The mission of the CCR LGI Flow Cytometry Core in Building 37 (FC37) is to offer up-to-date instrumentation and technical expertise to CCR investigators to assist cancer research. The core provides full-scale, state-of-the-art flow cytometry services including analytical sample acquisition, data analysis, fluorescent cell sorting and experimental planning and consultation. The LGI FC37 also provides training on analytical instrumentation and cell sorters to permit investigators to independently acquire samples and perform data analysis. The core is committed to the dissemination of novel flow cytometry-based technologies through continuous education of investigators, sponsoring application seminars and participating in the NCI-wide flow cytometry training course with active contributions from director Ferenc Livak staff members, Karen W. Wolcott and Caiyi (Cherry) Li. In addition, the LGI FC37 strives to become a leader of excellence in flow cytometry applications by constantly seeking opportunities to enhance and broaden its technological platform in support of cutting edge cancer research. The LGI FC37 provides instrumentation for flow cytometry sample acquisition, data analysis, high-throughput sample acquisition, imaging flow cytometry, spectral flow cytometry and high-speed and single-cell sorting. The core is equipped with six analytical instruments: a state-of-the-art BD FACSymphony A5 flow cytometer equipped with five lasers and the capacity to simultaneously identify 27 fluorochromes also equipped with a high throughput sampler (HTS) attachment that accommodates 96-well plates to further increase the capacity of the instrument. Two high end BD LSRFortessa Special Order Research Product (SORP1 and 2) cytometers with identical configuration, equipped with five lasers and the capacity to simultaneously identify 18 fluorochromes. One of these instruments, LSRFortessa SORP1 is also equipped with HTS attachment. The core also has one three-laser, digital BD FACSCanto II cytometer and one, 32-channel Sony SA3800 spectral analyzer. In addition, the Core operates an ImageStream MarkII imaging flow cytometer. With the addition of the latest, 26-color BD FACSymphony analyzer in FY19, the Core can offer the highest analytical capabilities currently possible in fluorescent cell analysis. The increased use of analytical flow cytometry is accompanied by increasing demand of professional support for advanced panel design and data analysis, which lead to the active involvement of the Core in published work with Michael Bustin's lab [LM]. In FY19 the Core installed the new, high-end BD FACSymphony A5 flow analyzer. This analyzer is the top analytical flow cytometer currently available commercially and expands not only capacity but also broadens the horizons of high-dimensional, multi-parameter flow cytometry analysis. Several laboratories (Trinchieri, Goldszmid, Bosselut) begun to use the instrument to expand analysis up to 20-24 color studies. The Core continued to expand the use of the ImageStream MarkII imaging flow cytometer (Luminex Corp). This instrument records high quality images of every cell in flow and combines the high throughput of flow cytometry with the high information content of microscopic details. Director, Dr. Ferenc Livak participated in an intense, one-week training course at Amnis headquarters in Seattle to gain more experience with advanced, complex imaging flow cytometry data analysis that will help expand the use of this novel technology for the benefit of several CCR labs (Ashwell, Samelson, Yang, Hunter, Taylor). The core completed 739 sort requests on four cell sorters so far in FY19. In FY19 one of the cell sorters, operated by Dr. Subhadra Banerjee, was equipped with a special, 445nm violet-blue laser, purchased entirely from funds of the Core. This laser enables us to perform chromosome sorting using Hoechst and Chromomycin A3 dyes in support of ongoing cytogenetics studies in Drs. Misteli andRied's laboratories at NCI. This is a unique capability that no other flow cytometry service can offer at NIH and thus can allow us to provide support also to other NIH institutes in their studies of chromosome structure and function. The Core operates four cell sorters. Two BD FACSAria II instruments are equipped with 3-4 lasers with the capacity to simultaneously identify 10-11 fluorochromes. Both of these sorters are enclosed in flexible, BioBubble Benchtop Biocontainment units. One Beckman Coulter MoFlo Astrios EQ instrument, housed in a custom-designed Class II biosafety cabinet, is equipped with five lasers and capable of simultaneously detecting 20 fluorochromes. One BD FACSAria Fusion cell sorter is also housed in a custom-designed Class II type A2 biosafety cabinet and is equipped with five lasers and has the capacity to simultaneously identify 18 fluorochromes. These two instruments have configurations similar to the two high-end LSRFortessa SORP flow cytometers. Further, the two Class II biosafety cabinets and two BioBubble Benchtop Biocontainment units, combined with the separate housing in a dedicated laboratory space in Room 6008A allow the LGI FC37 to operate in full compliance with NIH Policy for Biosafety of Cell Sorters (July 28, 2012), to perform all sorts at a BSL-2 with enhanced precautions aerosol containment level. The LGI FC37 served 275 users from 76 CCR laboratories as well as from 5 additional laboratories of four other NIH institutes in FY19. Several of these laboratories heavily depend on the services of the Core in conducting high-dimensional analyses aimed at better understanding of lymphocyte development (Ashwell, Bosselut (LICB] and Bhandoola [LGI] labs) and tumor-associated immune responses (Trinchieri lab [CIP] and Zhuang lab [NOB]). Single cell sorting helps the rapid generation of CRISPR-induced mutant cell lines (Hunter [LCBG], Lal [GB], Nussenzweig [LGI] and Stommel [ROB] labs among others) and is becoming an essential tool in single cell genomic analyses (Bosselut lab [LICB], Merlino and Yang labs [LCBG] and Herkenham lab [NIMHl]). The essential contribution of the Core to Michael Bustin's [LM] studies on iPS cell differentiation has resulted in a collaborative publication. The LGI FC37 provides critical help to the initial phases of translational research into glioblastoma (Gilbert, Chunzhang Yang [NOB] labs), liver cancer (X. Wang [LHC] lab) and tumor-specific immune responses (Waldman lab [LYMB] and Hodge labs [LTIB]). The LGI FC37 has trained 86 new users so far in FY19 on analytical instruments and has also trained 2 new users for cell sorting. New users are instructed to review instrument and software training tutorials before attending a 4-hor hands on training session that includes startup and shut down procedures for the cytometers, setting up the experiment, basic instrument troubleshooting, and data analysis. The Core staff provides follow-up assistance and helps the new user in developing confidence in using the technology correctly. Cell sorter training is offered to select users who need frequent, possibly after hour sorting time, and who have sufficiently mastered the BD FACS DIVA software using the analytical flow cytometers. The LGI FC37 employs an entirely online scheduling and record keeping platform using the NIH LabShare application. Appropriate policies are in place to ensure fair and equitable access of Core resources to all registered users. The LGI FC37 continues to be a leader of flow cytometry service at CCR by offering the highest quality, reliable support to the largest number of NCI investigators on campus and is dedicated to the introduction of innovative flow cytometry-based technologies to further advance the cutting-edge cancer research conducted at NIH.
