Ohio University

13 ARTICLES PUBLISHED IN JoVE

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 Benencia^1,2,3

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.

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.

Chemistry

LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

Ido Braslavsky^1,2, 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.

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. Burdick^1,2

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.

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

Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

Monica M. Burdick^1,2, Nathan M. Reynolds^1,2, 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, ³Department of Dermatology, Brigham and Women's Hospital, Harvard Medical School

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.

Chemistry

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Koli Basu¹, Christopher P. Garnham², Yoshiyuki Nishimiya³, Sakae Tsuda³, Ido Braslavsky⁴, Peter Davies¹

¹Department of Biomedical and Molecular Sciences, Queen's University, ²National Institute of Neurological Disorders and Stroke, Porter Neuroscience Research Center, ³Research Institute of Genome-Based Biofactory, National Institute of Advanced Industrial Science and Technology, ⁴The Robert H. Smith Faculty of Agriculture, Food and Environment, Institute of Biochemistry, Food Science, and Nutrition, The Hebrew University of Jerusalem

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

Immunology and Infection

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Arielle M. Bryan^*1, Amir M. Farnoud^*1, Visesato Mor¹, Maurizio Del Poeta¹

¹Department of Molecular Genetics and Microbiology, Stony Brook University

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

Medicine

Cloud-Based Phrase Mining and Analysis of User-Defined Phrase-Category Association in Biomedical Publications

Dibakar Sigdel^*1,2, Vincent Kyi^*1,2, Aiden Zhang^*1, Shaun P. Setty³, David A. Liem^1,2,4, Yu Shi⁵, Xuan Wang⁵, Jiaming Shen⁵, Wei Wang^1,6,7, JiaWei Han⁵, Peipei Ping^1,2,4,6

¹The NIH BD2K Center of Excellence in Biomedical Computing, University of California, Los Angeles, ²Department of Physiology, University of California, Los Angeles, ³Department of Pediatric and Adult Congenital Heart Surgery, Miller Children's and Women's Hospital and Long Beach Memorial Hospital, ⁴Department of Medicine/Cardiology, University of California, Los Angeles, ⁵NIH BD2K Program Centers of Excellence for Big Data Computing -- KnowEng Center, Department of Computer Science, University of Illinois at Urbana-Champaign (UIUC), ⁶Scalable Analytics Institute (ScAi), University of California, Los Angeles, ⁷Department of Computer Science, University of California, Los Angeles

We present a protocol and associated programming code as well as metadata samples to support a cloud-based automated identification of phrases-category association representing unique concepts in user selected knowledge domain in biomedical literature. The phrase-category association quantified by this protocol can facilitate in depth analysis in the selected knowledge domain.

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.

Biochemistry

Induction of Eryptosis in Red Blood Cells Using a Calcium Ionophore

Parnian Bigdelou¹, Amir M. Farnoud^1,2

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.

Neuroscience

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

Nicholas P. Boyer¹, Maite Azcorra^1,2, Peter Jung^3,4, 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.

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 Adhicary^1,5, Yunsheng Li⁴, Pratik Shriwas^1,2,4, Xuan Wang^1,2,4, Lindsey Bachmann⁶, Xiaozhuo Chen^1,2,4,7

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.