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;j) stem cell analyses and k) mechanisms of immune gene regulation.The following EIB/NCI/CCR Projects are supported by the core:PI: Alfred SingerBC 011106;Specification of T cell function during developmentBC 011111;Cytokine signaling in developing thymocytes and T cellsBC 011113;T cell SurvivalBC 011114;Role of microRNAs in T cell developmentBC 009273;T Cell Differentiation and Repertoire SelectionBC 011116;MHC-independent T cellsBC 011117;T cell receptor regulation of cytokine signalingBC 011112;Development and function of regulatory T cellsPI: Richard HodesBC 009265;Analysis of the T Cell repertoireBC 009281;Receptor Mediated T and B Cell ActivationBC 009405;Regulation of Lymphocyte Proliferation and Replicative CapacityPI: Stephen Shaw009257 Mechanisms of Cellular Immune Responses010272:Facilitating Access to Information on Human Proteins and Their Phosphorylation010993: Molecular architecture of lymphocyte cortex010994: Lymphocyte membrane-proximal basophilic kinases010995: ERM phosphorylation in lymphocytes010996: Molecular mechanisms of ERM regulationPI; Dinah SingerBC 009285;Responses of MHC Class I Genes to Exogeneous StimuliSC 010375;TAF7: A Check-point Regulator in Transcription InitiationBC 010927;Role of Chromatin Structure in Regulating MHC Class I ExpressionBC 10928;Boundary Elements Regulate MHC Class I Expression in VivoBC 009279;Regulation of Expression of MHC Class I GenesPI: David SegalBC 010879;Role of Toll-like receptors in the generation of acquired immunityBC 009254;Structure and Function of Toll-like ReceptorsPI: Paul RocheBC 009404;Regulation of MHC Class II Trafficking in Antigen Presenting CellsBC 011033;Mechanisms of MHC Class II Association with Plasma Membrane MicrodomainsBC 011035;Regulation of Exocytosis from Immune CellsPI: Hyun ParkZ1A BC 011214;Post-Transcriptional Regulation of Interleukin-7 Receptor ExpressionZ1A BC 011215;Immune Regulatory Roles of Suppressor of Cytokine Signaling (SOCS) MoleculesPI: Vanja LazarevicZIA BC 011431 Role of T-bet in the pathogenesis of experimental autoimmune encephalomyelitisZIA BC 011432 Regulation of T-bet expression in TH17 cells by microRNAs PI: Damian KovalovskyZIA BC 011429; Zbtb family members in lymphocyte differentiation and functionPI: 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 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), Kathleen Kelly (Cell and Cancer Biology Branch, CCR) and Paul Love (National Institute of Child Health and Development). The core has also continued to provide limited support to Andre Nussenzweig (Laboratory of Genome Integrity, CCR) subsequent to establishment of that laboratory.The facility operates and maintains two operator run multi-laser flow cytometers with cell sorting capabilities including a state-of-the-art 6-laser cell sorter and 5 user/operator flow cytometers with analysis only capabilities including two(2) state-of-the-art 5-laser flow cytometer analyzers. 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.
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