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Principal Investigator: Ferenc Livak
Organization: DIVISION OF BASIC SCIENCES - NCI
Fiscal Year: 2024
Award: $1,450,610
Funding agency: National Cancer Institute
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, imaging flow cytometry, fluorescent cell sorting and experimental planning and consultation. The core is committed to the dissemination of novel flow cytometry-based technologies through continuous education of investigators and participating in the NCI-wide flow cytometry training course. The LGI FC37 provides instrumentation for a wide variety of flow cytometry technologies. The core is equipped with six analytical instruments: a state-of-the-art 5-laser/29-detector BD FACSymphony A5 flow cytometer, which is 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 5-laser/20-detector BD LSRFortessa cytometers with identical configuration, one of which also has HTS attachment. The core also operates a 3-laser/10-detecrtor BD FACSCanto II cytometer. The LGI FC37 was a pioneer in introducing spectral flow cytometry to NCI and now offers the highest capacity such instrument in the World, a 5-laser/144 channel Sony ID7000 spectral analyzer with built-in 96/384-well plate attachment. In addition, the Core operates a dual-camera, 4-laser, 12-channel, Amnis ImageStream MarkII imaging flow cytometer equipped with HTS attachment. To support cell sorting the Core operates two BD FACSAria IIu instruments, equipped with 3-4 lasers to simultaneously identify 11-12 fluorochromes. Both of these sorters are enclosed in flexible, BioBubble Benchtop Biocontainment units. The Core also has one 5-laser/20-detector BD FACSAria Fusion cell sorter housed in a custom-designed Class II type A2 biosafety cabinet. In 2021 the Core acquired a Sony MA900 cell sorter with a custom-designed Class II biosafety cabinet from the CCR Single Cell Analysis Facility. These biosafety cabinets 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 Core faced two significant new challenges in 2024. In accordance with a well-planned modernization schedule discussed with CCR leadership in advance, director Ferenc Livak successfully applied for RRS funds to replace our oldest (>20-year-old) cell sorter with a new instrument in May 2023. This request was first delayed in FY23 and then denied of purchase in FY24. Since the manufacturer no longer offers annual service maintenance contract on these types of instruments, we must rely on short-term agreements or may have to purchase individual, higher priced service visits in the future which will cause delays and more expensive operating costs to CCR. At this point, with no decline in cell sorter requests, we cannot afford to retire this instrument without causing significant service disruptions. It should be mentioned that the manufacturer will also no longer offer annual service maintenance contract on our other FACSAria IIu cell sorter (15-years of age) from 2025 and we will face the same critical issue from next year if we are not able to replace that cell sorter. In May 2024, staff member Ms. Raghad Almofeez left the Core to pursue graduate studies at the University of Virginia. We immediately requested a replacement, but we have not been allowed to refill this position and the request was deferred until at least late 2025. Staff members at the Core primarily operate individual cell sorters. As our schedule involves requests for assisted cell sorting on 3-4 cell sorters every day, we are not able to utilize all cell sorters to full capacity with reduced staffing. To alleviate the problem, we have opted to offer more training on independent cell sorter use and restrict use of two cell sorters for trained users. It should be noted that this is not optimal for most of our clients, as independent operation of cell sorters is a complex, time-consuming job and most users prefer the sorts to be assisted by staff while they perform other tasks. Transferring the responsibilities of our essential duties to users, due to staff shortage, defies major mission-critical functions of the Core and places additional burden on laboratory personnel of CCR investigators. In FY24 the Core served 222 users from 79 CCR laboratories as well as from 4 additional laboratories of other NIH institutes. Major areas of supported research include better understanding of lymphocyte development and activation at the Bhandoola lab [LGI], Bosselut lab [LICB] and Samelson lab (LCMB]), tumor-associated immune responses (Gilbert and Zhuang labs [NOB]), developing novel models of breast cancer (Wakefield lab [LCBG]) and lung cancer (Thomas lab [LCBG]), and testing new tumor diagnostic approaches (Khan lab [Genetics] and anti-tumor biologics (Ho lab [LMB]). The LGI FC37 provides support in imaging flow cytometry technology by offering the only Amnis ImageStream instrument on campus to the study of cancer stem cells (Yang lab [LCBG]), phagocytosis assays (Appella lab [LCB]) and analysis of DNA damage response in neurons (Nussenzweig lab [LGI]). The LGI FC37 has trained 39 new users on analytical instruments and has also trained two users for cell sorting in FY24. New users are instructed to review instrument and software training tutorials before attending a 4-hour hands-on training session that includes startup and shut down procedures for the cytometers, setting up experiments, basic instrument troubleshooting, and data analysis. Staff member Karen Wolcott conducts a fully Webex-based remote training schedule and also provide follow-up support and periodic assistance to inexperienced users. This is a particularly essential work now with the on-site staff shortage. Cell sorter training is now offered to more users in order to alleviate scheduling pressure due to staff shortage (see above). The LGI FC37 has designed and maintained policies to ensure safe, fair and equitable access of Core resources to all registered users. Despite the challenges due to hiring and purchasing freeze, the LGI FC37 remains committed to be a leader of flow cytometry services at CCR by offering the highest quality, reliable support to the largest number of NCI investigators on campus The LGI FC37 is dedicated to the introduction of innovative flow cytometry-based technologies to further advance the cutting-edge cancer research conducted at NIH.
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