ORGANIZATION AND BUDGET OF THE CORE:? ? The Core became operational in June 2007 and is equipped with two FACSCaliburs (Ages: 2 years and 11 years old) and one FACSAria (Age: 4 years old). The Core operates from 2 small, noncontiguous, storage rooms totaling 137 square feet. The FACSCalibur workspace totals 77 square feet of which the machines' footprint accounts for 60% of the space. The FACSAria workspace totals 60 square feet of which the machine itself and its accessories use 80% of the space. The Core is operated by a Core Manager devoting 100% effort to the Core and is supervised by a PI devoting 7% effort to the Core.? ? 100% of the budget was used to support the goals and objectives of the core, including reagents, fluid and repairs, as well as to support the last lease payment on the FACS Aria. ? ? OPERATION OF THE CORE:? ? This year, thirty-four individuals received services from the facility. These services and collaborative services were performed for 8 Principal Investigators (PIs) from 5 NEI labs (LI, LRCMB, N-NRL, OSD and URN), plus 5 PIs from 4 other institutes at NIH (NCI, NIAMS, NICHD and NIDDK). Over 15,645 samples in total were analyzed by the instruments comprising the Core. Thirteen users required cell sorting for retrieval of highly purified cell populations. Over 145 sorts were carried out by the Core this year. Users who have the appropriate training (provided by the Core) are allowed to run the FACSCalibur machines under the general supervision of the Core Manager. The FACS Aria, which requires a higher level of training, is operated by the Core Manager only. ? ? In 2008, a state of the art web-based scheduling system was enabled to schedule and track instrumentation usage.? ? The scientific techniques presently in use by the Core include: methods for phenotyping live cells, measuring levels of gene expression, monitoring viability and apoptosis by changes in cell membrane and DNA content changes, measurement of intracellular proteins, and quantification of soluble proteins. An example of the research carried out by the core is the study of HtrA2/Omi in RPE cells under oxidative stress with respect to apoptosis. Another exciting technique is the identification of live cells expressing Interleukin 17.? ? The work involving human tissues is limited to the sorting of peripheral blood mononuclear cells to study their cytokine production, genotype, and RNA expression.? ? TRAINING:? ? The Core provides not only service, but also training to users in the theory and practice of flow cytometry. Ten frontal flow cytometry courses were taught by the Core manager to the NEI investigators. These courses include: Introduction to Flow Cytometry, Basics of Flow Cytometry, Analysis of Flow Cytometry Data using the FlowJo software, FACSCalibur Operation. In addition, the Core manager provides one-on-one training to FACSCalibur users who need training. Over the course of the year, seven users required extensive training for data acquisition and analysis. The Core also scheduled a lecture in intracellular staining by a guest speaker. ? ? The Core Manager completed 80 hours of continuing education in flow cytometry and eye research.? ? ? ? PUBLICATIONS GENERATED USING CORES SERVICES:? ? 1. Cox CA, Shi G, Yin H, Vistica BP, Wawrousek EF, Chan CC, Gery I. Both Th1 and Th17 are immunopathogenic but differ in other key biological activities. J Immunol. 2008 Jun 1;180(11):7414-22.? ? 2. Yu CR, Mahdi RM, Liu X, Zhang A, Naka T, Kishimoto T, Egwuagu CE. SOCS1 regulates CCR7 expression and migration of CD4+ T cells into peripheral tissues. J Immunol. 2008 Jul 15;181(2):1190-8.? ? 3. Liu X, Lee YS, Yu CR, Egwuagu CE. Loss of STAT3 in CD4+ T cells prevents development of experimental autoimmune diseases. J Immunol. 2008 May 1;180(9):6070-6.? ? 4. Liu X, Mameza MG, Lee YS, Eseonu CI, Yu CR, Kang Derwent JJ, Egwuagu CE. Suppressors of cytokine-signaling proteins induce insulin resistance in the retina and promote survival of retinal cells. Diabetes. 2008 Jun;57(6):1651-8. Epub 2008 Mar 20.? ? 5. Liu B, Li Z, Mahesh SP, Pantanelli S, Hwang FS, Siu WO, Nussenblatt RB. Glucocorticoid-induced tumor necrosis factor receptor negatively regulates activation of human primary natural killer (NK) cells by blocking proliferative signals and increasing NK cell apoptosis. J Biol Chem. 2008 Mar 28;283(13):8202-10. Epub 2008 Jan 29.? ? 6. Cortes LM, Mattapallil MJ, Silver PB, Donoso LA, Liou GI, Zhu W, Chan CC, Caspi RR (2008) Repertoire analysis and new pathogenic epitopes of IRBP in C57BL/6 (H-2b) and B10.RIII (H-2r) mice. Invest Ophthalmol Vis Sci 49:1946-56.? ? 7. Luger D, Silver PB, Tang J, Cua D, Chen Z, Iwakura Y, Bowman EP, Sgambellone NM, Chan CC, Caspi RR (2008) Either a Th17 or a Th1 effector response can drive autoimmunity: conditions of disease induction affect dominant effector category. J Exp Med 205:799-810.? ? 8. Rachitskaya AV, Hansen AM, Horai R, Li Z, Villasmil R, Luger D, Nussenblatt RB, Caspi RR (2008) Cutting edge: NKT cells constitutively express IL-23 receptor and RORgammat and rapidly produce IL-17 upon receptor ligation in an IL-6-independent fashion. J Immunol 180:5167-71.? ? 9. Silver PB, Agarwal RK, Su S, Suffia I, Grajewski RS, Luger D, Chan C, Mahdi RM, Nickerson JM, Caspi RR (2007) Hydrodynamic Vaccination with DNA Encoding an Immunologically Privileged Retinal Antigen Protects from Autoimmunity Through Induction of Regulatory T cells. J Immunol 179:5146-5158.? ? 10. Li R, Maminishkis A, Wang FE, Miller SS. PDGF-C and -D induced proliferation/migration of human RPE is abolished by inflammatory cytokines. Invest Ophthalmol Vis Sci. 2007 Dec;48(12):5722-32.Click here to read
