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Sichuan University and Collaborative Innovation Center for Biotherapy

11 ARTICLES PUBLISHED IN JoVE

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Biology

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)
Zhen Wang 1, Christoph Benning 1
1Department of Biochemistry and Molecular Biology, Michigan State University

Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.

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Bioengineering

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae
Bibhudatta Mishra 1, Mostafa Ghannad-Rezaie 2, Jiaxing Li 1, Xin Wang  1, Yan Hao 1, Bing Ye 3,4, Nikos Chronis 2,5, Catherine A. Collins 1
1Department of Molecular, Cellular and Developmental Biology, University of Michigan, 2Department of Biomedical Engineering, University of Michigan, 3Life Sciences Institute, University of Michigan, 4Department of Cell and Developmental Biology, University of Michigan, 5Department of Mechanical Engineering, University of Michigan

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the 'larva chip' for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

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Deriving De Novo Atomic Models by Cryo Electron Microscopy
Peng Ge 1, Nicole Poweleit 1,2, Robert P Gunsalus 2,3, Hong Zhou 1,2
1Electron Imaging Center for Nanomachines (EICN), California NanoSystems Institute, University of California, 2Immunology and Molecular Genetics, University of California, 3UCLA Institute of Genomics and Proteomics

Cryo electron microscopy (cryoEM) can be employed to derive de novo atomic models of macromolecular complexes in solution. The steps involved in high resolution cryoEM of biological molecules, from image recording, to data processing, to atomic modeling based on the resulting cryoEM density map, are illustrated.

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Medicine

A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury
Toshio Takayama 1, Xudong Shi 1, Bowen Wang 1, Sarah Franco 1, Yifan Zhou 1, Daniel DiRenzo 1, Alycia Kent 1, Peter Hartig 1, Joshua Zent 1, Lian-Wang Guo 1
1Department of Surgery, University of Wisconsin School of Medicine and Public Health

The mouse femoral artery wire injury model of restenosis is technically challenging. In this protocol we show the key technical details essential for successfully performing wire injury to induce consistent neointima for studies of restenosis.

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Neuroscience

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay
Wei Li 1,2, Chen Zhang 1, Xiulian Sun 3
1Department of Neurology, Qilu Hospital of Shandong University, 2Department of Neurology, Qingdao Municipal Hospital, 3Brain Research Institute, Qilu Hospital of Shandong University

This protocol aims to describe a method to examine the Ca2+ retention capacity and Ca2+- triggered mitochondrial swelling of isolated mitochondria of SH-SY5Y cells step-by-step.

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Developmental Biology

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation
Qinggang Dai *1, Yujiao Han *2, Furong Xie *1, Xuhui Ma 3, Zhan Xu 2, Xiao Liu 1, Weiguo Zou 2, Jun Wang 1
1Department of Pediatric Dentistry, Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai Key Laboratory of Stomatology & Shanghai Research Institute of Stomatology, National Clinical Research Center of Stomatology, 2State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, 3Department of Oral and Maxillofacial-Head and Neck Oncology, Ninth People's Hospital, Shanghai Jiaotong University School of Medicine

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 in osteoclast formation.

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Biology

Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse
Zhen Wang 1,4, Ramon Ocadiz-Ruiz 1, Sinju Sundaresan 1, Lin Ding 1, Michael Hayes 1, Nirakar Sahoo 3, Haoxing Xu 1,2, Juanita L. Merchant 1,2,5
1Department of Internal Medicine-Gastroenterology, University of Michigan, 2Department of Molecular and Integrative Physiology, University of Michigan, 3Department of Molecular, Cellular and Developmental Biology, University of Michigan, 4Department of Gastrointestinal Surgery, The First Affiliated Hospital of Guangxi Medical University, 5Division of Gastroenterology, University of Arizona College of Medicine

Here, we describe the isolation of enteric-glial cells from the intestinal-submucosa using sequential EDTA incubations to chelate divalent cations and then incubation in non-enzymatic cell recovery solution. Plating the resultant cell suspension on poly-D-lysine and laminin results in a highly enriched culture of submucosal glial cells for functional analysis.

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Biochemistry

A Semi-Quantitative Drug Affinity Responsive Target Stability (DARTS) assay for studying Rapamycin/mTOR interaction
Chen Zhang *1, Min Cui *1, Yazhou Cui 1, Aubryanna Hettinghouse 1, Chuan-ju Liu 1,2
1Department of Orthopaedic Surgery, New York University Medical Center, 2Department of Cell Biology, New York University School of Medicine

In this study, we enhanced the data analysis capabilities of the DARTS experiment by monitoring the changes in protein stability and estimating the affinity of protein-ligand interactions. The interactions can be plotted into two curves: a proteolytic curve and a dose-dependence curve. We have used mTOR-rapamycin interaction as an exemplary case.

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Immunology and Infection

Establishing Transcription Profile of Psoriasiform Cutaneous In Vitro using HaCaT Cells Stimulated with Combination of Cytokines
Huaping Zheng *1, Linna Gu *1, Zhen Wang *1, Hong Zhou *1, Chen Zhang 1, Xiu Teng 1, Zhonglan Hu 1, Xiaoqiong Wei 1, Xiao Liu 1, Fanlian Zeng 1, Qixiang Zhao 1, Yan Hao 1, Yawen Hu 1, Xiaoyan Wang 1, Jing Hu 1, Jiadong Yu 1, Wenlin Wu 1, Yifan Zhou 1, Kaijun Cui 2, Nongyu Huang 1, Jiong Li 1
1State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University and Collaborative Innovation Center for Biotherapy, 2Department of Cardiovascular Medicine, West China Hospital, Sichuan University

This paper presents a method of establishing an in vitro psoriasiform cutaneous inflammatory model at the transcription level using a combination of five cytokines (IL-17A, IL-22, IL-1α, TNF-α, OSM) on HaCaT cell line.

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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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Engineering

Interactive and Visualized Online Experimentation System for Engineering Education and Research
Zhongcheng Lei 1, Hong Zhou 1, Shengwang Ye 1, Wenshan Hu 1, Guo-Ping Liu 1, Zijie Wei 1
1Department of Artificial Intelligence and Automation, Wuhan University

This work describes an online experimentation system that provides visualized experiments, including the visualization of theories, concepts, and formulas, visualizing the experimental process with three-dimensional (3-D) virtual test rigs, and visualizing the control and monitoring system using widgets such as charts and cameras.

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