This cytometry core facility supports the research of the majority of NIAMS IRP investigators and is actively involved in instrument training and flow cytometric assay development for users unfamiliar with this technique. The Section currently houses and maintains the following instrumentation for the use of NIAMS : -BD Biosciences FACSAria Fusion cell sorter (5-laser excitation, 19-color detection, automated cell deposition unit, Biosafety Cabinet) -BD Biosciences FACSAria IIIu cell sorter (5-laser excitation, 19-color detection, automated cell deposition unit) -BD Biosciences Influx cell sorter (7-laser excitation, 20-color detection, automated cell deposition unit, spectral analyzer -BD Biosciences FACSCanto SORP analyzer w/HTS (4-laser excitation, 14-color detection, high-throughput sampler) -BD Biosciences LSR Fortessa SORP analyzer (7-laser excitation, 20-color detection, high-throughput sampler) -BD Biosciences FACSVerse analyzers, two (3-laser excitation, 8-color detection, universal high-throughput sampler (multi-well plates or tubes), volumetric measurement for absolute cell counts -Compucyte iCys laser scanning cytometer (3-laser excitation, 5-parameter detection), shared with NCI -Amnis ImageStreamX Mark II high resolution imaging cytometer (7-laser excitation, 3 imaging objectives, IdeasTM analysis software) In years past the FCS core has housed and utilized other cytometers (listed below) to support the NIAMS mission. These have since been retired and replaced with the more modern instruments listed above. -Beckman Coulter MoFlo high-speed cell sorter -Beckman Coulter CyAn ADP analyzer -BD Biosciences FACSCalibur -BD Biosciences FACS Vantage DiVa high-speed cell sorter -BD Biosciences LSR I In addition to projects from 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 readily served by flow cytometry such as bone, muscle and skin biology. The facility also has the capability of sorting patient specimens and human-derived cells with a higher degree of safety. A FACSAria Fusion cell sorter recently joined the facility, where it is housed in a laminar flow biosafety cabinet. The facilitys existing BD InFlux cell sorter also received its own Baker biosafety cabinet during the core facility relocation in June 2014. These sorters now provide better opportunities for NIAMS investigators to further the Bench to Bedside initiative promoted by the NIH Director, Dr. Francis Collins. Each equipped with seven lasers for excitation and 21 individual fluorescence detectors, the Influx cell sorter and LSR Fortessa analyzer continue to provide state-of-the-art tools that enable 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 utilize the Section's laser scanning cytometer, the Thorlabs (Compucyte) iCys, as well as the Amnis ImageStreamX Mark II. These microscope-based instruments provide fluorescent and morphological data on cells, tissue fixed to slides, multi-well plates (iCys), or cells in suspension as in traditional flow cytometry (ImageStreamX). 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 ImageStreamX Mark II allows higher throughput for more robust statistics, a characteristic of standard flow cytometers, while providing the image analysis advantage 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 Amnis ImageStreamX and will continue to promote and support usage of the instrument. Kevin Tinsley, PhD, joined the core facility in early January 2013 as a contractor and is now a Staff Scientist. Having completed his doctoral degree and postdoctoral fellowship in the field of immunology, he joined the Core with substantial experience with flow cytometry. He subsequently completed his FACSAria Operator training at BD Biosciences in 2013 and is now the primary operator of the FACSAria IIIu. Mr. James Simone, Section Leader, manages the day-to-day operations of the facility and 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 NIH 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. More than two hundred NIAMS flow cytometry users have been trained to date on the various cytometers available in the facility. We continue to use the multi-level training scheme introduced in 2011, which has proven to be very successful. Providing a more logical training structure, a progressive hybrid 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 instruments unassisted. In all cases, however, the FCS staff is available for troubleshooting and consultation. To further support of the NIAMS mission, the Flow Cytometry Section continues to investigate new techniques and applications in cytometry for the use of its investigators.
