The facility currently supports the research of the majority of NIAMS IRP investigators. In addition, it is actively involved in instrument training and flow cytometric assay development for all IRP users unfamiliar with this technique. The Facility currently supports the NIAMS IRP community with the following instrumentation: (One) BD Biosciences FACSAria IIIu cell sorter (5-laser excitation, 19-color detection, automated cell deposition unit) (One) BD Biosciences Influx cell sorter (7-laser excitation, 20-color detection, automated cell deposition unit, spectral analyzer (One) Beckman Coulter MoFlo cell sorter (3-laser excitation, 10-color detection, automated cell deposition unit) (One) BD Biosciences FACSCanto analyzer w/HTS (3-laser excitation, 8-color detection, high-throughput sampler) (One) BD Biosciences LSR Fortessa analyzer (7-laser excitation, 20-color detection, high-throughput sampler) (Two) BD Biosciences FACSVerse analyzers (3-laser excitation, 8-color detection, universal high-throughput sampler, volumetric measurement (for absolute cell counts) (One) Compucyte iCys research laser scanning cytometer (3-laser excitation, 5-parameter detection) (One) Amnis ImageStreamX Mark II high resolution microscope/cytometer (7-laser excitation, 3 imaging objectives, analysis software) (One) BeckmanCoulter Cyan analyzer. At 3-lasers, a true historical piece of analytical system of value. Retired in 2013 in light of incoming systems and core relocation More than two hundred NIAMS flow cytometry users have been trained to date on the different platforms available in the facility. In 2013, we continued to use the revised, multi-level training scheme that was introduced in 2011. To provide a more logical training structure, a progressive group and individual-based training system is used to provide investigators and their staff better access to the Flow Cytometry Sections equipment and services. All new users attend a general introduction and orientation to facility equipment and policies. Then, according to needs and previous experience, they progress to individualized or group instruction on the instruments most appropriate for their particular research needs. After approval by the section leader, users can then operate the instruments unassisted. In all cases, however, the FCS staff is available for troubleshooting and consultation. In addition to the projects from those laboratories focused on mechanisms and dysfunctions of the immune system, the facility has continued to provide flow cytometry and sorting services to NIAMS laboratories engaged in disciplines not often served by flow cytometry: the Developmental Skin Biology Unit;and the Laboratory of Muscle Stem Cells and Gene Regulation. The mission-critical projects in these laboratories continue to be quite productive. Usage of the facility continues to increase, taking advantage of the recently added Influx cell sorter, LSR Fortessa and FACSVerse cytometers. These instruments provide better tools for NIAMS investigators and support the newly established NIH Center for Regenerative Medicine (NIH CRM) Equipped with seven lasers and 20+ fluorescence detectors, both the Influx cell sorter and LSR Fortessa analyzer provide state-of-the-art tools, enabling investigators to take advantage of the latest fluorochromes and fluorescent protein reporters. Those investigators whose research involves specimens that are not well suited to flow cytometry can continue to take advantage of the Section's laser scanning cytometer, the Compucyte iCys, as well as the new Amnis ImageStreamx Mark II. These microscope-based instruments can provide fluorescent and morphological data on cells or tissue fixed to slides or multi-well plates (iCys) or cells in suspension as in traditional flow cytometry (ImageStream). Along with typical immunophenotyping, apoptosis assays and co-localization studies, the iCys can also re-analyze specific cells over time for kinetics-based assays. In contrast, the design of the ImageStream Mark II allows for higher throughput for more robust statistics, a characteristic of standard flow cytometers, while providing image analysis of a microscope-based system. The FCS has three dedicated personnel to support the need of NIAMS IRP investigators. Mr. Jeffrey Lay has been a part of the section since 2010. He has been recently trained to use the Amins ImageStream and will support future use of the instrument. Kevin Tinsley, PhD joined the core facility in early January 2013 as a contractor. Having completed his doctoral degree and postdoctoral fellowship in the field of immunology, he joined the Core with a substantial familiarity with flow cytometry. He was recently trained for the use of the ARIAIII at BD Biosciences and is becoming the point of reference for such instruments. While fulfilling his duties as Section Leader, Mr. James Simone continues to be involved in the promotion of flow cytometry at the NIH and within the Mid-Atlantic Region. As a co-chair of the Flow Cytometry Interest Group, he assists in organizing and promoting quarterly meetings that showcase leading researchers in the field and promote relationships with vendors of flow cytometry-related products. His longtime membership in the International Society for the Advancement of Cytometry (ISAC) and the ISAC Bio-Safety Committee helps facilitate the exchange of new ideas and information with peers from around the world.
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