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 newly developed NCI-wide flow cytometry training course with active contributions from director Ferenc Livak and two 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 five analytical instruments: 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 a high throughput sampler (HTS) attachment that accommodates 96-well plates to further increase the capacity of the instrument. The core also has one three-laser, digital BD FACSCanto II cytometer and one Sony SA3800 spectral analyzer. In FY18 the Core removed its oldest, two-laser, analog BD FACSCalibur cytometer from service. In addition, in FY18 the Core added an entirely new technology, imaging flow cytometry to its portfolio (see below). The two identical 18-color LSRFortessa SORP instruments ensure maximum utilization and flexibility for our users. Nonetheless, rapidly increasing usage in FY18 prompted the Director of the Core to apply for funds to purchase a new, high-end cell analyzer. After a successful RRS application, the core is scheduled to receive and install a new 27-color BD FACSymphony analyzer in FY19, which will 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 to a soon-to-be published work with Michael Bustin's lab [LM]. In FY18 the Core installed a new ImageStream MarkII imaging flow cytometer (MilliporSigma). This instrument represents an entirely novel technology previously not available to CCR investigators at NCI. It 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. It expands the analytical capabilities of traditional flow cytometry by adding hundreds of morphological features to the fluorescent intensity measurements and allows unprecedented depth of quantitative, statistical analysis based on these fluorescent and morphological parameters. Several CCR labs have already begun to use this novel technology to study nuclear translocation of transcription factors (Ashwell and Samelson labs [LICB] and Annuziata lab [WMB]), quantification of nuclear DNA damage foci (Nussenzweig lab [LGI]), enumeration of circulating tumor cells (Wang lab [LMGC] and Hunter lab [LCBG]) and for the characterization of inflammatory responses (Trinchieri lab [CIP]) and microparticles and exosomes (Roberts lab [LP]). The core completed 698 sort requests on four cell sorters so far in FY18. Two BD FACSAria II instruments are equipped with three lasers with the capacity to simultaneously identify 9-11 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. This instrument has similar capabilities to the MoFlo Astrios and these two sorters have configurations essentially identical to the two high-end LSRFortessa SORP analytical 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), which requires sorting of human cells and cell lines, and all cells transduced with lentivirus or retrovirus particles at a BSL-2 with enhanced precautions aerosol containment level. The LGI FC37 served 192 users from 71 CCR laboratories as well as from 5 additional laboratories of four other NIH institutes in FY18. 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 [CGB], Nussenzweig [LGI] and Stommel [ROB] labs among others) and is becoming an essential tool in single cell genomic analyses (Bosselut lab [LICB], Yang lab [LCBG] and Herkenham lab [NIMHl]). The essential contribution of the Core to Xin-Wei Wang's (LMGC) studies on cancer stem cell genomics in hepatocellular carcinoma 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 [LMGC] lab) and tumor-specific immune responses (Waldman lab [LMBR], Schlom and Hodge labs [LTIB]). The LGI FC37 has trained 102 users so far in FY18 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.
