GENERAL OVERVIEW: The Flow Cytometry Core at NEI provides flow cytometry analytical and sorting equipment and services to the NEI Intramural community. It utilizes and develops state-of-the-art sample preparation, data acquisition and analysis, and sorting procedures in collaborative research projects. Provides training to students, fellows, and principal investigators on sample preparation, staining, and post-sort handling. Assesses technical research needs and recommends recruitment of the appropriate staff and acquisition of the equipment needed to meet those needs. The core also supports NIH intramural research outside NEI by processing samples for laboratories without access to flow cytometry instrumentation and collaborations with the Foundation for Advanced Education in the Sciences SERVICES PROVIDED THE CORE: This year, fifty-one individuals from thirteen different laboratories used the facility. These services and collaborative services were performed for 12 Principal Investigators (PIs) from 8 NEI labs (ERPD, LI, LRCMB, MSF, N-NRL, RN, OGVFB and UNGIRD), plus 3 PIs from 2 other institutes at NIH (NICHD, and). Over 20,000 samples were analyzed. Seventeen users received training how to operate the analytical instrumentation. This year the core performed about 1,300 hours of sorting. Among the techniques now in use within the core are methods for phenotyping live cells, detecting gene expression, monitoring membrane and DNA content changes due to apoptosis or proliferation, measurement of intracellular proteins and quantification of soluble proteins. The work involving human tissues includes the sorting of peripheral blood mononuclear cells to study their cytokine production, genotype and DNA or RNA expression. The sources are blood, buffy coat, and white cells. Some analytical work had been done with eye fluids, eye tissue specimen, protein, and tears. No tissues were stored by the core. Among the techniques now in use within the core are methods for phenotyping live cells, detecting gene expression, monitoring membrane and DNA content changes due to apoptosis or proliferation, measurement of intracellular proteins and quantification of soluble proteins. The work involving human tissues includes the sorting of peripheral blood mononuclear cells to study their cytokine production, genotype and DNA or RNA expression. The sources are blood, buffy coat, and white cells. Some analytical work had been done with eye fluids, eye tissue specimen, protein, and tears. The National Eye Institute made a great investment in biosafety with the addition BD FACSAria Fusion flow cytometer equipped with a fully integrated biosafety cabinet. This sorter meets the recent NIH's operator and sample protection requirements as well as global standards for bioprotection for processing human samples. No human tissues were stored by the core. The Core encourages, but does not require, users to acknowledge the Core contribution in their publications. The following are examples of the publications that used NEI Flow Cytometry Core resources: St Leger AJ, Hansen AM, Karauzum H, Horai R, Yu CR, Laurence A, Mayer-Barber KD, Silver P, Villasmil R, Egwuagu C, Datta SK, Caspi RR. STAT-3-independent production of IL-17 by mouse innate-like T cells controls ocular infection. J Exp Med. 2018 Apr 2;215(4):1079-1090. doi: 10.1084/jem.20170369. Epub 2018 Feb 28. Choi JK, Dambuza IM, He C, Yu CR, Uche AN, Mattapallil MJ, Caspi RR, Egwuagu CE. IL-12p35 Inhibits Neuroinflammation and Ameliorates Autoimmune Encephalomyelitis. Front Immunol. 2017 Oct 5;8:1258. doi: 10.3389/fimmu.2017.01258. eCollection 2017. Yu CR, Choi JK, Uche AN, Egwuagu CE. Production of IL-35 by Bregs is mediated through binding of BATF-IRF-4-IRF-8 complex to il12a and ebi3 promoter elements. J Leukoc Biol. 2018 Aug 17. doi: 10.1002/JLB.3A0218-071RRR. Assawachananont J, Kim SY, Kaya KD, Fariss R, Roger JE, Swaroop A. Cone-rod homeobox CRX controls presynaptic active zone formation in photoreceptors of mammalian retina. Hum Mol Genet. 2018 Jul 31. doi: 10.1093/hmg/ddy272. May-Simera HL, Wan Q, Jha BS, Hartford J, Khristov V, Dejene R, Chang J, Patnaik S, Lu Q, Banerjee P, Silver J, Insinna-Kettenhofen C, Patel D, Lotfi M, Malicdan M, Hotaling N, Maminishkis A, Sridharan R, Brooks B, Miyagishima K, Gunay-Aygun M, Pal R, Westlake C, Miller S, Sharma R, Bharti K. Primary Cilium-Mediated Retinal Pigment Epithelium Maturation Is Disrupted in Ciliopathy Patient Cells. Cell Rep. 2018 Jan 2;22(1):189-205. doi: 10.1016/j.celrep.2017.12.038. TRAINING: Several formal training sessions were offered by the core to the NEI community. These courses included: Introduction to Flow Cytometry, Advanced Techniques (Ex. Apoptosis applications), Analytical Instrument Operation and Data Analysis with FloJo Software. The core stuff received about 50 hrs. or training a year in flow cytometry. The core stuff supported about 60 hrs of training through FAES.
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