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23 ARTICLES PUBLISHED IN JoVE

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Biology

The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF
Wen Xiong 1, Yan Gao 1, Xun Cheng 1, Charles Martin 1, Dongmei Wu 1, Shuyuan Yao 1, Min-Ju Kim 2, Yang Liu 1
1Research and Development, Stemgent, 2Product Marketing, Stemgent

SC1 functions through dual inhibition of Ras- GAP and ERK1. We tested the function of SC1 in supporting mouse ES cell self-renewal in the absence of LIF and showed that SC1 is able to maintain self-renewal of mouse ES cell cultures.

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Medicine

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth
Dalit Barkan 1, Jeffrey E. Green 2
1Department of Biology, University of Haifa, 2Transgenic Oncogenesis and Genomics Section, Laboratory of Cancer Biology and Genetics, National Cancer Institute

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

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Medicine

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model
Tamalee R. Kramp 1, Kevin Camphausen 1
1Radiation Oncology Branch, National Cancer Institute

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

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Biology

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis
Lyuba Khavrutskii 1,2, Joanna Yeh 1, Olga Timofeeva 3, Sergey G. Tarasov 4, Samuel Pritt 1, Karen Stefanisko 1, Nadya Tarasova 1
1Cancer and Inflammation Program, National Cancer Institute, 2Basic Science Program, SAIC-Frederick, Inc., 3Departments of Oncology and Radiation Medicine, Georgetown University Medical Center, 4Structural Biophysics Laboratory, National Cancer Institute

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

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Biology

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins
Andrius Masedunskas 1,2, Natalie Porat-Shliom 1, Muhibullah Tora 1, Oleg Milberg 1,3, Roberto Weigert 1
1Intracellular Membrane Trafficking Unit, Oral and Pharyngeal Cancer Branch National Institute of Dental and Craniofacial Research, National Institutes of Health, 2Department of Biology, University of North Carolina at Chapel Hill , 3Department of Chemical & Biochemical Engineering and Department of Biomedical Engineering, Rutgers University

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

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Immunology and Infection

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors
Steven J. Smith 1, Stephen H. Hughes 1
1HIV Drug Resistance Program, National Cancer Institute

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

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Bioengineering

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars
Mahtab Hassanpourfard 1, Xiaohui Sun 2, Amin Valiei 1, Partha Mukherjee 3, Thomas Thundat 1, Yang Liu 2, Aloke Kumar 4
1Department of Chemical and Material Engineering, University of Alberta, 2Department of Civil and Environmental Engineering, University of Alberta, 3Department of Mechanical Engineering, Texas A&M University, 4Department of Mechanical Engineering, University of Alberta

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

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Medicine

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury
Klaus Zweckberger 1, Yang Liu 1, Jian Wang 1, Nicole Forgione 1, Michael G. Fehlings 1,2,3
1Department of Genetics and Development, Toronto Western Research Institute and Spinal Program, University Health Network, Krembil Neuroscience Centre, 2Department of Surgery, University of Toronto, 3Institute of Medical Sciences, University of Toronto

Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established.

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Medicine

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells
Carrie D. House 1, Lidia Hernandez 1, Christina M. Annunziata 1
1Women's Malignancies Branch, National Cancer Institute

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

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Bioengineering

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii
Swayamdipta Bhaduri 1, Nandini Debnath 1, Sushanta Mitra 2, Yang Liu 3, Aloke Kumar 1
1Department of Mechanical Engineering, University of Alberta, 2Department of Mechanical Engineering, York University, 3Department of Civil and Environmental Engineering, University of Alberta

Protocols for microbiologically induced calcite precipitation (MICP) using the bacterium Sporosarcina pasteurii are presented here. The precipitated calcium carbonate was characterized through optical microscopy and scanning electron microscopy (SEM). It is also shown that exposure to MICP increases the compressive strength of sponge.

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Bioengineering

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique
Kazuhide Sato 1, Peter L. Choyke 1, Kobayashi Hisataka 1
1Molecular Imaging Program, National Cancer Institute

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

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JoVE Journal

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards
Joshua R. Porter 1, William G. Telford 2, Eric Batchelor 1
1Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, 2Experimental Transplantation and Immunology Branch, Center for Cancer Research, National Cancer Institute

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

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Genetics

Single Molecule Analysis of Laser Localized Psoralen Adducts
Jing Huang 1, Himabindu Gali 1, Julia Gichimu 1, Marina A. Bellani 1, Durga Pokharel 1, Manikandan Paramasivam 1, Michael M. Seidman 1
1Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health

Lasers are frequently used in studies of the cellular response to DNA damage. However, they generate lesions whose spacing, frequency, and collisions with replication forks are rarely characterized. Here, we describe an approach that enables the determination of these parameters with laser localized interstrand crosslinks.

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Cancer Research

Evaluating In Vitro DNA Damage Using Comet Assay
Yanxin Lu 1,2, Yang Liu 1, Chunzhang Yang 1
1Neuro-Oncology Branch, Center for Cancer Research, National Cancer Institute, 2Basic Medical Science Department, Zunyi Medical College-Zhuhai Campus

The comet assay is an efficient method to detect DNA damage including single and double-stranded DNA breaks. We describe alkaline and neutral comet assays to measure DNA damage in cancer cells to evaluate the therapeutic effect of chemotherapy.

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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

Merkel Cell Polyomavirus Infection and Detection
Wei Liu *1, Nathan A. Krump *1, Christopher B. Buck 2, Jianxin You 1
1Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, 2Laboratory of Cellular Oncology, National Cancer Institute

Here, we present a protocol to infect primary human dermal fibroblast with MCPyV. The protocol includes isolation of dermal fibroblasts, preparation of MCPyV virions, virus infection, immunofluorescence staining, and fluorescence in situ hybridization. This protocol can be extended for characterizing MCPyV-host interactions and discovering other cell types infectable by MCPyV.

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Behavior

Impact of High-intensity Interval Exercise and Moderate-Intensity Continuous Exercise on the Cardiac Troponin T Level at an Early Stage of Training
Haifeng Zhang 1,2, Jinlei Nie 3, Zhaowei Kong 4, Xiangui Zhu 1, Yang Liu 1, Qingde Shi 3
1Physical Education College, Hebei Normal University, 2Hebei Provincial Key Lab of Measurement and Evaluation in Human Movement and Bio-Information, 3School of Health Sciences and Sports, Macao Polytechnic Institute, 4Faculty of Education, University of Macau

Here, we present protocols of high-intensity interval and moderate-intensity continuous exercise to observe the response of circulating cardiac troponin T (cTnT) concentration to acute exercise over 10 days. The information may assist with clinical interpretations of post-exercise cTnT elevation and guide the prescription of exercise.

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Medicine

Near Infrared Photoimmunotherapy for Mouse Models of Pleural Dissemination
Hirotoshi Yasui 1, Yuko Nishinaga 1, Shunichi Taki 1, Kazuomi Takahashi 1, Yoshitaka Isobe 1, Kazuhide Sato 1,2,3
1Respiratory Medicine, Nagoya University Graduate School of Medicine, 2Nagoya University Institute for Advanced Research, 3B3-Unit, Advanced Analytical and Diagnostic Imaging Center (AADIC)/Medical Engineering Unit (MEU), Nagoya University Institute for Advanced Research

Near-infrared photoimmunotherapy (NIR-PIT) is an emerging cancer therapeutic strategy that utilizes an antibody-photoabsorber (IR700Dye) conjugate and NIR light to destroy cancer cells. Here, we present a method to evaluate the antitumor effect of NIR-PIT in a mouse model of pleural disseminated lung cancer and malignant pleural mesothelioma using bioluminescence imaging.

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Medicine

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model
Yuan Yuan 1, Meng-hua Chen 1, Jing Huang 2, Yuan Tian 2, Ke Qin 3, Zhang Yuan 1, Wen-yan Wang 1, Zhi-jiang Wu 1, Xin-yue Tian 1, Yubin Zhang 4
1Department of Intensive Care Unit, The Second Affiliated Hospital of Guangxi Medical University, 2Department of hepatobiliary surgery, The Affiliated Ningbo Medical Center Lihuili Hospital of Medical School of Ningbo University, 3Department of transplantation, The Second Affiliated Hospital of Guangxi Medical University, 4Digital Technology and Engineering college, Ningbo University of Finance & Economics

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

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Biology

Imaging Neutrophil Migration in the Mouse Skin to Investigate Subcellular Membrane Remodeling Under Physiological Conditions
Nicolas Melis *1, Bhagawat Subramanian *1,2, Desu Chen 1, Roberto Weigert 1
1Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, 2Lineberger Comprehensive Cancer Center, UNC-Chapel Hill

Neutrophil migration relies on the rapid and continuous remodeling of the plasma membrane in response to chemoattractant and its interactions with the extracellular microenvironment. Described herein is a procedure based on Intravital Subcellular Microscopy to investigate the dynamic of membrane remodeling in neutrophils injected in the ear of anesthetized mice.

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Cancer Research

Measurement of the Compressibility of Cell and Nucleus Based on Acoustofluidic Microdevice
Qibin Fu 1, Yan Zhang 1, Tuchen Huang 1, Yang Liu 1
1Sino-French Institute of Nuclear Engineering and Technology, Sun Yat-sen University

Here a protocol is presented to build a fast and non-destructive system for measuring cell or nucleus compressibility based on acoustofluidic microdevice. Changes in mechanical properties of tumor cells after epithelial-mesenchymal transition or ionizing radiation were investigated, demonstrating the application prospect of this method in scientific research and clinical practice.

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Cancer Research

Translational Orthotopic Models of Glioblastoma Multiforme
Rajaa El Meskini 1, Devon Atkinson 1, Zoë Weaver Ohler 1
1Center for Advanced Preclinical Research, Frederick National Laboratory for Cancer Research, National Cancer Institute

Here, we describe a preclinical orthotopic mouse model for GBM, established by intracranial injection of cells derived from genetically engineered mouse model tumors. This model displays the disease hallmarks of human GBM. For translational studies, the mouse brain tumor is tracked by in vivo MRI and histopathology.

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Engineering

A Teleoperated Robotic System-Assisted Percutaneous Transiliac-Transsacral Screw Fixation Technique
Binbin Liu 1, Fei Xu 1, Yang Liu 1, Tianqi Wang 2, Yajing Cao 2, Zehang Zheng 1, Hanqing Xu 1, Chuang Huang 1, Zhengqiang Luo 1
1Department of Orthopedics, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, 2Wuhan United Imaging Healthcare Surgical Technology Co. Ltd.

Teleoperated robotic system-assisted percutaneous transiliac-transsacral screw fixation is a feasible technique. Screw channels can be implemented with high accuracy owing to the excellent freedom of movement and stability of the robotic arms.

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