Ohio University View Institution's Website 10 articles published in JoVE Cancer Research Fluorescence Microscopy for ATP Internalization Mediated by Macropinocytosis in Human Tumor Cells and Tumor-xenografted Mice Corinne M. Nielsen*1,2,3, Yanrong Qian*4, Subhodip Adhicary1,5, Yunsheng Li4, Pratik Shriwas1,2,4, Xuan Wang1,2,4, Lindsey Bachmann6, Xiaozhuo Chen1,2,4,7 1Department of Biological Sciences, Ohio University, 2Molecular and Cellular Biology Program, Ohio University, 3Neuroscience Program, Ohio University, 4Edison Biotechnology Institute, Ohio University, 5Translational Biomedical Sciences Program, Ohio University, 6Honors Tutorial College, Ohio University, 7Department of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University We developed a reproducible method to visualize the internalization of nonhydrolyzable fluorescent adenosine triphosphate (ATP), an ATP surrogate, with high cellular resolution. We validated our method using independent in vitro and in vivo assays-human tumor cell lines and immunodeficient mice xenografted with human tumor tissue. Neuroscience Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve Nicholas P. Boyer1, Maite Azcorra1,2, Peter Jung3,4, Anthony Brown1 1Department of Neuroscience, The Ohio State University, 2Present address: Interdepartmental Neuroscience Graduate Program and Department of Neurobiology, Northwestern University, 3Quantitative Biology Institute, Ohio University, 4Department of Physics and Astronomy, Ohio University We describe fluorescence photoactivation methods to analyze the axonal transport of neurofilaments in single myelinated axons of peripheral nerves from transgenic mice that express a photoactivatable neurofilament protein. Biochemistry Induction of Eryptosis in Red Blood Cells Using a Calcium Ionophore Parnian Bigdelou1, Amir M. Farnoud1,2 1Biomedical Engineering Program, Ohio University, 2Department of Chemical and Biomolecular Engineering, Ohio University A protocol for the induction of eryptosis, programmed cell death in erythrocytes, using the calcium ionophore, ionomycin, is provided. Successful eryptosis is evaluated by monitoring the localization phosphatidylserine in the membrane outer leaflet. Factors affecting the success of the protocol have been examined and optimal conditions provided. Chemistry Performing Spectroscopy on Plasmonic Nanoparticles with Transmission-Based Nomarski-Type Differential Interference Contrast Microscopy Anthony S. Stender1 1Department of Chemistry and Biochemistry, Ohio University The goal of this protocol is to detail a proven approach for the preparation of plasmonic nanoparticle samples and for performing single particle spectroscopy on them with differential interference contrast (DIC) microscopy. Bioengineering Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow Monica M. Burdick1,2, Nathan M. Reynolds1,2, Eric W. Martin2, Jacquelyn V. Hawes1, Grady E. Carlson1, Chaz M. Cuckler1, Michael C. Bates1, Steven R. Barthel3, Charles J. Dimitroff3 1Department of Chemical and Biomolecular Engineering, Ohio University, 2Biomedical Engineering Program, Russ College of Engineering and Technology, Ohio University, 3 Compared with traditional affinity chromatography using protein A agarose bead-packed columns, protein A membrane adsorbers can significantly speed laboratory-scale isolation of antibodies and other Fc fragment-expressing proteins. Appropriate analysis and quantification methods can further accelerate protein processing, allowing isolation/characterization to be completed in one workday, instead of 20+ work hours. Biology Antibody Staining in C. Elegans Using "Freeze-Cracking" Janet S. Duerr1 1Department of Biological Sciences, Ohio University "Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated. Bioengineering Simultaneously Capturing Real-time Images in Two Emission Channels Using a Dual Camera Emission Splitting System: Applications to Cell Adhesion Grady E. Carlson1, Eric W. Martin2, Monica M. Burdick1,2 1Department of Chemical and Biomolecular Engineering, Russ College of Engineering and Technology, Ohio University, 2Biomedical Engineering Program, Ohio University Dual camera emission splitting systems for two-color fluorescence microscopy generate real-time image sequences with exceptional optical and temporal resolution, a requirement of certain live cell assays including parallel plate flow chamber adhesion assays. When software is employed to merge images from simultaneously acquired emission channels, pseudocolored image sequences are produced. Chemistry LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations Ido Braslavsky1,2, Ran Drori1 1Institute of Biochemistry, Food Science, and Nutrition , The Robert H. Smith Faculty of Agriculture, Food, and Environment, The Hebrew University of Jerusalem, 2Department of Physics and Astronomy, Ohio University Ice binding proteins (IBPs), also known as antifreeze proteins, inhibit ice growth and are a promising additive for use in the cryopreservation of tissues. The main tool used to investigate IBPs is the nanoliter osmometer. We developed a home-designed cooling stage mounted on an optical microscope and controlled using a custom-built LabVIEW routine. The nanoliter osmometer described here manipulated the sample temperature in an ultra-sensitive manner. Medicine Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System David A. Goss1, Richard L. Hoffman1, Brian C. Clark1 1Ohio Musculoskeletal and Neurological Institute (OMNI) and the Department of Biomedical Sciences, Ohio University Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature. Immunology and Infection Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies Maria Muccioli*1, Michelle Pate*2, Omowaleola Omosebi2, Fabian Benencia1,2,3 1Molecular and Cell Biology Program, Ohio University, 2Department of Biomedical Sciences, College of Osteopathic Medicine, Ohio University, 3Department of Biomedical Engineering, Russ College of Engineering and Technology, Ohio University Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.