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14 ARTICLES PUBLISHED IN JoVE

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Biology

Electron Cryotomography of Bacterial Cells
Songye Chen 1, Alasdair McDowall 1,2, Megan J. Dobro 1, Ariane Briegel 1,2, Mark Ladinsky 1,2, Jian Shi 2, Elitza I. Tocheva 1, Morgan Beeby 1,2, Martin Pilhofer 1,2, H. Jane Ding 1, Zhuo Li 1,2, Lu Gan 1, Dylan M. Morris 1, Grant J. Jensen 1,2
1Division of Biology, California Institute of Technology - Caltech, 2Howard Hughes Medical Institute, California Institute of Technology - Caltech

We illustrate here how to use electron cryotomography (ECT) to study the ultrastructure of bacterial cells in near-native states, to "macromolecular" (~4 nm) resolution.

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Biology

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain
Kerry Flegel *1, Dan Sun *1, Olga Grushko *1, Yiqin Ma 1, Laura Buttitta 1
1Molecular, Cellular and Developmental Biology, University of Michigan

A protocol for cell cycle analysis of live Drosophila tissues using the Attune Acoustic Focusing Cytometer is described. This protocol simultaneously provides information about relative cell size, cell number, DNA content and cell type via lineage tracing or tissue specific expression of fluorescent proteins in vivo.

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Biology

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method
Clarisse van der Feltz 1, Daniel Pomeranz Krummel 1,2
1Department of Biochemistry, Brandeis University, 2Winship Cancer Institute, Emory University School of Medicine

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

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Medicine

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo
Mingming Liu 1, Xiaoyan Zhang 1, Bingwei Li 1, Bing Wang 1, Qingbin Wu 1, Fei Shang 1, Ailing Li 1, Hongwei Li 1, Ruijuan Xiu 1
1Institute of Microcirculation, Chinese Academy of Medical Sciences & Peking Union Medical College

Pancreatic islet microvascular vasomotion regulates islet blood distribution and maintains the physiological function of islet β cells. This protocol describes using a laser Doppler monitor to determine the functional status of pancreatic islet microvascular vasomotion in vivo and to assess the contributions of pancreatic islet microcirculation to pancreatic-related diseases.

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Environment

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants
Zhuo Li 1,2, Fangting Ai 3, Jing Zhang 4, Zhenyang Yu 1,2, Daqiang Yin 1,2
1College of Environmental Sciences and Engineering, Tongji University, 2Shanghai Institute of Pollution Control and Ecological Security, 3Jiaxing Tongji Institute of Environment, 4College of Ecological Technique and Engineering, Shanghai Institute of Technology

Trans- and multi-generational effects of persistent chemicals are essential in judging their long-term consequences in the environment and on the human health. We provide novel detailed methods for studying trans- and multi-generational effects using free-living nematode Caenorhabditis elegans.

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Cancer Research

A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research
Zhuo Li *1, Xiao Yang *1, Yilu Zhang 1, Xiaolong Yang 1, Xinxin Cui 1, Yanjuan Zhang 1, Wenfeng Gong 1, Huichen Bai 1, Ning Liu 1, Zhiyu Tang 1, Mingming Guo 1, Kang Li 1, Tong Zhang 1, Lai Wang 1, Xiaomin Song 1
1BeiGene (Beijing) Co., Ltd.

We describe a human peripheral blood mononuclear cell (PBMC) — based humanized xenograft mouse model for translational immuno-oncology research. This protocol could serve as a general guideline for establishing and characterizing similar models for I-O therapy assessment.

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Bioengineering

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device
Bingchen Che *1, Jie Zhu *1, Dan Sun 1, Xiaoqiang Feng 1, Ce Zhang 1
1State Key Laboratory of Cultivation Base for Photoelectric Technology and Functional Materials, State Key Laboratory of Photon-Technology in Western China Energy, Institute of Photonics and Photon-Technology, Northwest University

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

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Behavior

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera (Hübner)
Dong Ai 1,2, Bing Wang 2, Zhennan Fan 1, Yuyao Fu 1, Caihong Yu 1, Guirong Wang 2,3
1School of Chemical and Environmental Engineering, China University of Mining and Technology, 2State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, 3Guangdong Laboratory of Lingnan Modern Agriculture, Shenzhen; Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences

Presented here is a protocol of Helicoverpa armigera (Hübner) embryo microinjection and knockout mutant identification created by CRISPR/Cas9 genome editing. Mutant insects enable further research of gene function and interaction among different genes in vivo.

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Genetics

ACT1-CUP1 Assays Determine the Substrate-Specific Sensitivities of Spliceosomal Mutants in Budding Yeast
Clarisse van der Feltz 1
1College of Arts and Sciences, Northwest University

The ACT1-CUP1 assay, a copper growth assay, provides a quick readout of precursor messenger RNA (pre-mRNA) splicing and the impact mutant splicing factors have on spliceosomal function. This study provides a protocol and highlights the customization possible to address the splicing question of interest.

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Bioengineering

Isolation and Culture of Bone Marrow Mesenchymal Stem Cells from the Human Mandible
Chunlin Zong *1, Lu Zhao *1, Chong Huang 2, Yuanli Chen 1, Lei Tian 1
1State Key Laboratory of Military Stomatology, National Clinical Research Center for Oral Diseases, Shaanxi Clinical Research Center for Oral Diseases, Department of Oral and Maxillofacial Surgery, School of Stomatology, The Fourth Military Medical University, 2College of Life Sciences, Northwest University

The present protocol describes an efficient procedure for isolating and culturing of human mandibular bone marrow-derived mesenchymal stem cells using the whole bone marrow adherence method. The cultured cells were identified by cell proliferation assays, flow cytometry, and multilineage differentiation induction.

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Biology

Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues
Yuan Cao *1,2, Ji-Yu Qiu *1,3, Da Chen 1,2, Chen-Yao Li 1,4, Shu-Juan Xing 1,5, Chen-Xi Zheng 1, Xin Liu 2, Yan Jin 1,6, Bing-Dong Sui 1
1State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi International Joint Research Center for Oral Diseases, Center for Tissue Engineering, School of Stomatology, The Fourth Military Medical University, 2Department of Orthodontics, School of Stomatology, The Fourth Military Medical University, 3Department of VIP Dental Care, School of Stomatology, The Fourth Military Medical University, 4School of Basic Medicine, The Fourth Military Medical University, 5College of Life Science, Northwest University, 6Xi'an Institute of Tissue Engineering and Regenerative Medicine

The present protocol describes a method to extract extracellular vesicles from the peripheral blood and solid tissues with subsequent profiling of surface antigens and protein cargos.

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Biology

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells
Shu-Juan Xing 1,2, Kai-Chao Zhang 2,3, Si-Yuan Tang 2,4, Lu Liu 2,5, Yuan Cao 2,5, Chen-Xi Zheng 2, Bing-Dong Sui 2, Yan Jin 2,3
1College of Life Science, Northwest University, 2State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi International Joint Research Center for Oral Diseases, Center for Tissue Engineering, School of Stomatology, Fourth Military Medical University, 3Xi'an Institute of Tissue Engineering and Regenerative Medicine, 4School of Basic Medicine, Fourth Military Medical University, 5Department of Orthodontics, School of Stomatology, Fourth Military Medical University

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

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Environment

A Complete Method for Evaluating the Performance of Photocatalysts for the Degradation of Antibiotics in Environmental Remediation
Bing Wang 1, XuXia Zhang 1, LeCheng Li 1, MengTing Ji 1, Zheng Zheng 1, ChuanHui Shi 1, Zhuo Li 1, Hong Hao 1
1School of Chemical Engineering, Northwest University

Presented here is a protocol to explore a universal set of experimental procedures for comprehensive laboratory evaluation of photocatalysts in the field of environmental purification, using the example of photocatalytic removal of antibiotic organic pollutant molecules from water by phthalocyanine sensitized silver phosphate composites.

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Bioengineering

Construction, Characterization, and Regenerative Application of Self-Assembled Human Mesenchymal Stem Cell Aggregates
Shijie Li 1,2, Peisheng Liu 1,2,3, Ting Zhu 1,2,3, Bingdong Sui 1, Yan Jin 1, Kun Xuan 2, Chenxi Zheng 1
1State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi International Joint Research Center for Oral Diseases, Center for Tissue Engineering, The Fourth Military Medical University, 2Department of Preventive Dentistry, School of Stomatology, The Fourth Military Medical University, 3College of Life Science, Northwest University

This study describes a method to construct aggregates based on the self-assembly of human mesenchymal stem cells and identifies the morphological and histological characteristics for the regenerative treatment of cranial bone defects.

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