PROJECT SUMMARY: FLOW CYTOMETRY SHARED RESOURCE The Sylvester Comprehensive Cancer Center (Sylvester) Flow Cytometry Shared Resource (FCSR) offers comprehensive, readily accessible, cost-effective, state-of-the-art analytical flow cytometry and cell sorting services that are critical to the success of a wide range of cancer research, including complex immunophenotyping, DNA content and cell cycle analysis, cell signaling and pathway activation analysis, and measurement of proliferation and apoptosis. The aims of the FCSR are to 1) provide state-of-the-art instrumentation and high-level expertise for fluorescence-activated cell sorting (FACS), standard and spectral flow cytometry, and mass cytometry and imaging mass cytometry (IMC); 2) develop, optimize and offer existing and emerging cytometric methods; and 3) provide training in all aspects of cytometry. The facility has nine major research instruments (four cell analyzers, three cell sorters, and two mass cytometry platforms) that facilitate studies of normal and cancer cells. The FCSR works closely with other Shared Resources to support genomics and spatial multiomics research. FCSR cell sorters can sort based on up to 17 fluorescence markers, into bulk tubes or multi-well plates for single-cell cloning under BSL-2 conditions. The FCSR also offers expertise and access to spectral cytometric analysis of up to 40 fluorescent parameters for both cell surface and intracellular antigen detection in complex mixtures of cancer cells and adjacent normal tissue. Moreover, the FCSR provides CyTOF mass cytometry and IMC services (with instruments funded by a competitive grant award from the State of Florida), with the capability of multiplexing up to 50 markers simultaneously in single-cell suspensions or in tissue sections at sub-cellular resolution for spatial analysis of the tumor microenvironment. In alignment with the 2019-2023 Sylvester Strategic Plan, Sylvester significantly strengthened the FCSR during the current reporting period (6/1/2018-5/31/2023) by investing in equipment (adding five major research instruments) and personnel (including a new Director). FCSR staff perform mass cytometry and cell sorting, assist with spectral and standard flow analysis, and regularly perform maintenance and QA/QC assessment on all instruments. They also provide training on all aspects of cytometry experiments, including experimental and panel design, hands-on training for analyzer instruments, self- operated sorting, data acquisition, and data analysis. During the reporting period, the FCSR supported 84 Sylvester members who comprised 84% of total usage by revenue, reflecting the critical importance of FCSR services to members. The FCSR supported 73 publications (48% were in high-impact journals (JIF≥10)) and facilitated 48 cancer-related grants awarded to members, including 14 NCI awards. Future plans include implementing technologies to expand multiomics support with spatial genomics service; improving cell...