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.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Scientific Cores Intramural Research (ZIC)
Project #
1ZICEY000457-11
Application #
9796770
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
11
Fiscal Year
2018
Total Cost
Indirect Cost
Name
U.S. National Eye Institute
Department
Type
DUNS #
City
State
Country
Zip Code
St Leger, Anthony J; Desai, Jigar V; Drummond, Rebecca A et al. (2017) An Ocular Commensal Protects against Corneal Infection by Driving an Interleukin-17 Response from Mucosal ?? T Cells. Immunity 47:148-158.e5
Zárate-Bladés, Carlos R; Horai, Reiko; Mattapallil, Mary J et al. (2017) Gut microbiota as a source of a surrogate antigen that triggers autoimmunity in an immune privileged site. Gut Microbes 8:59-66
Dillenburg-Pilla, Patricia; Zárate-Bladés, Carlos R; Silver, Phyllis B et al. (2016) Preparation of Protein-containing Extracts from Microbiota-rich Intestinal Contents. Bio Protoc 6:
Kielczewski, Jennifer L; Horai, Reiko; Jittayasothorn, Yingyos et al. (2016) Tertiary Lymphoid Tissue Forms in Retinas of Mice with Spontaneous Autoimmune Uveitis and Has Consequences on Visual Function. J Immunol 196:1013-25
Zárate-Bladés, Carlos R; Horai, Reiko; Caspi, Rachel R (2016) Regulation of Autoimmunity by the Microbiome. DNA Cell Biol 35:455-8
Ma, Wenxin; Cojocaru, Radu; Gotoh, Norimoto et al. (2013) Gene expression changes in aging retinal microglia: relationship to microglial support functions and regulation of activation. Neurobiol Aging 34:2310-21
Horai, Reiko; Silver, Phyllis B; Chen, Jun et al. (2013) Breakdown of immune privilege and spontaneous autoimmunity in mice expressing a transgenic T cell receptor specific for a retinal autoantigen. J Autoimmun 44:21-33
Chong, Wai Po; Ling, Man To; Liu, Yinping et al. (2013) Essential role of NK cells in IgG therapy for experimental autoimmune encephalomyelitis. PLoS One 8:e60862
Wang, Yujuan; Shen, Defen; Wang, Vinson M et al. (2012) Enhanced apoptosis in retinal pigment epithelium under inflammatory stimuli and oxidative stress. Apoptosis 17:1144-55
Shi, Guangpu; Lovaas, Jenna D; Tan, Cuiyan et al. (2012) Cell-cell interaction with APC, not IL-23, is required for naive CD4 cells to acquire pathogenicity during Th17 lineage commitment. J Immunol 189:1220-7

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