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

National Institute Of Arthritis And Musculoskeletal And Skin Diseases

Investigators

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Abstract

Summary 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 NFCS currently houses and maintains the following instrumentation for the use of NIAMS investigators: -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, Aerosol Management Option) -BD Biosciences Influx cell sorter (7-laser excitation, 20-color detection, automated cell deposition unit, spectral cytometer, Biosafety Cabinet) -BD Biosciences FACSCanto SORP cytometer with HTS (4-laser excitation, 13-color detection, high-throughput sampler) -BD Biosciences LSR Fortessa SORP cytometer with HTS (7-laser excitation, 20-color detection, high-throughput sampler) -BD Biosciences FACSymphony A5 cytometer (5-laser, 28-color detection, HTS-ready, advanced electronics) -BD FACSCelesta SORP cytometer with HTS (4-laser, 15-color detection, high-throughput sampler, advanced electronics) -Cytek Aurora spectral cytometer with AL (5-laser, 34-color full-spectrum detection, autoloader) The following instruments are no longer housed and maintained by NFCS 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) In years past the FCS core has utilized other cytometers to support the NIAMS mission. These have since been retired and replaced with the more modern instruments listed above. -BD Biosciences FACSVerse cytometer -Beckman Coulter MoFlo cell sorter -Beckman Coulter CyAn ADP cytometer -BD Biosciences FACSCalibur cytometer -BD Biosciences FACS Vantage 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. Both the FACSAria Fusion and the BD InFlux cell sorters are individually 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 provide the NFCS with even more capable tools, the 5-laser, 28-color BD FACSymphony A5 and 4-laser, 15-color BD FACSCelesta SORP cytometers were added to the core facility in 2018. Early this year, the addition of the recently upgraded 5-laser, 34-color Cytek Aurora further augmented the facilitys cytometer toolbox. Capable of simultaneously detecting highly overlapping dyes, it provides NIAMS investigators with greater flexibility for their experimental panel designs. These state-of-the-art cytometers with their better quality optics and more sensitive detection electronics will enable investigators to take advantage of the synergy of higher dimensional immunophenotyping and potentially discover previously undetectable rare cell populations. The NFCS has three dedicated personnel to support the needs of NIAMS IRP investigators: -Mr. Jeffrey Lay has been an integral part of the NFCS 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, equipment inventories, actively promotes the utilities of core operation, guidance and learning, and actively immerse himself with new technologies. -Kevin Tinsley, PhD Having completed postdoctoral fellowship in the field of immunology at NCI, he joined the NFCS with substantial experience with flow cytometry as a contractor in 2013. 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 NFCS in 2003 and continues to oversee the day-to-day operations of the facility. He is expert in the use of all the 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 from 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 NFCS staff for technical support and consultation. To advance the institutes mission, the NIAMS Flow Cytometry Section 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 NFCS can now make cytometry services available to investigators outside of NIAMS.