The Experimental Immunology Branch (EIB) Flow Cytometry Core Facility currently supports multiple research projects for more than 50 investigators from within the EIB and elsewhere in the Center for Cancer Research (CCR). These investigations involve multiparametric quantitative single cell analysis of, and electronic cell separation based upon, parameters associated with cells freshly prepared from different species and/or tissues, as well as a spectrum of in vitro cultured cells. Basic research support is provided to members of the EIB and to other investigators within the Center for Cancer Research, NCI. Currently supported projects include, but are not limited to, the following areas of study: a) in vivo and in vitro analyses of intra-cellular signaling via cell surface molecules;b) analyses of cellular processes and/or defects in animals and/or cells with genetic modifications;c) studies of the mechanisms and consequences of immune pathogenesis;d) analyses of the coordinate cell surface expression of a variety of molecules;e) investigations of T cell repertoire generation;g) analyses of expression of transplantation antigens;h) investigations of mechanisms involved in T cell lineage development;i) mechanisms of cell death;and j) stem cell analyses. The following EIB/NCI/CCR Projects are supported by the core: PI: Alfred Singer BC 011106;Specification of T cell function during development BC 011111;Cytokine signaling in developing thymocytes and T cells BC 011113;T cell Survival BC 011114;Role of microRNAs in T cell development BC 009273;T Cell Differentiation and Repertoire Selection BC 011116;MHC-independent T cells BC 011117;T cell receptor regulation of cytokine signaling BC 011112;Development and function of regulatory T cells PI: Richard Hodes BC 009265;Analysis of the T Cell repertoire BC 009281;Receptor Mediated T and B Cell Activation BC 009405;Regulation of Lymphocyte Proliferation and Replicative Capacity PI: Andre Nussenzweig BC 010283;Dna repair BC 010959;Relationship between dna damage detection and signaling BC 010961;High-throughput screen for activators of DNA strand break repair PI: Stephen Shaw 009257 Mechanisms of Cellular Immune Responses 010272:Facilitating Access to Information on Human Proteins and Their Phosphorylation 010993: Molecular architecture of lymphocyte cortex 010994: Lymphocyte membrane-proximal basophilic kinases 010995: ERM phosphorylation in lymphocytes 010996: Molecular mechanisms of ERM regulation PI;Dinah Singer BC 009285;Responses of MHC Class I Genes to Exogeneous Stimuli SC 010375;TAF7: A Check-point Regulator in Transcription Initiation BC 010927;Role of Chromatin Structure in Regulating MHC Class I Expression BC 10928;Boundary Elements Regulate MHC Class I Expression in Vivo BC 009279;Regulation of Expression of MHC Class I Genes PI: David Segal BC 010879;Role of Toll-like receptors in the generation of acquired immunity BC 009254; Structure and Function of Toll-like Receptors PI: Paul Roche BC 009404;Regulation of MHC Class II Trafficking in Antigen Presenting Cells BC 011033;Mechanisms of MHC Class II Association with Plasma Membrane Microdomains BC 011035;Regulation of Exocytosis from Immune Cells PI: Triantafyllos Chavakis BC 010663;The Role of JAM-C in Adhesive Interactions of Immune Cells BC 010664;Crosstalk between inflammation and Angiogenesis BC 010790;Regulation of Leukocyte Integrins in Inflammatory Cell Recruitment PI: Hyun Park Z1A BC 011214; Post-Transcriptional Regulation of Interleukin-7 Receptor Expression Z1A BC 011215;Immune Regulatory Roles of Suppressor of Cytokine Signaling (SOCS) Molecules During this reporting period, the core also provided limited research support to the following non-EIB PI: Thomas Waldmann (Metabolism Branch, CCR), Jay Berzofsky (Vaccine Branch, CCR), Genoveffa Franchini (Vaccine Branch, CCR) and Paul Love (National Institute of Child Health and Development). The facility operates and maintains 2 operator run 3-laser flow cytometers with cell sorting capabilities and 4 user-operator flow cytometers with analysis only capabilities. The facility has recently acquired a state-of-the-art 5-laser, analysis only, flow cytometer that will be used to extend and expand available applications. Facility staff provide consultation to investigators in the areas of: experimental design, problem-solving, reagent selection and data analysis and interpretation. The facility supports a wide variety of flow cytometric applications including: rare event analysis (including stem cell analysis) and cell sorting; multi-color phenotypic analyses, cell cycle analysis, proliferation analysis, metabolic analyses including calcium flux analysis, sterile cell sorting, and intra-cellular cytokine analyses. The facility also, as a cost-savings measure, maintains a reagent bank of over 150 commonly used flow cytometry reagents that are pre-titred and aliquoted by facility personnel for use by multiple EIB investigators. The reagent bank minimizes costs by buying in bulk and minimizing labor and effort involved in characterizing individual batches of reagents. The facility is developing WINDOWS-based pc software for flow cytometry analysis that will provide capabilities not currently available in software available from instrument manufacturers or 3rd party software sources.