteome.

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Cancer Research

Analysis of Side Population in Solid Tumor Cell Lines
Xiaoli Dong 1, Yingying Wei 1, Tao Xu 1, Xiaoyue Tan 1,2,3, Na Li 1,2,3
1School of Medicine, Nankai University, 2Tianjin Key Laboratory of Tumour Microenvironment and Neurovascular Regulation, 3Collaborative Innovation Center for Biotherapy, Nankai University

A convenient, fast, and cost-effective method to measure the proportion of side population cells in solid tumor cell lines is presented.

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Medicine

Diagnosis and Surgical Treatment of Human Brucellar Spondylodiscitis
Yakefu Abulizi *1, Xiaoyu Cai *1,2,3, Tao Xu 1, Chuanhui Xun 1, Weibin Sheng 1, Liang Gao 2,3, Maierdan Maimaiti 1
1Department of Spine Surgery, First Affiliated Hospital of Xinjiang Medical University, 2Center of Experimental Orthopaedics, Saarland University Medical Center, 3Sino Euro Orthopaedics Network (SEON)

We describe a clinical algorithm, based on decades of front-line experience of diagnosis and surgical treatment of human Brucellar spondylodiscitis in the largest medical center of the China's Xinjiang Pastoral Area.

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Immunology and Infection

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis
Linlin Zhang 1,2, Mengjia Liu 1,2, Meng Zhang 1,2, Junhong Ai 1,2, Jiao Tian 1,2, Ran Wang 1,2, Zhengde Xie 1,2
1Beijing Key Laboratory of Pediatric Respiratory Infectious Diseases, Key Laboratory of Major Diseases in Children, Ministry of Education, National Clinical Research Center for Respiratory Diseases, Laboratory of Infection and Virology, Beijing Pediatric Research Institute, Beijing Children's Hospital, Capital Medical University, National Center for Children's Health, 2Research Unit of Critical Infection in Children, 2019RU016, Chinese Academy of Medical Sciences

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

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Immunology and Infection

Detection of Polyfunctional T Cells in Children Vaccinated with Japanese Encephalitis Vaccine via the Flow Cytometry Technique
Linlin Zhang *1,2, Meng Zhang *1,2,3, Mengjia Liu 1,2, Junhong Ai 1,2, Jiao Tian 1,2, Huizheng Ge 4, Ran Wang 1,2, Zhengde Xie 1,2
1Beijing Key Laboratory of Pediatric Respiratory Infectious Diseases, Key Laboratory of Major Diseases in Children, Ministry of Education, National Clinical Research Center for Respiratory Diseases, Laboratory of Infection and Virology, Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, National Center for Children’s Health, 2Research Unit of Critical Infection in Children, 2019RU016, Chinese Academy of Medical Sciences, 3Department of Pediatric Rehabilitation, Beijing Boai Hospital, School of Rehabilitation Medicine, Capital Medical University, China Rehabilitation Research Center, 4Center of Excellence (Beijing), Becton Dickinson Medical Devices (Shanghai) Co Ltd

The present protocol combines ex vivo stimulation and flow cytometry to analyze polyfunctional T cell (TPF) profiles in peripheral blood mononuclear cells (PBMCs) within Japanese encephalitis virus (JEV)-vaccinated children. The detection method and flow cytometry color scheme of JEV-specific TPFs were tested to provide a reference for similar studies.

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Medicine

Purification and Characterization of Extracellular Vesicles from Human Adipose-Derived Mesenchymal Stem Cells
Yuting Wang 1,2, Yan Han 1, Yudi Han 1
1Department of Plastic and Reconstructive Surgery, the First Medical Centre, Chinese PLA General Hospital, 2School of Medicine, Nankai University

This protocol describes all procedures, from culturing human adipose-derived mesenchymal stem cells (ADSCs) and collecting supernatant to extracting extracellular vesicles (EVs) using ultracentrifugation.

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Biochemistry

Analyzing Telomeric Protein-DNA Interactions Using Single-Molecule Magnetic Tweezers
Han Gao 1, Yanling Liu 1, Zhongbo Yu 1
1State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Nankai University

This protocol demonstrates using single-molecule magnetic tweezers to study interactions between telomeric DNA-binding proteins (Telomere Repeat-binding Factor 1 [TRF1] and TRF2) and long telomeres extracted from human cells. It describes the preparatory steps for telomeres and telomeric repeat-binding factors, the execution of single-molecule experiments, and the data collection and analysis methods.

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