Application of Flow Cytometry to Cell Biology

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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 Singer; ZIA BC 009273 T Cell Differentiation and Repertoire Selection, ZIA BC 011106 Specification of T cell function during development, Z1A BC 011111 Cytokine signaling in developing thymocytes and T cells, ZIA BC 011112 Development and function of regulatory T cells, Z1A BC 011113 T cell Survival, Z1A BC 011114 Role of microRNAs in T cell development, Z1A BC 011116 MHC-independent T cells, Z1A BC 011117 T cell receptor regulation of cytokine signaling. PI: Richard Hodes: Z1A BC 009265; Analysis of the T Cell repertoire, Z1A BC 009281 Receptor Mediated T and B Cell Activation, Z1A BC 009405; Regulation of Lymphocyte Proliferation and Replicative Capacity. PI: Dinah Singer Z1A BC 009285 Responses of MHC Class I Genes to Exogeneous Stimuli, Z1A SC 010375 TAF7: A Check-point Regulator in Transcription Initiation, Z1A BC 009279 Regulation of Expression of MHC Class I Genes, ZIA BC 011381 Brd4 is an atypical kinase that regulates transcription, ZIA BC 011425 Expression of class I in Treg cells. PI: Paul Roche Z1A BC 009404 Regulation of MHC Class II Trafficking in Antigen Presenting Cells, Z1A BC 011033 Mechanisms of MHC Class II Association with Plasma Membrane Microdomains, Z1A BC 011035 Regulation of Exocytosis from Immune Cells. 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. PI: Vanja Lazarevic ZIA BC 011431 Role of T-bet in the pathogenesis of experimental autoimmune encephalomyelitis, ZIA BC 011432 Regulation of T-bet expression in TH17 cells by microRNAs. PI: Damian Kovalovsky ZIA BC 011429 Zbtb family members in lymphocyte differentiation and function. During this reporting period, the facility provided limited research support to the following non-EIB CCR PI: Thomas Waldmann (Metabolism Branch, CCR), Jay Berzofsky (Vaccine Branch, CCR), and David Levens (Laboratory of Pathology, CCR). In addition the facility has provided limited support to non-NCI PI Jeffrey Kopp, Kidney Diseases Branch, NIDDK and Paul Love, Section on Cellular and Developmental Biology, NICHD. 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 6 user/operator flow cytometers with analysis only capabilities including two (2) state-of-the-art 5-laser flow cytometer analyzers. Facility staff members 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.