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 2 sorters, FACS Aria II and FACS Aria Fusion, are largely operated by trained Core personnel. The analytical instruments, 2 MACS Quant instruments and the BD FACS Calibur, are largely operated by users who receive training from Core personnel. SERVICES PROVIDED THE CORE: This year, eighty 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, LRCMB, MSF, N-NRL, RN, OGVFB and UNGIRD), plus 3 PIs from 3 other institutes at NIH (NICHD, NIDCD and NIDDK). Over 10,000 samples were analyzed. This year the core performed about 1,000 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. 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: Holly Yu Chen, Koray Dogan Kaya, Lijin Dong, Anand Swaroop. Three-dimensional retinal organoids from mouse pluripotent stem cells mimic in vivo development with enhanced stratification and rod photoreceptor differentiation. Molecular Vision 2016; 22:1077-1094 Khan SY, Vasanth S, Kabir F, et al. FOXE3 contributes to Peters anomaly through transcriptional regulation of an autophagy-associated protein termed DNAJB1. Nature Communications. 2016;7:10953. doi:10.1038/ncomms10953. Chong WP, van Panhuys N, Chen J, et al. NK-DC crosstalk controls the autopathogenic Th17 response through an innate IFN-IL-27 axis. The Journal of Experimental Medicine. 2015;212(10):1739-1752. doi:10.1084/jem.20141678. TRAINING: The Core has supported the education program of the Foundation for Advancement of Education in Science. Core stuff have serve as lead trainer, technical trainer and class material developers. d More than twenty users from different labs received training how to operate the MACSQuant instrumentation. The availability of the Technical IRTA position continues to have 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 greatly enhanced 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 worked on Analysis of Exosomes in Ophthalmology Research Using Flow Cytometry and presented a poster at the NEI Summer Interns Poster Day. 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 Manager completed 40 hours of continuing education in management of core laboratories. The technical IRTA completed 20 hours of formal training outside the Institute.
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