The objective of the Cell Analysis Core, is to provide the provide the program with an integrated system of technical approaches to perform structural analyses of blood cells, particularly lymphocytes and platelets, of mutant mice, WAS patients, and experimental cell models. Specifically, Core C will provide confocal microscopy, electron microscopy and flow cytometry services to all the individual projects and to the Patient Mutation Analysis Core (Core B). The scanning electron microscopy services will be used to characterize the surface topography of lymphocytes of WASp, N-WASP, WIP and Vav deletion mutant mice and cells transfected with cDNA expression constructs. The high-resolution rapid freeze/freeze-drg and immunoelectron microscopy will be used to examine cytoskeletal organization in lymphocytes and platelets of mutant mice, before and after activation. Projects 1 and 2 will use confocal microscopy to determine the subcellular localization of WASp and WIP, respectively, b immunocytochemical approaches. Analytical flow cytometry, preparative cell sorting and kinetic, fluorocytometric measurements on viable blood cells and cultured cell models will be used by all the projects to: 1) characterize mutant mice, 2) characterize the development and function of lymphoid cells (peripheral blood lymphocytes in spleen, bone marrow, lymph nodes, thymus, and peritoneum) in WASP, N-WASP, WIP and Vav deletion mutants and in the respective chimeras generated in RAG-2 knockout mice, 3) screen knockout, transgenic and somatic chimeric mice and cells transfected with cDNA expression constructs by analysis of surface makers, 4) measure specific cell activation marker expression to follow intracellular signal transduction events in platelets and lymphocytes of mutant mice and transfected cell lines. Core B, the Patient Mutation Analysis Core, requires standard immunophenotypic characterization of patient blood cells, subpopulations and transformed cell lines. Core C contains, or has access to, all major equipment necessary to carry out these responsibilities.
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