The facility currently supports the research of numerous NIAMS IRP investigators. In addition, it is actively involved in training and assay support for all the IRP users who are unfamiliar with this technique. The Facility houses: (One) Beckman Coulter MoFlo high-speed sorter (3-laser excitation, 10-color detection, automated cell deposition unit) (One) BD Biosciences FACSAria IIIu high-speed sorter (5-laser excitation, 19-color detection, automated cell deposition unit) (One) Beckman Coulter CyAn analyzer (3-laser excitation, 9-color detection) (One) BD Biosciences FACSCanto II analyzer w/HTS (3-laser excitation, 8-color detection, high-throughput sampler) (Two) BD Biosciences FACSCalibur analyzers (2-laser excitation, 4-color detection) (One) Compucyte iCys Research Laser Scanning Cytometer (3-laser excitation, 5-parameter detection) More than one hundred NIAMS flow cytometry users have already been trained to date on the different platforms available in the facility. In 2010, a revised, multi-level training scheme was introduced. To provide a more logical training structure, a progressive group and individual-based training system is now be used to provide investigators and their staff better access to Flow Cytometry Section equipment and services. All new users first attend a general introduction and orientation to facility equipment and policies. Then, according to needs and previous experience, progress to individualized or group instruction on the instrument most appropriate for their particular research project. After approval by the section leader, users can then operate the instruments unassisted. However, the FCS staff is always available for troubleshooting and consultation. Besides the projects from those laboratories focused on immunology, the facility has successfully expanded flow cytometry and sorting services to other investigators within NIAMS engaged in disciplines not often associated with flow cytometry: Dr. Morassos Developmental Skin Biology Unit, Dr. Sartorellis Laboratory of Muscle Stem Cells and Gene Regulation and Dr. Wangs Laboratory of Muscle Biology. These projects are in areas not typically considered applicable to flow cytometry but have been initially quite encouraging. Usage of the facility has increased steadily over the last few years requiring the upgrade of our high-speed cell sorters as well as our FACSCanto analyzer. To take advantage of breakthroughs in both fluorochrome and laser technologies, two lasers 375 nm and 561 nm, have been added to the FACSAria II. The near-UV 375 nm laser along with additional system upgrades such as a more light efficient flow cell allow investigators to sort bone marrow stem cells using the Hchst side population labeling technique. The yellow-green 561 nm laser provides a more efficient excitation source (compared to the 488 nm blue laser) for phycoerythrin (PE), PE tandem dyes (PE-Cy5, PE-Cy5.5, PE-Cy7, etc.) and the DsRed fluorescent proteins like mCherry, mRaspberry and tdTomato. These upgrades allow the simultaneous detection of up to 19 fluorochromes and transform our FACSAria II to a state-of-the-art third generation FACSAria IIIu. Similarly, to enable our investigators to take advantage of the newer violet-excited dyes like Pacific Orange and Qdot nanocrystals, a 405 nm violet laser was added to our high-throughput FACSCanto analyzer. NIAMS investigators mentioned above whose research models are not well-suited to flow cytometry continue to take advantage of the sections state-of the-art laser scanning cytometer, the Compucyte iCys. This microscope-based instrument can provide fluorescent and morphological data on cells or tissue fixed to slides or multi-well plates. Along with typical immunophenotyping, the iCys can also re-analyze specific cells over time for kinetics-based assays. While fulfilling his duties as the core Section Leader, Mr. James Simone, continues to be involved in the promotion of flow cytometry at NIH-wide level. As a member and 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.
