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Beth Israel Deaconess Medical Center, Harvard Medical School

11 ARTICLES PUBLISHED IN JoVE

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Biology

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker
Yue Zhang *1, Luv Kashyap *2, Annabel A. Ferguson 2, Alfred L. Fisher 2
1Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, 2Department of Medicine, Division of Geriatric Medicine and Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

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Biology

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes
Evan Lee Graham 1, Cristina Balla 1,2, Hannabeth Franchino 1, Yonathan Melman 1, Federica del Monte *1, Saumya Das *1
1Cardiovascular Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, 2Division of Cardiology, Sapienza University

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

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Environment

Production and Measurement of Organic Particulate Matter in a Flow Tube Reactor
Yue Zhang 1,2, Pengfei Liu 1, Zhaoheng Gong 1, Franz M. Geiger 3, Scot T. Martin 1,4
1School of Engineering and Applied Sciences, Harvard University, 2Department of Environmental Science and Engineering, Gillings School of Global Public Health, University of North Carolina, Chapel Hill, 3Department of Chemistry, Northwestern University, 4Department of Earth and Planetary Sciences, Harvard University

This paper describes the operation procedure for the flow tube reactor and related data collection. It shows the protocols for setting the experiments, recording data and generating the number-diameter distribution as well as the particle mass information, which gives useful information about chemical and physical properties of the organic aerosols.

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Environment

Production and Measurement of Organic Particulate Matter in the Harvard Environmental Chamber
Yue Zhang 1,2, Zhaoheng Gong 1, Suzane de Sa 1, Adam P. Bateman 1, Yingjun Liu 1, Yongjie Li 1, Franz M. Geiger 3, Scot T. Martin 1,4
1School of Engineering and Applied Sciences, Harvard University, 2Department of Environmental Science and Engineering, Gillings School of Global Public Health, University of North Carolina, 3Department of Chemistry, Northwestern University, 4Department of Earth and Planetary Sciences, Harvard University

This paper describes operation procedures for the Harvard Environmental Chamber (HEC) and related instrumentation for measuring gaseous and particle species. The environmental chamber is used to produce and study secondary organic species produced from the organic precursors, especially related to atmospheric organic particulate matter.

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Immunology and Infection

Generation of Monoclonal Antibodies Against Natural Products
Yue Zhang 1, Peng Cao 2, Fang Lu 3, Xin Yan 4, Bingqian Jiang 3, Jinjun Cheng 3, Huihua Qu 5
1School of Life Science, Beijing University of Chinese Medicine, 2Third Affiliated Hospital, Beijing University of Chinese Medicine, 3School of Basic Medical Sciences, Beijing University of Chinese Medicine, 4School of Chinese Materia Medica, Beijing University of Chinese Medicine, 5Center of Scientific Experiment, Beijing University of Chinese Medicine

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

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Cancer Research

Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer's Patches
Yavuz F. Yazicioglu 1,2, Halil I. Aksoylar 1,2, Rinku Pal 1,2, Nikolaos Patsoukis 1,2, Vassiliki A. Boussiotis 1,2
1Division of Hematology-Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, 2Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School

In this study, we present a novel and effective protocol for the isolation of lymphocytes from Peyer's Patches (PPs), which can be subsequently used for in vivo and in vitro functional assays as well as flow cytometric studies of follicular T helper and germinal center B cells.

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Behavior

A Conflict Model of Reward-seeking Behavior in Male Rats
Shaofei Jiang 1,2, Yue Zhang 1,2, Xigeng Zheng 1,2, Haoshuang Luo 1,2, Zhengkui Liu 1,2, Yunjing Bai 1,2
1CAS Key Laboratory of Mental Health, Institute of Psychology, Chinese Academy of Sciences, 2Department of Psychology, University of Chinese Academy of Sciences

This conflict model is used to measure the impairment of inhibitory control after exposure to addictive drugs, or other factors that may influence inhibitory control. A sexual stimulus and an aversive obstacle are concurrently presented, thus male rats have to conquer the obstacle to approach the sexual reward.

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Biology

Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation
Lauren Thompson 1, Brandy Pinckney 1, Shulin Lu 2, Mark Gregory 2, John Tigges 1, Ionita Ghiran 2
1Nano Flow Core Facility, Beth Israel Deaconess Medical Center, Harvard Medical School, 2Department of Medicine, Division of Allergy and Inflammation, Beth Israel Deaconess Medical Center

This paper describes a magnetic levitation-based method that can specifically detect the presence of antigens, either soluble or membrane-bound, by quantifying changes in the levitation height of capture beads with fixed densities.

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Biochemistry

Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds
Yue Zhang *1, Peng Cao *2, Fang Lu 1, Jinjun Cheng 3, Huihua Qu 4
1School of Life Science, Beijing University of Chinese Medicine, 2Third Affiliated Hospital, Beijing University of Chinese Medicine, 3National Institute of TCM Constitution and Preventive Medicine, Beijing University of Chinese Medicine, 4Center of Scientific Experiment, Beijing University of Chinese Medicine

Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds

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Immunology and Infection

Flow Cytometric Analysis for Identification of the Innate and Adaptive Immune Cells of Murine Lung
Anthos Christofides 1,2, Carol Cao 1,2,3, Rinku Pal 1,2, Halil I. Aksoylar 1,2, Vassiliki A. Boussiotis 1,2
1Division of Hematology-Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, 2Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 3Harvard College

In this study, we present an effective and reproducible protocol to isolate the immune populations of the murine respiratory system. We also provide a method for the identification of all innate and adaptive immune cells that reside in the lungs of healthy mice, using a 9-color-based flow cytometry panel.

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Bioengineering

A Passive Ankle Dorsiflexion Testing System for an In Vivo Model of Overuse-induced Tendinopathy
Pooja H. Chainani 1,2, Patrick M. Williamson 1,2, Diana Yeritsyan 1, Kaveh Momenzadeh 1, Nadim Kheir 1, Joseph P. DeAngelis 1,3, Arun J. Ramappa 1,3, Ara Nazarian 1,2,3,4
1Musculoskeletal Translational Innovation Initiative, Carl J. Shapiro Department of Orthopaedic Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, 2Mechanical Engineering Department, Boston University, 3Carl J. Shapiro Department of Orthopaedic Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, 4Department of Orthopaedic Surgery, Yerevan State Medical University

This protocol presents a testing system used to induce quantifiable and controlled fatigue injuries in a rat Achilles tendon for an in-vivo model of overuse-induced tendinopathy. The procedure consists of securing the rat's ankle to a joint actuator that performs passive ankle dorsiflexion with a custom-written MATLAB script.

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