Flow Cytometry CORE

National Eye Institute

Investigators

Linked publications & trials

Abstract

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. The work of the cytometry core was tremendously impacted by the emergence of the COVID-19 pandemic. The laboratory was secured and closed in March 2020. In May 2020, the core partially restarted for a Covid related project. In August, the biologist in the core moved to NIAID. As of September, users of the core have not been able to restart their regular operations. SERVICES PROVIDED THE CORE: This year, fifty-fivindividuals from twentyfour different laboratories used the facility. These services and collaborative services were performed for 10 Principal Investigators (PIs) from 5 NEI labs (ERPD, LI, LRCMB, MSF, N-NRL, RN, OGVFB and UNGIRD), plus 15 other laboratories at NIH. This year the core performed 750 hours of sorting and over 9,000 samples were analyzed. Among the techniques now in use within the core are methods for phenotyping live cells, gene expression detection through fluorescent reporter genes, 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: Sharma R, Bose D, Maminishkis A, Bharti K. Retinal Pigment Epithelium Replacement Therapy for Age-Related Macular Degeneration: Are We There Yet? Annu Rev Pharmacol Toxicol. 2020 Jan 6;60:553-572. doi: 10.1146/annurev-pharmtox-010919-023245. PMID: 31914900. Kang M, Lee HS, Choi JK, Yu CR, Egwuagu CE. Deletion of Irf4 in T Cells Suppressed Autoimmune Uveitis and Dysregulated Transcriptional Programs Linked to CD4+ T Cell Differentiation and Metabolism. Int J Mol Sci. 2021 Mar 9;22(5):2775. doi: 10.3390/ijms22052775. PMID: 33803441; PMCID: PMC7967141. Choi JK, Egwuagu CE. Analysis of Regulatory B Cells in Experimental Autoimmune Uveitis. Methods Mol Biol. 2021;2270:437-450. doi: 10.1007/978-1-0716-1237-8_23. PMID: 33479912. 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 100 hrs. or training a year in flow cytometry. The Core stuff provided about 50 hrs of training to the NEI community and 60 hrs of training through FAES to the NIH community.