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, 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 older, two-laser, analog BD FACSCalibur cytometer. The two identical 18-color LSRFortessa SORP instruments ensure that despite heavy utilization users can perform the most demanding experiments with minimum wait time and maximum flexibility. In addition, the Core installed a new Sony SA3800 spectral flow analyzer during FY17. This instrument relies on the novel spectral flow cytometry technology which collects fluorescence data on the entire visible spectrum of 500-800nm range in real time for every single cell. An innovative software unmixes the spectral data to identify every each fluorescent reagent that is used in the experiment. The instrument utilizes a High Throughput Sampler (HTS) loading mechanism to enable investigators to analyze large number of samples either from FACS tubes of from 96 well plates. The core also operates four cell sorters: two BD FACSAria II instruments equipped with three lasers with the capacity to simultaneously identify 9-11 fluorochromes. 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. This instrument was upgraded with an additional, enhanced forward scatter photomultiplier tube that permits analysis and sorting of microparticles with sizes substantially below the typical resolution limit of other cell sorters. The 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. This conscious instrumentation planning has resulted in a core service where users can seamlessly transfer advanced, complex multi-parameter analytical panels to cell sorting without the need for extensive further optimization or compromising their experimental design. Further, the Class II biosafety cabinets, combined with the separate housing in a dedicated laboratory space in Room 6008A allow the LGI FC37 to operate in compliance with NIH Policy for Biosafety of Cell Sorters (July 28, 2012), which requires sorting of human cell lines, primary human and non-human primate cells and all cells transduced with 2nd or 3rd generation lentivirus or retrovirus particles at a BSL-2 with enhanced precautions aerosol containment level. Since these types of samples make up 70% of all sort requests at the LGI FC37, the Core purchased and installed a lower cost, flexible, BioBubble Benchtop Biocontainment unit to enclose one of the FACSAria cell sorters in 2016. In FY17 the Core purchased another BioBubble Benchtop Biocontainment unit to enclose the last remaining open FACSAria cell sorter to have all of its cell sorters placed in biocontainment enclosures to allow maximum flexibility in sort scheduling. The LGI FC37 served 75 principal investigators in FY17 from over 30 CCR Labs, Branches and Sections as well as 4 additional labs from four other NIH institutes. Several of these laboratories heavily depend on the services of the Core in conducting advanced] studies aimed at better understanding of lymphocyte development (Ashwell, Bosselut (LICB] and Bhandoola [LGI] labs) and tumor-specific innate immune (Trinchieri [CIP] lab) and NK-cell responses (Schlom and Hodge [LTIB] labs). Sorting for fluorescent protein-marked, genetically-engineered cancer cell lines allows investigators to generate novel models for in vivo tracking of tumor metastases (Li Yang and Merlino [LCBG] labs), to monitor breast cancer stem cell maintenance (Wakefiled [LCBG] lab) and to study the rare precursors of skin Merkel cell carcinoma (Brownell [DB] lab). 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 [LICB], Brownell [DB] and X. Wang [LMGC] labs). The LGI FC37 provides critical help to the intitial phases of translational research into glioblastoma (Gilbert, Chunzhang Yang [NOB] labs), liver cancer (X. Wang [LMGC] lab) and mesothelioma (Hassan [TGB] lab). The LGI FC37 has trained 116 users so far in FY17 on analytical instruments, more than ever in previous years, and has also trained three new users for cell sorting. New users are instructed to review instrument and software training tutorials before attending the hands on training which typically takes a 4 hour one-on-one session that includes startup and shut down procedures for the cytometers, setting up the experiment, basic instrument troubleshooting, and analyzing and exporting the data to a shared network for data archiving. The Core staff provides follow-up assistance for performing the next several experiments and assists 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 has fully transitioned in FY17 to an entirely online scheduling and record keeping platform using the NIH LabShare application. Users can now view and place requests online for analytical instruments, cell sorting and traning. 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 inverstigators 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/NCI.
