Flow cytometry support to research in immune, skin, muscle and bone diseases

National Institute Of Arthritis And Musculoskeletal And Skin Diseases

Investigators

Linked publications & trials

Abstract

This flow cytometry core facility supports the research of the majority of NIAMS IRP investigators. In addition, it is actively involved in cytometer training, data analysis and flow cytometric assay development for users new to this technique. The NIAMS FCS currently houses and maintains the following instrumentation for the use of NIAMS investigators and their collaborators: -BD Biosciences FACSAria Fusion cell sorter with ACDU and BSC (5-laser excitation, 17-color detection, Automated Cell Deposition Unit, BioSafety Cabinet) -BD Biosciences FACSAria IIIu cell sorter with ACDU (5-laser excitation, 17-color detection, Automated Cell Deposition Unit, Aerosol Management Option) -BD Biosciences Influx cell sorter with CCDU and BSC (7-laser excitation, 21-color detection, Computerized Cell Deposition Unit, BioSafety Cabinet) -BD Biosciences FACSMelody automated cell sorter with ACDU and BSC (3-laser excitation, 9-color detection, ACDU, BioSafety Cabinet) -BD Biosciences LSR Fortessa SORP cytometer with HTS (7-laser excitation, 18+3-color detection, high-throughput sampler) -BD Biosciences FACSymphony A5 cytometer (5-laser excitation, 28-color detection, HTS-ready, advanced electronics) -BD Biosciences FACSymphony A3 cytometer (5-laser excitation, 28-color detection, HTS-ready, advanced electronics) -BD Biosciences FACSCelesta SORP cytometer with HTS (4-laser excitation, 15-color detection, high-throughput sampler, advanced electronics) -Cytek Biosciences Aurora spectral cytometer with AL (5-laser excitation, 40-color full-spectrum detection, autoloader for plates) -Cytek Biosciences Aurora-Clinical spectral cytometer with ASL (5-laser excitation, 40-color full-spectrum detection, automated sample loader for tubes and plates) -ThermoFisher Scientific Attune NxT acoustic-assisted focusing cytometer with CytKick Max (4-laser excitation, 14-color detection, autoloader) The following instruments are no longer housed and maintained by NIAMS FCS but are still accessible to NIAMS investigators: -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) To support the NIAMS mission the FCS core has utilized other cytometers, which have since been retired and replaced with the more modern instruments listed above. -BD Biosciences FACSCanto SORP cytometer with HTS -BD Biosciences LSR II cytometer -BD Biosciences FACSVerse cytometer -Beckman Coulter MoFlo cell sorter -Beckman Coulter CyAn ADP cytometer -BD Biosciences FACSCalibur cytometer -BD Biosciences FACSVantage DiVa cell sorter -BD Biosciences LSR I cytometer In addition to the projects from those laboratories focused on mechanisms and dysfunctions of the immune system, the facility continues to provide flow cytometry and sorting services to NIAMS laboratories engaged in disciplines not typically served by flow cytometry such as bone, muscle and skin biology. The facility has the capability of sorting patient specimens and human-derived cells with a higher degree of safety. The FACSAria Fusion, the BD InFlux, and BD FACSMelody cell sorters are each housed in a laminar flow biosafety cabinet. This provides NIAMS investigators with greater opportunities to further the Bench to Bedside initiative promoted by the NIH Director, Dr. Francis Collins. To handle the increasing demand of NIAMS clinical research projects, a 4-laser, 14-color ThermoFisher Attune NxT acoustic-assisted focusing cytometer, a next generation 5-laser, 40-color Cytek Aurora and a 3-laser, 9-color BD FACSMelody automated cell sorter were added to the core facility in 2021. These instruments provide NIAMS clinical investigators with greater capability and flexibility for their experimental panel designs. Furthermore, a 5-laser, 28-color BD FACSymphony A3 was purchased to replace a legacy BD LSR II cytometer that was outdated and too costly to maintain. These state-of-the-art cytometers with their more powerful lasers, higher-quality optics, and more sensitive detection electronics will enable investigators to take advantage of higher dimensional immunophenotyping and potentially discover previously undetectable rare cell populations. The NIAMS FCS has three dedicated personnel to support the needs of NIAMS IRP investigators: -Mr. Jeffrey Lay has been an integral part of the NIAMS FCS since 2008. He is technically trained in the use of all cytometers and cell sorters and is the primary operator of the BD InFlux. He actively and routinely handles complete core operation and equipment inventories, actively promotes the utilities of core operation, guidance and learning, and actively immerse himself in new technologies. -Kevin Tinsley, PhD joined the NIAMS FCS as a contractor in 2013 with substantial experience in flow cytometry, having completed a postdoctoral fellowship in the field of immunology at NCI. He subsequently completed his FACSAria Operator training at BD Biosciences, was promoted to Staff Scientist and is the primary operator of the FACSAria Fusion. Additionally, he regularly takes courses through FAES in the area of Data Science to increase his understanding of much of the data analysis done in NIAMS. -Mr. James Simone, Section Leader, took over the management of the NIAMS FCS in 2003 and continues to oversee the day-to-day operations of the facility. He is expert in the use of all facility equipment 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 Biosafety Committee helps facilitate the exchange of new ideas and information with peers around the world. Greater than 400 NIAMS flow cytometry users have been trained to date on the various cytometers available in the facility. All new users attend a general introduction and orientation to the facility equipment and its policies. Then, according to needs and 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 cytometers unassisted. In any case, users can always rely on the NIAMS FCS staff for technical support and consultation. To advance the institutes mission, the NIAMS FCS continues to explore new techniques and applications of cytometry including sorting for ChIP-Seq, ATAC-Seq, RNA-Seq, and Single Cell RNA-Seq and single-cell Western Milo (Protein Simple). Moreover, through the NIH CREx (Collaborative Research Exchange) advocated by Drs. Collins and Gottesman, the NIAMS FCS can now make cytometry services available to investigators outside of NIAMS.