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 MoFlo Cell Sorter was transferred from the National Cancer Institute. OPERATION OF THE CORE: This year, forty eight individuals from thirteen different laboratories used the facility. These services and collaborative services were performed for 8 Principal Investigators (PIs) from 8 NEI labs (ERPD, LI, LMDB, LRCMB, MSF, N-NRL, OCD and OGVFB), plus 3 PIs from 3 other institutes at NIH (NCI, NICHD, and NINDS). Over 16,000 samples were analyzed. Several users received training on data acquisition using the FACSCaliburs. This year the core performed over 1,600 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, 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. Limited analytical work had been done with eye fluids, eye tissue specimen, protein, and tears. No tissues were stored by the core. TRAINING: The creation of the Technical IRTA position has had a very positive effect on the Core operation. There is a great need for well-trained flow cytometry operators. Through the TechIRTA, the hours of operation of the facility have been expanded from 40 hr/week to 60 hr/week. The expanded hours of operation helped to reduce the backlog on the sorter schedule. The TechIRTA position has had a crucial role in the ability of the Core to use the cell sorter for high throughput multicolor analysis. This year the core hosted a summer student from the Diversity In Vision Research and Ophthalmology (DIVRO) summer internship program. The student work in identification of stem cells in the eye. Several formal training sessions were offered by the core to the NEI community. These courses included: Introduction to Flow Cytometry, Basics of Flow Cytometry, Luminex sample preparation, data acquisition and analysis and FACSCalibur Operation. The Core Manager completed 48 hours of continuing education in flow cytometry and 12 hours of training in management of Core facilities. The technical IRTA completed 40 hours of training outside the Institute.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Scientific Cores Intramural Research (ZIC)
Project #
1ZICEY000457-05
Application #
8557108
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2012
Total Cost
$225,122
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
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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
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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
Zhou, Ru; Horai, Reiko; Silver, Phyllis B et al. (2012) The living eye ""disarms"" uncommitted autoreactive T cells by converting them to Foxp3(+) regulatory cells following local antigen recognition. J Immunol 188:1742-50

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