CCR, LGI, Flow Cytometry Core

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

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 seven analytical instruments: a state-of-the-art BD FACSymphony A5 flow cytometer equipped with five lasers and the capacity to identify 27 fluorochromes, 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 BD LSRFortessa cytometers with identical configuration, equipped with five lasers and the capacity to identify 18 fluorochromes. One of these instruments, LSRFortessa SORP1 also has HTS attachment. The core also operates a three-laser, 8-color BD FACSCanto II cytometer. The LGI FC37 was a pioneer in introducing spectral flow cytometry to NCI with the purchase of a 4-laser/32-channel Sony SA3800 analyzer in 2016. Later we purchased a 5-laser/144 channel Sony ID7000 spectral analyzer. At the end of last year we retired the SA3800 instrument, once the ID7000 instrument has become a major spectral analyzer of the Core during FY23. 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 BD FACSAria Fusion cell sorter housed in a custom-designed Class II type A2 biosafety cabinet which is equipped with five lasers with the capacity to identify 18 fluorochromes. In 2021 the Core acquired a Sony MA900 cell sorter, housed in a custom-designed Class II biosafety cabinet. 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. Ferenc Livak and Karen Wolcott continued working with James Galentine of NCI Office of Space and Facilities Management, to oversee the renovation and expansion of the Core in BG37. The construction has now reached its final phase and the Core is expected to move into the finished, expanded space on August 28-29, 2023, pending approval. The new, expanded space will allow users and staff to maintain minimum 6-foot physical distancing and would give the Core the opportunity to introduce another, high-end cell sorter in the coming years in a separate lab space that can continue to operate even in case of future emergency situations. In March 2023, staff member Dr. Caiyi Li announced her desire to leave the Core for a more benchwork-oriented job at another NCI branch. We immediately initiated a search to find a replacement for Dr. Li. After exploring possible candidates within NIH, the search was expanded nationwide. A total of 12 candidates were interviewed either online, or in some cases in-person. All of these interviews involved the entire staff of FC37 and assessments from all staff members were collected. Finally, Ms. Raghad Almofeez, a recent masters graduate from George Mason University was offered the position based on her personal qualities and hands-on experience in analytical flow cytometry. Ms. Almofeez began to work as a staff member at the Core in July 31, 2023. We have high hopes that she will be soon a fully competent, valuable member of our Core service. In FY23 utilization of the LGI FC37 returned to pre-pandemic levels. The Core served 203 users from 79 CCR laboratories as well as from 2 additional laboratories of two other NIH institutes in FY23. Many of these laboratories heavily depend on the services of the Core in conducting high-dimensional analyses aimed at better understanding of lymphocyte development and activation (Bosselut lab [LICB] and Samelson lab (LCMB]), thymus epithelium development (Bhandoola lab [LGI]) and tumor-associated immune responses (Gilbert and Zhuang labs [NOB]). Flow cytometry analyses are used to develop novel immunoassays and immunotherapeutic reagents (Ashwell lab [LICB], Ho and Pastan labs [LMB]). Cell sorting helps with the establishment and maintenance of fluorescent protein reporter-based models (Ruiz Macias lab [LGI] and Wakefield lab [LCBG]). Single cell sorting helps the rapid generation of CRISPR-induced mutant cell lines and is becoming an essential tool in single cell genomic analyses and CRSIPR library screening (Nussenzweig, Bhandoola and Lazzerining-Denchi labs [LGI], Bosselut lab [LICB]). The LGI FC37 provides unique support in imaging flow cytometry technology by offering the only Amnis ImageStream instrument on campus to the study of lymphoma drug targeting (Staudt lab [LMBr]), cancer stem cell studies (Yang lab [LCBG]), phagocytosis assays (Appella lab [LCB]) and DNA damage response (Nussenzweig lab [LGI]). The LGI FC37 has trained 57 new users in FY23 on analytical instruments and has also trained one new user for cell sorting. 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 introduced and continued with a fully Webex-based remote training schedule. She is also using Webex to provide follow-up support and periodic assistance to inexperienced users which limits the need for the rest of staff to on-site handling of occasional instrument malfunctions. Cell sorter training is offered to select users who need frequent, possibly after hour sorting time. The LGI FC37 has designed and maintained policies to ensure safe, fair and equitable access of Core resources to all registered users. Despite recent challenges associated with the ongoing renovations and move of the Core, 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.