CCR, LGI, Flow Cytometry Core

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

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 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, 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 also equipped with HTS attachment. To support cell sorting the Core operates two BD FACSAria IIu instruments, equipped with 3-4 lasers 11-12 fluorescent detectors. 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 continued to face significant challenges in 2025. Despite the urgent need of at least one new cell sorter, for the purchase of which director Ferenc Livak successfully applied for RRS funds in May 2023, this purchase has not been executed. Since the manufacturer, BD Biosciences no longer offers annual, or even short-term service maintenance contracts on this model, we are operating the two FACSAria IIu cell sorters without dedicated service contract. As a result, when one of the sorters broke down recently, we have not been able to repair it, and we effectively lost 25% of our cell sorting capacity. Given that the other FACSAria IIu cell sorter is even older, it is very likely that within the next year that sorter will also break down depriving the largest CCR flow cytometry service at NCI half of its cell sorting capacity. In addition, since May 2024, the Core operates with one unfilled staff position after the departure of a contractor, Ms. Raghad Almofeez. Although we requested a replacement, we have not been allowed to refill this position for the past more than a year. Staff members at the Core primarily operate individual cell sorters. Initially we opted to offer more training on independent cell sorter use and restrict use of two cell sorters for trained users. Since the breakdown of one of the cell sorters, some of this restriction has become irrelevant. However, it should be noted that 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. While the recently reduced instrument capacity "compensates' for the chronically reduced staffing, it is obviously not an optimal solution, it denies major mission-critical functions of the Core and places additional burden on laboratory personnel of CCR investigators. In FY25 the Core served 223 users from 73 CCR laboratories as well as from 2 additional laboratories of other NIH institutes. This utilization is comparable to last year's utilization, although we have noted small decline in usage in recent months, in part due to closure of some CCR labs (Pastan, Ashwell labs). Major users of the Core, however, continue to perform flow cytometry at the Core, including the Nussenzweig and Bhandoola labs (LGI), Bosselut lab (LICB), Ho lab (LMB), Lebensohn lab (LCMB) and Khan lab (Genetics). New users, such as Thomas lab (LCBG) significantly increased reservations for cell sorters or the Fuji lab (WMB) for the spectral analyzer. The LGI FC37 remains the only provider of imaging flow cytometry technology on campus supporting experiments for the Yang lab (LCBG]), Appella lab (LCB), Nussenzweig lab (LGI) and Staudt lab (LMBr). The LGI FC37 has trained 52 new users on analytical instruments and has also trained 3 experienced users for cell sorting in FY25. 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. Cell sorter training is now offered to more users and is performed on site as multi-day hands-on training. 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. However, accomplishing these goals is potentially at risk if chronic staff shortage and loss of instrument support continue to interfere with our work.