Immunology Core

Personnel

Location

Massachusetts General Hospital
Charlestown Navy Yard, Building 149, 13th Street
Center for Immunology and Inflammatory Diseases, 6th Floor
Division of Rheumatology, Allergy and Immunology

Overview

The Immunology Core offers CSIBD investigators service, consultative advice and in-depth instruction in a variety of immunologic methods to facilitate their ability to perform IBD-related research. The primary activities of this core are (1) multicolor flow cytometric analysis (2) immunological assays and (3) preparation of novel monoclonal antibodies to proteins of interest to IBD research.

The Immunology Core will move to an extension of the Center for Immunology and Inflammatory Diseases (CIID) adjacent to the new MGH Translational Immunology (TI) Center in December 2006. When fully operational the TI Center will allow the Immunology core to provide priority access to high-speed flow cytometric sorting and multi (8-12) color flow cytometric analysis to Center members at reduced rates. During the upcoming year the core plans to greatly expand its immunological assay services through the joint purchase (with MGH Translational Immunology) of a Luminex multiplex array bioassay system. This new service will allow us to offer CSIBD investigators arrays that allow for the analysis of as many as 30 cytokines in one 50 ml sample. Center member interest in other types of arrays (e.g. cell signaling/phosphoprotein assays) will be surveyed.

Services

Flow Cytometry Core Facilities (Jackson 7)

The Flow Cytometry Facility currently provides three color (5-parameter) flow cytometric analysis as well DNA/cell cycle analysis. We also have expertise in labeling cells with the vital dye 5, 6 carboxyfluorescein diacetate succinimidyl ester (CFSE) prior to their adoptive transfer into naove recipients. Cellular proliferation can then be measured directly ex vivo in response to antigen challenge as a reduction in CFSE fluorescence intensity. In addition, the Becton Dickinson trained research assistant is available to trouble-shoot staining techniques, background staining problems etc. If they prefer to do so, center investigators can also use the machine to run and analyze their own samples. Cell Quest analysis workstations are also available on CNY6.

Immunological Assays (CNY 149-6)

The core performs a large variety of ELISA assays for Center investigators at no cost. As mentioned above, the core's immunoassay capabilities will be greatly enhanced by the purchase of a multiplex array bioassay system this year. The Core also provides technical assistance for CSIBD investigators in the use of other immunological assays including [3H]-thymidine incorporation, TUNEL method, immunofluorescent staining and cytokine real time PCR for analysis of cytokine expression

Monoclonal Antibody (Mab) Core Facility (CNY 149-6)

The MAb facility provides Center Investigators with custom-made antibodies that are generally not available from other researchers or at a commercial base, and provides training in a wide variety of immunological techniques. The Mabs that are produced in this Core are used by Center Investigators for various techniques and purposes including Western blot analysis, immunoprecipitation, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, flow cytometric analysis and cell sorting, cDNA cloning, neutralization experiments, and signal transduction studies.

The Monoclonal Antibody Core performs the following procedures for the Investigators:

1. Immunization: This involves the administration of an antigen/adjuvant-mixture to an appropriate recipient animal (mice or hamsters). This Core collects the sera of immunized mice 5-6 weeks after three immunizations to set up and trouble-shoot a screening assay for hybridoma production.
2. Cell Fusion: Cell fusion is performed for the immortalization of lymphoid cells from immunized mice. A myeloma partner cell line is fused to immune splenocytes with PEG and standard HAT selection techniques are used to identify immortalized hybridoma cells. After screening for the antibody of interest, positive wells are cloned twice by limiting dilution to insure clonality.
3. Screening: In conjuction with the CSIBD investigator the Core assists in the development a rapid, reliable screening assay to select clones secreting the antibody of interest. This is the key step for successful monoclonal antibody production and is usually performed with an ELISA based assay. The Core typically uses a Molecular Devices Emax precision microplate reader system that has been optimized for this purpose. The Emax provides both single and dual wavelength endpoint reading over the 405-750nm range. With a measurement range to 4.0 O.D., resolution to 0.001 OD and linearity to 3.0 O.D. Emax provides accuracy for even the most demanding ELISA endpoint assays. It is also useful for protein assays and quantitation of cytoproliferation by MTT reduction. Our system uses Mac-based Soft Max Pro software. Other sorts of screening assays are also possible depending on the nature of the antigen being studied.

These services are being provided to Center investigators without any cost.

Moreover reagents of general interest are made available to all Center investigators as a service of the Center.

Training and Enrichment Activities

Dr. Nagler has taken over supervision of the Mass. General Hospital Immunology Seminar Series for the 2006-2007 academic year (see schedule below). This well attended seminar series features both national and local speakers on a large variety of topics in basic and clinical immunology. Dr. Nagler has initiated a monthly luncheon for graduate students, post-doctoral or clinical fellows and visiting faculty of interest. These luncheons are expected to provide opportunities for all of these trainees to interact with the speakers in a relaxed, casual setting.

Seminar series secretary: Michelle Angelo, mangelo@partners.org
Student/faculty luncheon coordinator: Bethany Mingle, Bethany_mingle@hms.harvard.edu

Drs. Nagler and Snapper (Co-Director, Molecular Biology Core) have also also designed a new quarter course for the Immunology and Biological and Biomedical Sciences graduate programs with a particular focus on mucosal recognition of commensal and pathogenic microbes (see course description below).

There are more bacteria resident in our gastrointestinal tracts than there are cells in our bodies! It is becoming clear that we exist in a symbiotic relationship with the billions of bacteria that comprise our commensal flora. In addition to performing vital physiological functions, recent evidence suggests that the flora plays a central role in directing the maturation of the immune system and the generation of immunoregulatory cells and mediators. Potential pathogens at mucosal surfaces must also be identified and effectively eliminated.

This course will examine how the mucosa associated lymphoid tissues (with emphasis on the gut) distinguish commensal from pathogenic microbes and mount an appropriate response to each. What are some of the mechanisms that maintain mucosal homeostasis? In some individuals these immunoregulatory controls fail. How does uncontrolled responsiveness to the contents of the intestinal lumen lead to inflammatory bowel disease or food allergy?

Each session will consist of a 45-60 minute overview of that week's topic presented by the faculty speaker. Prior to each session, students will be assigned a review article and 1-2 primary articles. The faculty speakers will facilitate an interactive, in depth discussion of the assigned articles, which will be led by selected students. Student evaluations will be based on participation in these discussions.

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