|Andrew D. Luster, MD, PhD
|Shiv Pillai, MD, PhD
|Shannon Bromley, PhD
|Thorsten Mempel, MD, PhD
Frederick Preffer, PhD
Although the CSIBD research base encompasses many investigators exploring mechanisms of immune responsiveness in their laboratories, the Immunology Core offers cost-effective service while providing access to these techniques (by service and/or training) to laboratories not focused on these approaches in their primary research orientation. The specific services offered to CSIBD investigators include:
- Flow cytometry
- DNA/Cell Cycle Analysis
- Analysis of Intracellular Cytokine Expression in Permeabilized Cells
- Leukocyte isolation and cell sorting
- Magnetic Bead Isolation
- High Speed Cell Sorting
- Multiplex cytokine assays
- Flow Cytometer Based Cytokine Bead Array
- Luminex Multiplex Cytokine Array
- T and B lymphocyte functional assays
- Intravital imaging of immune cells in vivo
- T and B lymphocyte function assays
- T and B lymphocyte function services/assays currently available in the core lab include:
- Measurement of mitogen and antigen-induced proliferation by 3H Tdr incorporation.
- ELISA and ELISPOT assays for cell associated cytokine production.
- Morphological and biochemical assays for antigen-induced cell death.
- Intravital imaging of immune cells
Specifically, as part of the Immunology core, Dr. Mempel and his laboratory and MP-IVM facility will:
- Provide instruction, equipment and assistance in the execution and analysis of intravital based cell migration studies in mouse peripheral lymph nodes (Fig. 2).
- Provide expertise in the planning, execution and analysis of cell migration protocols using novel intravital imaging models to be developed by individual CSIBD investigators.
|Figure 1: Steps involved in the analysis of in vivo cell motility. A pulsed beam of infrared (IR) laser light is raster-scanned via a high numerical objective over sequential optical planes within the specimen by rapid, synchronized movement of a pair of steering mirrors and graduated motion of the objective relative to the specimen (A). Serial stacks (15 seconds cycle time in this example) of optical sections (B) are rendered as three-dimensional volumes (C) and 2-dimensional renditions are exported as movie files for purposes of demonstration (D). Determination of cell centroids as representatives of cell position allows automated tracking of migration paths of three or more cell populations recorded in separate color channels by assigning track identities to serial images of cells (E). Tracks, consisting of serial sets of xyz coordinates of single cells (F) are used to compute various parameters of cell motility (G). Specialized software for automated cell tracking (if possible in 3 dimensions) and automated computing of the resulting tracks is essential for in-depth, large-scale analysis of migratory behavior of cell on a single cell basis.|
|Figure 2: The popliteal lymph node model for MP-IVM. A small skin incision gives access to the popliteal lymph node, which is immersed in normal saline and sealed with a cover glass. On top of the cover glass, a water-perfused circular metallic tube the objective lens immersion medium serve to regulate the LN ambient temperature through adjustment of flow of the heated perfusate. Temperature is monitored through a small thermocouple placed in close vicinity to the LN. Blood and efferent lymph flow occur at the LN hilus while several afferent lymph vessels enter the node at its distal and lateral poles.|
Immunology Core of the CSIBD runs weekly MGH Immunology Seminar Series. For more information, see our Education page.