ORGANIZATION AND BUDGET OF THE CORE: The NEI Flow Cytometry Core has been operational for 3 years. It is equipped with two FACSCaliburs (Ages: 3 years and 12 years old), one FACSAria II (Age: 1 years old), one Luminex 200 system (Age: 1 years old). The Core operates from 2 small, noncontiguous storage rooms totaling 137 square feet. The FACSCalibur workspace totals 77 square feet of which the machines'footprint accounts for 60% of the space. The FACSAria workspace totals 60 square feet of which the machine itself and its accessories use 80% of the space. The Core is operated by a Core Manager and a Technical IRTA tranee, each devoting 100% effort to the Core, and is supervised by a PI devoting 7% effort to the Core. The Core utilized 100% of its budget plus a $50,00 contribution from the NEI Office of Scientific Director for its routine operations. The Luminex 200 system (magnetic plate washer and reader) was provided by the Laboratory of Immunology. The stipend of the Technical IRTA trainee was funded by an equal contribution from the Neurobiology Neurodegeneration &Repair Laboratory and the Laboratory of Immunology. OPERATION OF THE CORE: This year, over fifty individuals from thirteen different laboratories used the facility. These services and collaborative services were performed for 9 Principal Investigators (PIs) from 5 NEI labs (LI, LRCMB, N-NRL, OSD and URN), plus 3 PIs from 3 other institutes at NIH (NCI, NICHD, and NIDDK). Over 13,800 samples were analyzed. Several users received training on data acquisition using the FACSCaliburs. This year over 1,600 hours of sorting were carried out. Among the techniques now in use within the core there 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 involves the sorting of peripheral blood mononuclear cells to study their cytokine production, genotype and DNA or RNA expression. The sources of these cells include 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 has had a crucial role in the ability to use the sorter for high throughput multicolor analysis. 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 80 hours of training outside the Institute.
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