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13 ARTICLES PUBLISHED IN JoVE

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Biology

Chromatographic Purification of Highly Active Yeast Ribosomes
Arturas Meskauskas 1,2, Jonathan A. Leshin 1, Jonathan D. Dinman 1
1Department of Cell Biology and Molecular Genetics, University of Maryland , 2Department of Biotechnology and Microbiology, Vilnius University

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

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Neuroscience

Derivation of Glial Restricted Precursors from E13 mice
André W. Phillips 1,2, Sina Falahati 1,2, Roshi DeSilva 1,3, Irina Shats 2, Joel Marx 1, Edwin Arauz 1, Douglas A. Kerr 4, Jeffrey D. Rothstein 2,5, Michael V. Johnston 1,2,6, Ali Fatemi 1,2,6
1Hugo W. Moser Research Institute at Kennedy Krieger, Johns Hopkins University, 2Department of Neurology, Johns Hopkins School of Medicine, 3University of Maryland , 4Experimental Neurology, Biogen Idec, 5The Brain Science Institute, Johns Hopkins School of Medicine, 6Department of Pediatrics, Johns Hopkins School of Medicine

This protocol outlines the derivation of Glial Restricted Precursors from fetal spinal cords and maintained in vitro either for transplantation or for the study of oligodendrocytic lineage.

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Neuroscience

The Mouse Forced Swim Test
Adem Can 1, David T. Dao 2, Michal Arad 1, Chantelle E. Terrillion 3, Sean C. Piantadosi 1, Todd D. Gould 1,3,4
1Department of Psychiatry, University of Maryland School of Medicine, 2Tulane University School of Medicine, 3Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, 4The Program in Neuroscience, University of Maryland

The forced swim test is validated as an experimental approach to assess potential antidepressant efficacy in rodents. Experimental animals are placed in a tank of water and escape-related mobility behavior is quantified. The common procedures for the mouse version of this test are described.

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Neuroscience

The Tail Suspension Test
Adem Can *1, David T. Dao *1,2, Chantelle E. Terrillion 3, Sean C. Piantadosi 1, Shambhu Bhat 1, Todd D. Gould 1,3,4
1Department of Psychiatry, University of Maryland School of Medicine, 2Tulane University School of Medicine, 3The Program in Neuroscience, University of Maryland , 4Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine

The tail-suspension test is validated as an experimental procedure to assess antidepressant efficacy of drug treatments in mice. Mice are suspended by their tails for six minutes and escape-related behaviors are assessed. We describe procedures used in conducting the tail suspension test.

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JoVE Journal

A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes
Ryan D. Heselpoth 1, Daniel C. Nelson 1
1Institute for Bioscience and Biotechnology Research, University of Maryland

A novel directed evolution method specific to the field of thermostability engineering was developed and consequently validated for bacteriolytic enzymes. After only one round of random mutagenesis, an evolved bacteriolytic enzyme, PlyC 29C3, displayed greater than twice the residual activity when compared to the wild-type protein after elevated temperature incubation.

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Bioengineering

Bridging the Bio-Electronic Interface with Biofabrication
Tanya Gordonov *1, Benjamin Liba *2, Jessica L. Terrell *1, Yi Cheng 3, Xiaolong Luo 2, Gregory F. Payne 1, William E. Bentley 1
1Fischell Department of Bioengineering, University of Maryland , 2Institute for Bioscience and Biotechnology Research, University of Maryland , 3Department of Materials Science and Engineering, University of Maryland

This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

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Immunology and Infection

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells
James E. East *1, Wenji Sun *1, Tonya J. Webb 1
1Department of Microbiology and Immunology, University of Maryland

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

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Advanced Biology

ELISPOT Assay: Detection of IFN-γ Secreting Splenocytes
Tonya J. Webb 1
1Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Comprehensive Cancer Center

ELISPOT Assay: Detection of IFN-γ Secreting Splenocytes

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Advanced Biology

Immunohistochemistry and Immunocytochemistry: Tissue Imaging via Light Microscopy
Michael S. Lee 1, Tonya J. Webb 1
1Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Comprehensive Cancer Center

Immunohistochemistry and Immunocytochemistry: Tissue Imaging via Light Microscopy

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Advanced Biology

Confocal Fluorescence Microscopy: A Technique to Determine the Localization of Proteins in Mouse Fibroblasts
Dominique R. Bollino 1, Eric A. Legenzov 2, Tonya J. Webb 1
1Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Comprehensive Cancer Center, 2Center for Biomedical Engineering and Technology, University of Maryland School of Medicine

Confocal Fluorescence Microscopy: A Technique to Determine the Localization of Proteins in Mouse Fibroblasts

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Advanced Biology

Immunoprecipitation-Based Techniques: Purification of Endogenous Proteins Using Agarose Beads
Susannah C. Shissler 1, Tonya J. Webb 1
1Department of Microbiology and Immunology, University of Maryland

Immunoprecipitation-Based Techniques: Purification of Endogenous Proteins Using Agarose Beads

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Biology

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds
Christina Tam 1, Andrew R. Flannery 1, Norma Andrews 1
1Department of Cell Biology and Molecular Genetics, University of Maryland

Live imaging of cells exposed to the lipophilic dye FM1-43 allows precise determination of the kinetics by which pore-forming toxins are removed from the plasma membrane. This is a sensitive assay that can be used to assess requirements for Ca2+, sphingomyelinase and other factors on plasma membrane repair.

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Bioengineering

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis
Hadar Ben-Yoav 1, Peter H. Dykstra 1, Tanya Gordonov 2, William E. Bentley 2, Reza Ghodssi 1
1MEMS Sensors and Actuators Laboratory (MSAL), Department of Electrical and Computer Engineering, Institute for Systems Research, University of Maryland, 2Institute for Bioscience and Biotechnology Research, Fischell Department of Bioengineering, University of Maryland

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

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