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20 ARTICLES PUBLISHED IN JoVE

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Biology

PuraMatrix Encapsulation of Cancer Cells
Adnan O. Abu-Yousif 1, Imran Rizvi 1,2, Conor L. Evans 1, Jonathan P. Celli 1, Tayyaba Hasan 1,3
1Wellman Center for Photomedicine Massachusetts General Hospital, Harvard Medical School, 2Thayer School of Engineering, Dartmouth College, 3Department of Dermatology, Harvard Medical School

This video demonstrates how to encapsulate and culture cancer cells in PuraMatrix, a commercially available self assembling peptide gel.

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Immunology and Infection

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells
Sophie Moreau-Marquis 1, Carly V. Redelman 2, Bruce A. Stanton 1, Gregory G. Anderson 2
1Department of Physiology, Dartmouth College, 2Department of Biology, Indiana University Purdue University Indianapolis

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

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Biology

Using SCOPE to Identify Potential Regulatory Motifs in Coregulated Genes
Viktor Martyanov 1, Robert H. Gross 1
1Department of Biology, Dartmouth College

A straight-forward and robust method to identify potential regulatory motifs in co-regulated genes is presented. SCOPE does not require any user parameters and returns motifs that represent excellent candidates for regulatory signals. The identification of such regulatory signals helps to understand the underlying biology.

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Immunology and Infection

Determining the Phagocytic Activity of Clinical Antibody Samples
Elizabeth G. McAndrew 1, Anne-Sophie Dugast 1, Anna F. Licht 1, Justin R. Eusebio 1, Galit Alter 1, Margaret E. Ackerman 2
1Massachusetts General Hospital, Ragon Institute of MGH, MIT, and Harvard, 2Thayer School of Engineering, Dartmouth College

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors—providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

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Medicine

Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers
Kenneth M. Tichauer 1, Robert W. Holt 2, Kimberley S. Samkoe 3, Fadi El-Ghussein 1, Jason R. Gunn 1, Michael Jermyn 1, Hamid Dehghani 4, Frederic Leblond 1, Brian W. Pogue 1,2
1Thayer School of Engineering, Dartmouth College, 2Department of Physics and Astronomy, Dartmouth College, 3Darmouth Medical School, Dartmouth College, 4School of Computer Science, University of Birmingham

Diffuse fluorescence tomography offers a relatively low-cost and potentially high-throughout approach to preclinical in vivo tumor imaging. The methodology of optical data collection, calibration, and image reconstruction is presented for a computed tomography-guided non-contact time-domain system using fluorescent targeting of the tumor biomarker epidermal growth factor receptor in a mouse glioma model.

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Biology

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes
Regina Salvat 1, Leonard Moise 2, Chris Bailey-Kellogg 3, Karl E. Griswold 1
1Thayer School of Engineering, Dartmouth College, 2Institute for Immunology and Informatics, University of Rhode Island, 3Department of Computer Science, Dartmouth College

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design.  Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

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Medicine

Catheterization of the Carotid Artery and Jugular Vein to Perform Hemodynamic Measures, Infusions and Blood Sampling in a Conscious Rat Model
Jing Feng 1, Yvonne Fitz 1, Yan Li 1, Melinda Fernandez 1, Irene Cortes Puch 1, Dong Wang 1, Stephanie Pazniokas 2, Brandon Bucher 3, Xizhong Cui 1, Steven B. Solomon 1
1Critical Care Medicine Department, Clinical Center, National Institutes of Health, 2Harvard Apparatus, 3ADInstruments

Vascular accesses to measure hemodynamics, provide fluids and perform blood sampling are important to any small animal model study. We present a technique for implanting catheters into the carotid artery and the common jugular vein in an anesthetized rat for connecting to a system to perform monitoring, infusions and sampling.

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Behavior

A Dual Task Procedure Combined with Rapid Serial Visual Presentation to Test Attentional Blink for Nontargets
Zhengang Lu *1, Jessica Goold *1, Ming Meng 1
1Department of Psychological and Brain Sciences, Dartmouth College

This paper describes a novel dual task procedure combined with Rapid Serial Visual Presentation to look at attentional blink at varied stimulus onset asynchronies. This experimental procedure differed from others in that it tested attentional blink for nontargets.

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Engineering

Using Synchrotron Radiation Microtomography to Investigate Multi-scale Three-dimensional Microelectronic Packages
Holly D. Carlton 1, John W. Elmer 1, Yan Li 2, Mario Pacheco 2, Deepak Goyal 2, Dilworth Y. Parkinson 3, Alastair A. MacDowell 3
1Materials Engineering Division, Lawrence Livermore National Laboratory, 2Assembly Test and Technology Development Failure Analysis Labs, Intel Corporation, 3Advanced Light Source, Lawrence Berkeley National Laboratory

For this study synchrotron radiation micro-tomography, a non-destructive three-dimensional imaging technique, is employed to investigate an entire microelectronic package with a cross-sectional area of 16 x 16 mm. Due to the synchrotron's high flux and brightness the sample was imaged in just 3 min with an 8.7 µm spatial resolution.

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JoVE Core

Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes
Adrienne T. Perkins 1, Sharon E. Bickel 1
1Department of Biological Sciences, Dartmouth College

This manuscript presents a detailed method for generating X-chromosome arm probes and performing fluorescence in situ hybridization (FISH) to examine the state of sister chromatid cohesion in prometaphase and metaphase I arrested Drosophila oocytes. This protocol is suitable for determining whether meiotic arm cohesion is intact or disrupted in different genotypes.

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Bioengineering

Probing the Roles of Physical Forces in Early Chick Embryonic Morphogenesis
Yan Li *1, Hannah Grover *1, Eric Dai 2, Kevin Yang 1, Zi Chen 1
1Thayer School of Engineering, Dartmouth College, 2Department of Bioengineering, University of Pennsylvania

Here, we present a protocol introducing a set of new ex-ovo experiments and physical modeling approaches for studying the mechanics of morphogenesis during early chick embryonic brain torsion.

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Immunology and Infection

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice
Ming Meng *1,2, Shengde Chen *1,2, Xiang Gao *1,2, Huifang Liu *1,2, Yuanyuan Wang *1,2, Jingnan Zhang *1,2, Haiyang Dou *1,2, Wenjuan Li *1,2, Dongzhi Chen *1,2
1Medical School of Hebei University, 2Key Laboratory of Pathogenesis Mechanism and Control of Inflammatory-Autoimmune Diseases in Hebei Province

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

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JoVE Journal

Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO
Adam H. Barczewski 1, Michael J. Ragusa 1, Dale F. Mierke 1, Maria Pellegrini 1
1Department of Chemistry, Dartmouth College

We describe protocols for the structure determination of the IKK-binding domain of NEMO by X-ray crystallography. The methods include protein expression, purification and characterization as well as strategies for successful crystal optimization and structure determination of the protein in its unbound form.

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Neuroscience

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice
Francesca Gilli 1, Nora C. Welsh 1,2, Michael R. Linzey 1,2, Darlene B. Royce 1, Krista D. DiSano 1, Andrew R. Pachner 1
1Department of Neurology, Geisel School of Medicine & Dartmouth-Hitchcock Medical Center, 2Program in Experimental and Molecular Medicine, Dartmouth College

Elevated spinal fluid protein levels can either be the result of diffusion of plasma protein across an altered blood-brain barrier or intrathecal synthesis. An optimized testing protocol is presented in this article that helps to discriminate both cases and provides quantitative measurements of intrathecally synthesized proteins.

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Neuroscience

Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro
Qianru He 1, Fanhui Yu 1, Yan Li 1, Junjie Sun 1, Fei Ding 1
1Key Laboratory of Neuroregeneration of Jiangsu and Ministry of Education, Co-Innovation Center of Neuroregeneration, Jiangsu Clinical Medicine Center of Tissue Engineering and Nerve Injury Repair, Nantong University

Here, we present a method to purify fibroblasts and Schwann cells from sensory and motor nerves in vitro.

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Immunology and Infection

Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells
Krista D. DiSano 1, Michael R. Linzey 1,2, Nora C. Welsh 1,2, Joshua S. Meier 1, Andrew R. Pachner 1, Francesca Gilli 1
1Department of Neurology, Geisel School of Medicine, Dartmouth-Hitchcock Medical Center, 2Program in Experimental and Molecular Medicine, Dartmouth College

This paper presents two optimized protocols for examining resident and peripherally derived immune cells within the central nervous system, including the brain, spinal cord, and meninges. Each of these protocols helps to ascertain the function and composition of the cells occupying these compartments under steady state and inflammatory conditions.

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Medicine

In Vitro and In Vivo Delivery of Magnetic Nanoparticle Hyperthermia Using a Custom-Built Delivery System
Kayla E. A. Duval 1, James D. Petryk 2, P. Jack Hoopes 1,2
1Thayer School of Engineering, Dartmouth College, 2Geisel School of Medicine, Dartmouth College

This protocol presents techniques and methodology necessary for the accurate delivery of magnetic nanoparticle hyperthermia using a sophisticated delivery and monitoring system.

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Biochemistry

Transplantation of Neonatal Mouse Cardiac Macrophages into Adult Mice
Yandong Li 1, Jie Feng 1, Yan Li 1, Jianqiu Pei 1, Shengshou Hu 1, Yu Nie 1,2
1Fuwai Hospital & Chinese Academy of Medical Sciences, State Key Laboratory of Cardiovascular Disease, Cardiovascular Institute, National Center for Cardiovascular Diseases, Peking Union Medical College, 2National Health Commission Key Laboratory of Cardiovascular Regenerative Medicine, Fuwai Central-China Hospital, Central-China Subcenter of National Center for Cardiovascular Diseases

We provide a protocol for neonatal cardiac macrophage separation and transplantation into an adult mouse heart, which could be a promising way to promote cardiac repair.

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Developmental Biology

Optogenetic Inhibition of Rho1-Mediated Actomyosin Contractility Coupled with Measurement of Epithelial Tension in Drosophila Embryos
Hanqing Guo 1,2, Michael Swan 3, Bing He 1
1Department of Biological Sciences, Dartmouth College, 2School of Life Sciences, Westlake University, 3Department of Molecular Biology, Princeton University

Actomyosin contractility plays an important role in cell and tissue morphogenesis. However, it is challenging to manipulate actomyosin contractility in vivo acutely. This protocol describes an optogenetic system that rapidly inhibits Rho1-mediated actomyosin contractility in Drosophila embryos, revealing the immediate loss of epithelial tension after the inactivation of actomyosin in vivo.

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Biology

Methods for Tattooing Xenopus laevis with a Rotary Tattoo Machine
Joanna R. Suber 1, Jennifer Landino 1
1Department of Biochemistry and Cell Biology, Geisel School of Medicine, Dartmouth College

Here, we describe methods for tattooing adult Xenopus laevis (African clawed frog) with a rotary tattoo machine. Proper tattooing results in dark, easily legible numerals that last for several months and make animals easily distinguishable for research and recordkeeping purposes.

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