Dartmouth College View Institution's Website 16 articles published in JoVE Developmental Biology Optogenetic Inhibition of Rho1-Mediated Actomyosin Contractility Coupled with Measurement of Epithelial Tension in Drosophila Embryos Hanqing Guo1,2, Michael Swan3, Bing He1 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. Medicine In Vitro and In Vivo Delivery of Magnetic Nanoparticle Hyperthermia Using a Custom-Built Delivery System Kayla E. A. Duval1, James D. Petryk2, P. Jack Hoopes1,2 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. Immunology and Infection Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells Krista D. DiSano1, Michael R. Linzey1,2, Nora C. Welsh1,2, Joshua S. Meier1, Andrew R. Pachner1, Francesca Gilli1 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. Biochemistry Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO Adam H. Barczewski1, Michael J. Ragusa1, Dale F. Mierke1, Maria Pellegrini1 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. Neuroscience Quantitative Measurement of Intrathecally Synthesized Proteins in Mice Francesca Gilli1, Nora C. Welsh1,2, Michael R. Linzey1,2, Darlene B. Royce1, Krista D. DiSano1, Andrew R. Pachner1 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. Bioengineering Probing the Roles of Physical Forces in Early Chick Embryonic Morphogenesis Yan Li*1, Hannah Grover*1, Eric Dai2, Kevin Yang1, Zi Chen1 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. Developmental Biology Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes Adrienne T. Perkins1, Sharon E. Bickel1 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. Bioengineering Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo Joshua R. Biller1,2, Deborah G. Mitchell1, Mark Tseytlin3,4, Hanan Elajaili1, George A. Rinard5, Richard W. Quine6, Sandra S. Eaton1, Gareth R. Eaton1 1Department of Chemistry and Biochemistry, University of Denver, 2Magnetic Imaging Group, Applied Physics Division, Physical Measurements Laboratory, National Institute of Standards and Technology, 3Department of Radiology, Geisel School of Medicine, Dartmouth University, 4Department of Biochemistry, West Virginia University, 5Department of Electrical and Computer Engineering, University of Denver, 6Department of Engineering, University of Denver A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency. Behavior A Dual Task Procedure Combined with Rapid Serial Visual Presentation to Test Attentional Blink for Nontargets Zhengang Lu*1, Jessica Goold*1, Ming Meng1 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. Biology A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes Regina Salvat1, Leonard Moise2, Chris Bailey-Kellogg3, Karl E. Griswold1 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. Medicine Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers Kenneth M. Tichauer1, Robert W. Holt2, Kimberley S. Samkoe3, Fadi El-Ghussein1, Jason R. Gunn1, Michael Jermyn1, Hamid Dehghani4, Frederic Leblond1, Brian W. Pogue1,2 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. Immunology and Infection Determining the Phagocytic Activity of Clinical Antibody Samples Elizabeth G. McAndrew1, Anne-Sophie Dugast1, Anna F. Licht1, Justin R. Eusebio1, Galit Alter1, Margaret E. Ackerman2 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. Biology Using SCOPE to Identify Potential Regulatory Motifs in Coregulated Genes Viktor Martyanov1, Robert H. Gross1 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. Immunology and Infection Microtiter Dish Biofilm Formation Assay George A. O'Toole1 1Microbiology and Immunology, Dartmouth Medical School The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation. Immunology and Infection Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells Sophie Moreau-Marquis1, Carly V. Redelman2, Bruce A. Stanton1, Gregory G. Anderson2 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. Biology PuraMatrix Encapsulation of Cancer Cells Adnan O. Abu-Yousif1, Imran Rizvi1,2, Conor L. Evans1, Jonathan P. Celli1, Tayyaba Hasan1,3 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.