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Medical College of Wisconsin

8 ARTICLES PUBLISHED IN JoVE

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Biology

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures
Maya Sieber-Blum 1,2, Yaofei Hu 2
1Institute of Human Genetics and Northeast England Stem Cell Institute, Newcastle University, 2Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

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Neuroscience

Vibratome Sectioning for Enhanced Preservation of the Cytoarchitecture of the Mammalian Organ of Corti
Katherine Shim 1
1Department of Pediatrics, Children’s Research Institute, Medical College of Wisconsin

A simple procedure of vibratome sectioning the organ of Corti, followed by immunohistochemistry and confocal microscopy is described. This procedure allows for improved preservation of the fine cytoarchitecture of the mammalian organ of Corti, and consequently allows for accurate quantification of cell types.

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Immunology and Infection

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance
Jill Waukau 1, Jeffrey Woodliff 2, Sanja Glisic 3
1Department of Pediatrics/Allergy, Medical College of Wisconsin , 2Flow Cytometry Core Facility, Medical College of Wisconsin , 3Max McGee National Research Center for Juvenile Diabetes and Human Molecular Genetics Center, Medical College of Wisconsin

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

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Biology

Pull-down of Calmodulin-binding Proteins
Kanwardeep S. Kaleka 1, Amber N. Petersen 1, Matthew A. Florence 1, Nashaat Z. Gerges 1
1Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin

Calmodulin (CaM) pull-down assay is an effective way to investigate the interaction of CaM with various proteins. This method uses CaM-sepharose beads for efficient and specific analysis of CaM-binding proteins. This provides an important tool to explore CaM signaling in cellular function.

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Immunology and Infection

High-throughput Detection Method for Influenza Virus
Pawan Kumar 1, Allison E. Bartoszek 1, Thomas M. Moran 2, Jack Gorski 3, Sanjib Bhattacharyya 4, Jose F. Navidad 4, Monica S. Thakar 1,5, Subramaniam Malarkannan 1,6
1Laboratory of Molecular Immunology and Immunotherapy, Blood Research Institute, 2Department of Microbiology, Mount Sinai School of Medicine , 3Laboratory of Molecular Genetics, Blood Research Institute, 4City of Milwaukee Health Department Laboratory, 5Division of Hematology-Oncology/BMT, Children's Hospital of Wisconsin, Medical College of Wisconsin , 6Division of Hematology and Oncology, Dept Medicine, Medical College of Wisconsin

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires <10 μl volume of test material and has the potential for concurrent screening of multiple pathogens.

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Behavior

How to Detect Amygdala Activity with Magnetoencephalography using Source Imaging
Nicholas L. Balderston 1, Douglas H. Schultz 1, Sylvain Baillet 2,3, Fred J. Helmstetter 1,3
1Department of Psychology, University of Wisconsin-Milwaukee, 2McConnell Brain Imaging Centre, Montreal Neurological Institute, McGill University, 3Department of Neurology, Medical College of Wisconsin

This article describes how to record amygdala activity with magnetoencephalography (MEG). In addition this article will describe how to conduct trace fear conditioning without awareness, a task that activates the amygdala. It will cover 3 topics: 1) Designing a trace conditioning paradigm using backward masking to manipulate awareness. 2) Recording brain activity during the task using magnetoencephalography. 3) Using source imaging to recover signal from subcortical structures.

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Neuroscience

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures
Ling Zhong 1, Joshua C. Brown 1, Clive Wells 2, Nashaat Z. Gerges 1
1Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin , 2Department of Microbiology and Molecular Genetics, Medical College of Wisconsin

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

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Immunology and Infection

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells
Arash Nanbakhsh 1, Brad Best 2, Matthew Riese 3, Sridhar Rao 4, Li Wang 5, Jeffrey Medin 6, Monica S. Thakar 6, Subramaniam Malarkannan 1,5,6,7
1Laboratory of Molecular Immunology and Immunotherapy, Blood Research Institute, The Blood Center of Wisconsin, 2Vector Core Lab, Blood Research Institute, The Blood Center of Wisconsin, 3Laboratory of Lymphocyte Biology, Blood Research Institute, The Blood Center of Wisconsin, 4Laboratory of Stem Cell Transcriptional Regulation, Blood Research Institute, The Blood Center of Wisconsin, 5Department of Microbiology and Immunology, The Medical College of Wisconsin, 6Department of Pediatrics, The Medical College of Wisconsin, 7Department of Medicine, The Medical College of Wisconsin

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

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