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Geisel School of Medicine at Dartmouth

10 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

Microtiter Dish Biofilm Formation Assay
George A. O'Toole 1
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.

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Medicine

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer
Melanie R. Rutkowski *1, Michael J. Allegrezza *1, Nikolaos Svoronos 1, Amelia J. Tesone 1, Tom L. Stephen 1, Alfredo Perales-Puchalt 1, Jenny Nguyen 1, Paul J. Zhang 2, Steven N. Fiering 3, Julia Tchou 4,5,6, Jose R. Conejo-Garcia 1
1Tumor Microenvironment and Metastasis Program, Wistar Institute, 2Department of Pathology and Lab Medicine, Perelman School of Medicine, University of Pennsylvania, 3Department of Microbiology and Immunology and Department of Genetics, Geisel School of Medicine at Dartmouth, 4Division of Endocrine and Oncologic Surgery, Department of Surgery, Perelman School of Medicine, University of Pennsylvania, 5Rena Rowan Breast Center, Abramson Cancer Center, University of Pennsylvania, 6Center for Advanced Medicine, University of Pennsylvania

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

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Neuroscience

Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis
Francesca Gilli 1, Darlene B. Royce 1, Andrew R. Pachner 1
1Department of Neurology, Geisel School of Medicine at Dartmouth

An optimized testing protocol is presented in this paper for the Rotarod performance test, used for measuring progressive neurological disability in TMEV-infected mice.

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Neuroscience

Large-scale Reconstructions and Independent, Unbiased Clustering Based on Morphological Metrics to Classify Neurons in Selective Populations
Elise M. Bragg 1, Farran Briggs 1
1Physiology & Neurobiology, Geisel School of Medicine at Dartmouth

This protocol describes large-scale reconstructions of selective neuronal populations, labeled following retrograde infection with a modified rabies virus expressing fluorescent markers, and independent, unbiased cluster analyses that enable comprehensive characterization of morphological metrics among distinct neuronal subclasses.

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Immunology and Infection

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies
Marta Rodriguez-Garcia 1, Jared M. Fortier 1, Fiona D. Barr 1, Charles R. Wira 1
1Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

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Neuroscience

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice
Francesca Gilli 1, Nora C. Welsh 1,2, Michael R. Linzey 1,2, Darlene B. Royce 1, Krista D. DiSano 1, Andrew R. Pachner 1
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.

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Immunology and Infection

Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells
Krista D. DiSano 1, Michael R. Linzey 1,2, Nora C. Welsh 1,2, Joshua S. Meier 1, Andrew R. Pachner 1, Francesca Gilli 1
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.

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Neuroscience

Acquisition of Resting-State Functional Magnetic Resonance Imaging Data in the Rat
Diana J. Wallin *1,2, Emily D. K. Sullivan *1,2, Elise M. Bragg 1, Jibran Y. Khokhar 2,4, Hanbing Lu 2,3, Wilder T. Doucette 1,2
1Dartmouth-Hitchcock Medical Center, 2Geisel School of Medicine at Dartmouth, 3National Institute on Drug Abuse, National Institutes of Health, 4University of Guelph

This protocol describes a method for obtaining stable resting-state functional magnetic resonance imaging (rs-fMRI) data from a rat using low dose isoflurane in combination with low dose dexmedetomidine.

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Medicine

Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome
Jacob Vieira 1, Tara Gallagher 2, Hui-Yu Sui 1, Sirus Jesudasen 3, Katrine Whiteson 2, George A. O'Toole 4, Kurt Hanselmann 5, Peggy S. Lai 1
1Division of Pulmonary and Critical Care Medicine, Massachusetts General Hospital, 2Department of Molecular Biology & Biochemistry, University of California, Irvine, 3Department of Medicine, Massachusetts General Hospital, 4Department of Microbiology & Immunology, Geisel School of Medicine at Dartmouth, 5Swiss i-research and teaching institute

The goal of this protocol is to develop a model system for the effect of hyperoxia on cystic fibrosis airway microbial communities. Artificial sputum medium emulates the composition of sputum, and hyperoxic culture conditions model the effects of supplemental oxygen on lung microbial communities.

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JoVE Core

A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors
Kali A. Smolen 1, Arminja N. Kettenbach 1,2
1Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, 2Norris Cotton Cancer Center

Here, we present a protocol for the enrichment of endogenous phosphoprotein phosphatases and their interacting proteins from cells and tissues and their identification and quantification by mass spectrometry-based proteomics.

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