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 with active contributions from director Ferenc Livak and staff members, Karen W. Wolcott and Caiyi Li. The Core also works together with other NCI Flow Cytometry cores and facilities to take advantage of a shared bioinformatics expert who was hired and started in FY22 to support high dimensional flow cytometry data analysis at NCI. Director Ferenc Livak and staff member Shafiuddin Siddiqui together wrote a chapter on design and execution of high dimensional flow cytometry for the upcoming edition of Methods in Molecular Biology, Vol 2580, T-Cell Development section. This chapter offers users a step-by-step guide in planning and setting up successful high dimensional flow cytometry analytical experiments. 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 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 is also equipped with HTS attachment. The core also has a three-laser, digital BD FACSCanto II cytometer. The LGI FC37 has been a pioneer in introducing the novel technology of spectral flow cytometry to NCI and currently operates a 4-laser/32-channel Sony SA3800 and a 5-laser/144 channel Sony ID7000 spectral analyzer. It is anticipated that the ID7000 instrument will fully replace the smaller and older SA3800 analyzer by the end of FY22. In addition, the Core operates a dual-camera, 4-laser 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 operates 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. The old Beckman Coulter MoFlo Astrios EQ instrument has been replaced with a newer, much more user-friendly Sony MA900 cell sorter, housed in a custom-designed Class II biosafety cabinet. The 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. In FY22 utilization of the Core began to return to pre-pandemic levels. The new Sony ID7000 spectral analyzer has been installed and with staff member Karen Wolcott's expert training exercises, it has now almost completely replaced the older Sony SA3800 analyzer. Ferenc Livak and Karen Wolcott working with James Galentine of NCI Office of Space and Facilities Management, developed a detailed plan to expand and renovate the Core's physical space in BG37. These plans have been approved, however work has not begun yet. It is hoped that this renovation can proceed urgently as the Core has continuously expanded its instrumentation repertoire and user base in the past years and now has reached the limits of its physical space. 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 FY22 the LGI FC7 purchased and installed a new online calendar and tracking software, iLab from Agilent Technologies. This software replaces the suddenly discontinued LabShare system. After several weeks of intense training and user support, the Core has successfully switched to this modern and more versatile online scheduling platform which will hopefully provide our more than 200 users with more robust and reliable online reservation mechanism than the previous LabShare-based system did. The LGI FC37 served 286 users from 85 CCR laboratories as well as from 3 additional laboratories of three other NIH institutes in FY22. 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 (Ashwell, Samelson labs (LICB], thymus epithelium development (Bhandoola lab [LGI]) and tumor-associated immune responses (Goldszmid lab [CIP], Gilbert and Zhuang labs [NOB]). 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 and Lazzerining-Denchi labs [LGI], Bosselut lab [LICB], Casellas lab, [NIAMS/NCI]). 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]), thymic selection and innate-like cell development (Takahama and Lazarovic labs [EIB]), advanced AI-assisted image-based T-cell characterization (Jiang lab [CDSL]) and DNA damage response (Nussenzweig lab [LGI]). The LGI FC37 has trained 70 new users in FY22 on analytical instruments and has also trained three 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 experiments, basic instrument troubleshooting, and data analysis. Using a completely Webex-based remote training schedule allows the Core to offer training while observing the NIH guidelines on physical distancing. Core staff also provides follow-up assistance and helps new users develop confidence in using the technology correctly. Cell sorter training is offered to select users who need frequent, possibly after hour sorting time. The LGI FC37 has designed policies to ensure safe, fair and equitable access of Core resources to all registered users. Despite recent challenges associated with the immediate aftermath of the COVID-19 pandemic, 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.