Shared Resources Laboratories (SRL) are fundamental cornerstones of the 2003 NIH Roadmap. Often referred to as core laboratories, SRLs are the most cost-effective approach to provide a broader range of scientific expertise from skilled technology scientists and advanced instrumentation than would be feasible or economically-viable for individual laboratories. Best practices for a Flow Cytometry SRL include: 1) stable and consistent financial support, 2) a highly qualified scientific director (head), 3) policies conducive to recruitment and retention of highly skilled technical staff, 4) user training and staff continuing education, 5) a quality control program for the instruments, 6) support for research and development, 6) overview of satellite facilities), and 7) at a minimum, a 5-year plan for future technology expansion and operation. The NIA Flow Cytometry Unit (LMG) functions as a Shared Resource Laboratory (SRL) to provide analytical flow cytometric analysis and cell sorting services to the entire NIA IRP. The core was judged to be exceptional from the 2011 BSC review. The core is used by investigators from every bench-science based laboratory in the Baltimore IRP. 99.5% of the core usage, facilities, and publications coming from the core are in support of NIA intramural scientists. Laboratory usage has increased over the years as sorting capacity has increased along with new research initiatives by IRP labs, such as sorting murine and human muscle stem cells. Core usuage continues to be at or exceeding capacity. Sorting is heavily scheduled by users requiring multiple instruments (2-3) for the full day or greater in order to process >300 million cells. Some investigators experimental protocols require specific instruments. Besides the current core users, two additional laboratories have projected substantial sorting needs (50-100 sorts and >400 million cells for processing) in the near future, obligating full use of the sorting capacity for 4 days per week with 1 day open for other laboratories. Because of need for high volume sorting, the core added a 4th staff member half-time in 2014 who became full time on March 1 2015. This individual is responsible for extended hours sorting,from 6PM to 4AM. The flow core is an integral part of long-term, longitudinal study (Gestalt) inter-laboratory examining changes blood cell subpopulations (T, B, monocyte) at the immunological, functional and epigenetic levels. This project, initiated by the SD, NIA IRP, utilizes a substantial portion of the core resources. The core provides cutting edge, state-of-the-art flow cytometry for multi-parameter (polychromatic) analysis and sorting. Flow cytometry instrumentation has a 5 7 year lifespan and to plan for the future, the IRP has made major instrument purchases in 2008 with the acquisition of an iCyt Reflection HAPS (Highly Automated Parallel Sorting) instrument that is housed in a Baker BL-2 hood for sorting adeno- and lentivirus-infected animal and human cells. Each HAPS is configured differently to provide the maximum flexibility for investigators experimental design. In 2013 with the move to the BRC, the obsolete (purchased in 2001 with parts no longer widely available) MoFlo high speed cell sorter was upgraded to an digital version incorporating 405nm, 532nm, and 561nm lasers in addition to an 640nm laser and the 90-5 water-cooled argon and krypton-ion lasers that were retained from the analog instrument. In 2015, we retired the 90-5 Argon ion-laser and replaced it with a Coherent 488nm 200mW Sapphire solid state laser. This was done because Coherent does not stock replacement plasma tubes and we experienced down time of almost 3 months on the instrument waiting for a replacement tube. The problematic issues with this instrument that we had in 2014 have been resolved and turned out to be a computer virus that was on the instruments server installed during manufacturing. With the removal of the 90-5 laser, we added a Coherent Genesis MX 460nm to retain functional, as the only instrument in a 50 mile radius able to perform chromosome analysis and sorting. Our MoFlo is the only 7-laser instrument in existence. The MoFlo is also equipped with a Propel NanoView forward scatter detector, enabling detection and sorting of submicron particles in the down to 100nm, i.e exosomes. The laboratory space for the BRC facility was designed to house the Aria SORP (4 lasers) in a dedicated BL-3 room to be able to sort live non-human primate cells. Service contract costs account for approximately 80% of the annual operating budget and will increase in 2014 as the MoFlo comes out of its warranty period. Also in 2015, we added a Propel (BioRad) semi-automated S3 sorter, uniquely configured with both 488 and 640nm excitation. The mission of the NIA flow cytometry shared resource lab is to enhance the scope and quality of the research within the IRP by providing all investigators with the expertise necessary to employ its advanced technologies optimally for their research. This includes having a highly qualified director (with 35yrs flow cytometry core experience), active consultation on experimental design, implementation, and data interpretation, originating new procedures, suggesting or performing the critical experiments that take their research in new directions, and in advancing technological developments in the field. Users can visit the core web page, in use since 1999. The NIA Flow Cytometry Unit is organized and managed in a manner consistent with the policies and staffing levels of flow cytometry cores at extramural academic institutions and at NIH. A survey (updated March 2011) of these sites (9 NIH, 133 academic, total 142) indicates that in 91.4% of the cores, sorting is performed only by core personnel. The reason for this is twofold: 1) it is the most efficient way to provide reliable services to the IRP (i.e. minimizing the possibility of instrument abuse by users and thus removing the cascading effect that this has on the ability to complete the next scheduled users sort, especially when the core is heavily booked). 2) neither the MoFlo nor iCyt are turnkey black box, menu-driven instruments (nor is the Aria II in the case of the ability to troubleshoot). Relevance to the NIA IRP Program In addition to the level of core utilization by investigators, measurement of the effectiveness and value of the core to IRP productivity is evident from the 157 publications generated by NIA investigators using flow cytometry/sorting as an integral part of their research since the cores inception in 1999. A partial list of these includes J Immunology (10), BMC Immunology (2), J Exp Med (1), Cellular Immunology (2), Immunity (1), Nature Immunology (1), Blood (2), Cancer Res (1), PNAS USA (3), Endocrinology (1), PLOSone (3), DNA Repair (3), Mol Cell Biol (3), EMBO J (1), and Nature (1), other journals (43). 100% of research papers using core services are from NIA IRP investigators.
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